Plant development is a multiphasic process, with new organ initiation and elaboration occurring throughout the life cycle. According to the traditional view point in plant developmental biology, development is inextricably coupled with growth.

Growth is the irreversible change in the size of cells and plant organs due to both cell division and cell expansion. Plant development is punctuated by physiological transitions, such as seed maturation, dormancy and germination separating embryogenesis from vegetative development. Moreover, flowering distinguishes vegetative growth from reproductive growth.

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. During the morphogenesis phase, the basic body plan of the plant is established with the specification of the shoot-root axis and the formation of the embryonic organ and tissue systems. A seed containing a full-size embryo undergoes maturation. Major characteristics of the maturation phase include the arrest of embryo morphogenesis

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I would like to express my sincerest gratitude to my supervisor, Prof. Wen-hui SHEN for his rigorous training, heartful support for my research in his lab, for his guidance, suggestions, and encouragement, as well as for his help throughout my studies and composition of this thesis. I will benefit for the rest of my life as an independent and creative scientist from the logical reasoning, critical reading and interest-driven attitude I learned from him. I also would like to express my appreciation to Dr.

Alexandre BERR for his care and help in my daily life here at Strasbourg. I am especially grateful to Dr. Donghong CHEN for teaching me experiments containing the methods and how to think and control the direction of my research. At the same time, he gave me advice and suggestions for my life when I first came here.

Moreover, I would like to show my sincere appreciation to Prof. Léon OTTEN for kindly helping me.

Needless to say, it was difficult for me to survive in France due to my poor knowledge of French. Many more persons helped me in various ways during my study here.

Thanks to all of them. I am deeply grateful to my parents, for their greatest love, abiding support, encouragement, and understanding. I am also grateful to the China Scholarship Council (CSC) for funding my PhD program. and activate downstream genes involved in the accumulation of storage proteins and lipids [START_REF] Ikeda | Embryogenesis-related genes; Its expression and roles during somatic and zygotic embryogenesis in carrot and Arabidopsis[END_REF]. Plants ectopically expressing LEC2 accumulate seed proteins and lipids in vegetative and reproductive tissues, and trigger somatic embryo formation [START_REF] Lotan | Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells[END_REF][START_REF] Stone | LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[END_REF]. All four abi3, lec1, lec2 and fus3 mutants are severely affected in seed maturation and share some common phenotypes, such as decreased dormancy at maturation and reduced expression of seed storage proteins [START_REF] Gutierrez | Combined networks regulating seed maturation[END_REF].

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A dormant seed does not have the capacity to germinate in a specified period of time under any combination of normal physical environmental factors that are otherwise favourable to its germination. Thus, the transition of the seed from dormancy to germination is a critical step in the life cycle of plants. Dormancy is a complex trait that is controlled by a large number of genes which are affected by both developmental and environmental factors. It is known that the relative levels of plant hormones control seed dormancy and germination. Several studies have shown that ethylene, gibberellic acid and brassinosteroids promote the germination of dormant seeds, but there is now a general agreement that abscisic acid (ABA) is the primary mediator of seed dormancy [START_REF] Koornneef | Seed dormancy and germination[END_REF]. Moreover, other mechanisms, which might be independent of hormones or specific to the seed dormancy pathway, are also emerging from genetic analysis of "seed dormancy mutants".

Seed dormancy is induced during the seed maturation phase simultaneously with the accumulation of storage compounds, the acquisition of desiccation tolerance and, finally, the quiescence of metabolic activity. Thus, seed dormancy is controlled by four major seed maturation regulators: ABI3, LEC1, LEC2 and FUS3. In addition, DELAY OF GERMINATION 1 (DOG1) is a key player specific for the induction of seed dormancy in Arabidopsis [START_REF] Bentsink | Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis[END_REF]. Loss-of-function mutant alleles of DOG1 are completely nondormant and do not show any other phenotypes [START_REF] Bentsink | Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis[END_REF].

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A meristem is a tissue containing undifferentiated cells (meristematic cells) which give rise to various organs of the plant and keep the plant growing. Plants possess different types of meristems that control both primary and secondary organ growth. Both roots and shoots have meristematic tissues at their tips. These tissues are called apical meristems and are responsible for the lengthening of roots and shoots.

The shoot apical meristem (SAM) comprises a small, dome-shaped population of undifferentiated cells, which is formed during embryonic development and after seed germination gives rise to the stem, leaves, and flowers. The root apical meristem (RAM) is also formed during embryo development, but after seed germination it gives rise to the root system. In order to fulfill these functions, the meristem must maintain a balance between the self-renewal of a reservoir of central stem cells and organ initiation from peripheral cells. Throughout the life of the plant, the rate of cell division and cell elongation in the meristems is regulated by complex, overlapping signaling networks that include the feedback regulation of meristem maintenance genes and the signaling of plant hormones.

I.1.3.1 Shoot meristem maintenance by WUS-CLV feedback loop

The shoot meristem is composed of three zones exhibiting different functions.

The central zone (CZ) at the tip of SAM contains the slowly dividing stem cells, which are necessary for the indeterminate growth and development of the plant. The peripheral zone (PZ) surrounds CZ and ultimately gives rise to lateral organs. The rib meristem (RM) is located beneath CZ; division and elongation of rib meristem cells give rise to the stem of the plant. The organizing center (OC) residing in RM acts as the stem cell niche; it specifically expresses the homeodomain transcription factor WUSCHEL (WUS) [START_REF] Mayer | Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem[END_REF]. WUS is both necessary and sufficient for stem cell specification [START_REF] Laux | The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis[END_REF]. Mutations in WUS result in the mis-specification of stem cells and premature termination of the shoot. Thus, restriction of WUS transcription to cells of the OC is critical for maintaining a constant number of stem cells, and this is mediated by the CLAVATA (CLV) signaling pathway [START_REF] Brand | Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity[END_REF][START_REF] Fletcher | Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems[END_REF][START_REF] Mayer | Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem[END_REF].

In the WUS-CLV pathway, the expression of WUS is controlled by the three CLV genes (CLV1, CLV2, and CLV3) that act together in a signal transduction pathway and restrict stem cell fate [START_REF] Brand | Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity[END_REF]. CLV3 encodes a putative signalling peptide and is expressed in the CZ cells. CLV1 represents a putative receptor kinase [START_REF] Clark | The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis[END_REF], and CLV2 is a presumed accessory protein of the signalling complex which lacks the kinase domain and contributes to the stability of CLV1 [START_REF] Jeong | The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase[END_REF]. The emerging mechanism comprises three steps: firstly, CLV3 protein is secreted from the CZ cells into the extracellular space; secondly, it acts as a signaling molecule that binds to and activates a heterodimeric receptor complex containing CLV1 and CLV2; lastly, the activated complex restricts the expression of WUS to a small domain in the deeper regions of the meristem, the OC [START_REF] Brand | Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity[END_REF]. As a consequence, wus mutants lack the meristem, and loss of CLV1, CLV2, or CLV3 activity leads to an accumulation of meristem cells and to a gradual increase in size of the shoot meristem dome [START_REF] Fletcher | Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems[END_REF]. Moreover, in this pathway, WUS not only specifies stem cell fate in overlaying cells of the CZ, it also activates its own negative regulator CLV3 by binding to the genomic regions of CLV3 to activate its transcription [START_REF] Yadav | WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex[END_REF]. Thus, the WUS-CLV feedback system forms a self-correcting mechanism for maintaining a constant number of stem cells and the SAM size.

I.1.3.2 Shoot meristem maintenance by KNOX transcription factors

In parallel to the WUS-CLV signaling pathway, equally essential for SAM maintenance is the Class-I KNOTTED1-like homeobox (KNOX) genes, which encode homeodomain transcriptional regulators, including BREVIPEDICELLUS (BP)/KNAT1, KNAT2, KNTA6 and SHOOT MERISTEMLESS (STM). They have been identified to play an essential role in the development and to be complementary to WUS in the maintenance of the stem cell niche in the SAM [START_REF] Endrizzi | The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE[END_REF][START_REF] Hake | The role of knox genes in plant development[END_REF][START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF][START_REF] Tsuda | Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice[END_REF]. Among these genes, STM is required for both the establishment and maintenance of SAM and is expressed throughout the SAM, but not in lateral organ primordia. BP/KNAT1, KNAT2, and KNAT6 are also specifically expressed in SAM and have partially redundant roles with STM in SAM maintenance (reviewed in [START_REF] Scofield | KNOX gene function in plant stem cell niches[END_REF]. Loss-of-function stm weak mutants show meristem defects in maintaining SAM organisation, and the stm strong mutants totally fail to establish the SAM during embryogenesis. Moreover, overexpression of STM can lead to the formation of ectopic meristems [START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF][START_REF] Scofield | STM sustains stem cell function in the Arabidopsis shoot apical meristem and controls KNOX gene expression independently of the transcriptional repressor AS1[END_REF]. In contrast, mutations in KNAT1/BP, KNAT2 or KNAT6 alone do not obviously a ect shoot meristem development or function [START_REF] Byrne | ASYMMETRIC LEAVES1 reveals knox gene redundancy in Arabidopsis[END_REF]Douglas et al., 2002[START_REF] Venglat | The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis[END_REF][START_REF] Dean | KNAT6 gene of Arabidopsis is expressed in roots and is required for correct lateral root formation[END_REF]. KNAT1/BP and KNAT2 expression levels are increased by STM induction while in STM-RNAi lines, KNAT1/BP and KNAT2 are down-regulated. But overexpression of neither KNAT1/BP nor KNAT2 causes an increase in STM mRNA [START_REF] Gallois | Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis[END_REF][START_REF] Lenhard | The WUSCHEL and SHOOT MERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation[END_REF][START_REF] Scofield | The Arabidopsis homeobox gene SHOOT MERISTEMLESS has cellular and meristemorganisational roles with differential requirements for cytokinin and CYCD3 activity[END_REF]. This indicates that STM can regulate BP and KNAT2, but BP and KNAT2 have no action in the regulation of STM. Moreover, STM plays a major role in maintaining shoot meristems. BP regulates internode development (Douglas et al., 2002;[START_REF] Venglat | The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis[END_REF] and contributes, together with STM, to SAM maintenance [START_REF] Byrne | ASYMMETRIC LEAVES1 reveals knox gene redundancy in Arabidopsis[END_REF]. In addition, KNAT6 function is integrated in a network comprising STM and the CUC genes to regulate organ separation and to maintain the SAM. KNAT2, the member closest to KNAT6, did not display such a role [START_REF] Belles-Boix | KNAT6: an Arabidopsis homeobox gene involved in meristem activity and organ separation[END_REF].

I.1.3.3 Phytohormones in shoot meristem development

Many studies showed that phytohormones and transcription factors cooperate to balance meristem maintenance and organ formation (Figure I.1). KNOX transcription factors promote meristem function partly through repression of biosynthesis of gibberellin (GA) [START_REF] Chen | The tandem complex of BEL and KNOX partners is required for transcriptional repression of ga20ox1[END_REF][START_REF] Hay | The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans[END_REF][START_REF] Sakamoto | KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem[END_REF]. They target and repress the transcription of genes encoding GA20 oxidase enzymes, which are required for GA biosynthesis [START_REF] Chen | The tandem complex of BEL and KNOX partners is required for transcriptional repression of ga20ox1[END_REF][START_REF] Sakamoto | KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem[END_REF]. Moreover, GA reduction enhances phenotypes associated with KNOX overexpression. However, KNOX-mediated repression of GA biosynthesis would not be sufficient to maintain reduced GA levels. Thus, there is a synergistic action with another pathway by which KNOX proteins activate transcription of GA2 oxidase genes, which encode GA catabolic enzymes, at the leaf-meristem boundary [START_REF] Hay | The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans[END_REF][START_REF] Sakamoto | KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem[END_REF]. Altering GA levels is not sufficient to rescue KNOX mutant phenotypes [START_REF] Hay | The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans[END_REF], indicating that KNOX proteins control additional processes in the SAM. Indeed, STM induces cytokinin (CK) synthesis to inhibit cellular differentiation; in addition it functions to organize undifferentiated cells into a selfsustaining meristem [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF][START_REF] Yanai | Arabidopsis KNOXI proteins activate cytokinin biosynthesis[END_REF] (Figure I.1). In CK biosynthesis pathway, adenosine phosphate-isopentenyltransferases (IPTs) catalyze the transfer of an isopentenyl group from dimethylallyl diphosphate to an adenine nucleotide (ATP, ADP, or AMP) [START_REF] Kakimoto | Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases[END_REF][START_REF] Takei | Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana[END_REF]. STM promotes induction of IPT gene expression [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF][START_REF] Yanai | Arabidopsis KNOXI proteins activate cytokinin biosynthesis[END_REF].

Conversely, expression of IPT under control of the STM promoter can partially rescue some traits of the stm mutant phenotypes [START_REF] Yanai | Arabidopsis KNOXI proteins activate cytokinin biosynthesis[END_REF]. Furthermore, CK triggers a rapid increase in mRNA levels of the KNOX genes [START_REF] Rupp | Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem[END_REF]. It appears that a positive feedback loop exists between STM and CK signalling to coordinately control SAM activity. CK also stimulates the expression of genes involved in GA catabolism to reinforce the low GA levels established by the KNOX proteins within the SAM [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF]Wolters and Jürgens, 2009).

In addition, CK signaling has also been associated with the WUS-CLV pathway (Figure 1). CK acts as a downstream signaling network of the WUS-CLV feedback loop. In Arabidopsis, CK is perceived by a multi-step phosphorelay pathway.

Three transmembrane histidine kinases have been identified as CK receptors: they are the ARABIDOPSIS HIS KINASE 2 (AHK2), AHK3, and CYTOKININ RESPONSE1 (CRE1)/AHK4 [START_REF] Inoue | Identification of CRE1 as a cytokinin receptor from Arabidopsis[END_REF][START_REF] Riefler | Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism[END_REF][START_REF] Su | Auxin-cytokinin interaction regulates meristem development[END_REF].

Upon CK perception, AHKs could activate the ARABIDOPSIS RESPONSE REGULATOR (ARR) proteins, for example ARR7/15, through the phosphorelay system [START_REF] Hwang | Cytokinin signaling networks[END_REF]. The WUS-CLV feedback loop interacts with CK through perceiving CK signalling to positively regulate the shoot meristem. Type-A ARR7 and ARR15 have been validated as negative regulators of CK signalling (To et al., 2004;[START_REF] To | Cytokinin signaling: two-components and more[END_REF], and are required for CLV3 expression [START_REF] Zhao | Hormonal control of the shoot stem-cell niche[END_REF].

Ectopic expression of WUS represses the negative A-type ARRs [START_REF] Leibfried | WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators[END_REF]. Moreover, overexpression of an A-type ARR inhibits WUS expression and can mimic the wus mutant phenotype. It thus appears that CK not only maintains shoot meristem function, but is also involved in regulating the size of the stem cells.

Auxin also plays a critical role in the maintenance of the shoot meristem. There is much evidence for an extensive cross-talk between auxin and cytokinin during shoot meristem development [START_REF] Cheng | Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by AUXIN RESPONSE FACTOR3[END_REF][START_REF] Su | Auxin-cytokinin interaction regulates meristem development[END_REF].

YUCCA genes encode key enzymes which catalyse a rate-limiting step of auxin biosynthesis [START_REF] Cheng | Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis[END_REF]. In yucca mutants with reduced auxin levels, the expression levels of ARR7 and ARR15 are dramatically increased in SAM. Similar results were observed in pin1 mutants [START_REF] Zhao | Hormonal control of the shoot stem-cell niche[END_REF]. These observations suggest that ARR7 and ARR15 activation can be directly induced by the loss of local auxin accumulation. It is likely that auxin and CK signalling converge on shoot meristem function regulation by controlling A-type ARR activity.

Figure I.1. Participation of hormones in meristem identity

This promotion is reinforced by at least one member of the KNOX family. Gibberellin (GA) function is repressed at the meristem by CK and KNOX by inhibition of GA biosynthesis. On the other hand, emerging primordia experience an increase in GA activity because the product of the AS1 gene and auxin repress the expression of the KNOX gene that inhibits GA biosynthesis. Beige colour represents nascent primordia. (From Alabadi et al., 2009).

I.1.3.4 SHR/SCR pathway in maintenance of the root stem cell niche

The root is composed of three main regions: the meristematic zone (MZ), the elongation zone (EZ), and the differentiation zone (DZ). The quiescent center (QC) cells (four in Arabidopsis thaliana), whose function resembles those of the OC in the shoot meristem, are located in the region at the tip of MZ. QC is essential for the maintenance of the stem cell fate of the surrounding cells (van den [START_REF] Van Den Berg | Short-range control of cell differentiation in the Arabidopsis root meristem[END_REF]. QC, together with the surrounding stem cells, constitute the root stem cell niche (SCN) which provides the source of cells for the formation of all root tissues [START_REF] Dinneny | Plant stem cell niches: standing the test of time[END_REF][START_REF] Van Den Berg | Cell fate in the Arabidopsis root meristem determined by directional signalling[END_REF].

SHORT ROOT (SHR) and SCARECROW (SCR), encode members of the GRAS family of transcription factors, and they play essential roles in QC establishment and stem cell maintenance [START_REF] Helariutta | The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling[END_REF][START_REF] Sabatini | SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem[END_REF]. The SHR protein is expressed in the stele, but moves out of the stele into the endodermis and QC, where it upregulates SCR [START_REF] Nakajima | Intercellular movement of the putative transcription factor SHR in root patterning[END_REF]. The expression of SCR in the QC was shown to be both necessary and sufficient for the specification of the QC and the maintenance of the stem cells [START_REF] Sabatini | SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem[END_REF].

However, the expression of SCR in the QC region could not rescue the root meristem defects of SHR mutant seedlings. Moreover, SCR has a role for restricting SHR movement. Disruption of either SHR or SCR expression results in the formation of a short root that fails to maintain the QC and meristem [START_REF] Helariutta | The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling[END_REF][START_REF] Lucas | Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis[END_REF][START_REF] Sabatini | SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem[END_REF].

I.1.3.5 WOX5-IAA17 feedback circuit in root development

The homeobox gene WUSCHEL-RELATED HOMEOBOX 5 (WOX5), a homologue of WUS, is a major regulator of the root stem cell activity. WOX5 is expressed exclusively in the QC cells and is required for maintenance of distal stem cell (DSC) fate [START_REF] Sarkar | Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers[END_REF]. In the wox5 mutant, root tips show more DSC differentiation, and overexpression of WOX5 inhibited DSC differentiation [START_REF] Ding | Auxin regulates distal stem cell differentiation in Arabidopsis roots[END_REF]. Auxin and WOX5 have opposite effects on DSC activity. Both exogenous auxin application and stimulation of auxin biosynthesis enhance DSC differentiation. This indicates that auxin acts as a positive signal for the differentiation of DSC. Genetic experiments suggest that auxin enhances DSC differentiation through downstream transcriptional repression of the WOX5 homeobox regulator of stem cell activity [START_REF] Ding | Auxin regulates distal stem cell differentiation in Arabidopsis roots[END_REF].

Auxin signaling requires IAA17/AUXIN RESISTANT3 (AXR3) as well as auxin response factors (ARF10 and ARF16). Both ARF10 and ARF16 negatively regulate WOX5 transcription and restrict WOX5 transcripts to the QC center [START_REF] Ding | Auxin regulates distal stem cell differentiation in Arabidopsis roots[END_REF]. Moreover, WOX5 modulates free auxin production and restricts its own expression via IAA17-dependent feedback regulation (Tian et al., 2014). The WOX5-IAA17 feedback circuit assures the maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of DSC populations.

I.1.3.6 Auxin and PLT in maintenance of root meristem

PLETHORA1 (PLT1) and PLT2 genes, which encode members of the AP2 class of transcription factors, are essential for QC and stem cell activity. Accordingly, PLT expression is detected in the stem cell niche [START_REF] Aida | The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche[END_REF][START_REF] Galinha | PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development[END_REF]. PLT proteins have been shown to act in a dosage-dependent manner, high levels of PLT being required to maintain stem cell fates, whereas low PLT activity promotes their differentiation [START_REF] Galinha | PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development[END_REF]. PLT expression is regulated by auxin and is dependent on auxin response factors [START_REF] Aida | The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche[END_REF]). An interaction network of PINs and PLTs functions in controlling auxin-mediated root patterning: PIN proteins restrict PLT expression in the basal embryo region to initiate the root primordium; in turn, PLT genes maintain PIN transcription, which stabilizes the position of the stem cell niche [START_REF] Blilou | The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots[END_REF][START_REF] Dinneny | Plant stem cell niches: standing the test of time[END_REF][START_REF] Grieneisen | Auxin transport is sufficient to generate a maximum and gradient guiding root growth[END_REF].

I.1.3.7 Auxin and cytokinin cross-talk in maintenance of root meristem

A genetic framework has shown that cytokinin and auxin interact antagonistically to control the balance of cell division and differentiation in the root meristem. On the one hand, CK stimulates cell differentiation by suppressing auxin signalling and transport. On the other hand, auxin promotes cell division by inactivating CK signalling [START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF][START_REF] Moubayidin | Cytokinin-auxin crosstalk[END_REF]. During this interaction, CK and auxin regulate the size of root meristem tissue by means of the effect on the expression of SHORT HYPOCOTYL 2 (SHY2/IAA3), a member of the Aux/IAA gene family [START_REF] Tian | Regulation of Arabidopsis SHY2/IAA3 protein turnover[END_REF][START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF], which suppresses the expression of PINFORMED (PIN) auxin transport facilitator genes inducible by auxin.

The mechanism is described as follows: in the transition zone (TZ), CK activates SHY2 transcription factor by means of ARR1, a member of cytokinin signaling regulators, which directly binds to the promoter of SHY2 [START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF].

Then, activation of SHY2 inhibits PIN genes expressed in the TZ, causing the redistribution of auxin for cell differentiation [START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF][START_REF] Moubayidin | Cytokinin-auxin crosstalk[END_REF]. On the other hand, auxin mediates degradation of SHY2 protein and thereby stabilizes PIN expression levels [START_REF] Tian | Regulation of Arabidopsis SHY2/IAA3 protein turnover[END_REF][START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF]. Auxin influences the CK level because SHY2 down-regulates IPT, which is the rate-limiting enzyme in CK biosynthesis [START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF].

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Flowering is a central event in the life cycle of plants, representing the transition from vegetative growth to reproductive development. The process is accompanied by the transformation from SAM into an inflorescence meristem (IM). This transition is a result of responses to various endogenous and exogenous signals that later integrate to result in flowering. In Arabidopsis, flowering time regulation occurs through two main pathways mediating environmental responses (photoperiod pathway and vernalization pathways) and two pathways that function independently of environmental cues: the autonomous pathway, which promotes flowering under all conditions, and the gibberellin (GA) pathway, which is needed for flowering under non-inductive short-day conditions. Additionally, light quality, ambient temperature, and biotic as well as abiotic stresses can also contribute to floral induction in plants [START_REF] Srikanth | Regulation of flowering time: all roads lead to Rome[END_REF].

A number of signals controlling flowering converge in the regulation of the FLOWERING LOCUS C (FLC) gene, which encodes a MADS-box transcription factor and represses flowering through the repression of flowering time integrators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) (Michaels andAmasino, 1999, 2001;[START_REF] Searle | The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis[END_REF]. There are five close homologues of FLC in the Arabidopsis genome, and these are called MADS AFFECTING FLOWERING1 (MAF1) to MAF5 [START_REF] Ratcliffe | Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold[END_REF][START_REF] Ratcliffe | Regulation of flowering in Arabidopsis by an FLC homologue[END_REF]. In the vernalization (long exposure to low temperature) pathway, FLC and MAF1 to MAF4 act as floral repressors and might contribute to the maintenance of a vernalization requirement, while MAF5 may play an opposite role to FLC [START_REF] Ratcliffe | Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold[END_REF]. The autonomous pathway promotes flowering, independently of environmental conditions, by endogenous regulators such as FLD, FVE, FCA and FPA which act to repress the expression of FLC to accelerate flowering (Michaels andAmasino, 1999, 2001;[START_REF] Veley | Functional redundancy and new roles for genes of the autonomous floral-promotion pathway[END_REF]. However, in the photoperiod pathway, long-day (LD) conditions accelerate flowering through the function of FT protein, and gibberellic acid signals play a major role in promoting flowering under short days (SDs) by regulating both LFY and SOC1 expression [START_REF] Lee | The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis[END_REF][START_REF] Moon | The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis[END_REF]

(Figure I.2).
FT protein is a major component of florigen, which is synthesized in the leaf vasculature and moves through the phloem to SAM [START_REF] Corbesier | FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis[END_REF].

Mutations in FT cause a considerable delay in flowering, and overexpression of FT causes precocious flowering. This indicates that FT is necessary and sufficient to accelerate the floral transition [START_REF] Kobayashi | A pair of related genes with antagonistic roles in mediating flowering signals[END_REF]. The activation of FT requires the expression of CONSTANS (CO) and GI, CO encoding the zinc finger transcriptional regulator of the FT promoter [START_REF] Tiwari | The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element[END_REF]. The activity of CO is responsive to light and the circadian clock. CO protein is stable in the light and rapidly degraded in the dark. And GI is a large plant-specific protein involved in circadian clock function [START_REF] Fowler | GIGANTEA: a circadian clockcontrolled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains[END_REF]. However, how CO regulates FT expression remains largely unknown. Recent research indicates that Arabidopsis Morf Related Gene (MRG) group proteins MRG1 and MRG2 interact with CO to activate FT expression in leaves [START_REF] Bu | Regulation of Arabidopsis flowering by the histone mark readers MRG1/2 via interaction with CONSTANS to modulate FT expression[END_REF]. In the SAM, FT, by binding to the transcription factor FD, activates the expression of LFY and AP1, and thereby induces flowering [START_REF] Corbesier | FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis[END_REF][START_REF] Kobayashi | Move on up, it's time for change--mobile signals controlling photoperiod-dependent flowering[END_REF][START_REF] Wigge | FT, a mobile developmental signal in plants[END_REF].

Figure I.2. A simplified schematic shows flowering time gene interactions in

Arabidopsis thaliana [START_REF] Ballerini | Environmental and molecular analysis of the floral transition in the lower eudicot Aquilegia formosa[END_REF] 1317 !$,' +!$%"#) Flowers are the reproductive structures of angiosperms. They are composed of four distinct types of organs: sepals, petals, stamens, and carpels. Floral organs are generated by a flower meristem (FM) [START_REF] Jenik | Regulation of cell proliferation patterns by homeotic genes during Arabidopsis floral development[END_REF][START_REF] Kwiatkowska | Flower primordium formation at the Arabidopsis shoot apex: quantitative analysis of surface geometry and growth[END_REF], which is produced by IM. FMs arise from the main SAM and they are able to transform from one to another meristem [START_REF] Nardmann | The evolution of plant regulatory networks: what Arabidopsis cannot say for itself[END_REF][START_REF] Prunet | Time to stop: flower meristem termination[END_REF]. KNOX homeodomain transcription factors keep meristematic cells in an undifferentiated state, while the WUS-CLV negative feedback loop maintains a constant population of stem cells in the SAM. These genes are expressed in a similar way in the FM compared to the SAM. Thus, during its first developmental stages, FM homeostasis seems to be achieved by roughly the same molecular mechanisms as it is in the SAM [START_REF] Prunet | Time to stop: flower meristem termination[END_REF].

However, the FM also differs from the SAM. The FM growth pattern is determinate; stem cells are only transiently maintained within the FM. At stage 6 of flower development [START_REF] Smyth | Early flower development in Arabidopsis[END_REF], WUS expression is shut off, which results in the disruption of floral stem cell maintenance. That is to say, the activity of the FM stops and floral meristems only form a fixed complement of organs. The differences between the SAM and FMs are determined by meristem identity genes, for example, APETALA1 (AP1) or LEAFY (LFY) [START_REF] Irish | Function of the APETALA1 gene during Arabidopsis floral development[END_REF][START_REF] Schultz | LEAFY, a homeotic gene that regulates inflorescence development in Arabidopsis[END_REF][START_REF] Weigel | LEAFY controls floral meristem identity in Arabidopsis[END_REF].

The MADS-box protein AG, which serves as a key factor in specifying the identities of stamens and carpels [START_REF] Bowman | Genes directing flower development in Arabidopsis[END_REF], plays an essential role in terminating the floral meristem [START_REF] Lenhard | Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS[END_REF][START_REF] Lohmann | A molecular link between stem cell regulation and floral patterning in Arabidopsis[END_REF]. In ag mutant flowers, the expression of WUS and CLV3 is not down-regulated, but is rather continually expressed during the formation of many whorls of floral organs [START_REF] Lenhard | Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS[END_REF]. Moreover, WUS can induce AG expression. Thus, AG and WUS form a negative feedback loop to terminate stem cell activity in flower buds. There are two parallel mechanisms: early in floral development, AG directly represses WUS expression by recruiting Polycomb group (PcG) complexes (Liu et al., 2011); later, AG activates the C2H2 zinc-finger-encoding KNUCKLES (KNU) gene, which in turn directly or indirectly represses WUS expression [START_REF] Sun | A timing mechanism for stem cell maintenance and differentiation in the Arabidopsis floral meristem[END_REF].

In addition to AG-WUS pathway, a number of other genes are also known for their functions in floral meristem regulation. These include ULTRAPETALA1 (ULT1),

SUPERMAN (SUP), CRABS CLAW (CRC).

The ULT1 gene encodes a SAND domain-containing protein, which is a negative regulator of stem cell accumulation in the floral meristem and maintains floral meristem determinacy [START_REF] Carles | ULTRAPETALA1 encodes a SAND domain putative transcriptional regulator that controls shoot and floral meristem activity in Arabidopsis[END_REF]. Loss-of-function of ULT1 results in larger floral meristems with more floral organs than wild-type flowers and a decrease in floral meristem determinacy. Genetic and molecular studies revealed that ULT1 negatively regulates the size of the WUS-expressing domain in the floral meristem. This repression may act upstream of AG and establish the proper floral meristem determinacy, acting through the WUS-AG temporal feedback loop [START_REF] Carles | ULTRAPETALA1 encodes a SAND domain putative transcriptional regulator that controls shoot and floral meristem activity in Arabidopsis[END_REF].

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Chromatin is a highly ordered structure found in cells, consisting of DNA, protein and RNA. The primary protein components of chromatin are histones.

Histones are highly basic proteins, found in the nuclei of eukaryotic cells, which package and order the DNA into structural units named nucleosomes. A nucleosome is the most fundamental unit of chromatin and is composed of roughly 146 bp of DNA wrapped around the histone octamer comprising two molecules each of the four core histones H2A, H2B, H3 and H4 (Luger et al., 1997) (Figure I.3a).

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These core histones are predominantly globular except for the flexible, protruding, highly basic amino-terminal tails (histone H2A and H2B also have a carboxy-terminal tail). These histone tails are essential for the higher-order folding of chromatin fibres, and they also provide binding sites for non-histone regulatory proteins. They are subject to a vast array of post-translational modifications, such as: methylation, phosphorylation, acetylation and ubiquitination (Figure I.3b). In addition, modifications also occur in their globular domains [START_REF] Marks | Histone deacetylases and cancer: causes and therapies[END_REF]. These modifications can occur at many sites and have different biochemical functions, but not all will be on the same histone at the same time. The timing of the appearance of a modification will depend on the signaling conditions within the cell. Modifications on histones are dynamic and rapidly changing. They can appear and disappear on chromatin within minutes following a stimulus arriving at the cell surface. Histone modifications can affect genome function via at least two distinct mechanisms: the first by disrupting contacts between nucleosomes, thereby loosening chromatin structure and promoting transcriptional activity; the second by serving as docking sites for recruiting nonhistone proteins to relevant genomic loci [START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Laugesen | Chromatin repressive complexes in stem cells, development, and cancer[END_REF].

I.2.1.1 Histone methylation/demethylation

Histone methylation is a process by which methyl groups are transferred to amino acids of histone proteins of chromosomes . This reaction is catalyzed by histone methyltransferases (HMTs) which can be classified into three types: the lysinespecific SET domain containing HMTs, the non-SET domain-containing lysine HMTs, and the arginine HMTs. Histones can be methylated on lysine (K) and arginine (R)

residues, but methylation is most commonly observed on lysine residues of the tails of histones H3 and H4. In particular, lysine methylation at H3K4, H3K9, H3K27, H3K36, and H4K20 is mediated by lysine methyltransferases (KMTs) that contain a SET domain. The SET domain was first identified as a shared sequence motif in three Drosophila proteins, suppressor of variegation , enhancer of zeste [E(z)], and homeobox gene regulator trithorax [Trx] [START_REF] Martin | The diverse functions of histone lysine methylation[END_REF]. These lysines can be either mono-(K me1 ), or di-(K me2 ), or tri-methylated (K me3 ). These incremental methylation states can lead to diverse outcomes. According to recent findings, H3K9, H3K27, and H4K20 methylation is associated mainly with repressed transcription, whereas methylation of H3K4 and H3K36 is associated with activated transcription [START_REF] Guenther | A chromatin landmark and transcription initiation at most promoters in human cells[END_REF]. Histone methylation is a process that can be reversed by histone lysine demethylases (KDMs) to eliminate methylation. So far, there are more than 50 human KMTs and 30 KDMs that have been identified [START_REF] Arrowsmith | Epigenetic protein families: a new frontier for drug discovery[END_REF][START_REF] Spannhoff | The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors[END_REF]. KDMs contain two major families: the KDM1 family including KDM1A/LSD1 and KDM1B/LSD2, and the Jumonji C (JmjC) domain-containing protein family including 14 members of KDMs.

KDMs and KMTs work coordinately to maintain normal global histone lysine methylation levels and to regulate gene expression patterns.

I.2.1.2 Histone acetylation/deacetylation

Acetylation was the first histone modification to beidentified. Acetylation and deacetylation of lysine residues on histone 3 and histone 4 at the N-terminal tail have been shown to play a regulatory role in gene activation and repression, respectively. This reversible modification is the result of the fine-tuned balance of the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs).

Histone acetylation is usually carried out by protein complexes involving HATs utilizing acetyl Coenzyme-A (acetyl-CoA) as a cofactor. In histone acetylation, HAT molecules facilitate the transfer of an acetyl group from a molecule of acetyl-CoA to the NH3 + group on Lysine. In histone deacetylation the acetyl group can be transferred back to CoA or to ADP-ribose by NAD-dependent deacetylases [START_REF] Denu | Linking chromatin function with metabolic network: Sir2 family of NAD+-dependent deacetylases[END_REF].

The effect of acetylation is to change the overall charge of the histone tail from positive to neutral, thus decreasing its affinity for DNA. This leads to a change in nucleosomal conformation thereby increasing the accessibility of transcriptional regulatory proteins to the chromatin template. Thus, acetylation of histones is known to increase the expression of genes through transcription activation [START_REF] Fukuda | Simple histone acetylation plays a complex role in the regulation of gene expression[END_REF]. Following deacetylation of the histone tails, the DNA becomes more tightly wrapped around the histone cores, making it more difficult for transcription factors to bind to the DNA. This leads to decreased levels of gene expression and is known as gene silencing.

HATs are classified into two categories based on their subcellular distribution [START_REF] Roth | Histone acetyltransferases[END_REF]. The type A HATs, including the Gcn5-related N-acetyltransferases (GNAT), MYST (MOZ, Ybf2/Sas3, Sas2, Tip60), p300/CBP and basal transcription factors (including TFIID), are responsible for acetylation of nuclear histones and thus are directly involved in regulating chromatin assembly and gene transcription [START_REF] Carrozza | The diverse functions of histone acetyltransferase complexes[END_REF]. Type B HATs contains nuclear receptor cofactors. They act on newly synthesized histones before incorporation. The HDAC family consists of 18 members in humans which are grouped into four classes [START_REF] Gregoretti | Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis[END_REF]

I.2.1.3 Histone phosphorylation/dephosphorylation

Phosphorylation of histones is also highly dynamic. All four core histones have been shown to be phosphorylated, at their N-terminal tails, on specific serine, threonine and tyrosine residues by a number of protein kinases and dephosphorylated by phosphatases [START_REF] Oki | Role of histone phosphorylation in chromatin dynamics and its implications in diseases[END_REF]. Phosphorylation on serine is the most common.

The phosphorylation reaction transfers a phosphate group from ATP to the hydroxyl group of the target amino-acid side chain by histone kinases. This adds a significant negative charge to the histone and influences the chromatin structure.

Histone phosphorylation is correlated with various cell activities, such as mitosis, meiosis, cell death, DNA repair, recombination, replication and transcription.

In these processes, so far only phosphorylation on serine 10 of histone H3 has been linked with transcriptional activation [START_REF] Prigent | Phosphorylation of serine 10 in histone H3, what for?[END_REF]. Moreover, research indicates that it has a role opposite to transcriptional activation. During mitosis, phosphorylation of this serine residue condenses chromosomes. All this suggests that its effect is context-dependent and might be influenced by other histone modifications [START_REF] Johansen | Regulation of chromatin structure by histone H3S10 phosphorylation[END_REF].

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Although an individual histone modification may have its own downstream effectors or specific roles, the development of organisms is a complex network, which usually requires that the various epigenetic marks work together. Cross regulation between different modifications can produce different outcomes: either in a compatible or a mutually exclusive manner, at the single histone tail level or in the context of the nucleosome or even the chromatin level.

Flowering time is the best studied process for crosstalks, with interactions between several kinds of histone modifications. For instance, FLC is a key player for flowering; its expression level is regulated by histone H2B monoubiquitination (H2Bub1) and H3 methylation at the FLC locus. In Arabidopsis, the loss of H2Bub1 on FLC chromatin results in a decrease in H3K4me3 and H3K36me2/3 (Cao et al., 2008;Gu et al., 2009;Schmitz et al., 2009). FLOWERING LOCUS D (FLD) is known to influence histone methylation and acetylation in the autonomous pathway [START_REF] He | Regulation of flowering time by histone acetylation in Arabidopsis[END_REF]Liu et al., 2007;[START_REF] Yu | HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis[END_REF]. Lesions in FLD result in hyperacetylation of histones and a decreased level of H3K27me3 on FLC chromatin [START_REF] He | Regulation of flowering time by histone acetylation in Arabidopsis[END_REF]. In addition, crosstalks between methylated residues also happen in flowering time regulation. Deletion of FLD increases H3K4me3 levels and reduces H3K27me3 levels (Liu et al., 2007;[START_REF] Yu | HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis[END_REF]. And reduction of H3K4me3 in atx1 or sdg25 mutants results in an increase of H3K27me3 on FLC [START_REF] Pien | Arabidopsis TRITHORAX1 dynamically regulates FLOWERING LOCUS C activation via histone 3 lysine 4 trimethylation[END_REF].

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Histone modifications influence almost every process in plant development.

In the earliest phase of plant development, histone modifications are required for establishing the correct body plan during embryogenesis (Köhler and Makarewitch, 2006;[START_REF] Tai | Dynamic histone acetylation of late embryonic genes during seed germination[END_REF]. During later stages of the plant life cycle, histone modifications influence patterning of down-ground or the overground structures [START_REF] Xu | Histone acetylation affects expression of cellular patterning genes in the Arabidopsis root epidermis[END_REF]Xu and Shen, 2008), flowering time (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009) and fertilization (Köhler and Makarewitch, 2006) At the cellular level, there is much evidence that the cell cycle (Fleury et al., 2007;Sanchez et al., 2008), cell division [START_REF] Alatzas | Distribution of ubiquitinated histone H2A during plant cell differentiation in maize root and dedifferentiation in callus culture[END_REF], cell expansion and cell differentiation [START_REF] Shen | Ectopic expression of the NtSET1 histone methyltransferase inhibits cell expansion, and affects cell division and differentiation in tobacco plants[END_REF]Xu and Shen, 2008) are partly regulated by histone modifications. During these regulations, not only different histone modifications cross-talk, but histone modifications are also correlated with the action of most plant hormones.

For instance, the first identified plant PcG protein, CURLY LEAF (CLF), a methyltransferase with specificity for H3K27, is involved in many aspects of development processes. The clf mutant causes pleiotropic effects on leaf and flower morphology as well as on flowering time. CLF controls these processes via repressing AG and STM by H3K27me3 [START_REF] Goodrich | A Polycomb-group gene regulates homeotic gene expression in Arabidopsis[END_REF][START_REF] Schubert | Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27[END_REF]. Moreover, silencing of AG and STM is reflected in reduced enrichment of H3K4me2 [START_REF] Gendrel | Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1[END_REF] In addition, STM has been shown to be a positive factor of cytokinin biosynthesis [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF][START_REF] Yanai | Arabidopsis KNOXI proteins activate cytokinin biosynthesis[END_REF]; therefore silencing STM affects the concentration of cytokinin.

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Histone ubiquitination has a particular interest. This modification is diverse, as it can involve one single ubiquitin molecule (monoubiquitination) or one ubiquitin at multiple sites of the same substrate (multi-monoubiquitination), or chains of ubiquitin (polyubiquitination). The most famous fate of ubiquitinated proteins is their degradation by the 26S proteasome, an ATP-dependent proteolytic machinery that degrades the target protein with concomitant release of the ubiquitin moieties for reuse.

Lysine 48-linked chains on the target proteins serve as a signal which is recognized by specific subunits of the 26S proteasome (Smalle and Viestra, 2004). However, there are other modifications such as polyubiquitination with different linkages (Lysine 63linked chains) or monoubiquitination. Ubiquitination is highly controlled and can be reversed by the action of deubiquitinating enzymes or deubiquitinases [START_REF] Nijman | A genomic and functional inventory of deubiquitinating enzymes[END_REF].

INTRODUCTION

Ubiquitin (Ub) and Ub-like (e.g., SUMO) proteins constitute a family of modifiers that are linked covalently to target pro-teins. Although ubiquitination (also called ubiquitylation or ubiquitinylation) first came to light in the context of protein destruction, it is now clear that ubiquitination can also carry out proteolysis-independent functions. Ubiquitination can alter biochemical, molecular and/or subcellular localization activi-ties of a target protein. The first ubiquitinated protein to be described was histone H2A in calf thymus, a finding dated more than 36 years ago (Goldknopf et al., 1975;Hunt and Dayhoff, 1977). Yet, only more recently have the underlying mechanisms and regulatory functions of histone ubiquitination begun to emerge (reviewed in Zhang, 2003;Shilatifard, 2006;Weake and Workman, 2008;Braun and Madhani, 2012;Pin-der et al., 2013). Histones are highly alkaline proteins, found in the nuclei of eukaryotic cell, which package and order the DNA into structural units named nucleosomes. A nucle-osome is composed of roughly 146 bp of DNA wrapping around the histone octamer comprising two molecules each of the four core histones H2A, H2B, H3, and H4 (Luger et al., 1997). Histone monoubiquitination together with other types of posttranslational modifications, e.g., acetylation, methylation, phosphorylation, and SUMOylation, can modulate nucleo-some/chromatin structure and DNA accessibility and thus regulate diverse DNA-dependent processes, such as genome replication, repair, and transcription (Zhang, 2003;Shilatifard, 2006;Weake and Workman, 2008;Braun and Madhani, 2012;Pinder et al., 2013).

Ubiquitination occurs via conjugation of the C-terminal substrate/acceptor protein, a reaction involving three coordinated enzymatic activities (reviewed in Hershko and Ciechanover, 1998). Ub is first activated by an ATP-dependent reaction involving the Ub-activating enzyme E1, then conjugated to the active site cys-teine residue of the Ub-conjugating (UBC) enzyme E2, and finally transferred to the target K residue of the substrate protein by the Ub-protein isopeptide ligase E3. Most organisms have only one E1, but dozens of different E2 and hundreds up to thousands of different E3 enzymes, providing the need in coping with effec-tive substrate specificity (Hua and Vierstra, 2011;Braun and Madhani, 2012). Identification and characterization of E3s and some E2s involved in histone ubiquitination had been a key for understanding biological functions of histone ubiquitination in various organisms. Because of its suitability for genomics, genetics, and cellular and molecular biological approaches, Arabidopsis thaliana is an ideal model to investigate histone ubiquitination functions. In this review, we focus on this reference plant to expose current progress made on

H2B MONOUBIQUITINATION IN Arabidopsis

GENOME-WIDE DISTRIBUTION OF H2Bub1

Monoubiquitinated H2B (H2Bub1) was first discovered in mouse cells and was estimated to represent about 1-2% of total cellular H2B (West and Bonner, 1980). Later, H2Bub1 was detected widely throughout eukaryotes spanning from yeast to humans and plants (Zhang, 2003;Shilatifard, 2006;Srid-har et al., 2007;Zhang et al., 2007a;Weake and Workman, 2008). The ubiquitination site is mapped to a highly con-served K residue, H2BK123 in budding yeast, H2BK119 in fission yeast, H2BK120 in humans, and H2BK143 in Arabidopsis.

Genome-wide analysis revealed that in Arabidopsis as in animals H2Bub1 is associated with active genes distributed throughout the genome and marks chromatin regions notably in combina-tion with histone H3 trimethylated on K4 (H3K4me3) and/or with H3K36me3 (Roudier et al., 2011). During early photomor-phogenesis, gene upregulation was found to be associated with H2Bub1 enrichment whereas gene downregulation did not show detectable correlation with any H2Bub1 level changes (Bourbousse et al., 2012). In general, H2Bub1 is considered to represent an active chromatin mark broadly involved in genome transcription regulation. activation of the FLC (FLOWERING LOCUS C) gene (Schmitz et al., 2009). More recently, the otubain-like deubiquitinase OTLD1 was reported as implicated in deubiquitination of H2BUb1 and repression of At5g39160, a gene of unknown function (Krichevsky et al., 2011).

ROLE OF H2Bub1 IN FLOWERING TIME REGULATION

The timing of flowering is critical for the reproductive success of plants. As compared to wild type, the hub1 and hub2 sin-gle mutants as well as the hub1 hub2 and ubc1 ubc2 double mutants exhibit an early flowering phenotype whereas but the ubc1, ubc2, and ubc3 single mutants and the ubc1 ubc3 and ubc2 ubc3 double mutants have a normal phenotype (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009). This early flowering phenotype is detectable under both longday and short-day photoperiod plant growth conditions. Molecular analyses of the mutants indicate that H2Bub1 controls flowering time primarily through transcriptional activation of FLC (Figure 1). FLC encodes a key transcrip-tion repressor involved in both the autonomous/developmental and vernalization flowering pathways, and its active transcription is associated with several histone marks, e.g., H3K4me3, H3K36me2/3 and H2Bub1 (reviewed in Berr et al., 2011). In the early flowering mutants hub1, hub2, hub1

ENZYMES INVOLVED IN REGULATION OF H2Bub1 LEVELS

The budding yeast Rad6 (radiation sensitivity protein 6) was the first factor identified and shown to work as an E2 enzyme involved in catalyzing H2Bub1 formation both in vitro and in vivo (Robzyk et al., 2000). It contains a highly conserved catalytic UBC domain of approximately 150 amino acids in length with an active-site cysteine for linking Ub. The E3 enzyme working together with Rad6 in catalyzing H2Bub1 formation in budding yeast is Bre1 (Brefeldin-A sensitivity protein 1), which contains a C3HC4-type RING finger domain typical for all E3s (Hwang et al., 2003;Wood et al., 2003). The depletion of either Rad6 or Bre1 eliminates genome-wide H2Bub1 and causes yeast cell growth defects (Robzyk et al., 2000;Hwang et al., 2003;Wood et al., 2003). Human contains at least two homologs of Rad6, namely hHR6A and hHR6B, and two homologs of Bre1, namely RNF20/hBRE1A and RNF40/hBRE1B (Kim et al., 2005;Zhu et al., 2005). In Arabidopsis, three homologs of Rad6, namely UBC1, UBC2, and UBC3, were identified and UBC1 and UBC2 but not UBC3 were shown to be redundantly responsible for H2Bub1 formation in planta (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009). The two Bre1 homologs HUB1 (HISTONE MONOUBIQUITINATION 1) and HUB2 work non-redundantly, possibly as a hetero-tetramer composed of two copies of HUB1 and two copies of HUB2, in catalyzing H2Bub1 formation in Arabidopsis (Fleury et al., 2007;Liu et al., 2007;Cao et al., 2008). H2Bub1 levels are drastically reduced or undetectable in Western blot analysis in the loss-offunction hub1 and hub2 single mutants as well as in the hub1 hub2 and ubc1 ubc2 double mutants, but are unaffected in the ubc1, ubc2, and ubc3 single mutants or in the ubc1 ubc3 and ubc2 ubc3 double mutants (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009).

H2Bub1 levels are also regulated by deubiquitination enzymes. Two Ub-specific proteases, Ubp8 and Ubp10, are involved in deubiquitination of H2Bub1 in budding yeast. Strik-ingly, while Ubp8 acts as a component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex specifically in H2Bub1 deubiquitination in transcription activation, Ubp10 functions independently of SAGA and primarily acts in Sir-mediated silenc-ing of telomeric and rDNA regions (reviewed in Weake and Workman, 2008). In human, USP22 acts as Ubp8 ortholog in a SAGA complex in H2Bub1 deubiquitination (Weake and Workman, 2008). In Arabidopsis, although a SAGA complex remains uncharacterized so far, the Ub protease UBP26/SUP32 has been shown to deubiquitinate H2Bub1 involved in both heterochromatic silencing (Sridhar et al., 2007) and ubc2, FLC expression levels are reduced and the FLC chromatin shows reduced H2Bub1 levels (Cao et al., 2008;Gu et al., 2009). The loss-of-function mutant ubp26/sup32 showed also an early flowering phenotype and reduced FLC expression but an elevated level of H2Bub1 in the FLC chromatin (Schmitz et al., 2009), indicating that not only H2Bub1 formation but also H2Bub1 removal are necessary for FLC transcription. Accompa-nying H2Bub1 reduction compromised levels of H3K4me3 and to a less extent H3K36me2 were detected at FLC in hub1 and ubc1 ubc2 (Cao et al., 2008), and reduced level of H3K36me3 but ele-vated level of H3K27me3 was observed at FLC in ubp26/sup32 (Schmitz et al., 2009). On parallels to the knowledge in yeast, it was proposed that the UBC-HUB-mediated H2Bub1 for-mation is necessary for H3K4me3 deposition at transcription initiation whereas UBP26/SUP32-mediated H2Bub1 removal is required for H3K36me3 deposition during transcription elon-gation (Cao et al., 2008;Schmitz et al., 2009). Nonetheless, this hierarchy of histone modifications needs to be cautioned because multiple factors are involved in H3K4me3 and H3K36me2/3 depositions and the SDG8 (SET DOMAIN GROUP 8)-mediated H3K36me2/3 deposition remarkably override H3K4me2/3 depo-sition in FLC transcription (Yao and Shen, 2011;Shafiq et al., 2014). Besides FLC, Arabidopsis has five FLC paralogs, namely MAF1 circadian clock genes (Himanen et al., 2012;Malapeira et al., 2012).

During photomorphogenesis, hundreds of genes show upreg-ulation associated with H2Bub1 enrichment in their chromatin in response to light exposure (Bourbousse et al., 2012). Strikingly, over 50% of these genes gain H2Bub1 enrichment upon the 1 h of illumination, illustrating the highly dynamic nature of H2Bub1 deposition during a likely cell division-independent genome regrogramming process. In contrast to the above discussed cases, in this study the H2Bub1 changes is neither accompanied by any detectable changes of H3K36me3 nor required for H3K4me3 enrichment following six hours of light exposure (Bourbousse et al., 2012). In line with the function of H2Bub1 in gene activation in response to light, the hub1-3 mutant seedlings are overly light sensitive, exhibiting a photobleaching phenotype (Bourbousse et al., 2012).

The hub1 mutants also show increased susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola (Dhawan et al., 2009). Precise role of H2Bub1 in plant defense against pathogens still remains largely unclear. Structure defects, e.g., thinner cell walls and altered surface cutin and wax compositions, together with impaired induction of some defense genes might have partly contributed to the increased suscepti-bility to pathogen infection in the hub mutant plants (Dhawan et al., 2009;Ménard et al., 2014). It is worthy noting that the sdg8 mutants impaired in H3K36me3 deposition also display reduced resistance to necrotrophic fungal pathogen infection (Berr et al., 2010(Berr et al., , 2012;;Palma et al., 2010). It will be interesting to study in future research whether a trans-histone crosstalk between H2Bub1 and H3K36me3 acts MAF4 and MAF5. Some MAFs are also downregulated in the early flowering mutants hub1, hub2, hub1 hub2, ubc1 ubc2, and ubp26/sup32 (Cao et al., 2008;Gu et al., 2009;Schmitz et al., 2009;Xu et al., 2009). Thus, H2Bub1 may also regulate flowering time through control of MAF gene expression under some plant growth conditions.

H2Bub1 FUNCTION IN OTHER PROCESSES

In addition to flowering, many other H2Bub1 as evidenced by studies of and hub2 mutants. The hub mutants processes also involve the Arabidopsis hub1 display reduced seed

MECHANISMS OF H2Bub1 IN TRANSCRIPTION REGULATION

So far only limited information is available concerning how H2Bub1 enzymes are recruited to the target chromatin. The evolutionarily conserved PAF1 (Polymerase Associated Factor 1) complex interacts with Pol II (RNA polymerase II) and plays a role as a "platform" for association of enzymes involved in H2bub1, H3K4me3, and H3K36me2/3 deposition, linking his-tone modifications with active transcription (Shilatifard, 2006;Weake and Workman, 2008;Berr et al., 2011;Braun and Mad-hani, 2012). A direct interaction between PAF1 complex and Rad6-Bre1 has been detected and shown as required for cat-alyzing H2Bub1 formation (Xiao et al., 2005). As in yeast and animals, deletion or knockdown of PAF1 components markedly reduces H2Bub1 in Arabidopsis (Schmitz et al., 2009). Genetic analysis shows that HUB2 and ELF8 encoding a PAF1 subunit act in a same floral-repression pathway in Arabidopsis flowering time regulation (Gu et al., 2009). Although physical interac-tion between UBC-HUB and PAF1 needs future investigation, interactions were observed between UBC and HUB (Cao et al., 2008) and between HUB and MED21 (mediator complex subunit 21), a subunit of the evolutionarily conserved Mediator com-plex (Dhawan et al., 2009). Mediator complex is associated with both general transcription factors and Pol II and is essential for activator-dependent transcription in all eukaryotes (for a recent review, see Carlsten et al., 2013). dormancy associated with reduced expression of several dormancy-related genes, including DOG1 (DELAY OF GERMI-NATION 1), ATS2 (ACYLTRANSFERASE 2), NCED9 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 9), PER1 (CYSTEINE PEROXIREDOXIN 1), and CYP707A2 (Liu et al., 2007). At vegeta-tive growth stages, the hub mutants exhibit pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited root growth (Fleury et al., 2007). Cell cycle genes, particularly some key regulators of the G2-to-M transition, are downreg-ulated, which could largely explain the plant growth defects of the hub mutants (Fleury et al., 2007). A more recent study shows that several circadian clock genes, including CCA1 (CIR-CADIAN CLOCK ASSOCIATED 1), ELF4 (EARLY FLOWERING 4) and TOC1 (TIMING OF CAB EXPRESSION 1), are down-regulated and their chromatin regions contain lower levels of H2Bub1 in the hub mutants, suggesting that H2Bub1 may con-tribute to the regulation of plant growth fitness to environment through expression modulation of some circadian clock genes (Himanen et al., 2012). It is worth to note that SDG2-mediated H3K4me3 deposition is also required for expression of several circadian clock genes (e.g., CCA1, TOC1) and the hub mutants exhibit reduced levels of interactors are generally involved in Pol II transcribed genes and thus cannot fully explain why UBC-HUB targets some but not all active genes. It is reasonable to speculate that UBC-HUB recruitment might also involve some gene-specific yet uncharacterized factors.

The next question is how H2Bub1 affects transcription. In yeast and animals, H2Bub1 can promote transcription elongation by enhancing the recruitment of RNA Pol II and by facilitat-ing nucleosome removal through interplay with FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone complex (Pavri et al., 2006;Tanny et al., 2007). FACT acts on displacement of H2A/H2B dimer from a nucleosome core, facilitating transcription elongation on chromatin template. In Arabidopsis, FACT genetically interacts with HUB1 and plays criti-cal roles in multiple plant developmental processes (Lolas et al., 2010). Yet its precise interplay with H2Bub1 in transcription regulation needs future investigations.

Alternatively or additionally, H2Bub1 may regulate transcription indirectly through crosstalk with H3K4me3 and H3K36me2/3 (Shilatifard, 2006;Weake and Workman, 2008;Berr et al., 2011;Braun and Madhani, 2012). In line with this idea, lack of H2Bub1 in Arabidopsis impairs H3K4me3 and H3K36me2 formation in chromatin at FLC and clock genes (Cao et al., 2008;Himanen et al., 2012), and elevated H2Bub1 inhibits H3K36me3 formation in the FLC chromatin (Schmitz et al., 2009). Nevertheless, in contrast to the requirement of H2Bub1 for genome-wide H3K4me3 formation in yeast, lack of H2Bub1 in Arabidopsis barely affects global H3K4me2/3 and H3K36me2/3 levels, as evidenced by Western blot analysis (Cao et al., 2008;Dhawan et al., 2009;Gu et al., 2009) as well as by ChIP (chromatin immunoprecipitation) analysis of light responsive genes during photomorphogenesis (Bourbousse et al., 2012). It is currently unclear to which extent applies the crosstalk of H2Bub1 with H3K4me2/3 and H3K36me2/3 in Arabidopsis gene transcription regulation and what are the molecular mechanisms underlying the crosstalk.

Finally, while H2Bub1 is generally associated with active gene transcription, it can also regulate transcription repression in a chromatin context-dependent manner. The ubp26/sup32 mutant shows release of transgene and transposon silencing (Sridhar et al., 2007) as well as elevated expression of PHE1 (PHERES1) associated with seed developmental defects (Luo et al., 2008). It has been shown that the silencing release is accompanied by reduction of H3K9me2 and of siRNA-mediated DNA methylation and the PHE1 expression elevation is associated with a reduced level of H3K27me3. Nevertheless, whether these changes of repressive marks are directly linked with H2Bub1 still need to be investigated.

of the human PRC1 (Polycomb repressive complex 1) component Ring1B (also known as Ring2 and RNF2) as a E3 involved in catalyzing H2Aub1 formation (Wang et al., 2004). In Arabidopsis, H2Aub1 was undetectable in a large-scale analysis of histone post-translational modifications by mass spectrometry (Sridhar et al., 2007;Zhang et al., 2007a) and had been thought for a long time to be non-existent (Weake and Workman, 2008). However, five PRC1-like RING-finger proteins, namely AtRING1a, AtRING1b, AtBMI1a, AtBMI1b, and AtBMI1c, have been identified in Ara-bidopsis (Sanchez-Pulido et al., 2008;Xu and Shen, 2008). More recent immunodetection and in vitro enzyme activity assays have revealed that these RINGfinger proteins are effectively involved in catalyzing H2Aub1 formation in Arabidopsis (Bratzel et al., 2010;Li et al., 2011;Yang et al., 2013).

PRC2 AND PRC1 IN H2Aub1 DEPOSITION

Polycomb group (PcG) proteins, first identified in Drosophila as repressors of homeotic (Hox) genes, are nowadays known to act in multiprotein complexes in transcription repression of a large number of genes in many multicellular organisms including plants (Bemer and Grossniklaus, 2012;Molitor and Shen, 2013;Schwartz and Pirrotta, 2013;Simon and Kingston, 2013). The most intensively studied complexes are PRC1 and PRC2. In Drosophila, PRC2 is composed of four core subunits, namely Ez (Enhancer of zeste), Suz12 (Suppressor of zeste 12), Esc (Extra sex combs) and N55 (a 55 kDa WD40 repeat protein), and PRC1 also contains four main subunits, namely Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs) and Ring1 (also known as dRing). In mammals, alternate subunit compositions create larger families of related PRC2type and PRC1-type complexes (Schwartz and Pirrotta, 2013;Simon and Kingston, 2013). Nevertheless, defined biochemical activities of PRC2 and PRC1 are conserved from flies to humans. The clas-sical model proposes a sequential mode of action of the two complexes: PRC2 catalyzes H3K27me3 formation, and PRC1 rec-ognizes the H3K27me3 mark and further mediates downstream H2Aub1 deposition. The PRC1 components, acting as E3 ligases in H2Aub1 formation, are RING-finger proteins: Ring1 in Drosophila and Ring1A and Ring1B in human (Braun and Madhani, 2012;Schwartz and Pirrotta, 2013).

In Arabidopsis, the four PRC2 core components are highly con-served (Figure 2) and encoded by small gene families, and their function in H3K27me3 deposition and transcription repression have been intensively studied (Bemer and Grossniklaus, 2012). In contrast, PRC1 compositions are drastically diverged in plants as compared to animals (Molitor and Shen, 2013). No sequence homologue of Ph could be identified in plants so far. LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), also known as TFL2 (TER-MINAL FLOWER 2), binds H3K27me3 and may play a Pclike function (Turck et al., 2007;Zhang et al., 2007b). This remark-ably differs from the distinct roles of HP1 and Pc in animals, where HP1 binds H3K9me3 involved in hetereochromatin for-mation whereas Pc binds H3K27me3 involved in PRC1-mediated silencing in euchromatin. The best conservations found about PRC1 core components are from

H2A MONOUBIQUITINATION IN Arabidopsis

PRESENCE OF H2Aub1

In contrast to H2Bub1, H2Aub1 has not been found in yeast and has been generally implicated in transcription repression in ani-mal cells (Weake and Workman, 2008;Braun and Madhani, 2012). Albeit its early discovery and high abundance (about 5-15% of the total H2A) in animal cells (Goldknopf et al., 1975;Hunt and Dayhoff, 1977;Zhang, 2003), H2Aub1 function has only more recently begun to be elucidated, thanking to the first identification

Frontiers in Plant Science | Plant Genetics and Genomics

These RING-finger proteins can be classified into two phylogenic groups: the first group comprises Drosophila Ring1, human Ring1A and Ring1B, and Arabidopsis AtRING1a and AtRING1b; the second group comprises Drosophila Psc, human Bmi1, and Arabidopsis AtBMI1a, AtBMI1b, and AtBMI1c. Consistent with their sequence conservation, AtRING1a, AtRING1b, AtBMI1a, and AtBMI1b each can ubiquitinate H2A in vitro, and loss of function of AtBMI1a and AtBMI1b causes H2Aub1 reduction in planta (Bratzel et al., 2010;Yang et al., 2013).

and embryonic callus formation in somatic tissues of the Atring1a Atring1b and Atbmi1a Atbmi1b mutants (Figure 2B). The VAL (VP1/ABI3-LIKE) transcription factors can physi-cally interact with AtBMI1 proteins and the val1 val2 mutant exhibits comparable phenotype to Atbmi1a Atbmi1b, suggest-ing that VAL and AtBMI1 proteins may form complexes in repression of embryonic regulatory genes during vegetative development (Yang et al., 2013). Notably, loss of VAL or AtBMI1 causes H2Aub1 reduction in chromatin regions at ABI3, BBM, FUS3 and LEC1 but not STM (Yang et al., 2013). Future investigation is necessary to clarify whether AtBMI1 and AtRING1 proteins repress KNOX transcription via H2Aub1 deposition or other independent chromatin remodeling mecha-nisms.

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN STEM CELL MAINTENANCE

Plant growth and development largely depend on stem cells located in SAM (shoot apical meristem) and RAM (root apical meristem), whose activities are fine-tuned by multiple families of chromatin factors (Sang et al., 2009;Shen and Xu, 2009).

The first uncovered biological role of the Arabidopsis PRC1like RING-finger proteins are on the regulation of SAM activity (Xu and Shen, 2008) (Xu and Shen, 2008). Ectopic expression of KNOX genes colocalizes with and precedes ectopic meristem formation. It has been proposed that AtRING1a/b acts as a crucial PRC1 component in conjunction with PRC2 in repression of KNOX genes to promote lateral organ formation in the SAM (Figure 2A).

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN SEED GERMINATION

Seed germination defines the entry into a new generation of the plant life cycle. It is generally accepted that the process of germination starts with water uptake followed by seed coat rup-ture and is completed following radicle protrusion (Bentsink and Koornneef, 2008). During the very early phase, the embry-onic growth program remains latent and can be reinstated in response to unfavorable environmental cues.

With the attain-ment of photosynthetic competence, the irreversible transition to autotrophic growth is accomplished and embryonic program is stably suppressed. A recent study (Molitor et al., 2014) has identified the Arabidopsis PHDdomain H3K4me3-binding AL (ALFIN1-like) proteins as interactors of AtBMI1 and AtRING1 proteins and has demonstrated a crucial function of chromatin state switch in establishment of seed developmental gene repres-sion during seed germination (Figure 2C). Loss of AL6 and AL7 as well as loss of AtBMI1a and AtBMI1b retards seed ger-mination and causes transcriptional derepression and a delayed chromatin state switch from H3K4me3 to H3K27me3 enrichment of seed developmental genes, including ABI3 and DOG1. The ger-mination delay phenotype of the al6 al7 and Atbmi1a Atbmi1b mutants is more pronounced under osmotic stress (Molitor et al., 2014), suggesting that AL PHD-PRC1 complexes may participate in regulation of seed germination in response to environmental cues.

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN EMBRYONIC CELL FATE DETERMINACY

Further characterization of the ectopic meristem structures observed in Atring1a Atring1b unravels that these callus structures exhibit embryonic traits (Chen et al., 2010). The Atbmi1a Atbmi1b mutant also displays derepression of embryonic traits (Bratzel et al., 2010;Chen et al., 2010). Embryonic callus formation has been observed broadly in somatic tissues of cotyledons, leaves, shoots and roots of the mutant plants. Treatment with an auxin transport inhibitor can inhibit embryonic callus formation in Atring1a Atring1b, indicating that a normal auxin gradient is required for somatic embryo formation in the mutant (Chen et al., 2010). Both Atring1a Atring1b and Atbmi1a Atbmi1b mutants exhibit elevated expression of several key embryonic regulatory genes, including ABI3 (ABSCISIC ACID INSENSITIVE 3), AGL15 (AGAMOUS LIKE 15), BBM (BABYBOOM), FUS3 (FUSCA 3), LEC1 (LEAFY COTYLEDON 1), and LEC2 (Bratzel et al., 2010;Chen et al., 2010). It is likely that derepression of these regulatory genes together with KNOX has contributed to the ectopic meristem

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN OTHER PROCESSES

AtBMI1a and AtBMI1b, also named DRIP1 (DREB2A-INTERACTING PROTEIN 1) and DRIP2, had been reported first as E3 ligases involved in ubiquitination of DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING PRO-TEIN 2A), a transcription factor controlling water deficitinducible gene expression (Qin et al., 2008). The drip1 drip2 mutant shows enhanced expression of water deficit-inducible genes and more tolerance to drought (Qin et al., 2008). Overexpression of AtBMI1c accelerates flowering time, which is associated with reduction of FLC expression (Li et al., 2011). In addition to SAM maintenance defects and derepression of embryonic traits, the Atring1a Atring1b mutant also displays homeotic conversions of floral tissues (Xu and Shen, 2008). Therefore, more precise functions and underlying molecular mechanisms for the PRC1-like RING-finger proteins are still waiting to be uncovered during plant development and in plant response to environmental changes.

nicely in vitro as E3 ligases, their in vivo functions in H2Aub1 deposition are still poorly documented. H2Aub1 level in Ara-bidopsis seems very low because large-scale analyses of either the histone-enriched or the Ub-affinity-purified protein preparations fail to detect H2Aub1 (Maor et al., 2007;Sridhar et al., 2007;Zhang et al., 2007a;Manzano et al., 2008;Saracco et al., 2009). H2Aub1 has been detected only by using specific antibodies, and in this case AtBMI1 genes have been shown to act as positive regula-tors for H2Aub1 deposition in Arabidopsis plants (Bratzel et al.,

MECHANISMS OF PRC1-LIKE RING-FINGER PROTEINS IN TRANSCRIPTION REPRESSION

H2Aub1 function in plants is primarily evidenced through investigation of roles of the Arabidopsis PRC1-like RING-finger proteins (Xu and Shen, 2008;Bratzel et al., 2010;Chen et al., 2010;Li et al., 2011;Yang et al., 2013). Although these RINGfinger proteins act 2010; Li et al., 2011;Yang et al., 2013). It is unknown whether any deubiquitinases might cause low levels of H2Aub1 in Arabidopsis.

In animal cells, several deubiquitinases are characterized as specific for H2Aub1 (Weake and Workman, 2008;Simon and Kingston, 2013). Future characterization of Arabidopsis H2Aub1 deubiquitinases may provide useful information regarding regulatory mechanisms of H2Aub1 dynamics. AtRING1 and AtBMI1 proteins physically interact each other and with the H3K27me3-binding protein LHP1 (Xu and Shen, 2008;Bratzel et al., 2010;Chen et al., 2010), providing a pos-sible recruitment mechanism similar to the classical sequential PRC2 then PRC1 silencing pathway in animal cells. However, the Atring1a Atring1b, Atbmi1a Atbmi1b, or Atbmi1a Atbmi1b Atbmi1c mutant exhibits much more severe phenotypic defects than the lhp1 mutant does, and lhp1 enhances the Atring1a Atring1b mutant defects. It is thus apparent that AtRING1 and AtBMI1 proteins also act independently from LHP1. Recent iden-tification of the transcriptional regulator VAL as AtBMI1-binding protein and of AL as AtRING1 and AtBMI1 interactor provides some novel insight about recruitment mechanisms (Yang et al., 2013;Molitor et al., 2014). It is particular intriguing that loss of AtBMI1 impairs H3K27me3 enrichment at seed developmental genes during seed germination and vegetative growth (Yang et al., 2013;Molitor et al., 2014). It has also been reported that loss of LHP1 impairs H3K27me3 enrichment at flower gene loci in roots (Derkacheva et al., 2013). These recent findings challenge the classic hierarchical paradigm where PRC2-mediated H3K27me3 deposition precedes PRC1 recruitment (Figure 2). It is obvious that future investigations are necessary to better understand the composition and function of different PRC1-like complexes In addition to H2Aub1 and H2Bub1, ubiquitinated H1, H3, and H4 are also found in Arabidopsis (Maor et al., 2007;Man-zano et al., 2008;Saracco et al., 2009). H3 ubiquitination catalyzed by Rtt101-Mms1 in yeast and by Cul4-DDB1 in human has been recently shown to play an important role in the histone chaperone Asf1-mediated nucleosome assembly (Han et al., 2013). Arabidop-sis contains a conserved family of CULLINs and CUL4-DDB1 complexes are reported (Shen et al., 2002;Hua and Vierstra, 2011). The Asf1 homologues in Arabidopsis are also identified (Zhu et al., 2011). It remains to be investigated whether CUL4-DDB and AtASF1 collaboratively act on nucleosome assembly via H3 ubiquitination in epigenetic regulation in Arabidopsis.

CONCLUSIONS AND PERSPECTIVES

Studies over the last few years in the model plant Arabidop-sis have greatly advanced our knowledge about the roles of H2Aub1 and H2Bub1 in transcription regulation in plant growth and development. In view of additional functions described in animal cells for both H2Aub1 and H2Bub1 in DNA damage repair (Bergink et al., 2006;Marteijn et al., 2009;Chernikova et al., 2010;Ginjala et al., 2011;Moyal et al., 2011;Nakamura et al., 2011), it is anticipated that more roles of H2Aub1 and H2Bub1 in plant response to environmental stresses are waiting to be uncovered. Mutagenesis of enzymes involved in H2Aub1 and H2Bub1 deposition or removal is required to address the question whether these enzymes effectively exert their biological functions via H2Aub1 and H2Bub1. Identification and char-acterization of factors associated with these different enzymes will be essential for understanding molecular mechanisms of their recruitment and function at specific targets within the genome. We need to know whether and how their function is spatially and temporally integrated with plant development. Genome-wide tools need to be further explored to provide a global view of links among enzyme or associated factor bind-ing, H2Aub1/H2Bub1 enrichment, H3 methylation, and Pol II occupation. Crosstalks between H2Aub1 or H2Bub1 and different H3 methylations need to be addressed for

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Readers are proteins with specific domains that recognize and bind to particular modifications [START_REF] Taverna | How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers[END_REF][START_REF] Plass | Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer[END_REF]. Chromatin readers are able to identify different modified amino acids and also different modification states of the same amino acid. H2Bub1 has various functions, and it probably exerts molecular and cellular functions by recruiting various H2Bub1-specific readers or preventing the binding of others. There are two types of readers (reviewed in [START_REF] Fuchs | Writing and reading H2B monoubiquitylation[END_REF]. One type comprises unmodified H2B readers; H2Bub1 may actually prevent the binding of other factors. Examples are yeast cyclin dependent kinase Ctk1 [START_REF] Wyce | H2B ubiquitylation acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8 within a SAGA-related complex[END_REF], elongation factor TFIIS [START_REF] Shema | RNF20 inhibits TFIIS-facilitated transcriptional elongation to suppress pro-oncogenic gene expression[END_REF] and the splicing factors UIA/U2B [START_REF] Zhang | USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing[END_REF]. H2Bub1 disrupts the interaction between Ctk1 and histone H2A to prevent premature phosphorylation of RNA Pol II on Serine 2.

H2Bub1 inhibits the loading of TFIIS elongation factor onto chromatin to repress gene expression. And the increased levels of H2Bub1 upon USP49 knockdown prevent the association of U1A and U2B with the chromatin, which results in impaired splicing.

Another type comprises H2Bub1-specific readers, such as yeast proteasomal ATPases Rpt4/ Rpt6 [START_REF] Ezhkova | Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3[END_REF], Dot1 [START_REF] Oh | A lysine-rich region in Dot1p is crucial for direct interaction with H2B ubiquitylation and high level methylation of H3K79[END_REF], Cps35 [START_REF] Lee | Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS[END_REF], WDR82, the human orthologue of yeast Cps35 [START_REF] Shema-Yaacoby | Systematic identification of proteins binding to chromatin-embedded ubiquitylated H2B reveals recruitment of SWI/SNF to regulate transcription[END_REF][START_REF] Wu | Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS[END_REF], and ASH2L [START_REF] Wu | ASH2L regulates ubiquitylation signaling to MLL: trans-regulation of H3 K4 methylation in higher eukaryotes[END_REF]. These readers play a key role in the crosstalk between H2Bub1 and H3 methylation. In addition, the H2Bub1-specific reader SKIP [START_REF] Bres | SKIP interacts with c-Myc and Menin to promote HIV-1 Tat transactivation[END_REF] links H2Bub1 to viral infection. MRG15 [START_REF] Wu | Chfr and RNF8 synergistically regulate ATM activation[END_REF], a common subunit of both the MOF and Tip60 complexes, mediates the contribution of H2Bub1 to DNA damage response. BRG1, or BAF155 [START_REF] Shema-Yaacoby | Systematic identification of proteins binding to chromatin-embedded ubiquitylated H2B reveals recruitment of SWI/SNF to regulate transcription[END_REF], is a component of the SWI/SNF complex, which, as a novel H2Bub1 reader, helped to shed light on the role of H2Bub1 as a positive transcription regulator.

I.3.2.2 ZRF1, a reader of histone H2A monoubiquitination?

Characteristics of ZRF1

Zuotin related factor 1 (ZRF1) is evolutionary conserved in most species. It is a member of the M-phase phosphoprotein (MPP) family. It localizes to both the nucleus and the cytosol. It contains a tRNA and Z-DNA binding (Zuotin) domain at the N-terminus, and was isolated as a Z-DNA binding protein in yeast [START_REF] Zhang | Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae[END_REF]Wilhelm et al., 1994). Z-DNA is a left-handed DNA double helical structure which is correlated with some important biological processes, such as transcription, replication, and recombination of DNA [START_REF] Naylor | d(TG)n.d(CA)n sequences upstream of the rat prolactin gene form Z-DNA and inhibit gene transcription[END_REF]Wahls et al., 1990;Witting et al., 1991). Zuotin contains a DnaJ motif, which is similar to mammalian HSP-40 (heat shock protein 40) chaperone. DnaJ/Hsp40 proteins all contain the J domain. The DnaJ domain is composed of a 70-amino acid sequence consisting of four helices and a loop region. Between helices II and III it contains a highly conserved tripeptide of histidine, proline, and aspartic acid (the HPD motif) [START_REF] Qian | Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain[END_REF]. The DnaJ protein serves to recruit Hsp70 to substrate polypeptides as well as to stimulate Hsp70's adenosine triphosphatase (ATPase) activity, thus stabilizing Hsp70's interaction with the substrate.

DnaJ/Hsp40 proteins have been preserved throughout evolution. In addition, they are important for protein translation, folding, unfolding, translocation and degradation [START_REF] Qiu | The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones[END_REF]. Zuotin also binds to ribosomes, in part via interaction with ribosomal RNA (Yan et al., 1998). The Zuotin domain was first identified as [START_REF] Aasland | The SANT domain: a putative DNA-binding domain in the SWI-SNF and ADA complexes, the transcriptional co-repressor N-CoR and TFIIIB[END_REF][START_REF] Boyer | The SANT domain: a unique histone-tail-binding module?[END_REF][START_REF] Chen | Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution[END_REF]. The two SANT domains can carry out distinct functions; SANT2 may be more conserved than SANT1. But they may have coevolved as an intact functional unit within ZRF proteins. The ZRF1 SANT domain might function as a molecular sensor, which couples substrate-binding to enzyme catalysis through a compulsory conformational change [START_REF] Boyer | The SANT domain: a unique histone-tail-binding module?[END_REF]. A single SANT domain has been identified in Micromonas pusilla MpZRF1 [START_REF] Chen | Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution[END_REF].

Recently, GST pulldown experiments showed that the SANT domains are not required for binding ubiquitin [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. The SANT domain was found to be essential for asymmetric cell division [START_REF] Pappas | Functional analysis of the Volvox carteri asymmetric division protein GlsA[END_REF]. With these domains, ZRF1 is a multifunctional protein involved in transcriptional control through -interaction with multiple factors. The human protein was identified as a leukemiaassociated antigen and expression of the gene is upregulated in leukemic blasts.

Figure I.4. Schematic diagram of ZRF1 orthologues indicating the DnaJ domain and SANT domains

The numbers along the right-hand side of panels indicate the number of amino acids of the proteins (from [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF].

Functions of ZRF1 homologs

In animals, ZUO1-related factor (ZRF) homologs mainly focus on nematodes, mice, and humans.

In Caenorhabditis elegans, DNJ11, a ZRF ortholog, exhibits a wide expression pattern and functions in asymmetric cell division and subsequent apoptosis [START_REF] Chen | Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution[END_REF][START_REF] Hatzold | Control of apoptosis by asymmetric cell division[END_REF].

MIDA1 (mouse Id associate 1) is the mouse Zuotin ortholog. Like DNJ11, MIDA1 also shows a ubiquitous expression pattern. GST pull-down experiments showed that MIDA1 associates with the HLH (helix-loop-helix) region of the inhibitor of differentiation (Id) [START_REF] Shoji | MIDA1, a protein associated with Id, regulates cell growth[END_REF]. Id was also shown to act as a positive growth regulator. MIDA1 has two different domains containing DNA binding activities. One is the Zuotin domain with a Z-DNA binding activity, the other is SANT domain containing a specific DNA binding activity. In growth promotion, Id interacts with MIDA1 to stimulate the sequence-specific DNA binding activity and interrupt Z-DNA binding activity [START_REF] Inoue | MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein[END_REF][START_REF] Inoue | MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties[END_REF]. Antisense oligonucleotides for MIDA1 inhibit the growth of murine leukemia cells and the loss of MIDA1 also strongly interfered with the growth of MEL cells. This growth suppression is consistent with the slow growing phenotype of Zuotin null mutant yeast [START_REF] Zhang | Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae[END_REF]. Furthermore, loss of MIDA1 seemed not to interfere with entry into S phase, but delayed DNA synthesis especially at S phase [START_REF] Shoji | MIDA1, a protein associated with Id, regulates cell growth[END_REF]. These results demonstrated that MIDA1 regulates cell growth.

Using an antibody recognizing a specific set of phosphopeptides, MPP11/ ZRF1 was identified as a homolog of human Zuotin. This protein is involved in mitotic division [START_REF] Matsumoto-Taniura | Identification of novel M phase phosphoproteins by expression cloning[END_REF] and its knockdown leads to pronounced slow growth [START_REF] Jaiswal | The chaperone network connected to human ribosome-associated complex[END_REF]. Immunofluorescence experiments indicated that MPP11 is localized to the cytosol. Purification of MPP11 revealed that, together with Hsp70L1, it forms a mammalian ribosome-associated complex (mRAC). In vivo complementation data demonstrate that the C-terminal domain of MPP11 is not required for complex formation with Hsp70L1. However, complementary experiments demonstrated that mRAC can interact with the yeast ribosome, and can partly complement the yeast RAC mutant in the presence of the C-terminal domain of MPP11 [START_REF] Otto | The chaperones MPP11 and Hsp70L1 form the mammalian ribosome-associated complex[END_REF]. Recently, ZRF1 was shown to localize to both the cytoplasm and the nuclei in mammalian cells [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. Current research indicates that MPP11/MIDA1 is a multifunctional protein involved in transcriptional control through interaction with multiple factors.

ZRF1 has been identified as a novel H2A-ubiquitin binding protein [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. It is known that PcG proteins catalyze H2A monoubiquitination. To understand the relationship between ZRF1 and PcG proteins Richly et al. performed pull-down assays using recombinant fusion protein His-RING1B and H2A-FLAG mononucleosome complexes. These assays showed that RING1B was efficiently released from nucleosomes following incubation with ZRF1. Furthermore, when GSTubiquitin was incubated with constant amounts of His-RING1B and increasing amounts of His-ZRF1 finally reaching equimolar levels, immunoblot analysis indicated that the level of RING1B decreased. These results show that ZRF1 can compete with RING1B at H2Aubi. Consistent with the previous results, and using the UBD domain of ZRF1, Richly showed that RING1B was replaced by the UBD of ZRF1 [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. These results showed that ZRF1 can directly antagonize gene silencing. In addition, ZRF1 can interact with USP21 to promote deubiquitination, which facilitates transcriptional activation. Considering these data they proposed a model (Figure I.5) in which ZRF1 is a chromatin-associated protein that recognizes the H2A mono-ubiquitin mark at lysine 119 (H2AK119ub1) and displaces RING1B (PRC1) from chromatin [START_REF] Richly | The flip side of the coin: role of ZRF1 and histone H2A ubiquitination in transcriptional activation[END_REF].

MPP11 knockdown results in slow growth and sensitivity to the aminoglycoside G418. Furthermore, MPP11 affects the fidelity of translation [START_REF] Jaiswal | The chaperone network connected to human ribosome-associated complex[END_REF]. In addition, Demajo I. had obtained consistent results. Knockdown of ZRF1 in five different human AML cell lines led to a strong decrease in cell proliferation and an increase in apoptosis [START_REF] Demajo | ZRF1 controls the retinoic acid pathway and regulates leukemogenic potential in acute myeloid leukemia[END_REF]. However, strikingly, in RA-induced conditions, ZRF1 deletion leads to reduced differentiation. Overexpression of ZRF1 increased the cell differentiation potential following RA treatment.

They also found that the ZRF1 effect was dose dependent. Taken together, ZRF1 seems to have a dual role, as a differentiation repressor in basal conditions but then switching to an activator following RA induction. As a repressor, ZRF1 could interact with RARα and control histone acetylation [START_REF] Demajo | ZRF1 controls the retinoic acid pathway and regulates leukemogenic potential in acute myeloid leukemia[END_REF].

Recent preliminary work suggests that ZRF1 could be involved in embryonic development. ZRF1 was also found to be a key player required for first inducing neural progenitor cell (NPCs) specification from ESCs and then maintaining NPC identity [START_REF] Aloia | Zrf1 is required to establish and maintain neural progenitor identity[END_REF]. Deletion of ZRF1 did not affect neither mesodermal and endodermal specification from embryonic stem cells (ESCs) nor the stem cell features of several non-neural stem cell lineages, but it led to a significant reduction of neuroectodermal markers. Among these down-regulated genes, several are involved in maintaining NPC identify. Moreover, ZRF1 re-expression restored the expression of the reduced neuroectodermal markers.

Figure I.5. Hypothetical model for polycomb action at chromatin

In promoter regions (purple nucleosomes), PRC2 carries out specific methylations of H3K27 (red circles). After ubiquitination of histone H2A (yellow circles) at promoter regions, PRC1 propagates into the gene body (light nucleosomes) to carry out ubiquitination. ZRF1 displaces PRC1 complexes by interacting with mono-ubiquitinated chromatin. After PRC1 removal, ZRF1 acts in concert with specific deubiquitinases (USP21) to facilitate deubiquitination. The enzyme might then either propagate to an adjacent nucleosome bound by ZRF1, or propagate together with ZRF1, which could confer multi-substrate binding since it is an oligomer. [START_REF] Richly | The flip side of the coin: role of ZRF1 and histone H2A ubiquitination in transcriptional activation[END_REF] The Arabidopsis PRC1-like RING-finger homologs (AtRNIG1A/B and AtBMI1A/B/C) have been characterized and shown to catalyze monoubiquitination of H2AK119 (reviewed in Molitor and Shen, 2013;[START_REF] Feng | Dynamic regulation and function of histone monoubiquitination in plants[END_REF]. Studies in animals showed that ZRF1 has a H2AK119ub1 reader-like function in the derepression of polycomb-repressed genes [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. ZRF1 specifically binds to H2AK119ub1 and then displaces PRC1 from chromatin. The depletion of PRC1 subsequently causes the loss of PRC2 from the chromatin, consequently switching polycomb-repressed genes from a repressive to an active state [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF].

Two homologs of human ZRF1 have been identified on the Arabidopsis genome [START_REF] Chen | Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution[END_REF], and hereinafter are named as AtZRF1a and AtZRF1b. A function for ZRF1 homologues in plants has not been reported so far. Thus my PhD work focuses on the functional characterization of AtZRF1a and AtZRF1b.

Our first objective was to study the gene expression patterns, the subcellular localization, as well as histone-binding activities of AtZRF1a and AtZRF1b. We found that AtZRF1a and AtZRF1b are broadly expressed in Arabidopsis plants and that the AtZRF1b protein binds H2Aub with characteristics similar to those previously reported for the human ZRF1 protein.

While the mammalian ZRF1 function has been studied in cultured cell lines, knowledge of ZRF1 function in the development of the whole organism is still lacking.

We used the powerful genetic tool available in Arabidopsis to investigate the functions of AtZRF1a and AtZRF1b. Several independent T-DNA insertion mutant lines were identified. Because of functional redundancy of the two genes, my study subsequently focused on the characterization of two independent double mutants exhibiting simultaneous loss of function of both AtZRF1a and AtZRF1b; these double mutants are named Atzrf1a-1 Atzrf1b-1 andAtzrf1a-2 Atzrf1b-1 I further investigated the roles of AtZRF1a and AtZRF1b in transcriptional regulation of genes. I studied the expression levels of selected genes in association with mutant phenotypes of Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1, as well as genes at the whole genome level by transcriptome analysis of the mutants. This allowed the identification of perturbed genes in the two double mutants, and showed an overlap of perturbed genes between these mutants and PRC1 defective mutants.

Lastly, to get insight into the mechanisms of AtZRF1 in transcriptional regulation, I investigated the H3K4me3, H3K27me3 and H2Aub1 levels in chromatin regions of some expression-perturbed genes in the Atzrf1a- 1 Atzrf1b-1 andAtzrf1a-2 Atzrf1b-1 mutants. My results showed that loss of AtZRF1 reduces H3K27me3 and H2Aub1 levels to varied degrees depending on the genes examined. Most strikingly, in all examined cases no increase of H2Aub1 could be detected, suggesting that ZRF1mediated deubiquitination of H2Aub1 is not a major event in transcriptional regulation in Arabidopsis.

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To identify genes with homology to human ZRF1 in the Arabidopsis genome, we performed BLAST searches with full-length human ZRF1 nucleotide and protein sequences as a query. The sequence analysis revealed that AtZRF1a (gene locus The AtZRF1a and AtZRF1b genes each contain one intron in their 5′ untranslated region (5'-UTR); no intron was found within the gene body or the 3'-UTR.

The AtZRF1a and AtZRF1b proteins share a conserved Zuotin domain at their Nterminus; this domain consists of a DnaJ domain and a potential ubiquitin-binding motif. In addition, they contain a pair of SANT domains at their C-terminus, a feature characteristic for this group of proteins in eukaryotes which is not found in prokaryotes [START_REF] Chen | Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution[END_REF]. The SANT domain is proposed to function as a histone modification reader in chromatin remodeling by coupling histone binding with enzyme catalysis [START_REF] Boyer | The SANT domain: a unique histone-tail-binding module?[END_REF]).

To understand the function of AtZRF1b, the pGEX-4T-1 plasmids and the target fragments (full length ubiquitin and ZRF1bSANT, ZRF1bUBD cDNAs), as well as the pET30a plasmids and AtZRF1b truncated cDNAs were digested by restriction enzymes, and then isolated and purified. Each target fragment was ligated into the corresponding vector with DNA ligase. The recombinant plasmids obtained were then introduced into E. coli cell line BL21 (DE3) via electroporation. Glutathione Stransferase (GST)-tagged ubiquitin proteins and different recombinant AtZRF1b truncated proteins with a His-tag were produced in E. coli and used in in vitro pulldown assays. In these assays GST and GST-Ubi beads were incubated with total protein extracts of E. coli expressing recombinant His-tagged proteins. By western blot with an anti-His antibody, His-AtZRF1bUBD (a ZRF1 protein only containing the UBD domain) and His-AtZRF1bSANT (a ZRF1 protein lacking the C-terminal SANT domain) fusion proteins were found to bind GST-Ubi but not GST alone (Figure

III.3).

To further confirm this interaction, the Arabidopsis H2A.1 isoform (At1g51060) cDNA was N-terminally tagged with the FLAG epitope and introduced into a modified pCAMBIA1300 vector under the control of the CaMV35s promoter. The construct was introduced into Agrobacterium strain GV3101 and subsequently transformed into WT Arabidopsis by the floral-dip method. Then, for pull-down assays, GST, GST-

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In contrast to single mutants, the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 are highly similar: they show not only defects in the vegetative phase of development (Figure III.7C), but also developmental aberrations in the inflorescence and siliques. For example, we found that the length of wild-type siliques is 1.50.2 cm (n=10), while the length of the siliques of Atzrf1a-1 Atzrf1b-1 double mutant plants is 0.490.14 cm (n=10) and the length of the siliques of Atzrf1a-2

Atzrf1b-1 double mutant plants is 0.520.1 cm (n=10). Thus, the silique length of the mutants is significantly shorter than that of wild-type plants.

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We further investigated the association of mutant phenotype with loss-of- Taken together, our molecular data, transgenetic complementation, and identification of multiple loss-of-function allelic mutants firmly establish that AtZRF1a

function
and AtZRF1b have redundant functions and that simultaneous loss of function of both genes caused the Atzrf1a -/-Atzrf1b -/-mutant phenotype. Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1.

-2 Atzrf1b-1 Col Atzrf1a-1 Atzrf1b-2 Atzrf1a-2 Atzrf1b-2 Atzrf1a-1 Atzrf1b-1 C o l A t z r f 1 a -1 A t z r f 1 a -2 A t z r f 1 b -1 A t z r f 1 b -2 A t z r f 1 a -1 A t z r f 1 b -1 A t z r f 1 a -2 A t z r f 1 b -1 A t z r f 1 a -1 A t z r f 1 b -2 A t z r f 1 a -2 A t z
To investigate cell cycle progression, we compared the ploidy levels of Trichomes are specialized epidermal cells. Their distribution is spatially and temporally regulated and can serve as a trait to distinguish between juvenile and adult leaves. Trichome density and age of leaf are associated [START_REF] Telfer | Phase change and the regulation of trichome distribution in Arabidopsis thaliana[END_REF].

The cell cycle is defined by a series of complex events, and is normally divided into phases with a defined temporal order. Each transition phase is controlled by cyclins and co-factors. To investigate the regulation of cell cycle progression in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants, we performed a qRT-PCR analysis on transcripts from cell cycle-related genes in 2-week-old wild-type and Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants seedlings. These genes include CDKA, CYCB, CYCD, RBR, E2F, and KRP2 which are key components of the pathway regulating entry into the cell cycle (Dewitte and Murray, 2003;[START_REF] Francis | The plant cell cycle--15 years on[END_REF].

qRT-PCR analysis indicates that, in the Atzrf1a Arzrf1b double mutant, KRP2

and CyclinB1;1 showed a significant reduction in their expression level as compared to wild-type plants (Figure III.15). CyclinB1;1 controls cell cycle progression at the G2to-M transition, while KRP2 specifically inhibits CDKA;1 [START_REF] Verkest | The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during Arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes[END_REF].

CDKA;1 acts in both the mitotic cell cycle and the endoreduplication cycle. Distinct CDKA;1/cyclin complexes have been shown to regulate the mitotic cell cycle and the endoreduplication cycle [START_REF] Verkest | The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during Arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes[END_REF]. The expression of CDKA;1 in the double mutants shows no obvious difference with the wild-type. These results suggest that deletion of AtZRF1a and AtZRF1b may affect G2-M transition. The expression levels of E2Fa and E2Fc were increased in the double mutant, compared to wild-type.

In Arabidopsis, the CYCD3-RBR-E2F pathway acts as a key regulator that controls Atzrf1b-1 double mutants seedlings. STM and BP are important for meristem maintenance and inflorescence architecture [START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF][START_REF] Venglat | The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis[END_REF].

Both STM and BP expression levels were higher (6-fold for STM and 4-fold for BP) in the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants than in the wild-type.

In contrast, expression of WUS, AG, and ULT1 was detected down-regulated in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants. It thus appears that loss of function of both AtZRF1a and AtZRF1b specifically induces ectopic expression of Class I KNOX genes.

To further confirm that loss of both AtZRF1a and AtZRF1b caused ectopic expression of KNOX genes, we used the pSTM promoter to drive β-glucuronidase (GUS) expression in the Atzrf1a-1 Atzrf1b-1 double mutant and wild-type backgrounds.

In the seedling stage, GUS activity was detected in an enlarged zone containing the axis cell fate [START_REF] Breuninger | Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo[END_REF][START_REF] Laux | The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis[END_REF], are drastic down-regulated and up-regulated, respectively. WOX5 and WOX8 are crucial for RAM function and basal embryo-axis cell fate termination [START_REF] Breuninger | Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo[END_REF], are upregulated by BABY BOOM (BBM) encoding an AP2/ERF transcription factor [START_REF] Boutilier | Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth[END_REF], LEAFY COTYLEDON 1 (LEC1) encoding a CCAAT-binding transcription factor [START_REF] Lotan | Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells[END_REF], as well as LEC2. It was reported that overexpression of LEC1 triggers spontaneous somatic embryo formation in plants [START_REF] Stone | LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[END_REF].

ABI3 encoding B3 domain factors [START_REF] Giraudat | Isolation of the Arabidopsis ABI3 gene by positional cloning[END_REF][START_REF] Stone | LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[END_REF]. It is known that the phytohormone auxin plays an important role in embryogenesis and somatic embryo formation [START_REF] Verdeil | Pluripotent versus totipotent plant stem cells: dependence versus autonomy[END_REF]. We detected a 2to 3-fold upregulation of PIN1 in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants, but neither PIN4 nor PIN7 expression was down-regulated, all from a gene family To examine gametophyte function under normal sporophytic growth, we investigated the inheritance of Atzrf1a Atzrf1b mutant alleles in heterozygous mutant plants. Growth tests on seeds produced by self-pollination of heterozygous plants revealed that the ratio of wildtype phenotype plants to double mutant plants is significantly higher than the expected ratio of 3:1 (Table III.1). The Atzrf1a-1

-/+ Atzrf1b-1 -/-, Atzrf1a-2 -/+ Atzrf1b-1 -/-, Atzrf1a-1 -/-Atzrf1b-1 -/+ and Atzrf1a-2 -/-
Atzrf1b-1 -/+ lines behaved very similarly, but we found a reduced transmission of the Atzrf1b-1 -/-gamete in the Atzrf1a -/-background; this reduced transmission is more obvious in the Atzrf1a-1 -/-background than in the Atzrf1a-2 -/-background (Table III.1).

As no seed abortion could be observed, this suggested that male and/or female transmission of the Atzrf1a Atzrf1b mutant alleles was decreased. In order to further

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determine the inheritance of the Atzrf1a Atzrf1b mutant alleles in the male and female gametes, reciprocal backcrosses of heterozygous mutant plants with the wild-type Col-0 plants (pollination of mutant pistils with Col pollen grains or pollination of Col pistils with mutant pollen grains) were performed. Genotyping by PCR analysis revealed that the inheritance of each of the Atzrf1a-1 -/+ Atzrf1b-1 -/-, Atzrf1a-2 -/+

Atzrf1b-1 -/-, Atzrf1a-1 -/-Atzrf1b-1 -/+ and Atzrf1a-2 -/-Atzrf1b-1 -/+ double mutants was reduced drastically through male and female gametes; when they are used as the pollen donor, the transmission rate is lower than when they are used as the egg donor (Table III.1). Taken together, these genetic data establish a gametophytic function of AtZRF1a/b, which is largely independent from its sporophytic function. It affects both male and female gametophytes, with a stronger effect on the male than on the female. double mutant for a more detailed analysis.

Auxin is important for many aspects of root development, including initiation and emergence, patterning of apical meristem, gravitropism, and root elongation. In order to investigate whether ZRF impaired auxin regulation of the primary root, we introgressed DR5GFP into Atzrf1a-1 Atzrf1b-1 double mutant by genetic crossing.

The auxin-sensitive reporter DR5GFP reveals auxin signaling in single cells [START_REF] Friml | Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis[END_REF][START_REF] Grieneisen | Auxin transport is sufficient to generate a maximum and gradient guiding root growth[END_REF]. negatively regulate root growth [START_REF] Fukaki | Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis[END_REF][START_REF] Tatematsu | MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana[END_REF]; their upregulation is consistent with the root growth suppression phenotype of the Atzrf1a-1

Atzrf1b-1 double mutant. In addition to auxin, other phytohormones such as brassinosteroids (BRs) are also involved in the regulation of root meristem activity [START_REF] Perilli | Growth and development of the root apical meristem[END_REF]. A downregulation of BES1, which encodes a key transcription factor of the BR signaling pathway [START_REF] Li | Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression[END_REF], was observed in Atzrf1a-1

Atzrf1b-1 double mutant (Figure III.24B).
We wanted to address whether auxin supply would rescue the Atzrf1a-1

Atzrf1b-1 double mutant phenotype. Root growth was investigated in the presence of various concentrations of exogenous 1-naphthalene acetic acid (NAA). We measured root length of 8-day-old wild-type and double mutants supplemented with 0, 1, 10, 100 nM NAA, respectively. We found that root growth is less responsive to NAA inhibition in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants compared to wild-type The Atzrf1a-1 Atzrf1b-1 double mutant showed a similar phenotype as the shr mutant. They both have severely reduced primary root growth; nevertheless, their mutants' seedlings are able to grow and complete their life cycle. The Arabidopsis root is composed of single layers of epidermis, cortex, endodermis and pericycle [START_REF] Dolan | Cellular organisation of the Arabidopsis thaliana root[END_REF]. However, in the shr-1 mutant, the root lacks the endodermal cell layer [START_REF] Benfey | Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis[END_REF]. We therefore wanted to check whether there was a loss of cell layers in Atzrf1a-1 Atzrf1b-1 double mutant. The down-regulation of FLC activates FT and SOC1 and promotes flowering [START_REF] Helliwell | The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex[END_REF][START_REF] Li | A repressor complex governs the integration of flowering signals in Arabidopsis[END_REF]. The floral integrator FT is a major target of multiple flowering pathways and of the photoperiod pathway in particular [START_REF] Samach | Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis[END_REF]. AGL24 is a dosage-dependent promoter of flowering [START_REF] Yu | AGAMOUS-LIKE 24, a dosagedependent mediator of the flowering signals[END_REF].

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qRT-PCR results showed that the expression of FLC, FLM, MAF2 and MAF4

FT I II

Seeds of wild-type (Col), single mutants Atzrf1a -1, Atzrf1a-2 andAtzrf1b-1 anddouble mutants Atzrf1a-1 Atzrf1b-1 andAtzrf1a-2 Atzrf1b- Atbmi1b quadruple mutant also displayed a significantly decreased germination efficiency. Among these mutants, the decrease level of the Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b quadruple mutant was highest while the decrease level of Atbmi1a

Atbmi1b was lower than in Atzrf1a- 1 Atzrf1b-1 andAtzrf1a-2 Atzrf1b- Atzrf1a-2 Atzrf1b-1 plated on supplemented with different concentrations of GA (0.5, 1.0 and 2.0µmol/L, respectively). All data represent average germination percentages ±SD of three biological replicates, each 80 seeds, observed daily for 12 days after stratification.

To investigate which seed germination genes are responsible for the germination defective phenotypes in the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1, we harvested the seedlings at 5 DAS, and then compared the there is an increase of H3K27me3 levels at DOG1 in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants, these levels are not significantly affected at DOG1 in Atring1a Atring1b mutants PRC1 RING-finger proteins as E3 ligase enzymes specifically catalyze H2A.1 monoubiquitination in Arabidopsis (Bratzel et al., 2010). We used a commercial ChIPgrade anti-human H2Aub antibody to recognize H2Aub1. The levels of H2Aub1 were strongly down-regulated at the examined seed development genes in Atring1a Atring1b, Atbmi1a Atbmi1b and Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants.

For ABI3, the level of H2Aub1 is lower in Atring1a Atring1b than in Atbmi1a Atbmi1b. For DOG1 and CRC, the level of H2Aub1 is drasticly lower in Atbmi1a Atbmi1b than in Atring1a Atring1b. Interestingly, in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants, the levels of H2Aub1 were also strongly reduced at the examined

2

Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 genome-wide expression levels were profiled by microarray in comparison to wild-type. Total RNA were isolated from 15-days-old in vitro seedlings, three independent biological replicates for each sample, and analyzed by Agilent single channel arrays. Loci were considered significantly deregulated compared to Col-0 when the expression fold-change exceeded 2 with a pvalue inferior to 0.05.

Transcriptome analysis showed a total of 11116 and 11291 mis-regulated genes in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants, respectively. Among the 11116 mis-regulated genes, the analysis identified 5235 genes (47.09%) that were upregulated and 5881 genes (52.91%) that were down-regulated in Atzrf1a-1 Atzrf1b-1 double mutant. In the Atzrf1a-2 Atzrf1b-1 double mutant, among the 11291 misregulated genes we identified 5240 genes (46.41%) that were up-regulated and 6051 genes (53.59%) that were down-regulated. A comparison showed that the Atzrf1a-1

Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants exhibited a very high degree of overlap.

The statistical significance of the overlap between two gene sets was qualified by a representation factor (RF) and P-value. The RF characterizes the fold increase of overlapping genes compared to the expected overlap of two random gene populations.

It takes into account the size of the two analyzed data sets and the global gene sets (i.e.

number of protein-encoding loci according to The Arabidopsis Information Resource 10 database (TAIR10)). Thus, the overlap between two random gene populations is qualified by a RF of one, while the RF of an overlap between two populations enriched in common members is superior to one.

The significance of the overlaps between the commonly mis-regulated genes in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants was calculated considering the entire populations of commonly mis-regulated genes or solely up-or down-regulated gene sets. The total number of commonly mis-regulated genes is 8956 (RF=1.95, P<3.54e-10). Among them, our analysis identified that 4103 genes (RF=4. and between Atzrf1a Atzrf1b-up and Atbmi1a Atbmi1b-up it is 104 (24.07% of Atbmi1a

Atbmi1b total). Even though these percentages may not appear to be very high, however, the insertions for both microarray data sets were more than two-fold increased compared to a random distribution (Figure III.37). Some of the Atzrf1a Atzrf1b double mutant defects are similar to those previously reported for the PRC1 mutants Atring1a Atring1b and Atbmi1a Atbmi1b.

Our microarray analysis (behind section) showed that there are significant overlaps of the perturbed genes between Atzrf1a Atzrf1b and Atring1a Atring1b or Atbmi1a

Atbmi1b. We tested the physical interaction of AtZRF1b with AtRING1 and AtBMI1

proteins. For this, we carried out pull-down experiments. Recombinant plasmids of GST-RING1A, GST-BMI1A, GST-BMI1B and GST-BMI1C have been produced in our lab. Following plasmid transformation, protein expression, isolation and purification, we obtained the recombinant proteins GST-RING1A, GST-BMI1A, GST- Atzrf1a -1 Atzrf1b-1 Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b Atzrf1a-1Atzrf1b-1 Atbring1a Atring1b

BMI1B

$#!*($# # (*(($#

In Arabidopsis, AtZRF1a and AtZRF1b are the homologs of human ZRF1.

They contain a tandem repeat of the SANT domain and a Zuotin domain. The Zuotin homology region includes a DnaJ motif, considered to interact with Hsp70s. Our results indicate that ZRF1, which contains a ubiquitin-binding domain, can interact with H2A-ubiquitin. These properties are comparable with those described for mammalian ZRF1 [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. The human chromatin-binding factor ZRF1 has a strong link with PRC1. In differentiation conditions, ZRF1 can displace the PRC1 complex from chromatin by competing for the binding to mono-ubiquitinated H2A; it can directly antagonize gene silencing [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF]. During development, a block of the G2-to-M transition may also result in an increased endoreduplication. Endoreduplication occurs after cells have ceased the mitotic cycles, and endoreduplicated cells do not reenter the mitotic cell cycle. Thus, endoreduplication is characteristic of a switch between cell proliferation and differentiation. It is also believed to be essential for enhancing metabolic capacity and supporting cell growth and for maintaining an optimal balance between cell volume and nuclear DNA content (reviewed in [START_REF] Kondorosi | Plant cell-size control: growing by ploidy?[END_REF]Inzé and De Veylder, 2006). Interestingly, the double mutant Atzrf1a Atzrf1b shows slightly elevated polyploidy levels but reduced cell size. Ploidy-dependent epigenetic regulation has been reported to be involved in differential reprogramming of orthologous gene expression and in stable silencing of epialleles [START_REF] Lee | Protein-coding genes are epigenetically regulated in Arabidopsis polyploids[END_REF][START_REF] Baubec | Cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic states in Arabidopsis[END_REF]. Based on its global effect on H2Aub1 deposition, it is reasonable to speculate that AtZRF1a and AtZRF1b are involved in the regulation of chromatin structure and gene expression in diploid and polyploid cells, playing important roles in the coordination of cell division, differentiation, and expansion to determinate organ size.

Arabidopsis
The Atring1a Atring1b and Atbmi1a Atbmi1b (drip1-1 drip2-1) mutants show pleiotropic phenotypes (Qin et al., 2008;Xu and Shen, 2008). During seedling growth these mutants reveal a crucial function of PRC1-like complexes in the repression of embryogenesis and stem cell activities for proper vegetative growth. Some defects of the Atzrf1a Atzrf1b mutant are similar to the previously reported phenotype of the Atring1a Atring1b or Atbmi1a Atbmi1b mutant. In the Atzrf1a Atzrf1b mutant, we observed callus-like plants. By qRT-PCR, we found many regulatory genes involved in embryogenesis and stem cell maintenance were upregulated in the Atzrf1a Atzrf1b mutant. These include the key embryonic regulatory genes LEC1, LEC2, ABI3 and BBM [START_REF] Boutilier | Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth[END_REF][START_REF] Giraudat | Isolation of the Arabidopsis ABI3 gene by positional cloning[END_REF][START_REF] Lotan | Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells[END_REF][START_REF] Stone | LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[END_REF], the embryonic competence-enhanced gene AGL15 [START_REF] Harding | Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15[END_REF], the key RAM-regulatory and basal embryo-axis cell fate genes WOX5 and WOX8 [START_REF] Breuninger | Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo[END_REF], the key SAM-regulatory genes STM, BP, KNAT2 and KNAT6 (Xu and Shen, 2008), the organ boundary regulatory genes CUC1, CUC2 and CUC3 [START_REF] Vroemen | The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in Arabidopsis[END_REF] and the auxin transporter gene PIN1 [START_REF] Blilou | The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots[END_REF]. The LEC1, LEC2, ABI3, BBM, AGL15, WOX5, WOX8 and CUC1 genes were also found to be upregulated to varying extents in the Atring1a Atring1b and Atbmi1a

Atbmi1b mutants. Interestingly, like in Atring1a Atring1b, the Class I KNOX genes (STM, BP, KNAT2 and KNAT6) were upregulated in the Atzrf1a Atzrf1b double mutant, but barely changed in Atbmi1a Atbmi1b. This is consistent with the highly fasciated stem phenotype observed in Atzrf1a Atzrf1b and Atring1a Atring1b but not in Atbmi1a

Atbmi1b (drip1-1 drip2-1).

AtZRF1a and AtZRF1b transcripts were detected in the inflorescence meristem and in floral organs. We found that Atzrf1a Atzrf1b mutant flowers showed

abnormal

IV

The root system is the main organ of the plant responsible for nutrient and water uptake. In this study, we have demonstrated that loss-of-function mutant Atzrf1a

Atzrf1b exhibits SCN disorganization and stem cell termination, causing primary root growth arrest. The auxin gradient maximum which appeared in the QC cells in WT is almost lost in Atzrf1a Atzrf1b roots. Previous genetic analysis showed that auxin acts upstream of the major regulators of stem cell activity [START_REF] Ding | Auxin regulates distal stem cell differentiation in Arabidopsis roots[END_REF][START_REF] Sabatini | An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root[END_REF], and QC ablation experiments demonstrated that reestablishment of auxin maximum is earlier than the re-specification of a new QC in root [START_REF] Grieneisen | Auxin transport is sufficient to generate a maximum and gradient guiding root growth[END_REF]. We therefore believe that the loss of auxin accumulation and gradient is a potent cause of the irregular cell shape and position of QC in Atzrf1a Atzrf1b. High levels of auxin promote proteasome-mediated degradation of IAA proteins, which act as repressors of auxin response by binding Auxin Response Factors (ARFs) to regulate downstream gene transcription. Several IAA genes, including the previously characterized ones IAA14 and IAA19 [START_REF] Fukaki | Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis[END_REF][START_REF] Tatematsu | MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana[END_REF], are upregulated in Atzrf1a Atzrf1b, further supporting a perturbed auxin pathway by loss of AtZRF1a and AtZRF1b function.

In addition to QC, we have demonstrated that the surrounding stem cells of QC also exhibited varied degrees of impairment in the Atzrf1a Atzrf1b double mutant.

Development of the Arabidopsis root is a dynamic process that involves a complex interplay between transcriptional regulators and plant hormones. Understanding how AtZRF1 regulates root growth and development will require the integration of many different types of data.

IV

In this study, we found that loss-of-function mutants of AtZRF1a or AtZRF1b displayed a weak early-flowering phenotype. However, the simultaneous loss of Enrichment of the H3K27me3 repressive mark at the FLC and MAF4 loci is up-regulated by AtZRF1. However, in the Atring1a Atring1b mutant, enrichment of H3K27me3 at the FLC locus is not affected and is down-regulated at the MAF4 locus.

In the Atbmi1a Atbmi1b (drip1-1 drip2-1) mutant, enrichment of H3K27me3 at the FLC and MAF4 loci is down-regulated. These findings suggest that the function of and PER1. The expression of these seed developmental genes was previously shown to negatively regulate seed germination [START_REF] Bentsink | Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis[END_REF][START_REF] Haslekas | Seed 1-cysteine peroxiredoxin antioxidants are not involved in dormancy, but contribute to inhibition of germination during stress[END_REF][START_REF] Parcy | Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid[END_REF][START_REF] Yamagishi | CHOTTO1, a double AP2 domain protein of Arabidopsis thaliana, regulates germination and seedling growth under excess supply of glucose and nitrate[END_REF]. Quantitative differences in gene expression and the stress-inducible nature of these genes in seed germination regulation might explain the Atzrf1a Atzrf1b mutant phenotype observable under osmotic stress conditions. The detected gene derepression was more severe and persisting in Atzrf1a

Atzrf1b than in Atbmi1a Atbmi1b.

Some defects of the Atzrf1a Atzrf1b mutant are similar to the previously reported phenotypes of the Atbmi1a Atbmi1b or Atring1a Atring1b mutants; and there are significant overlaps of genome-wide perturbed genes between these mutants.

Genetic interaction tests revealed that Atzrf1a Atzrf1b is epistatic to Atbmi1a Atbmi1b

and Atring1a Atring1b, suggesting that AtZRF1 acts downstream of AtRING1 and AtBMI1.

Chromatin analysis at seed developmental genes revealed that their upregulation is associated with reduced levels of H2Aub1 and H3K27me3 in Atzrf1a

Atzrf1b as in Atbmi1a Atbmi1b and Atring1a Atring1b to varied degrees.

Some phenotypic defects observed in the double mutant Atzrf1a Atzrf1b are similar to those previously observed in PRC1 mutants Atring1a Atring1b and Atbmi1a

Atbmi1b. Our microarray date are consistent with a genetic interaction, since they reveal a significant overlap of deregulated genes between Atzrf1a Atzrf1b and Atring1a

Atring1b or Atbmi1a Atbmi1b. I further analyzed the physical interaction of AtZRF1b with AtRING1 or AtBMI1. Beads of agarose coupled to GST, or to GST-fusion proteins RING1A, GST-BMI1a, GST-BMI1B or GST-BMI1C were incubated with total extracts of nuclear proteins from Arabidopsis expressing the fusion protein FLAG-AtZRF1b. GST pull-down followed by Western blot analysis using anti-FLAG antibodies allowed me to demonstrate the interaction between AtZRF1B and AtBMI1A, AtBMI1B or AtBMI1C but not AtRING1A.

My results allowed the first functional characterization of the genes AtZRF1a and AtZRF1b. My data have shown that AtZRF1a and AtZRF1b play roles in part related with PRC1 but also with specific aspects. Their role in the kidnapping of H2Aub1, as proposed for ZRF1 in animals [START_REF] Richly | Transcriptional activation of polycomb-repressed genes by ZRF1[END_REF] has not been observed in plants. Recent data suggest that ZRF1 also exerts a Polycomb-independent role [START_REF] Aloia | Zrf1 is required to establish and maintain neural progenitor identity[END_REF]. The physical interaction between AtZRF1b and AtBMI1a/AtBMI1b is novel and has crucial importance for understanding AtZRF1 function. This needs to be confirmed by additional methods.

AtZRF1 has very important roles in plant development. Our microarray results show that, of the total of misexpressed genes in AtZRF1a and AtZRF1b knock out mutants, half were down-regulated of and half were up-regulated. These data are consistent with tthose observed in human cells (Demajo et al., 2013). The microarray data indicate that AtZRF1a/b may act as a bifunctional protein in plant development.

But the mechanism remains unclear, and therefore AtZRF1-binding at target chromatin regions requires further studies.

V. Materials and Methods

Ⅴ Ⅴ

The Atring1a Atring1b mutant was previously described (Xu and Shen, 2008). The Atbmi1a Atbmi1b mutant was reported by Calonje [START_REF] Calonje | EMBRYONIC FLOWER1 participates in polycomb group-mediated AG gene silencing in Arabidopsis[END_REF].

The Columbia (Col-0) ecotype was used as genetic background for both wild-type The steps are as follows:

and
(Q-PCR) actin-F AAGTCATAACCATCGGAGCTG actin-R ACCAGATAAGACAAGACACAC EXP-Q1 GAGCTGAAGTGGCTTCCATGA EXP-Q2 GGATCATGGGTATGTCGGACC PP2AA3-Q1 TAACGTGGCCAAAATGATGC PP2AA3-Q2 GTTCTCCACAACCGCTTGGT Tip4.1-Q1 GTGAAAACTGTTGGAGAGAAGCAA Tip4.1-Q2 TCAACTGGATACCCTTTCGCA ZRF1a-Q1 CCTCTCGTGGCTCAGCGTCT ZRF1a-Q2 GTCCTTCTTTGTTTCCCATTTT ZRF1b-Q1 AAAGGCGAAAAGAAGAAGAAGC ZRF1b-Q2 GAACAGGGGCGGAGAGAACT RING1a1283Q1 ATCTCTGTTGCCGACCCACT RING1a1406Q2 GCCGCATCTTCTCCTACTCT RING1b789Q1 TGAGAGGCAACGAAAAAAGC RING1b928Q2 AGTTCCACACAAGCACAGGT DRIP1-Q1 GGTCCCGTTTGGTTCTCACT DRIP1-Q2 TGTATTTCCATCCCTTATTCTC DRIP2-Q1 AGTTGTGTCCTCCATCTCATT DRIP2-Q2 GCTTTTGTTCTTTTTCCTGTT BMI1c-Q1 AACTTCACTGCGGGTCTCTTCA BMI1c-Q2 ACGGTCTCCCTATGTTTCTCCT CDKA;1-Q1 ACTGGCCAGAGCATTCGGTATC CDKA;1-Q2 TCGGTACCAGAGAGTAACAACCTC CYCD3;1-Q1 CCAAACTAATCTCCTCGGTGTCC CYCD3;1-Q2 TGATGATGAATCGTGACTCTTGCG CYCB1;1-Q1 TCAGCAATGGAAGCAACAAG CYCB1;1-Q2 AGCAGATTCAGTTCCGGTCA RBR1-Q1 CGCTTCCATTTTGGTTTTGA RBR1-Q2 TGAACAACAGCAGCAGCAAC E2Fa-Q1 CGAAGCCTTAACTGTTGACAACC E2Fa-Q2 GCATTTGAGAGAAGCCAGTAGTCC E2Fb-Q1 CCGATGAAAGAGGAAAGCACCG E2Fb-Q2 CGCCTACCTCTGATCGAAACC E2FC-Q1 TGCCGTTATGACAGTTCTTTAGGG E2FC-Q2 AGTGTTCCATCCTCAGCTTCCT KRP2-Q1 TCGTCGGTTTCGTGTTGTTC KRP2-Q2 CCTGCGGCGAGACTCTAC IAA2-F CGT TGG TTG GCC ACC AGT GA IAA2-R ACG CTT TGA GAA GCT CGG GGT IAA14-F CAG CTC CTT TAC CAT GGG GAG IAA14-R ACC AAC GAG CAT CCA GTC AC IAA16-F TGG GAT GGC CAC CGG TAC GA IAA16-R CAC GGT GGC ACA TGC GGA GG IAA19-F CGT GGC ATC GGT GTG GCC TT IAA19-R GCT GCA GCC CAA ACC CGG TA IAA28-F GCT CCT CCT TGT CAC CAA TTC ACT IAA28-R ACT GGA GCT ACC TCA ACC CTG TTA IAA29-F TGT GCG ATC GAG GGT GCT GC IAA29-R CGT CTT CCT CGT TGG GCT GGC IAA30-F GAG ACT CGG GCT CAG CTT CGG A IAA30-R CTC TGC CGC ACC GAC TCC AT IAA34-F GCA GCG ATC CTC CCC ATC CCT IAA34-R ACG CCA CCA AAC TCC GTG GTC BES1-F CGC CAG TTC CAT GCT CCG GC BES1-R GGT AGG CGA GGT TGG CAC CAT LEC1-Q1 AAATCCATCTCTGAATTGAACTT LEC1-Q2 CACGATACCATTGTTCTTGT LEC2-Q1 TACGAGGACGAAAGCAAGAA LEC2-Q2 CGTTAGGGATGGGATAGTGA ABI3-Q1 ATGTATCTCCTCGAGAACAC ABI3-Q2 CCCTCGTATCAAATATTTGCC AG-Q1 ACGGAATTATTTCCAAGTCGC AG-Q2 GCCTATATTACACTAACTGGAGAG BBM-Q1 GGTGGTTATCAAGGATTCGC BBM-Q2 TCTCAGCAGCAGTAAAGGGA AGL15-Q1 CGAAAGGAACGATTGCTGAC AGL15-Q2 GGATGGAACATAGTGGGTGAA PIN1-Q1 GCAACAAAACGACGCAGGCT PIN1-Q2 TGAAGGAAATGAGGGACCAG PIN2-Q1 TAGGAGGACAAAACAAGGAG PIN2-Q2 CGTGAGGAGGAATAGAAACT PIN4-Q1 TGATAATGGTGTGGAGAAAGC PIN4-Q2 TCTGAGAGTATGGAGATGGAT PIN7-Q1 GGAAACTCATAAGAAACCCA PIN7-Q2 CATCCCACCTGAAAGCAACA LHP1-Q1 GAGGAAGTCTGGTTCTGTGA LHP1-Q2 TCTGTAGGTGCTGTGTGGTT WOX2-Q1 CGCCAAAAGCAGAAACAGGA WOX2-Q2 TTGAGCAAGGAGGGGGGTAG WOX5-Q1 CCAAGGTGGACAAAATGAGAG WOX5-Q2 ATGATGAGTATGGAGAAAACG WOX8-Q1 GGTGGTAACGGAAGAAGGGA WOX8-Q2 TAATGGAACAGTCAAAGGAG CUC1-Q1 ACATTCCTTCCCGCTCCACC CUC1-Q2 AACTGACCAAACGCCACGCC CUC2-Q1 GAGCAACTGTGAGCGTAAGC CUC2-Q2 GGAGTGAGACGGAGGAAGGA CUC3-Q1 GGAACAACAACAACGACGAAG CUC3-Q2 AGACGAAAAACCCAACAGACC SERK1-Q1 GGAAGAGCGAACTCCAGGTG SERK1-Q2 GGTCGGTGTCATACAGAAAC SERK2-Q1 TTTAGCGGAGAAATGGGACG SERK2-Q2 CAGAGGTGGGGTGAGAAGAG WUS-Q1 CAGTTCGGAAAGATTGAGGG WUS-Q2 GGTGATGAAGATGGTGTGGT CRC-Q1 TGGCGTTCTCCAGGGTAAT CRC-Q2 TGACCACTTGGATCCTTCCT DOG1-Q1 TAGGCTCGTTTATGCTTTGTGTGG DOG1-Q2 CGCACTTAAGTCGCTAAGTGATGC AIL5-Q1 CTCCATGTACAGAGGCGTCA AIL5-Q2 GCAGCTTCCTCTTGAGTGCTA CRA1-Q1 CCGTGGATCTATCCGTCAAA CRA1-Q2 CAAACACTCTGTTACCATTGTCG PER1-Q1 CATATTGTTGGTCCTGACAGTAAGA PER1-Q2 GGCGATCTTGTTATTGTGCTT FLC-Q1 TGTGGATAGCAAGCTTGTGG FLC-Q2 TAGTCACGGAGAGGGCAGTC FLM-F GGAAAGAATACGTTGCTGGCAACA FLM-R CCGTTGATGATGGTGGCTAATTGA MAF2-F GGCTCCGGAAAACTCTACAA MAF2-R TTCTGCAAGATCTAAGGCTTCA MAF4-Q1 ATGGGAAGAAGAAAAGTAGAG MAF4-Q2 AGAGATGATGATAAGAGCGAC MAF5-Q1 ATTTTGGAAACAGGGGATGA MAF5-Q2 TTACTTGAGAAGCGGGAGAG SOC1-F CGAGAAGCTCTCTGAAAAGTGG SOC1-R TCAGAACTTGGGCTACTCTCTTC FT-F GGTGGAGAAGACCTCAGGAA FT-R GGTTGCTAGGACTTGGAACATC AGL24-F AGCCGTGTGTCTGAAAAGAAG AGL24-R AATTCCGATCCCCGTTTCT SVP-F GGACAAGAGCCACCGACTAA SVP-R TGCTGAAGCTCTTCAATGTCA STM-Q1 GCAACACATCCTCACCATTACTTCA STM-Q2 ATCAAAGCATGGTGGAGGAGA BP-Q1 TCCCATTCACATCCTCAACA BP-Q2 CCCCTCCGCTGTTATTCTCT KNAT2-Q1 AAACGCCATTGGAAGCCT KNAT2-Q2 ACAATGCACAATTTCATGTCTCTCT KNAT6-Q1 CCAAGAGAAGCAAGACAAGCTC KNAT6-Q2 CAGCTAATGCTATCTTATCTCCTTCAG LFY-F TTGATGCTCTCTCCCAAGAAG LFY-R TTGACCTGCGTCCCAGTAA AP1-F CCATCTCCTTTTCTCAACATGG AP1-R CGGGTTCAAGAGTCAGTTCG PI-F AACATGGCCTCGACAAAGTC PI-R CGCCATCATCTTCTCATTTCT SEP3-F CAACAACAACACTCCCAAGC SEP3-R TTGTTGCCCCTGATACCC CAL-F TCAGACTTCTCCTTTCCTAAATATGG CAL-R TCCAGATTGTTCCTCCTCATC GIK-F CGGAGGTAACGTAGTTGGTGA GIK-R TGTAAAAGACGCTGCCATGA ULT1-F TCAGATTTCCCATACGACAAGAT ULT1-R TGTGCACCCTCTGTACACCT STK-F GGGTGAAGCAAATTCTCAGG STK-R CGATTTGTTGAGTTCTCTATCCTCT UFO-F TCAGCCGCTCTACACACAGT UFO-R CCGACACACTCGAATCCTTT Name Sequences 5'-3' (Gateway Clone) ZRF1a-1658F GGGGACAAGTTTGTACAAAAAAGCAGGCTCCCAAGAT GGGCACAAGAAG ACA GAA CAC ZRF1a-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGCCG AGC CGG AGA AGTGAGTC ZRF1a-R GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTCCGG TTTTCTTGTTTCT AAT GAT TTC ZRF1b-1403F GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTGAGCA CAGAAAACGGAA ACA GTA AGGAG ZRF1b-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGCAG AGTTGGGGAATTAAC TCT GCT AT Name Sequences 5'-3' (ChIP-QPCR) qChABI3-1F GTTTAAGAACCACCGCTTGG qChABI3-1R CTC CTC GTG CCG CTA GTA TC qChABI3-2F TCGGATCTTTTCATATGCTTTG qChABI3-2R GAGATTCAAAAAGAACTCTTGATAAGG qChABI3-3F CAAAGAAGACGCACCACCA qChABI3-3R TGGATCTTGTTGGAATGATTGT qChABI3-4F GCTGGCTCAGCTTCTGCTAT qChABI3-4R AAAGATGATTGTGCATGTCTACCT qChABI3-5F CAACCGAGCGGACAAAAG qChABI3-5R TGTTCCTTTGCGACTTGTTTT qChDOG1-1F TGGAACAACAACTCGCACTC qChDOG1-1R GTG CTT TCC GAG CAA ATA AAA qChDOG1-2F TCTCGAGTGGATGAGTTTGC qChDOG1-2R TCTTCATCACCGTGAGAT CG ChDOG1-3F AACATCGACGGCTACGAATC qChDOG1-3R GCACCGTACTGACTACCGAAC ChDOG1-4F TCAAGCTCTCGACAAGCAAG qChDOG1-4R AGAAATCCGCTCCTTGTACC ChDOG1-5F TCACGTCGTGGCATTTTG qChDOG1-5R TCG AGA CGA GAT CAT GTT GC qChCRC-F1 GCTCTGCCCACTGGATCTAC qChCRC-R1 CGAGAAGAGCGATGATGACA qChCRC-F2 TCAGCAGCTTCAGAACCAAC qChCRC-R2 TGGAAAGGTCCCTTAACACG qChAIL5-F1 CTACAGCCACCGCTTCATC qChAIL5-R1 GCTAGCGGCTATTGACTTGAG qChAIL5-F2 CTAACCACACCGTCCCTCAC qChAIL5-R2 GTAAAAAGTTCCTCCATGGTCATT qChFT-F1 CCA AGA GTT GAG ATT GGT GGA qChFT-
1. From the inflorescence of the mother plant, remove mature siliques as well as open flowers and buds that have already a white tip, with fine scissors or forceps.

2. Remove the meristem with those buds that are too small: usually 3-5 flower buds have the right size and should remain.

3. Open one flower bud by inserting the tip of one pair of forceps between petals and sepals. And remove all immature anthers with the other pair of forceps.

4. Repeat this for all remaining buds of the inflorescence.

5. Mark the emasculated inflorescence with a piece of thread around its stem.

6. Let the plant grow for 2-3 days.

7. The stigmas have by now developed a rough, sticky surface. Take an open, mature flower (but not yet yellow) from the father plant with one pair of forceps and bring it under the binocular. With the other forceps, take hold of the filament of an anther with visible pollen shedding. Tap the anther on the stigma and cover it with pollen grains as much as possible. Repeat for all stigmas. Take a second male flower if necessary.

8. Mark the pollinated inflorescence with a colored thread and document the cross (mother, father, date, color code, number of pollinated flowers). 9. Depending on the growth conditions, siliques with the hybrid seeds will be mature after 15-25 days. They are harvested by cutting them into a paper bag when the siliques are a little yellow, but prior to opening. They should be kept for a couple of days at room temperature for further maturation.

V.2.1.2 Seed germination tests

Dispose around 100 seeds in a 2 ml tube (eppendorf), add 1.5 ml 70% ethanol and shake or vortex for 5-8 min (be sure not to wipe off the label with the ethanol). Pipet off ethanol, add 1.5 ml 96% ethanol and shake or vortex for 5-8 min.

Use 1 ml tip to transfer seeds to sterilized filter paper, and then wait for seeds to dry completely. Sow the seeds carefully on petri dishes containing the growth media:

Murashige and Skoog (MS) salts, 0.8% agar with or without addition of 100 mM NaCl or 200 mM mannitol. To synchronize germination, stratify the seeds after sowing for 3 days at 4 o C and subsequently transfer to a growth chamber (22 o C, photoperiod 16 h light, 8 h dark). Score germination rates daily for 12 days following stratification. Seeds are considered to have germinated when radicle emergence is visible under a dissecting microscope.

V.2.1.3 Arabidopsis transformation using the floral dip method

Grow healthy Arabidopsis plants until they are flowering. Clip first bolts to encourage proliferation of many secondary bolts. Plants will be ready roughly 4-6 days after clipping. Optimal plants have many immature flower clusters and not many fertilized siliques, although a range of plant stages can be successfully transformed. Prepare an Agrobacterium tumefaciens strain carrying gene of interest on a binary vector. A single colony is inoculated in 3 ml LB with antibiotics and incubated at 28 °C overnight. This 3 ml overnight culture is then diluted in 300 ml LB supplemented with the same antibiotics and incubated at 28 o C for 16-24 hours. The bacterial cells are harvested by centrifugation at 5000 rpm for 10 min. Spin down Agrobacterium, resuspend to OD 600 = 0.8 (can be higher or lower) in 5% Sucrose solution (if made fresh, no need to autoclave). You will need 100-200 ml for each two or three small pots to be dipped. Before dipping, add Silwet L-77 to a concentration of 0.05% (500 ul/L) and mix well. Dip above-ground parts of plant in Agrobacterium solution for 1 min, with gentle agitation. You should then see a film of liquid coating the plant. Some investigators dip the inflorescence only, while others also dip the rosette to hit the shorter axillary inflorescences. Place dipped plants under a dome or cover for 16 to 24 hours to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight (air under dome can get hot). Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes, twist-ties, or other means. Stop watering as seeds become mature.

V.2.1.4 Transient expression using tobacco leaf infiltration

Inoculate one single colony of Agrobacterium (GV3103) in 5 ml LB with appropriate antibiotics. Grow overnight at 28-30°C. Using 1 ml of the overnight culture to inoculate 25 ml LB (with same antibiotics, plus 20 µM acetosyringone added after autoclaving and immediately before use Resuspension solution: 10 mM MgCl 2; 10 mM MES-KpH 5.6 (First make 0.5 M MES, adjust pH with KOH to 5.6); Autoclave 15 min. 100 µM acetosyringone, added after autoclaving and immediately before using.

.

Genotyping

Seeds (F1) from crosses were collected from individual siliques on the parent plants and these seeds were then grown and self-pollinated to obtain the F2 generation.

These F2 or later generation plants were genotyped by PCR. For the PCR reaction, at first, collect leaves for PCR sample and place them in 96-well plastic plate. Then add 200 µL Extraction Buffer to the tube. Crush leaf with plastic rod against the tube wall.

The solution turns transparent green, and visible tissue residue is left in the solution.

If you want to remove tissue residue, centrifuge tube at 14,000 rpm for 5 min and recover supernatant. This solution is stable at -20 °C for several months with or without tissue residue. Add 1 µL of this solution to a total volume of 20 µL of the PCR reaction.

Extraction Buffer: 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, and 0.5% SDS; dilute 10-fold with TE Buffer to obtain Extraction Buffer TE Buffer: 10 mM Tris-HCl (pH 8) and 1 mM EDTA

V.2.2.2 Gateway cloning

The Gateway cloning technology is a universal system for cloning and subcloning DNA sequences, facilitating gene functional analysis, and protein expression. In this operating system, DNA segments are transferred between vectors using site-specific recombination.

This technology contains two reactions: the BP reaction and the LR reaction.

The BP reaction is a recombination reaction between an expression clone (and an attB-flanked PCR product) and a donor (pDONR™) vector to create an entry clone.

The LR reaction is a recombination reaction between an entry clone and a destination vector, mediated by a cocktail of recombination proteins, to create an expression clone. It is used to move the sequence of interest to one or more destination vectors in parallel reactions.

BP reaction: Amplify PCR products containing the attB site and purify them. twice. After that reactions are incubated at 25°C for 60 min or overnight. Finally, add 1 µl of proteinase K solution and incubate for 10 min at 37°C to stop the reaction.

V.2.2.3 RNA isolation

15-days-old in vitro-grown seedlings or soil-grown other young tissues (100-200 mg) were inserted into an eppendorf tube containing glass beads (diameter 1 mm) and frozen in liquid nitrogen. Samples were ground using the Silama S5 apparatus (Ivoclar, Vivadent). Total RNA was extracted by using the Nucleospin RNA kit (Macherey-Nagel) according to the manufacturer's instructions. RNA concentration was evaluated by measuring the absorbance at 260 nm (1 unit of OD 260 = 40 µg/ml of RNA) using Nanodrop 2000.

V.2.2.4 Reverse transcription

In a 200 µl PCR tube, 11 µl of RNA (5 µg total RNA) with 5 µl of Rxn buffer (Promega) and 5 µl MgCl 2 (25 mM) were treated with 1 µl of RQ1 Dnase (Promega) at 37°C for 10 min and 65°C for 10 min to inactivate Dnase. 1 µl of oligo dT (100 mM) was added to the reaction. The reaction was incubated at 70°C for 5 min, after that it was put on ice immediately for 5 min. 2.5 µl of dNTPs (10 mM) and 1µl of ImProm-II Reverse Transcriptase (Promega) were added into the mixture. The reaction mixture was incubated at 42°C for 70 min, followed by heat inactivation at 70°C for 15 min. The synthesized cDNA was used as template for quantitative PCR.

V.2.2.5 Quantitative PCR

Quantitative PCR is a method used to detect relative or absolute gene expression levels. It was performed in 384-wells optical plates on a light cycler 480 (Roche) apparatus, according to the manufacturer's instructions. Each PCR reaction total volume was scaled to 10 µl. At first, pipette 2µl of primer mix (containing 2.5 mM forward and reverse gene specific primers) into each well. Then add 8 µl of PCR master mix (Roche) containing 5 µl of 480 SYBER Greenfluorescent reporter, 2 µl water and 1 µl template into each well. For each sample, PCR was performed in triplicate using fixed amounts of cDNA template and PP2A, EXP and Tip4.1 which were used as internal reference genes. The PCR was carried out using the following conditions: pre-heating at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C, 30 sec at 60°C and 15 sec at 72°C. Melting curves of PCR reactions were checked to ensure the quality of the PCR reaction and to avoid any DNA contamination. The threshold cycle value (CT) was set so that the fluorescent signal was above the baseline noise but as low as possible in the exponential amplification phase.

6-days-old wild type and Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 homozygous mutants seedlings were harvested. Three independently derived sets of 6-days-old seedlings (30 to 40 plants per set) were pooled for each genotype. Total RNA was isolated from each sample and used for hybridization on Agilent microarray slides. The microarray analysis was performed using Affymetrix Gene-Chips by Biochip Company (Shanghai, China). Data analysis was performed using GeneSpring 5 software (Silicon Genetics, Redwood City, CA). Genes were considered significantly mis-expressed in a mutant when the change in expression was at least 2fold compared to the wild type control and the P-values inferior to 0.05 in the three independent biological replicates.

For histochemical GUS activity assays, Arabidopsis seedlings were fixed for 30 min in ice-cold 90% acetone, then washed in 50 mM sodium phosphate buffer (pH7.2) for 15 min at room temperature and subsequently incubated for various periods (30 min to overnight) at 37°C in GUS staining solution (0.1 M sodium phosphate buffer pH7.2, 0.5 mM Fe(CN) 2 , 0.5 mM Fe(CN) 3 , 0.1% Tween-20 and 2 mM 5-bromo-4-choro-3-indolyl-3β-d-glucuronide). The length of the incubation period depends on the activity of each reporter gene construct. Seedlings were cleared in 70% ethanol overnight at 4°C.

V.2.5.1 Nuclear protein extraction

Grow Arabidopsis seeds on MS for 2 weeks or on soil for 4 weeks. Collect approximately 5 g of Arabidopsis tissues, freeze in liquid nitrogen, and then follow the steps listed below. Note: always keep the sample on ice.

1. Grind the tissues to a fine powder in liquid nitrogen (3-5 ml materials) using a cold mortar and pestle. Collect the powder into a 50 mL Falcon tube.

2. Add 20 mL cold Lysis buffer into the powder, vortex and place on a rotation wheel for 30 min at 4°C.

3. Filter the solution through a 100 µm nylon mesh.

4. Centrifuge the filtered homogenate at 4000 x g at 4°C for 20 min to pellet the nuclei.

5. Discard the supernatant and add 2 mL Lysis buffer to the pellet. Re-suspend the nuclei by pipetting and transfer to a 2 ml tube.

6. Centrifuge the sample at 4000 x g at 4°C for 20 min.

7. Discard the supernatant and add 150 to 400 µl (depending on the starting powder quantity) 1xSDS loading buffer to the pellet and vortex.

8. Incubate at 95°C, 10 min. 9. Centrifuge 5 min at 12000 rpm at room temperature.

10. Remove the supernatant to a new tube. Use 10 µl to load for western blot.

Material can be stored at -20°C for no more than 2-3 weeks.

11. Test with corresponding antibody and/or @ H3 to adjust quantities between samples.

Low salt wash buffer (200 ml): 20 ml 0.5 M HEPES pH 7.5 + 6 ml 5 M NaCl + 400 µl 500 mM EDTA ( keep at 4°C) Lysis Buffer (50 ml): 45 ml Low salt wash buffer + 500 µl Triton X-100 + 5 ml glycerol + 50 µl 100 mM PMSF + 20 µl β-mercaptoethanol (on ice) (freshly prepared)

PMSF should be kept at RT for 5-10 min and added last!

V.2.5.2 Protein quantification

Three times 10 µL of each sample are pipetted in three tubes. 90 µL of water and 500 µL Amidoblack staining solution are added and well mixed before 10 min centrifugation at full speed. The supernatant is removed, and the pellet is washed with 750 µL washing buffer by inverting and following centrifugation 10 min at full speed.

Washed amidoblack pellets are let dry under the hood for 30 min. Coomassie destaining solution (1L): 400 mL Ethanol 100%; 100 mL Acetic Acid; 500 mL H2O

V.2.5.4 Western blot

After protein samples are separated by 10%-15% SDS-PAGE, the gel is equilibrated in transfer buffer for at least 10 min before transfer. Immobilon-P PVDF transfer membrane (Millipore) is pre-wetted by 100% methanol, rinsed in water for 5 min and equilibrated in transfer buffer for at least 10 min before use. Then proteins are transferred onto the immobilon-P PVDF membrane in the transfer buffer at 300 mA for 2 hours at 4°C. The membrane is subsequently washed in 1xTTBS for 5 min, blocked in 5% non-fat milk in TTBS for 1 hour, and incubated in diluted primary antibody (1:500-1:5000) at 4°C overnight. After washing 3 times with the milk-TTBS, the membrane is incubated in the diluted secondary antibody (1:5000-1:10000) for 1 hour at room temperature. Then the membrane is washed once with milk-TTBS, 3 times with TTBS, and each time for 10 min. Finally the membrane is detected using the ECL western blot detection kit (Amersham Biosciences).

Transfer buffer: 25 mM Tris; 192 mM glycine; 15% methanol TBS buffer: 20 mM Tris-HCl pH7.4; 150 mM NaCl TTBS buffer: TBS buffer plus 0.1% Triton X-100

V.2.5.5 Recombinant protein expression in E. coli

Complete ORFs of target genes were subcloned into an appropriate expression vector. All constructs were introduced into E. coli Rosetta (DE3) strain.

The next day, the bacteria on the plate are transferred to liquid LB medium for an overnight culture. The overnight culture is transferred into auto-induction medium at a dilution 1: 300, incubated for 3 hours at 37°C, then the temperature is changed to 25°C and the culture is incubated overnight. Cells are harvested and resuspended in 1xSDS loading buffer. Protein expression is detected by SDS-PAGE.

Auto-induction medium (1 L): Tryptone 10 g; yeast extract 5 g; 50 X M 40 ml; 50 X 5052 40 ml; 1 M MgSO 4 2 ml; adjust pH to 7.2 50 X M: 1.25M Na 2 HPO 4 ; 1.25M KH 2 PO 4 ; 2.5M NH 4 Cl; 0.25M Na 2 SO 4 50 X 5052: 25% glycerol; 2.5% glucose; 10% alpha-lactose monohydrate

V.2.5.6 GST fusion protein purification

GST-fusion protein purification was carried out by harvesting and resuspending the cell in 20 ml of ice-cold GST lysis buffer plus 100 µg/ml lysozyme and protease inhibitor cocktail (Roche). After disrupting the cells by sonification, the cell lysate was centrifuged at 13200 rpm for 20 min at 4°C. Then the supernatant was mixed with settled glutathione-Sepharose-4B beads (Amersham Bioscences), which was pre-washed 3 times with water and 3 times with 1x PBS. The suspension was then incubated on a rotation wheel for 2 hours or overnight at 4°C. Beads are spinned down, washed once in 1x PBS plus 0.1% Triton X-100 and 3 times in 1x PBS plus 10% glycerol. Finally, 1x PBS plus 10% glycerol was added to make 10% slurry. Proteins fixed to the beads were determined by SDS-PAGE.

PBS: 140 mM NaCl; 2.7 mM KCl; 10 mM Na 2 HPO 4 ; 1.8 mM KH 2 PO 4 ; pH7.4

GST lysis buffer: 1X PBS; 0.1% Triton X-100; 1 mM DTT; 10% glycerol

V.2.5.7 GST pull-down assay

15-day-old stably transformed Arabidopsis seedlings were harvested. After grinding in liquid nitrogen, 5 g fine powder was homogenized in 30 ml pull-down buffer plus protease inhibitor cocktail (Roche). Dnase(Roche) was added to 10 µg/ml as a final concentration to release the chromatin proteins. The whole cell lysate was centrifuged at 20000 g for 20 min at 4°C. The supernatant was collected and mixed with GST or GST fusion proteins. After rotating for 2 hours or overnight at 4°C on a wheel, the beads were washed four times with pull-down buffer. After washing, specifically bound proteins were eluted from the beads by pull-down buffer containing 1 M NaCl and then precipitated by 10% TCA. A quarter of each pull-down fraction was analyzed by SDS-PAGE and western blot using relative polyclone antibody.

Pull-down buffer: 20 mM Tris-Cl (pH 8.0); 200 mM NaCl; 1 mM EDTA (pH 8.0); 0.5% Nonidet P-40; 25 µg/mL PMSF

V.2.5.8 Fluorescence lifetime imaging (FLIM) assay

The AtZRF1b, AtRING1a, AtBMI1a, AtBMI1b and AtBMI1c cDNAs were PCR-amplified and introduced into the Gateway system and cloned as 39 or 59 inframe fusions to RFP or GFP sequences in plant expression vectors downstream of the 35S promoter (pB7WGF2; pB7FWG2; pH7WGR2; pH7RWG2; http:// gateway. psb.ugent.be/). Plasmids were introduced into A. tumefaciens (GV3101). Bacterial cultures grown overnight were centrifuged and pellets resuspended in 10 mM MgCl 2 to an optical density of 0.5 at 600 nm and incubated 2-3 h at RT. Leaves of 2-3 week old Nicotiana benthamiana plants were co-infiltrated with an equimolar bacterial suspension of the two constructs to be tested. Confocal laser scanning images of protein co-localization and FLIM data were recorded 2 days post-infiltration (LSM-700, Carl Zeiss; LIFA frequency domain fluorescence lifetime imaging system, Lambert Instruments). The percentage of GFP fluorescence lifetime decay was calculated relative to the absence of RFP fusion protein as an average of 3 biological replicates, each recording over 30 nuclei. Proteins were considered to interact if the presence of RFP-tagged proteins decreased GFP fluorescence lifetime by more than 5%, a reference value established according to the negative control: RFP with GFP.

Day 1: Arabidopsis two weeks old seedlings were harvested in 100 ml fixation buffer. Following vacuum infiltration for 10 min for cross-linking, the fix buffer should boil (too much cross-linking may mask epitope while too little crosslinking leads to incomplete fixation). Subsequently 5 ml freshly prepared 2.5 M glycine were added per 100 ml fix buffer and infiltration in vacuum was continued for a further 5 min to stop crosslinking. Seedlings were rinsed with MilliQ water 5 times and as much as possible water was removed (seedlings after fixation should be transparent and dark-green. They should sink to the bottom of beaker after merged into water). The dried seedlings were subsequently ground in liquid nitrogen to a fine powder and kept at -80°C (remember DO NOT over-grind! Grinding should be done only twice per material, which means the powder may not be very fine).

Day 2: To 5 ml power of each sample 30 ml of ChIP lysis buffer was added and the mixture was incubated on a rotation wheel for 40 min at 4°C. Then it was filtered through 100 µm nylon mesh and centrifuged for 20 min at 4000 rpm at 4°C.

After removing the supernatant, the pellet may appear grey; otherwise the pellet was resuspended again in ChIP lysis buffer and centrifuged again. The pellet was then resuspended in 700 µl of ChIP lysis buffer and the solution was transferred to a new 1.5 ml tube. The solution was centrifuged 20 min at 4000 rpm at 4°C. As much as possible of the supernatant was removed and the pellet was resuspended in 180 µl lysis buffer plus 0.8% SDS (the final SDS concentration should be between 0.5% and 1%. The more SDS added, the smaller the DNA fragment would be after sonication).

Chromatin in ice water was sonicated four times with a Bioruptor (Diagenode), for every cycle work 30 sec and pulse 30 sec for 5 min. The chromatin sample was then centrifuged for 10 min at 13000 rpm at 4°C and the supernatant was transferred to a new tube (chromatin can be frozen at -80°C at this point). To check the sonication efficiency, 40 µl of solution was taken and 360 µl ChIP elution buffer and 16 µl of 5 M NaCl were added and incubated at 65°C least 6 hour or overnight. DNA was recovered by adding equal volume of phenol:chloroform and precipitated following addition of 1/10 volume of 3 M sodium acetate pH5.2, 2 volumes of 100% ethanol and 1 µl of glycogen. After washing with 70% ethanol, the pellet was resuspended in 30 µl of water. Then 5 µl DNA was treated with 1 µl RNase and used for gel electrophoresis to control the quality of the sonication (DNA fragments should be 100-500 bp). Later, this DNA was used as an input for qPCR analysis.

If the sonication was correct, the chromatin solution was diluted with adequate ChIP lysis buffer to make the final volume enough for the following experiments (generally speaking, 100 µl chromatin should be taken for input and 200 µl for every sample). For every sample, combine 1.2 ml antibody binding buffer with 200 µl chromatin in one 1.5 ml tube. Add antibodies to each tube (from 1 to 3 µl depending on the antibody, but usually using 2 µl is fine) and incubate these tubes on a rotating mixer wheel overnight at 4°C. In parallel, chromatin without any antibody was used as a mock control.

Day 3: The chromatin antibody complex was collected by adding 20 µl slurry of magnetic Protein A beads (Millipore) for one reaction and incubated at 4°C for 1-3 hours under rotation. The beads were pelleted using a Magana GrIP racks (Millipore) and washed successively with 1 ml low salt wash buffer, and rotated at 4°C for 10 min. Then the supernatant was removed, the pellet was washed with 1 ml high salt wash buffer, and rotated at 4°C for 10 min. Then it was washed with 1 ml LiCl wash buffer, and rotated again at 4°C for 10 min. Finally it was washed with 1 ml TE buffer, and rotated at room temperature for 10 min. After the last wash, as much as poossible TE buffer was removed and immune complexes were eluted by adding 400 µl freshly prepared Elution buffer and incubation at 65°C for 30 min with agitation. The supernatant was transferred to another 1.5 ml EP tube. 300 µl Elution buffer were added to the prepared input. Then add 16 µl elution and input. Reverse protein-DNA cross-links at 65°C overnight. The Nikon E800 microscope was used for GUS staining observations. The LSM 700 Laser Scanning Microscopy (Carl Zeiss) was used for root cell observations.

The images were processed with ZEN software (Carl Zeiss).

Seedlings were grown in 1/2MS agar plates vertically for 5 days. Roots were incubated in the dark for 10 min in 15 µM (10 µg/mL) propidium iodide (PI) (Invitrogen). Then rinsed twice in water. Following transfer of the roots onto a microscopic slide, they were covered with a cover slip and observed under the LSM 700 microscope.

A small quantity of rosette leaves were roughly chopped Gently resuspend each pellet in 200 ml ice-cold dH 2 0. Centrifuge at 4000 rpm, 25 min at 4°C. Place bottles on ice. Remove supernatant. Gently resuspend each pellet in 100 ml of ice-cold dH 2 0. Centrifuge at 4000 rpm, 25 min at 4°C. Place bottles on ice.

Remove supernatant. Gently resuspend each pellet in 20 ml ice-cold 10% glycerol.

For each pair of 250 ml Corning bottles, transfer both 20 ml cell suspension into one chilled 50 ml conical tube. Therefore one should end up with two 50 ml conical tubes on ice where each tube contains ~40 ml of cells in 10% glycerol. Centrifuge at 4000 rpm, 10 min at 4°C. Place tubes on ice. Remove supernatant. Gently resuspend each cell pellet in 1 ml of ice-cold 10% glycerol. Finally prepare 70 µl aliquots of cells in pre-chilled 1.5 ml eppendorf tubes. Snap freeze tubes containing cells in liquid N2.

Store frozen cells at -80°C. Finalement, j'ai étudié les niveaux de H3K4me3, H3K27me3 et H2Aub1 dans la chromatine de certains gènes dont l'expression est perturbée dans les doubles mutants. Les résultats montrent que la dé-ubiquitination de H2Aubi1 n'est pas un événement majeur dans la régulation de la transcription chez Arabidopsis.

V.2.10.2 Heat shock transformation

SUMMARY

Studies in animals showed that ZRF1 can read the histone H2AK119ub1 modification in the derepression of polycomb-repressed genes. Two homologs of human ZRF1 have been identified in the Arabidopsis genome, and hereinafter are named AtZRF1a and AtZRF1b. So far, their functional characterization had not been reported yet.

My first objective was to acquire basic knowledge about AtZRF1a and AtZRF1b. Both genes are broadly expressed in Arabidopsis plants and the AtZRF1b protein is localized in the nucleus and the cytoplasm. Moreover, we found that AtZRF1b binds H2Aub1 with characteristics similar to those previously reported for the human ZRF1 protein.

I subsequently used the powerful genetic tools available in Arabidopsis to investigate the functions of AtZRF1a and AtZRF1b. Several independent T-DNA insertion Arabidopsis mutant lines were identified. Because of functional redundancy, single mutants have no obvious developmental defects. I therefore focused on double mutants displaying loss of function of both AtZRF1a and AtZRF1b. The study of a double mutant revealed important roles for these genes in plant growth and development ranging from cell proliferation and differentiation to flowering time control.

I then investigated the roles of AtZRF1a and AtZRF1b in gene transcriptional regulation and found that AtZRF1a and AtZRF1b function in a way that is partially similar to PRC1 function. Lastly, I investigated H3K4me3, H3K27me3 and H2Aub1 levels in the chromatin regions of some expression-perturbed genes in double mutants. The results show that ZRF1-mediated deubiquitination of H2Aub1 is not a major event in transcriptional regulation in Arabidopsis.

INTRODUCTION

Ubiquitin (Ub) and Ub-like (e.g., SUMO) proteins constitute a family of modifiers that are linked covalently to target proteins. Although ubiquitination (also called ubiquitylation or ubiquitinylation) first came to light in the context of protein destruction, it is now clear that ubiquitination can also carry out proteolysis-independent functions. Ubiquitination can alter biochemical, molecular and/or subcellular localization activities of a target protein. The first ubiquitinated protein to be described was histone H2A in calf thymus, a finding dated more than 36 years ago (Goldknopf et al., 1975;Hunt and Dayhoff, 1977). Yet, only more recently have the underlying mechanisms and regulatory functions of histone ubiquitination begun to emerge (reviewed in Zhang, 2003;Shilatifard, 2006;Weake and Workman, 2008;Braun and Madhani, 2012;Pinder et al., 2013). Histones are highly alkaline proteins, found in the nuclei of eukaryotic cell, which package and order the DNA into structural units named nucleosomes. A nucleosome is composed of roughly 146 bp of DNA wrapping around the histone octamer comprising two molecules each of the four core histones H2A, H2B, H3, and H4 (Luger et al., 1997). Histone monoubiquitination together with other types of posttranslational modifications, e.g., acetylation, methylation, phosphorylation, and SUMOylation, can modulate nucleosome/chromatin structure and DNA accessibility and thus regulate diverse DNA-dependent processes, such as genome replication, repair, and transcription (Zhang, 2003;Shilatifard, 2006;Weake and Workman, 2008;Braun and Madhani, 2012;Pinder et al., 2013).

Ubiquitination occurs via conjugation of the C-terminal residue of Ub to the side chain of a lysine (K) residue of the substrate/acceptor protein, a reaction involving three coordinated enzymatic activities (reviewed in Hershko and Ciechanover, 1998). Ub is first activated by an ATP-dependent reaction involving the Ub-activating enzyme E1, then conjugated to the active site cysteine residue of the Ub-conjugating (UBC) enzyme E2, and finally transferred to the target K residue of the substrate protein by the Ub-protein isopeptide ligase E3. Most organisms have only one E1, but dozens of different E2 and hundreds up to thousands of different E3 enzymes, providing the need in coping with effective substrate specificity (Hua and Vierstra, 2011;Braun and Madhani, 2012). Identification and characterization of E3s and some E2s involved in histone ubiquitination had been a key for understanding biological functions of histone ubiquitination in various organisms. Because of its suitability for genomics, genetics, and cellular and molecular biological approaches, Arabidopsis thaliana is an ideal model to investigate histone ubiquitination functions. In this review, we focus on this reference plant to expose current progress made on ubiquitination of different types of histones.

H2B MONOUBIQUITINATION IN Arabidopsis

GENOME-WIDE DISTRIBUTION OF H2Bub1

Monoubiquitinated H2B (H2Bub1) was first discovered in mouse cells and was estimated to represent about 1-2% of total cellular H2B (West and Bonner, 1980). Later, H2Bub1 was detected widely throughout eukaryotes spanning from yeast to humans and plants (Zhang, 2003;Shilatifard, 2006;Sridhar et al., 2007;Zhang et al., 2007a;Weake and Workman, 2008). The ubiquitination site is mapped to a highly conserved K residue, H2BK123 in budding yeast, H2BK119 in fission yeast, H2BK120 in humans, and H2BK143 in Arabidopsis.

Feng and Shen

Histone monoubiquitination

Genome-wide analysis revealed that in Arabidopsis as in animals H2Bub1 is associated with active genes distributed throughout the genome and marks chromatin regions notably in combination with histone H3 trimethylated on K4 (H3K4me3) and/or with H3K36me3 (Roudier et al., 2011). During early photomorphogenesis, gene upregulation was found to be associated with H2Bub1 enrichment whereas gene downregulation did not show detectable correlation with any H2Bub1 level changes (Bourbousse et al., 2012). In general, H2Bub1 is considered to represent an active chromatin mark broadly involved in genome transcription regulation.

ENZYMES INVOLVED IN REGULATION OF H2Bub1 LEVELS

The budding yeast Rad6 (radiation sensitivity protein 6) was the first factor identified and shown to work as an E2 enzyme involved in catalyzing H2Bub1 formation both in vitro and in vivo (Robzyk et al., 2000). It contains a highly conserved catalytic UBC domain of approximately 150 amino acids in length with an active-site cysteine for linking Ub. The E3 enzyme working together with Rad6 in catalyzing H2Bub1 formation in budding yeast is Bre1 (Brefeldin-A sensitivity protein 1), which contains a C3HC4-type RING finger domain typical for all E3s (Hwang et al., 2003;Wood et al., 2003). The depletion of either Rad6 or Bre1 eliminates genome-wide H2Bub1 and causes yeast cell growth defects (Robzyk et al., 2000;Hwang et al., 2003;Wood et al., 2003). Human contains at least two homologs of Rad6, namely hHR6A and hHR6B, and two homologs of Bre1, namely RNF20/hBRE1A and RNF40/hBRE1B (Kim et al., 2005;Zhu et al., 2005). In Arabidopsis, three homologs of Rad6, namely UBC1, UBC2, and UBC3, were identified and UBC1 and UBC2 but not UBC3 were shown to be redundantly responsible for H2Bub1 formation in planta (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009). The two Bre1 homologs HUB1 (HISTONE MONOUBIQUITINATION 1) and HUB2 work non-redundantly, possibly as a hetero-tetramer composed of two copies of HUB1 and two copies of HUB2, in catalyzing H2Bub1 formation in Arabidopsis (Fleury et al., 2007;Liu et al., 2007;Cao et al., 2008). H2Bub1 levels are drastically reduced or undetectable in Western blot analysis in the loss-offunction hub1 and hub2 single mutants as well as in the hub1 hub2 and ubc1 ubc2 double mutants, but are unaffected in the ubc1, ubc2, and ubc3 single mutants or in the ubc1 ubc3 and ubc2 ubc3 double mutants (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009).

H2Bub1 levels are also regulated by deubiquitination enzymes. Two Ub-specific proteases, Ubp8 and Ubp10, are involved in deubiquitination of H2Bub1 in budding yeast. Strikingly, while Ubp8 acts as a component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex specifically in H2Bub1 deubiquitination in transcription activation, Ubp10 functions independently of SAGA and primarily acts in Sir-mediated silencing of telomeric and rDNA regions (reviewed in Weake and Workman, 2008). In human, USP22 acts as Ubp8 ortholog in a SAGA complex in H2Bub1 deubiquitination (Weake and Workman, 2008). In Arabidopsis, although a SAGA complex remains uncharacterized so far, the Ub protease UBP26/SUP32 has been shown to deubiquitinate H2Bub1 involved in both heterochromatic silencing (Sridhar et al., 2007) and transcription activation of the FLC (FLOWERING LOCUS C) gene (Schmitz et al., 2009). More recently, the otubain-like deubiquitinase OTLD1 was reported as implicated in deubiquitination of H2BUb1 and repression of At5g39160, a gene of unknown function (Krichevsky et al., 2011).

ROLE OF H2Bub1 IN FLOWERING TIME REGULATION

The timing of flowering is critical for the reproductive success of plants. As compared to wild type, the hub1 and hub2 single mutants as well as the hub1 hub2 and ubc1 ubc2 double mutants exhibit an early flowering phenotype whereas but the ubc1, ubc2, and ubc3 single mutants and the ubc1 ubc3 and ubc2 ubc3 double mutants have a normal phenotype (Cao et al., 2008;Gu et al., 2009;Xu et al., 2009). This early flowering phenotype is detectable under both long-day and short-day photoperiod plant growth conditions. Molecular analyses of the mutants indicate that H2Bub1 controls flowering time primarily through transcriptional activation of FLC (Figure 1). FLC encodes a key transcription repressor involved in both the autonomous/developmental and vernalization flowering pathways, and its active transcription is associated with several histone marks, e.g., H3K4me3, H3K36me2/3 and H2Bub1 (reviewed in Berr et al., 2011). In the early flowering mutants hub1, hub2, hub1 hub2, and ubc1 Frontiers in Plant Science | Plant Genetics and Genomics ubc2, FLC expression levels are reduced and the FLC chromatin shows reduced H2Bub1 levels (Cao et al., 2008;Gu et al., 2009). The loss-of-function mutant ubp26/sup32 showed also an early flowering phenotype and reduced FLC expression but an elevated level of H2Bub1 in the FLC chromatin (Schmitz et al., 2009), indicating that not only H2Bub1 formation but also H2Bub1 removal are necessary for FLC transcription. Accompanying H2Bub1 reduction compromised levels of H3K4me3 and to a less extent H3K36me2 were detected at FLC in hub1 and ubc1 ubc2 (Cao et al., 2008), and reduced level of H3K36me3 but elevated level of H3K27me3 was observed at FLC in ubp26/sup32 (Schmitz et al., 2009). On parallels to the knowledge in yeast, it was proposed that the UBC-HUB-mediated H2Bub1 formation is necessary for H3K4me3 deposition at transcription initiation whereas UBP26/SUP32-mediated H2Bub1 removal is required for H3K36me3 deposition during transcription elongation (Cao et al., 2008;Schmitz et al., 2009). Nonetheless, this hierarchy of histone modifications needs to be cautioned because multiple factors are involved in H3K4me3 and H3K36me2/3 depositions and the SDG8 (SET DOMAIN GROUP 8)-mediated H3K36me2/3 deposition remarkably override H3K4me2/3 deposition in FLC transcription (Yao and Shen, 2011;Shafiq et al., 2014). Besides FLC, Arabidopsis has five FLC paralogs, namely MAF1 (MADS AFFECTING FLOWERING 1), MAF2, MAF3, MAF4 and MAF5. Some MAFs are also downregulated in the early flowering mutants hub1, hub2, hub1 hub2, ubc1 ubc2, and ubp26/sup32 (Cao et al., 2008;Gu et al., 2009;Schmitz et al., 2009;Xu et al., 2009). Thus, H2Bub1 may also regulate flowering time through control of MAF gene expression under some plant growth conditions.

H2Bub1 FUNCTION IN OTHER PROCESSES

In addition to flowering, many other processes also involve H2Bub1 as evidenced by studies of the Arabidopsis hub1 and hub2 mutants. The hub mutants display reduced seed dormancy associated with reduced expression of several dormancy-related genes, including DOG1 (DELAY OF GERMI-NATION 1), ATS2 (ACYLTRANSFERASE 2), NCED9 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 9), PER1 (CYSTEINE PEROXIREDOXIN 1), and CYP707A2 (Liu et al., 2007). At vegetative growth stages, the hub mutants exhibit pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited root growth (Fleury et al., 2007). Cell cycle genes, particularly some key regulators of the G2-to-M transition, are downregulated, which could largely explain the plant growth defects of the hub mutants (Fleury et al., 2007). A more recent study shows that several circadian clock genes, including CCA1 (CIR-CADIAN CLOCK ASSOCIATED 1), ELF4 (EARLY FLOWERING 4) and TOC1 (TIMING OF CAB EXPRESSION 1), are downregulated and their chromatin regions contain lower levels of H2Bub1 in the hub mutants, suggesting that H2Bub1 may contribute to the regulation of plant growth fitness to environment through expression modulation of some circadian clock genes (Himanen et al., 2012). It is worth to note that SDG2-mediated H3K4me3 deposition is also required for expression of several circadian clock genes (e.g., CCA1, TOC1) and the hub mutants exhibit reduced levels of H3K4me3 in chromatin regions of the circadian clock genes (Himanen et al., 2012;Malapeira et al., 2012).

During photomorphogenesis, hundreds of genes show upregulation associated with H2Bub1 enrichment in their chromatin in response to light exposure (Bourbousse et al., 2012). Strikingly, over 50% of these genes gain H2Bub1 enrichment upon the 1 h of illumination, illustrating the highly dynamic nature of H2Bub1 deposition during a likely cell division-independent genome regrogramming process. In contrast to the above discussed cases, in this study the H2Bub1 changes is neither accompanied by any detectable changes of H3K36me3 nor required for H3K4me3 enrichment following six hours of light exposure (Bourbousse et al., 2012). In line with the function of H2Bub1 in gene activation in response to light, the hub1-3 mutant seedlings are overly light sensitive, exhibiting a photobleaching phenotype (Bourbousse et al., 2012).

The hub1 mutants also show increased susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola (Dhawan et al., 2009). Precise role of H2Bub1 in plant defense against pathogens still remains largely unclear. Structure defects, e.g., thinner cell walls and altered surface cutin and wax compositions, together with impaired induction of some defense genes might have partly contributed to the increased susceptibility to pathogen infection in the hub mutant plants (Dhawan et al., 2009;Ménard et al., 2014). It is worthy noting that the sdg8 mutants impaired in H3K36me3 deposition also display reduced resistance to necrotrophic fungal pathogen infection (Berr et al., 2010(Berr et al., , 2012;;Palma et al., 2010). It will be interesting to study in future research whether a trans-histone crosstalk between H2Bub1 and H3K36me3 acts on transcription induction in plant response to pathogens.

MECHANISMS OF H2Bub1 IN TRANSCRIPTION REGULATION

So far only limited information is available concerning how H2Bub1 enzymes are recruited to the target chromatin. The evolutionarily conserved PAF1 (Polymerase Associated Factor 1) complex interacts with Pol II (RNA polymerase II) and plays a role as a "platform" for association of enzymes involved in H2bub1, H3K4me3, and H3K36me2/3 deposition, linking histone modifications with active transcription (Shilatifard, 2006;Weake and Workman, 2008;Berr et al., 2011;Braun and Madhani, 2012). A direct interaction between PAF1 complex and Rad6-Bre1 has been detected and shown as required for catalyzing H2Bub1 formation (Xiao et al., 2005). As in yeast and animals, deletion or knockdown of PAF1 components markedly reduces H2Bub1 in Arabidopsis (Schmitz et al., 2009). Genetic analysis shows that HUB2 and ELF8 encoding a PAF1 subunit act in a same floral-repression pathway in Arabidopsis flowering time regulation (Gu et al., 2009). Although physical interaction between UBC-HUB and PAF1 needs future investigation, interactions were observed between UBC and HUB (Cao et al., 2008) and between HUB and MED21 (mediator complex subunit 21), a subunit of the evolutionarily conserved Mediator complex (Dhawan et al., 2009). Mediator complex is associated with both general transcription factors and Pol II and is essential for activator-dependent transcription in all eukaryotes (for a recent review, see Carlsten et al., 2013). Nevertheless, the aforementioned www.frontiersin.org interactors are generally involved in Pol II transcribed genes and thus cannot fully explain why UBC-HUB targets some but not all active genes. It is reasonable to speculate that UBC-HUB recruitment might also involve some gene-specific yet uncharacterized factors.

The next question is how H2Bub1 affects transcription. In yeast and animals, H2Bub1 can promote transcription elongation by enhancing the recruitment of RNA Pol II and by facilitating nucleosome removal through interplay with FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone complex (Pavri et al., 2006;Tanny et al., 2007). FACT acts on displacement of H2A/H2B dimer from a nucleosome core, facilitating transcription elongation on chromatin template. In Arabidopsis, FACT genetically interacts with HUB1 and plays critical roles in multiple plant developmental processes (Lolas et al., 2010). Yet its precise interplay with H2Bub1 in transcription regulation needs future investigations.

Alternatively or additionally, H2Bub1 may regulate transcription indirectly through crosstalk with H3K4me3 and H3K36me2/3 (Shilatifard, 2006;Weake and Workman, 2008;Berr et al., 2011;Braun and Madhani, 2012). In line with this idea, lack of H2Bub1 in Arabidopsis impairs H3K4me3 and H3K36me2 formation in chromatin at FLC and clock genes (Cao et al., 2008;Himanen et al., 2012), and elevated H2Bub1 inhibits H3K36me3 formation in the FLC chromatin (Schmitz et al., 2009). Nevertheless, in contrast to the requirement of H2Bub1 for genome-wide H3K4me3 formation in yeast, lack of H2Bub1 in Arabidopsis barely affects global H3K4me2/3 and H3K36me2/3 levels, as evidenced by Western blot analysis (Cao et al., 2008;Dhawan et al., 2009;Gu et al., 2009) as well as by ChIP (chromatin immunoprecipitation) analysis of light responsive genes during photomorphogenesis (Bourbousse et al., 2012). It is currently unclear to which extent applies the crosstalk of H2Bub1 with H3K4me2/3 and H3K36me2/3 in Arabidopsis gene transcription regulation and what are the molecular mechanisms underlying the crosstalk.

Finally, while H2Bub1 is generally associated with active gene transcription, it can also regulate transcription repression in a chromatin context-dependent manner. The ubp26/sup32 mutant shows release of transgene and transposon silencing (Sridhar et al., 2007) as well as elevated expression of PHE1 (PHERES1) associated with seed developmental defects (Luo et al., 2008). It has been shown that the silencing release is accompanied by reduction of H3K9me2 and of siRNA-mediated DNA methylation and the PHE1 expression elevation is associated with a reduced level of H3K27me3. Nevertheless, whether these changes of repressive marks are directly linked with H2Bub1 still need to be investigated.

H2A MONOUBIQUITINATION IN Arabidopsis

PRESENCE OF H2Aub1

In contrast to H2Bub1, H2Aub1 has not been found in yeast and has been generally implicated in transcription repression in animal cells (Weake and Workman, 2008;Braun and Madhani, 2012). Albeit its early discovery and high abundance (about 5-15% of the total H2A) in animal cells (Goldknopf et al., 1975;Hunt and Dayhoff, 1977;Zhang, 2003), H2Aub1 function has only more recently begun to be elucidated, thanking to the first identification of the human PRC1 (Polycomb repressive complex 1) component Ring1B (also known as Ring2 and RNF2) as a E3 involved in catalyzing H2Aub1 formation (Wang et al., 2004). In Arabidopsis, H2Aub1 was undetectable in a large-scale analysis of histone posttranslational modifications by mass spectrometry (Sridhar et al., 2007;Zhang et al., 2007a) and had been thought for a long time to be non-existent (Weake and Workman, 2008). However, five PRC1-like RING-finger proteins, namely AtRING1a, AtRING1b, AtBMI1a, AtBMI1b, and AtBMI1c, have been identified in Arabidopsis (Sanchez-Pulido et al., 2008;Xu and Shen, 2008). More recent immunodetection and in vitro enzyme activity assays have revealed that these RING-finger proteins are effectively involved in catalyzing H2Aub1 formation in Arabidopsis (Bratzel et al., 2010;Li et al., 2011;Yang et al., 2013).

PRC2 AND PRC1 IN H2Aub1 DEPOSITION

Polycomb group (PcG) proteins, first identified in Drosophila as repressors of homeotic (Hox) genes, are nowadays known to act in multiprotein complexes in transcription repression of a large number of genes in many multicellular organisms including plants (Bemer and Grossniklaus, 2012;Molitor and Shen, 2013;Schwartz and Pirrotta, 2013;Simon and Kingston, 2013). The most intensively studied complexes are PRC1 and PRC2. In Drosophila, PRC2 is composed of four core subunits, namely Ez (Enhancer of zeste), Suz12 (Suppressor of zeste 12), Esc (Extra sex combs) and N55 (a 55 kDa WD40 repeat protein), and PRC1 also contains four main subunits, namely Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs) and Ring1 (also known as dRing). In mammals, alternate subunit compositions create larger families of related PRC2-type and PRC1-type complexes (Schwartz and Pirrotta, 2013;Simon and Kingston, 2013). Nevertheless, defined biochemical activities of PRC2 and PRC1 are conserved from flies to humans. The classical model proposes a sequential mode of action of the two complexes: PRC2 catalyzes H3K27me3 formation, and PRC1 recognizes the H3K27me3 mark and further mediates downstream H2Aub1 deposition. The PRC1 components, acting as E3 ligases in H2Aub1 formation, are RING-finger proteins: Ring1 in Drosophila and Ring1A and Ring1B in human (Braun and Madhani, 2012;Schwartz and Pirrotta, 2013).

In Arabidopsis, the four PRC2 core components are highly conserved (Figure 2) and encoded by small gene families, and their function in H3K27me3 deposition and transcription repression have been intensively studied (Bemer and Grossniklaus, 2012). In contrast, PRC1 compositions are drastically diverged in plants as compared to animals (Molitor and Shen, 2013). No sequence homologue of Ph could be identified in plants so far. LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), also known as TFL2 (TER-MINAL FLOWER 2), binds H3K27me3 and may play a Pc-like function (Turck et al., 2007;Zhang et al., 2007b). This remarkably differs from the distinct roles of HP1 and Pc in animals, where HP1 binds H3K9me3 involved in hetereochromatin formation whereas Pc binds H3K27me3 involved in PRC1-mediated silencing in euchromatin. The best conservations found about PRC1 core components are from RING-finger proteins structured by a RING domain at N-terminus and a Ub-like RAWUL domain at C-terminus (Sanchez-Pulido et al., 2008;Xu and Shen, 2008).

These RING-finger proteins can be classified into two phylogenic groups: the first group comprises Drosophila Ring1, human Ring1A and Ring1B, and Arabidopsis AtRING1a and AtRING1b; the second group comprises Drosophila Psc, human Bmi1, and Arabidopsis AtBMI1a, AtBMI1b, and AtBMI1c. Consistent with their sequence conservation, AtRING1a, AtRING1b, AtBMI1a, and AtBMI1b each can ubiquitinate H2A in vitro, and loss of function of AtBMI1a and AtBMI1b causes H2Aub1 reduction in planta (Bratzel et al., 2010;Yang et al., 2013).

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN STEM CELL MAINTENANCE

Plant growth and development largely depend on stem cells located in SAM (shoot apical meristem) and RAM (root apical meristem), whose activities are fine-tuned by multiple families of chromatin factors (Sang et al., 2009;Shen and Xu, 2009). The first uncovered biological role of the Arabidopsis PRC1like RING-finger proteins are on the regulation of SAM activity (Xu and Shen, 2008). While the single loss-of-function mutants Atring1a and Atring1b have a normal phenotype, the double mutant Atring1a Atring1b exhibits enlarged SAM, fasciated stem, and ectopic-meristem formation in cotyledons and leaves. This indicates that AtRING1a and AtRING1b play a redundant role in stable repression of stem cell activity to allow appropriate lateral organ differentiation. The balances between stem cell maintenance and cell differentiation for organ formation are controlled by specific transcription factors, including KNOX (Class I KNOTTED1-like homeobox) proteins. Strikingly, several KNOX genes, e.g., STM (SHOOT-MERISTEMLESS), BP (BRE-VIPEDICELLUS)/KNAT1, KNAT2 and KNAT6, are upregulated in Atring1a Atring1b (Xu and Shen, 2008). Ectopic expression of KNOX genes colocalizes with and precedes ectopic meristem formation. It has been proposed that AtRING1a/b acts as a crucial PRC1 component in conjunction with PRC2 in repression of KNOX genes to promote lateral organ formation in the SAM (Figure 2A).

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN EMBRYONIC CELL FATE DETERMINACY

Further characterization of the ectopic meristem structures observed in Atring1a Atring1b unravels that these callus structures exhibit embryonic traits (Chen et al., 2010). The Atbmi1a Atbmi1b mutant also displays derepression of embryonic traits (Bratzel et al., 2010;Chen et al., 2010). Embryonic callus formation has been observed broadly in somatic tissues of cotyledons, leaves, shoots and roots of the mutant plants. Treatment with an auxin transport inhibitor can inhibit embryonic callus formation in Atring1a Atring1b, indicating that a normal auxin gradient is required for somatic embryo formation in the mutant (Chen et al., 2010). Both Atring1a Atring1b and Atbmi1a Atbmi1b mutants exhibit elevated expression of several key embryonic regulatory genes, including ABI3 (ABSCISIC ACID INSENSITIVE 3), AGL15 (AGAMOUS LIKE 15), BBM (BABYBOOM), FUS3 (FUSCA 3), LEC1 (LEAFY COTYLEDON 1), and LEC2 (Bratzel et al., 2010;Chen et al., 2010). It is likely that derepression of these regulatory genes together with KNOX has contributed to the ectopic meristem and embryonic callus formation in somatic tissues of the Atring1a Atring1b and Atbmi1a Atbmi1b mutants (Figure 2B). The VAL (VP1/ABI3-LIKE) transcription factors can physically interact with AtBMI1 proteins and the val1 val2 mutant exhibits comparable phenotype to Atbmi1a Atbmi1b, suggesting that VAL and AtBMI1 proteins may form complexes in repression of embryonic regulatory genes during vegetative development (Yang et al., 2013). Notably, loss of VAL or AtBMI1 causes H2Aub1 reduction in chromatin regions at ABI3, BBM, FUS3 and LEC1 but not STM (Yang et al., 2013). Future investigation is necessary to clarify whether AtBMI1 and AtRING1 proteins repress KNOX transcription via H2Aub1 deposition or other independent chromatin remodeling mechanisms.

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN SEED GERMINATION

Seed germination defines the entry into a new generation of the plant life cycle. It is generally accepted that the process of germination starts with water uptake followed by seed coat rupture and is completed following radicle protrusion (Bentsink and Koornneef, 2008). During the very early phase, the embryonic growth program remains latent and can be reinstated in response to unfavorable environmental cues. With the attainment of photosynthetic competence, the irreversible transition to autotrophic growth is accomplished and embryonic program is stably suppressed. A recent study (Molitor et al., 2014) has identified the Arabidopsis PHD-domain H3K4me3-binding AL (ALFIN1-like) proteins as interactors of AtBMI1 and AtRING1 proteins and has demonstrated a crucial function of chromatin state switch in establishment of seed developmental gene repression during seed germination (Figure 2C). Loss of AL6 and AL7 as well as loss of AtBMI1a and AtBMI1b retards seed germination and causes transcriptional derepression and a delayed chromatin state switch from H3K4me3 to H3K27me3 enrichment of seed developmental genes, including ABI3 and DOG1. The germination delay phenotype of the al6 al7 and Atbmi1a Atbmi1b mutants is more pronounced under osmotic stress (Molitor et al., 2014), suggesting that AL PHD-PRC1 complexes may participate in regulation of seed germination in response to environmental cues.

ROLE OF PRC1-LIKE RING-FINGER PROTEINS IN OTHER PROCESSES

AtBMI1a and AtBMI1b, also named DRIP1 (DREB2A-INTERACTING PROTEIN 1) and DRIP2, had been reported first as E3 ligases involved in ubiquitination of DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING PRO-TEIN 2A), a transcription factor controlling water deficitinducible gene expression (Qin et al., 2008). The drip1 drip2 mutant shows enhanced expression of water deficit-inducible genes and more tolerance to drought (Qin et al., 2008). Overexpression of AtBMI1c accelerates flowering time, which is associated with reduction of FLC expression (Li et al., 2011). In addition to SAM maintenance defects and derepression of embryonic traits, the Atring1a Atring1b mutant also displays homeotic conversions of floral tissues (Xu and Shen, 2008). Therefore, more precise functions and underlying molecular mechanisms for the PRC1-like RING-finger proteins are still waiting to be uncovered during plant development and in plant response to environmental changes.

MECHANISMS OF PRC1-LIKE RING-FINGER PROTEINS IN TRANSCRIPTION REPRESSION

H2Aub1 function in plants is primarily evidenced through investigation of roles of the Arabidopsis PRC1-like RING-finger proteins (Xu and Shen, 2008;Bratzel et al., 2010;Chen et al., 2010;Li et al., 2011;Yang et al., 2013). Although these RING-finger proteins act nicely in vitro as E3 ligases, their in vivo functions in H2Aub1 deposition are still poorly documented. H2Aub1 level in Arabidopsis seems very low because large-scale analyses of either the histone-enriched or the Ub-affinity-purified protein preparations fail to detect H2Aub1 (Maor et al., 2007;Sridhar et al., 2007;Zhang et al., 2007a;Manzano et al., 2008;Saracco et al., 2009). H2Aub1 has been detected only by using specific antibodies, and in this case AtBMI1 genes have been shown to act as positive regulators for H2Aub1 deposition in Arabidopsis plants (Bratzel et al., Frontiers in Plant Science | Plant Genetics and Genomics 2010; Li et al., 2011;Yang et al., 2013). It is unknown whether any deubiquitinases might cause low levels of H2Aub1 in Arabidopsis. In animal cells, several deubiquitinases are characterized as specific for H2Aub1 (Weake and Workman, 2008;Simon and Kingston, 2013). Future characterization of Arabidopsis H2Aub1 deubiquitinases may provide useful information regarding regulatory mechanisms of H2Aub1 dynamics.

AtRING1 and AtBMI1 proteins physically interact each other and with the H3K27me3-binding protein LHP1 (Xu and Shen, 2008;Bratzel et al., 2010;Chen et al., 2010), providing a possible recruitment mechanism similar to the classical sequential PRC2 then PRC1 silencing pathway in animal cells. However, the Atring1a Atring1b, Atbmi1a Atbmi1b, or Atbmi1a Atbmi1b Atbmi1c mutant exhibits much more severe phenotypic defects than the lhp1 mutant does, and lhp1 enhances the Atring1a Atring1b mutant defects. It is thus apparent that AtRING1 and AtBMI1 proteins also act independently from LHP1. Recent identification of the transcriptional regulator VAL as AtBMI1-binding protein and of AL as AtRING1 and AtBMI1 interactor provides some novel insight about recruitment mechanisms (Yang et al., 2013;Molitor et al., 2014). It is particular intriguing that loss of AtBMI1 impairs H3K27me3 enrichment at seed developmental genes during seed germination and vegetative growth (Yang et al., 2013;Molitor et al., 2014). It has also been reported that loss of LHP1 impairs H3K27me3 enrichment at flower gene loci in roots (Derkacheva et al., 2013). These recent findings challenge the classic hierarchical paradigm where PRC2-mediated H3K27me3 deposition precedes PRC1 recruitment (Figure 2). It is obvious that future investigations are necessary to better understand the composition and function of different PRC1-like complexes in Arabidopsis.

CONCLUSIONS AND PERSPECTIVES

Studies over the last few years in the model plant Arabidopsis have greatly advanced our knowledge about the roles of H2Aub1 and H2Bub1 in transcription regulation in plant growth and development. In view of additional functions described in animal cells for both H2Aub1 and H2Bub1 in DNA damage repair (Bergink et al., 2006;Marteijn et al., 2009;Chernikova et al., 2010;Ginjala et al., 2011;Moyal et al., 2011;Nakamura et al., 2011), it is anticipated that more roles of H2Aub1 and H2Bub1 in plant response to environmental stresses are waiting to be uncovered. Mutagenesis of enzymes involved in H2Aub1 and H2Bub1 deposition or removal is required to address the question whether these enzymes effectively exert their biological functions via H2Aub1 and H2Bub1. Identification and characterization of factors associated with these different enzymes will be essential for understanding molecular mechanisms of their recruitment and function at specific targets within the genome. We need to know whether and how their function is spatially and temporally integrated with plant development. Genome-wide tools need to be further explored to provide a global view of links among enzyme or associated factor binding, H2Aub1/H2Bub1 enrichment, H3 methylation, and Pol II occupation. Crosstalks between H2Aub1 or H2Bub1 and different H3 methylations need to be addressed for chromatin context specificity.

In addition to H2Aub1 and H2Bub1, ubiquitinated H1, H3, and H4 are also found in Arabidopsis (Maor et al., 2007;Manzano et al., 2008;Saracco et al., 2009). H3 ubiquitination catalyzed by Rtt101-Mms1 in yeast and by Cul4-DDB1 in human has been recently shown to play an important role in the histone chaperone Asf1-mediated nucleosome assembly (Han et al., 2013). Arabidopsis contains a conserved family of CULLINs and CUL4-DDB1 complexes are reported (Shen et al., 2002;Hua and Vierstra, 2011). The Asf1 homologues in Arabidopsis are also identified (Zhu et al., 2011). It remains to be investigated whether CUL4-DDB and AtASF1 collaboratively act on nucleosome assembly via H3 ubiquitination in epigenetic regulation in Arabidopsis.

Caractérisation fonctionnelle des régulateurs chromatiniens ZRF1-like chez

Arabidopsis thaliana

Le

AtZRF1b interacts with H2Aub

In pull-down experiments, the His-AtZRF1b fusion protein can bind GST-Ub but not GST alone. Mutagenesis analysis revealed that the conserved UBD-domain is responsible for Ub binding. GST-fused UBD-domain fragment of AtZRF1b also can bind H2Aub as well as H2A and H3. These observed AtZRF1b properties are similar to those previously reported for human ZRF1. Atzrf1b-1 were on plates, stratified and germination rates were scored by counting the radical emergence for 12 days after stratification (DAS). Under standard growth conditions (MS medium), germination kinetics were not significantly affected in the Atzrf1a-1,Atzrf1a-2 and Atzrf1b-1 single mutants. However, under the same conditions, germination efficiency of the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants was found reduced. In Atbmi1a Atbmi1b double mutant, we found the germination rate also reduced, but it is not as strong as in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants. Gibberellin acid 3 (GA3) is generally known to effectively stimulate the breaking of seed dormancy and promote germination.

However, at different tested concentrations (0.5, 1.0 and 2.0ȝ mol/L) GA3 could not rescue the germination defects of the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants.

Next, mutants were challenged with salt and mannitol, which two stresses known to have a negative impact on seed germination. At 100 mM NaCl or 200 mM mannitol, the germination efficiency of the single mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1

was similar to that of Col, whereas the Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants displayed a significantly decreased germination efficiency. And the decrease is stronger in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants than in Atbmi1a Atbmi1b. Indeed, under the tested stress conditions, all wild-type seeds had germination after 5 days, while germination rates were reduced to ̚40% and ̚50% for Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants on 100 mM NaCl and 200 mM mannitol, respectively. And for Atbmi1a Atbmi1b double mutant the germination rates were reduced to ̚70% and ̚85% on 100 mM NaCl and 200 mM mannitol, respectively.

Next, I investigated molecular mechanisms underlying the seed germination defects of the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. I first analyzed expression levels of several seed development related genes including ABI3, DOG1, CRA1, CRC, PER and AIL5. As expected, all the examined genes except DOG1 displayed de-repression in the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2

Atzrf1b-1 seedlings.

To further understand the mechanism, I performed ChIP experiments using antibodies specific to H3K27me3, H3K4me3 or H2Aub. As results, we found that H3K4me3 level was slightly up-regulated at some gene regions whereas the levels of H3K27me3 and H2Aub were broadly down-regulated at the examined seed development genes in Nevertheless, when supplied with different concentrations of exogenous NAA the mutant root growth defects could not be rescued.

The Atzrf1a Atzrf1b double mutants also showed flowering defects. By counting the rosette leaf number, we found the flowering time of double mutant was obviously later than wild-type. Then we analyzed expression levels of the flowering genes FLC, MAFs, FT, SOC1, AGL24 and SVP. In double mutant, the expression level of FLC and MAFs was reduced strongly. This expression pattern is opposite to that of the above described seed development genes in the mutants. AtZRF1a and AtZRF1b functions are partially related to PRC1 Some of the Atzrf1a Atzrf1b double mutant defects are similar to those previously reported for the PRC1 mutants Atring1a Atring1b and Atbmi1a Atbmi1b. Our microarray analysis showed that there are significant overlaps of the perturbed genes between Atzrf1a Atzrf1b and Atring1a Atring1b or Atbmi1a Atbmi1b. I investigated physical interaction of AtZRF1b with AtRING1 and AtBMI1 proteins. Agarose beads coated with GST, GST-RING1A, GST-BMI1A, GST-BMI1B or GST-BMI1C were incubated with an equal aliquot of total nuclear protein extracts of Arabidopsis plants expressing FLAG-AtZRF1b. Then the pulldown fractions were analyzed by Western blot using antibodies against FLAG. We found that AtZRF1b can interact with AtBMI1A, AtBMI1B and AtBMI1C but not with AtRING1A. In order to confirm the observed interaction, we performed FLIM analysis to examine GFP-AtZRF1b interaction with RFP-AtRING1A, RFP-AtBMI1A, RFP-AtBMI1B or RFP-AtBMI1C, that are coexpressed in Nicotiana benthamiana leaves. We confirmed interaction between AtZRF1b and AtBMI1A or AtBMI1B.

All of my results allowed the first functional characterization of genes AtZRF1a and AtZRF1b. My data have shown that AtZRF1a and AtZRF1b play roles in part related PRC1 but also with specific aspects. Their role in the removal of H2Aub1, as was proposed for ZRF1 in animals has not been observed in plants.

Keywords :

Chromatin regulator ; Epigenetics ; Ubiquitin ; H2Aub1 ; Transcription regulation ; Plant development ; ZRF1 ; Seed germination

Figure I. 3 .

 3 Figure I.3. Schematic of histone structure in nucleosomesA. The core proteins of nucleosomes are designated H2A (histone 2A), H2B (histone 2B), H3(histone 3) and H4 (histone 4). Each histone is present in two copies, so the DNA (black) wraps around an octamer of histones -the core nucleosome. B. The amino-terminal tails of core histones. Lysines (K) in the amino-terminal tails of histones H2A, H2B, H3 and H4 are potential acetylation/deacetylation sites for histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation neutralizes the charge on lysines. A, acetyl; C, carboxyl terminus; E, glutamic acid; M, methyl; N, amino terminus; P, phosphate; S, serine; Ub, ubiquitin. (Adapted from[START_REF] Marks | Histone deacetylases and cancer: causes and therapies[END_REF]

  : the class I includes HDACs 1, 2, 3, and 8. These enzymes are closely related to the yeast transcriptional regulator Rpd3. The class II is divided into two subgroups, class IIA and class IIB. Class IIA includes HDACs 4, 5, 7, and 9, while Class IIB includes HDACs 6 and 10. All these enzymes are closely related to the yeast Hda1. The class III HDACs are sirtuin family enzymes with 7 members. They are related to the yeast transcriptional repressor Sir2 (Silent information regulator 2) and are NAD +dependent. Class IV contains only HDAC11. Although it is related to HDACs 3 and 8, its overall sequence is quite different from the other HDACs.

FIGURE 1 |

 1 FIGURE 1 | A proposed model for deposition and removal of histone H2B monoubiquitination in transcriptional activation of FLC and MAFs in flowering time regulation. In this model, HUB1 and HUB2 form a heterotetramer and recruit UBC1 or UBC2 to FLC /MAFs chromatin, leading to transfer of a ubiquitin (ub) monomer from UBC1 or UBC2 onto H2B. H2Bub1 formation enhances H3K4me3 deposition by methyltransferases, together promoting transcription initiation. UBP26 removes ubiquitin on H2B, favoring H3K36me3 deposition in promoting transcription elongation. Active transcription of FLC/MAFs represses Arabidopsis flowering, a transition from vegetative to reproductive plant development.

FrontiersFIGURE 2 |

 2 FIGURE 2 | Proposed models for histone H2A monoubiquitination suppression of numerous genes, including the key stem cell regulatory KNOX deposition in transcriptional repression of varied target genes. The genes that need to be stably repressed during lateral organ development. The Arabidopsis PRC1-like RING-finger proteins AtRING1a/b (RING1) and transcription factor VAL is involved in recruitment of BMI1 and RING1 in AtBMI1a/b/c (BMI1) have the E3 ligase activity in catalyzing H2A suppression of embryonic trait genes in somatic cells (B). AL proteins bind monoubiquitination (H2Aub1). Comparable to the classical model of BMI1 and RING1 and play important roles in suppression of several key seed sequential PRC2 then PRC1 action in Polycomb silencing in animal cells, the dormancy regulatory genes to promote germination (C). H3K27me3 Arabidopsis PRC1-like protein LHP1 binds H3K27me3 pre-deposited by the deposition at embryonic/seed genes is enhanced by VAL/AL-PRC1 (B,C), evolutionarily conserved PRC2 complexes and recruits RING1, BMI1 and unraveling a non-canonical crosstalk between H3K27me3 and H2Aub1. The possibly also EMF1 through protein-protein interactions (A). This question marks indicate that H2Aub1 deposition in the specified target gene combinatorial action by PRC2 then PRC1 likely plays a broad role in chromatin still requires future investigation.

  containing an ubiquitin-binding domain (UBD) close to the DnaJ domain at the Nterminus in humans. At the C terminus, ZRF1 contains two tandem repeats of SANT (Swi3, Ada2, NcoR1, and TFIIIB) domains, which are c-Myb-like repeats. The SANT domain consists of three -helices, each of them containing a corresponding, bulky aromatic residue. The region exhibits a sequence-specific DNA binding activity. It was found to exist only in higher eukaryotes (Figure I.4). The SANT domain is commonly associated with a number of chromatin remodeling factors involved in the recruitment of histone acetylases (HAT) or histone deacetylases (HDAC)

.

  My results showed that AtZRF1a and AtZRF1b have important roles in cell proliferation and differentiation, flowering time control, and seed germination.

  At3g11450) and AtZRF1b (At5g06110) encode Arabidopsis proteins showing high homologies to human ZRF1 (Figure III.1). The AtZRF1a mRNA was predicted to encode a protein with 647 amino acid residues and AtZRF1b mRNA a protein with 663 amino acid residues. They show 81% identity and 96% similarity to each other at the amino acid sequence level (Figure III.2).

Figure III. 1 .

 1 Figure III.1. Phylogram of ZRF1 homologs in several organismsOn the basis of amino acid sequence of the full-length protein, the phylogenetic analysis was performed using MEGA5.0 package with bootstrapping set at 500 replicates.

Figure III. 2 .

 2 Figure III.2. Alignment of AtZRF1a and AtZRF1b amino acid sequences White letters in black boxes indicate 100% sequence similarity, white letters in grey boxes indicate 80 to 100% similarity, black letters in grey boxes indicate 60 to 80% similarity, and grey letters on white background indicate <60% similarity. The alignment was generated using CLUSTALW program. The Zuotin domain starts at position 100 and extends to position 431 The SANT1 domain starts at position 484 and extends to position 524 The SANT2 domain starts at position 615 and extends to position 661

Figure III. 4 .

 4 Figure III.4. RT-PCR and Q-PCR analysis of AtZRF1a and AtZRF1b expression in different organs of the wild-type Col plants

  study the biological roles of AtZRF1a and AtZRF1b, we first obtained from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org/) two T-DNA insertion mutant lines for each of AtZRF1a and AtZRF1b. They are named Atzrf1a-1, Atzrf1a-2, Atzrf1b-1 and Atzrf1b-2. The mutant Atzrf1a-1 (SAIL_786_F09) harbors a T-DNA insertion 486 bp downstream of the translational start codon of AtZRF1a and the T-DNA carries the selection marker for BASTA (PPT, phosphinothricin) resistance. The mutant Atzrf1a-2 (SALK_070956. 55.25. x) harbors a T-DNA insertion 426 bp upstream of the translational stop codon of AtZRF1a and the T-DNA carries the selection marker for kanamycin (Kan) resistance. The mutant Atzrf1b-1 (FLAG_110A05) has a T-DNA insertion 849 bp downstream of the translational start codon of AtZRF1b and the T-DNA carries the selection marker for BASTA resistance. The mutant Atzrf1b-2 (SAIL_716_D04) harbors a T-DNA insertion in the AtZRF1b 3'-UTR, 159 bp downstream of the translational stop codon of AtZRF1b and the T-DNA carries the selection marker for BASTA resistance (Figure III.7A). For all these single mutants we confirmed the location of the T-DNA byPCR (polymerase chain reaction) amplification using the T-DNA-specific oligonucleotide primer LB1 and two gene-specific primers located at each side of T-DNA insertion site (Figure III.7A). Homozygous mutant plants were obtained for Atzrf1a-1, Atzrf1a-2, Atzrf1b-1 and Atzrf1b-2 by self-pollination; each line was identified by genotyping 30 antibiotic-resistant plants in PCR reactions. To further confirm these mutants, we examined the AtZRF1a and AtZRF1b mRNA transcript levels in wild type and mutants by reverse transcription PCR (RT-PCR). The analysis revealed that full-length transcripts of AtZRF1a and AtZRF1b are undetectable in homozygous mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1, indicating that T-DNA insertion caused a knockout of the respective gene in these mutants. Under standard laboratory growth conditions, none of the four mutants showed any obvious growth or developmental defect (Figure III.6A), probably due to the functional redundancy of the two AtZRF1 genes. Considering the high homology of AtZRF1a and AtZRF1b and their similar expression profiles, we generated double mutants through crosses of different single mutants (Figure III.6B) to investigate the possible redundant function of these genes.

Figure III. 6 .

 6 Figure III.6. Phenotype of single mutants and generation of Atzrf1a -/-Atzrf1b -/- mutant (A) Phenotype of single mutants Atzrf1a -/-and Atzrf1b -/-in the Columbia background. (B) A schematic representation of the procedure used to generate the double mutant Atzrf1a -/-Atzrf1b -/-. The two homozygous mutant alleles Atzrf1a -/-and Atzrf1b -/-were combined together by crossing. Then, genotyping by PCR was performed on F2 progenies. For the WT allele, PCR was carried out by gene-specific forward and reverse primers on the genomic sequence. For the T-DNA insertion, PCR was carried out using one primer on the left border of the T-DNA insertion and another gene-specific primer on the flanking genomic sequence.

  of the genes using the complementation test. A full-length cDNA of AtZRF1b was ligated by Gateway cloning into the binary plant-transformation vector pGWB11 which contains 35S promoter upstream of the cloning site and the hygromycin (Hyg) selection marker gene in the T-DNA. Agrobacterium tumefaciens-mediated plant transformation was used to introduce the 35-promoter-AtZRF1b transgene into Atzrf1a Atzrf1b double mutant plants. Because of low fertility of homozygous double mutants, we used double mutant Atzrf1a-1 -/-Atzrf1b-1 +/-heterozygote transformation, whose genotype was verified by PCR-based genotyping before transformation. The F 1 seeds post transformation were collected and plated on MS medium containing the antibiotics PPT and Hyg. The growing plants were transferred into soil and analyzed to identify the background of double mutant homozygotes by PCR-based genotyping using gene-specific primers. We found that introduction of the 35S:AtZRF1b completely rescued the Atzrf1a -/-Atzrf1b -/-mutant to the wild-type phenotype (Figure III.9), demonstrating that AtZRF1 gene knockout is indeed responsible for the phenotypic defects observed in the Atzrf1a Atzrf1b mutant. In parallel, we identified novel T-DNA insertion mutant lines and performed an allelism test. First, we crossed Atzrf1a-1 +/-Atzrf1b-1 -/-with Atzrf1a-3 -/-(SALK_ 070965.50.20.x), which contains a T-DNA insertion in the 3'-coding region of AtZRF1a (Figure III.10A) and the T-DNA carries the kanamycin resistance selection marker. The F 2 seeds resulting from the cross were plated on MS medium containingPPT and Kan, and the growing plants were transferred onto soil for further genotyping and phenotype analysis. We found that plants with the Atzrf1a-1 +/-Atzrf1a-3 +/-Atzrf1b-1 -/-genotype or with the Atzrf1a-3 -/-Atzrf1b-1 -/-genotype display a growth phenotype similar to that of Atzrf1a-1 -/-Atzrf1b-1 -/-(Figure III.10B). This indicates that Atzrf1a-3 is also a loss-of-function mutant allele of AtZRF1a. In a similar way, we found that Atzrf1b-3 (SAIL_625_B03.v2 , Atzrf1b-4 (SAIL_629_F09.v1) and Atzrf1b-5 (FLAG-099c10) are allelic to Atzrf1b-1 and represent novel loss-of-function mutant alleles of AtZRF1b (Figure III.10).

Figure III. 7 .

 7 Figure III.7. Structures and expression patterns of Atzrf1a -/-and Atzrf1b -/-mutant (A) (A) Diagram of the gene structures of Atzrf1a -/-and Atzrf1b -/-mutant alleles. Black boxes represent exons; Blue boxes represent UTR; White boxes represent introns; and triangles indicate T-DNA insertions. Primers of same colors represent primer pairs used together. The primer number (1 to 19) corresponds to the position in the primer list in Materials and Mehtods (Genotyping). (B) RT-PCR analysis of AtZRF1a and AtZRF1b expression in rosette leaves of the single mutants Atzrf1a -/-and Atzrf1b -/-, and in the double mutant Atzrf1a -/-Atzrf1b -/-(Atzrf1a-1 Atzrf1b-1; Atzrf1a-1 Atzrf1b-2; Atzrf1a-2 Atzrf1b-1; Atzrf1a-2 Atzrf1b-2). Full-length AtZRF1a, and AtZRF1b sequences were amplified from wild-type (Col), single and double mutant cDNAs, ACTIN serves as an internal control. (C) 35-day-old Col and double mutants grown under 12h light/ 12h dark at 21°C

Atzrf1a

Figure

  Figure III.8. Production and mature plant phenotype of Atzrf1a -/-Atzrf1b -/-mutant A. Comparison of a 45-day-old wild-type Col plant and 81-day-old mutant Atzrf1a -/-Atzrf1b -/- plants (Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1) (top of Figure A)). At the bottom of the figure, Siliques of wild-type and double mutants (bottom of Figure A). B. Representative photograph of wild-type (Col) and Atzrf1a Atzrf1b double mutant siliques. An abortion event, apparently frequent on Atzrf1a Atzrf1b double mutant siliques, is highlighted (arrow). Comparison between wild-type (Col) and Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 homozygous lines: C. length of siliques D. number of seeds per silique E. seeds aborted per silique. The error bars represent SE from 10 siliques along the stem of 5 independent plants (n= 50 for each).

Figure

  Figure III.9. Rescue of the Atzrf1a-1 -/-Atzrf1b-1 -/-mutant phenotype 35S:AtZRF1b was used to rescue of the Atzrf1a-1 -/-Atzrf1b-1 -/-mutant. Phenotypes of 5-weekold Col, Atzrf1a-1 -/-Atzrf1b-1 -/-mutants and rescued plants. 12h light/12h dark, 21.

Figure

  Figure III.10. Allelism test of Atzrf1a -/-and Atzrf1b -/-mutant (B) Schematic representation of the AtZRF1a gene and AtZRF1b gene. Black boxes represent exons; Blue boxes represent UTRs; White boxes represent introns; triangles indicate T-DNA insertions. Primers of same colors represent primer pairs used together. The primer number (1 to 19) corresponds to the position in the primer list in Materials and Mehtods (Genotyping). (C) Top left: allelism test between Atzrf1a-3 and Atzrf1a as scored by Atzrf1a-1 -/-Atzrf1b-1 -/- mutant phenotype. Bottom: allelism test between Atzrf1b-3, Arzrf1b-4, Atzrf1b-5 and Atzrf1b as scored by Atzrf1a-1 -/-Atzrf1b-1 -/-mutant phenotype. All plants (5-week-old) grown under long day conditions (16h light and 8h dark). Top right: RT-PCR analysis of AtZRF1a and AtZRF1b expression in the single mutants Atzrf1a-3, Atzrf1b-3, Arzrf1b-4, Atzrf1b-5 and wild-type (Col). Full-length of AtZRF1a, and AtZRF1b sequences were amplified from single mutants and Col using gene specific primers, ACTIN served as an internal control

Atzrf1a- 1

 1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 and Col-0 leaves by measurement of the relative nuclear DNA content via flow cytometry analysis. DNA was isolated from the first true leaf on three different 2-week-old plantlets. The cell cycle consists of four phases: the postmitotic interphase (G1), with 2C nuclear DNA content; the S phase, meaning DNA synthetic phase, with an intermediate 2C and 4C nuclear DNA content; the postsynthetic interphase (G2), with a 4C nuclear DNA content; and finally the M phase, meaning mitosis. I observed a slightly lower proportion of 2C cells in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants as compared to Col (Figure III.12E), suggesting a relatively shorter duration of G1 in the mutant. Higher ploidy levels ( 8C) are the result of endoreduplication cycles in which nuclear DNA is replicated without subsequent mitotic division. The relative proportion of cells with higher ploidy levels is slightly increased in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants as compared with Col (Figure III.12E). In addition, the leaf shape, leaf margin and leaf vein of Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants displayed an abnormal phenotype. Compared to wild-type, the double mutant displayed twisted blades. The leaf was folded from the midrib of the blade (Figure III.13). While the leaf margin of the wild-type is serrated, the leaf margin of double mutants is smooth. Moreover, the leaf vein of wild-type plants is netted while in the the double mutant it is very special in that there is a primary vein in the middle of leaf and the two sides of the leaf have a net-like venation(Figure III.13). Observation of the first true leaves of two-week-old Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 double mutants and wild-type seedlings showed that the number of trichomes on the leaf epidermis of double mutants decreased compared to wild-type. Also the trichomes of double mutants mainly have only one or two branches, while the trichomes of wild-type rosette leaves typically have three branches (Figure III.14).

  G1-S transition[START_REF] De Jager | Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc[END_REF] Dewitte et al. 2003;[START_REF] Menges | The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis[END_REF]. There are three typical E2Fs: E2Fa, E2Fb and E2Fc. Both E2Fa and E2Fb are transcriptional activators of the cell cycle, and they positively regulate the S phase[START_REF] De | Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor[END_REF][START_REF] Sozzani | Interplay between Arabidopsis activating factors E2Fb and E2Fa in cell cycle progression and development[END_REF]; in contrast, E2Fc serves as a repressor[START_REF] Del Pozo | The balance between cell division and endoreplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis[END_REF]. The qRT-PCR results showed a reduction of CYCD3;1 expression level, suggesting that the absence of AtZRF1a and AtZRF1b delays progression of the cell cycle into S-phase. The expression level of E2Fa is not consistent with our conclusion.However, some experimental data suggest that CYCD3;1 and E2Fa have divergent effects on the cell cycle[START_REF] De Jager | Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc[END_REF].

Figure III. 11 .

 11 Figure III.11. Abnormal cotyledons in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants A. Weak mutant of 4-week old plants grown under 12h light and 12h dark conditions. B-C. Fleshy cotyledons (4-week-old, under medium day conditions). D-F. Asymmetrical cotyledons (2-week-old, under medium day conditions).

Figure

  Figure III.13. Leaf shape, leaf margin and the distribution of leaf vein A.Comparison of leaf shape between wild-type and Atzrf1a-1 Atzrf1b-1 double mutant (4week-old, under medium day conditions).B-C. Comparison of leaf margin and the distribution of leaf vein between wild-type (B) andAtzrf1a-1 Atzrf1b-1 double mutant (C).

Figure III. 14 .Figure

 14 Figure III.14. Trichome phenotype of wild-type and Atzrf1a Atzrf1b double mutants (A-B) Scanning electron micrographs of trichomes from the first true leaves of two-week-old Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 double mutants and wild-type seedlings. (A) Wild-type. (B) Double mutant. (C-D) Scanning electron micrographs of wild-type and mutant trichomes branch. (C) Wildtype. (D) Double mutant.

  SAM in the mutant(Figure III.18), indicating that suppression of KNOX gene expression has been released.

Figure III. 16 .

 16 Figure III.16. Simultaneous knockout of AtZRF1a and AtZRF1b causes ectopicmeristem formation A-C: Fasciated stem and hook-like apex on primary Atzrf1a-1 Atzrf1b-1 inflorescence. E-G: Flower reversions, Atzrf1a-1 Atzrf1b-1 flowers producing one or more secondary flowers (E, F). Atzrf1a-1 Atzrf1b-1 floral organ reversion, with pistil (G). H-J: Terminal flowers of the double mutant. K-M: Abnormal inflorescence of the double mutant. (K) Florescence branch. The left is wildtype and the right is double mutant; (L-M) Cauline leaf. Inflorescences of double mutants.

Figure

  Figure III.17. Quantitative RT-PCR analysis of genes expression in 2-week-old wild-type and double mutants seedlings A: Relative expression levels are shown as induction fold in 2-week-old Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 seedlings compared with 2-week-old wild-type (set as 1) seedlings. B: Relative expression levels are shown as induction fold in Atring1a Atring1b and Atbmi1a Atbmi1b compared with wild-type (set as 1). Error bars represent standard deviation from triplicate repeats.

Figure

  Figure III.18. STM-GUS expression in wild-type and Atzrf1a-1 Atzrf1b-1 double mutant Histochemical GUS staining of pSTM:GUS in double mutant Atzrf1a-1 Atzrf1b-1 and wildtype Col. Seedlings at 10 DAS were stained for overnight. Blue staining indicates the reporter GUS activity.

  more than 5-to 10-fold in the mutants(Figure III.20). The embryonic competenceenhanced factor gene AGAMOUS-LIKE15 (AGL15)[START_REF] Harding | Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15[END_REF] is upregulated by 2-fold, whereas expression of the somatic embryogenesis receptor-like kinase genes SERK1 and SERK2[START_REF] Schmidt | A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos[END_REF] is almost unaffected in the mutant (Figure III.20). Drastic upregulation of expression was observed for several key embryonic regulatory genes (Figure III.20), including the root stem cell regulator

  encoding polar auxin transporters(Blilou et al., 2005) (Figure III.20). Taken together, our results show that some but not all stem cell and embryonic regulatory genes are ectopically derepressed in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants.

Figure 1 Atzrf1b- 1 1 and 1 and

 1111 Figure III.19. Phenotypic variability of Atzrf1a-1 Atzrf1b-1 double mutant seedlings A. Weak mutant B-C: Dedifferentiation mutants

Figure III. 21 .

 21 Figure III.21. Abnormal flower organs in Atzrf1a Atzrf1b mutant (A-B) Wild-type. Arabidopsis WT flowers usually have 4 sepals, 4 petals, 6 stamens, and two fused carpels. (C-I) Defective flower organs of Atzrf1a Atzrf1b weak double mutant. (D) has 3 normal petals and 1 smaller petal. (E) Flower no opened, but pistil and petal have appeared. Disruption of AtZRF1a and AtZRF1b leads to very short stamens (I). And produce sterile flowers with dramatically reduced number of floral organs (F). Statistic analysis of simple flowers from 10 wild-type plants or 10 weak double mutant plants and 10 single flowers of each plant.

Figure III. 22 .

 22 Figure III.22. Relative expression levels of flower developmental genes in Atzrf1a Atzrf1b mutants flower buds.In young flower buds around stage 6 of flower development, expression levels of flower developmental genes were determined by qRT-PCR in wild-type (Col), Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants. Expression levels are relative to Col and normalized to internal reference genes (Tip4.1, PP2A and Exp). Data shown are meansSD of technical replicates. Similar results were obtained in three independent experiments.

  Wild-type phenotype of heterozygous Atzrf1a +/-Atzrf1b -/-or Atzrf1a -/-Atzrf1b +/-mutant (WT) and mutant phenotype of Atzrf1a -/-Atzrf1b -/-(s) alleles were determined by PCR analysis. The ratios (WT: m) obtained from experimental data are higher than those expected from normal segregation, indicating reduced transmission efficiency of mutant alleles. Statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05.The root is a very important organ of plant development. During embryogenesis, the Arabidopsis primary root formation is initiated by hypophysis specification. The hypophysis is a single extraembryonic suspensor cell. The suspensor cell generates the quiescent centre (QC) after several rounds of asymmetrical cell divisions and expansions, leading to the generation of the root meristem of the primary root. A larger basal cell generates the lower tier of stem cells for the columella. During this process, it is very important that the hypophysis is specified properly, otherwise, a root meristem is not formed, which eventually results in rootless seedlings[START_REF] De Smet | Embryogenesis -the humble beginnings of plant life[END_REF] Tian et al., 2014). Compared to wild-type, we found that the primary root growth of the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 was strongly impaired. The mutants exhibit almost a rootless or short-root phenotype (Figure III.23A). Primary root growth was measured between 1 and 12 d after stratification (DAS). Compared to wild-type, the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1double mutant showed clear primary root growth retardation, and the difference became increasingly evident along with plant age, e.g. the primary root length at 12 DAS of the Atzrf1a-1 Atzrf1b-1 (4.880.83 mm, n=30) and Atzrf1a-2 Atzrf1b-1 (5.271.02 mm, n=30) double mutants only reached about 10% of that of wild-type plants (55.4 5.27 mm, n=30) (Figure III.23B). Subsequently we focused on the Atzrf1a-1 Atzrf1b-1

  Figure 33B, expression of IAA2, IAA16, IAA28, IAA29 and IAA30 was drastically decreased whereas expression of IAA14, IAA19 and IAA34 was increased in the Atzrf1a-1 Atzrf1b-1 double mutant. IAA14 and IAA19 genes are known to

  Figure III.24C). Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants root growth weren't obviously affected by different concentration of auxin. Nevertheless, in no case Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants roots growth could reach that of WT. Taken together, our data indicate that loss of AtZRF1a and AtZRF1b function affects auxin regulation and exogenous auxin supply could not rescue the mutant root defects. To determine to what extent the cell proliferation activity of the RAM was affected in the Atzrf1a-1 Atzrf1b-1 mutant, various parameters related to RAM activity were analyzed. Previous studies in the Atzrf1a-1 Atzrf1b-1 double mutant showed that the DR5::GFP expression level obviously decreased in QC. The QC is crucial for maintaining the identities of the surrounding stem cells which have the highest rate of cell division. To assess whether the QC was correctly specified in the primary root of the Atzrf1a-1 Atzrf1b-1 double mutant, we introgressed the WOX5GFP marker into the Atzrf1a-1 Atzrf1b-1 double mutant by genetic crossing. WOX5 is specifically expressed and functions in root QC cells to regulate the balance between cell division and differentiation of the adjacent stem cells. Microscopy analysis revealed GFP fluorescence in the QC cells of Atzrf1a-1 Atzrf1b-1 pWOX5::GFP, however, compared with wild-type, in 23 out of 25 (92%) of the Atzrf1a-1 Atzrf1b-1 double mutant roots examined, we found that the Atzrf1a-1 Atzrf1b-1 double mutant contains a disorganized stem cell niche (SCN) with a reduced number of QC cells (Figure III.25). Moreover, in order to investigate whether surrounding stem cells were destroyed in Atzrf1a-1 Atzrf1b-1 double mutant, we introgressed the enhancer-trap line J1092 and the columella stem cell-specific enhancer trap line J2341 into the Atzrf1a-1 Atzrf1b-1 double mutant by genetic crosses. The J1092 enhancer trap line showed weak GFP expression in the columella, including the columella stem cells, and a strong GFP signal in the lateral root cap of the wild-type seedlings (Figure III.26A). However, in 19 out of 21 (90.5%) of the Atzrf1a-1 Atzrf1b-1 double mutant roots examined, the GFP expression level was reduced in the root cap (Figure III.26). The J2341 enhancer trap line showed that GFP was specifically expressed in columella stem cells (CSCs) in the wild type root (Figure III.26), whereas in 25 out of 26 (96%) of the Atzrf1a-1 Atzrf1b-1 double mutant roots examined, GFP was detected in the cells at the position of CSCs, and the number of CSCs was reduced (Figure III.26B). Columella cells function in gravity sensing; we found a few Atzrf1a Atzrf1b double mutants that had lost geotropism when germinating. Taken together, this indicates that deletion of both AtZRF1a and AtZRF1b leads to a defective cellular organization in the root stem cell niche.

  To study this, we introgressed SCR SCR-GFP and CO2::GFP into the Atzrf1a-1 Atzrf1b-1 double mutant by genetic crosses. SCARECROW (SCR), a GRAS family transcription factor, is involved in RAM maintenance and radial patterning. While SCR SCR-GFP is specifically expressed in the endodermis and QC, CO2::GFP is specifically expressed in the cortex. The results showed that in the Atzrf1a-1 Atzrf1b-1 double mutant, the cortex layer and endodermis layer were abnormal. In 23 out of 27 (85.2%) of the Atzrf1a-1 Atzrf1b-1 double mutant roots examined, the cortex layer was partially lost. And in 19 out of 23 (82.6%) of the Atzrf1a-1 Atzrf1b-1 double mutant roots examined, the endodermis layer was disorganized (Figure III.27).Collectively, the results indicated that AtZRF1a and AtZRF1b are required for the maintenance of QC identity, for RAM organization, and for cell patterning.

Figure III. 23 .

 23 Figure III.23. Atzrf1a Atzrf1b double mutants exhibit altered primary root development (A) Representative photograph of wild-type and double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 12 DAG seedlings. (B) Primary root length changes with time. Starting from 1 DAG, observed between wild-type (Col) and double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. Error bars represent the mean±SE from30 seedlings analyzed at each indicated DAG. The experiment was repeated three times with similar results.

Figure III. 25 .

 25 Figure III.25. Loss of AtZRF1a and AtZRF1b impairs the primary root stem cell niche maintenance Confocal fluorescence micrographs of PI-stained root tips taken from five days seedlings from wild-type Col (left) and the double mutant Atzrf1a-1 Atzrf1b-1 (right), respectively. Comparison of the expression pattern of WOX5:GFP reporter, which is the quiescent center specific marker. Images are representative of 12-23 plants in four replicate experiments. GFP signal is shown in green, propidium iodide signal in red.

Figure III. 26 .

 26 Figure III.26. Cell-marker gene expression in wild-type and Atzrf1a-1 Atzrf1b-1 double mutant root tips Confocal fluorescence micrographs of PI-stained root tips taken at five days. Expression of root cap marker J1092 in wild-type roots (A left) and in Atzrf1a-1 Atzrf1b-1 double mutant (A right). Expression of the columella initials marker J2341 in wild-type roots (B left) and in Atzrf1a-1 Atzrf1b-1 double mutant (B right). Images are representative of 12-16 plants in four replicate experiments. GFP signal is shown in green, propidium iodide signal in red.

  the flowering time in the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 was strongly delayed both in LD and SD (Figure III.28C-D). Flowering is a central event in the life cycle of plants; proper flowering time ensures reproductive success. Thus, flowering is a highly regulated biological process in Arabidopsis. In order to explore the molecular mechanisms responsible for the change in flowering time, we performed quantitative PCR to analyse the expression levels of some endogenous flowering-related genes in seedlings. These include FLC, and FLC close homologs, MADS AFFECTING FLOWERING1 (MAF1/FLM), MAF2, MAF4, MAF5, and SHORT VEGETATIVE PHASE (SVP). We also studied promoters of flowering such as SOC1, FT, and AGAMOUS-like 24 (AGL24). FLC is a central floral repressor working in a dose-dependent manner, which is delicately controlled by various activators and repressors. FLC blocks the expression of floral activators such as FT and SOC1 to prevent the initiation of flowering during vegetative development.

  was strongly decreased compared to wild-type, while MAF5 expression remained unchanged both in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants (Figure III.30). These results indicate that deletion of AtZRF1a and AtZRF1b should exhibit early flowering. Instead, the phenotype of double mutants showed late flowering. Therefore, to further explore the flowering time regulatory pathway in the double mutants, I compared the expression level of several floral integrators between mutant and wild-type. Consistent with the decrease in FLC expression, an increase in the expression of FT compared to wild-type level was observed both in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants. And a strong decrease in SVP expression was observed in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants (Figure III.30). In both Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants, the SOC1 expression level did not change significantly. These results indicate that the alteration in flowering time was caused by loss of functions of both Atzrf1a and Atzrf1b. However, AtZRF1a and AtZRF1b may participate in repression of flowering time. The double mutants show late flowering, which may be caused by a delay in growth development.

Figure III. 28 .

 28 Figure III.28. Flowering time of single mutants and double mutants (A) Phenotypes of 56-day-old double mutants Atzrf1a-1 Atzrf1b-1 and wild-type Col plants, under long-day (16 h light and 8 h dark) conditions. (B) Comparison of branch number of 56-day-old double mutant with wild-type Col plants. Values shown represent the means and standard deviations for at least 10 plants of each genotype. (C) Flowering time of wild-type, single mutants and double mutants grown under LDs. Values were scored from at least 15 plants of each genotype. Error bars indicate s.d. Flowering time was measured by counting days when the first flower emerged under long-day conditions. (D) Flowering time of wild-type, single mutants and double mutants grown under SDs. Values were scored from at least 15 plants of each genotype. Error bars indicate s.d. Flowering time was measured by counting days when the first flower emerged under short-day conditions.

Figure

  Figure III.29. Phenotypes of heterozygous mutants Atzrf1a-1 +/-Atzrf1b-1 -/-, Atzrf1a-1 - /-Atzrf1b-1 +/-and Col 4-week-old heterozygous mutants Atzrf1a-1 +/-Atzrf1b-1 -/-, Atzrf1a-1 -/-Atzrf1b-1 +/-and Col plants grown in long day condition. 16h light and 8h dark.

Figure III. 30 .

 30 Figure III.30. Quantitative RT-PCR analysis of gene expression in double mutants (A) Relative expression level of FLC and FLC-related genes (MAF genes) (B) Relative expression level of floral integrators SOC1, FT, AGL24 and SVP are shown. A pool of 2-week old seedlings was used for RNA extraction and the averages of three biological replicates are shown. Each experiment was normalized to Tip4.1, Exp and PP2A expression. Error bar indicates standard error (SE).

Figure

  Figure III.31. Western blot analysis of global H3K4me3, H3K27me3, H3K36me3 and ubiquitination levels in the Atzrf1a-1 Atzrf1b-1, Atring1a Atring1b, Atbmi1a Atbmi1b mutants and wild-type plants.Histone-enriched protein extracts from plants 14 days after germination grown under mediumday (12 h light and 12 h dark) conditions were analyzed by Western blotting using antibodies that specifically recognize the indicated forms of histones.

Figure III. 32 .

 32 Figure III.32. Relative enrichments of H3K4me3, H3K27me3 and H2Aub1 at flowering time genes in Col-0 and mutantsRelative levels of histone modifications on FLC, MAF4 and FT chromatin were analyzed by ChIP using antibodies against H3K4me3, H3K27me3 and anti-hH2Aub. Chromatin from Col, Atbmi1a Atbmi1b, Atring1a Atring1b, Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 and clf was prepared from 2-week-old seedlings. The immunoprecipitated DNA fragments were quantified by qRT-PCR and normalized to internal controls (relative to Input and normalized to TUB2). Data shown are meansSD of three technical replicates. Similar results were obtained in three independent experiments. Amplified regions are numbered and indicated on the schematic representation of the FLC, MAF4 and FT genomic structure. Exons are represented by black boxes, untranslated regions by dashed boxes and introns by black lines.

1

 1 were put on plates, stratified and the germination rates were scored by counting the radical emergence for 12 days after stratification (DAS). Under standard growth conditions (MS medium), germination kinetics were not significantly affected in Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1 single mutants (Figure III.28A and B), whereas under the same conditions, the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants displayed a significantly decreased germination efficiency (Figure III.33D-E). The Atbmi1a Atbmi1b mutant also displayed a delay in seed germination in our assays (Figure III.33E). Interestingly, compared to that of Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1, the exponential phase of the Atbmi1a Atbmi1b germination rate curve started earlier after stratification, but reached a comparatively maximum percentage value. AtBMI1a/b may be involved primarily in the maintenance of the germination process (Molitor et al., 2014). These results indicate that AtZRF1a/b may be involved primarily in initiation of the germination process. We also obtained the quadruple mutant Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b and showed that it is drastically impaired in both germination initiation time and maximum percentage of germination rate (Figure III.33E). Gibberellic acid 3 (GA3) is generally known to effectively stimulate the breaking of seed dormancy and promote germination. However, at different tested concentrations (0.5, 1.0 and 2.0 µmol/L) GA3 could not rescue the germination defects of the Atzrf1a-1 Atzrf1b-1and Atzrf1a-2 Atzrf1b-1 double mutants (Figure III.33H).Next, the mutants were challenged under osmotic treatments with salt or mannitol, two stresses known to have a negative impact on seed germination. At 100 mM NaCl or 200 mM mannitol, the Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants but not the single mutants showed a delay in seed germination compared to the wild-type Col-0 (Figure III.33F and G). Interestingly, like on MS medium, in comparison to wild-type, the Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants as well as the Atzrf1a-1 Atzrf1b-1 Atbmi1a

1

 1 double mutants(Figure III.33F and G). Indeed, under the tested stress conditions, all wild-type seeds had germinated after 5 days, while germination rates were reduced to 40% and50% for Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants on 100 mM NaCl and 200 mM mannitol, respectively. Moreover for the Atbmi1a Atbmi1b double mutant the germination rates were reduced to 70% and 85% on 100 mM NaCl and 200 mM mannitol, respectively (Figure III.33F and G). While germination rates were reduced by 40% for the Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b quadruple mutant on 100 mM NaCl or 200 mM mannitol (Figure III.33F and G).

Figure III. 33 . 1 .

 331 Figure III.33. Germination rate of single mutants and double mutants (A) Representative seed germination images of Col-0, single mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1. Images were taken five days after stratification from plates containing MS media or MS supplemented with 100 mM NaCl. (B) Germination rate of Col-0 and single mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1 plated on MS. (C) Germination rate of Col-0 andsingle mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1plated on MS supplemented with 100 mM NaCl. (D) Representative seed germination images of Col-0 and double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. Images were taken five days after stratification from plates containing MS media or MS supplemented with 100 mM NaCl. (E) Germination rate of Col-0 and double mutants Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1and Atzrf1a-2 Atzrf1b-1 and Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b quadruple mutant plated on MS. (F) Germination rate of Col-0 and double mutants Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 and Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b quadruple mutant plated on MS supplemented with 100 mM NaCl. (G) Germination rate of Col-0 and double mutants Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1and Atzrf1a-2 Atzrf1b-1 and Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b quadruple mutant plated on MS supplemented with 200 mM mannitol. (H) Germination rate of Col-0 plated on MS and double mutants Atzrf1a-1 Atzrf1b-1andAtzrf1a-2 Atzrf1b-1 plated on supplemented with different concentrations of GA (0.5, 1.0 and 2.0µmol/L, respectively). All data represent average germination percentages ±SD of three biological replicates, each 80 seeds, observed daily for 12 days after stratification.

Atzrf1b- 1 .

 1 expression levels of several seed development related genes including ABI3, DOG1, CRA1, CRC, PER and AIL5 between WT and double mutants Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1, Atring1a Atring1b and Atbmi1a Atbmi1b. As expected, all the examined genes except DOG1 displayed derepression in the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 seedlings (Figure III.34). Moreover, we investigated the expression levels of the six seed developmental genes in the Atring1a Atring1b and Atbmi1a Atbmi1b mutants. As shown in Figure III.29, all six seed developmental genes showed higher expression levels in the Atring1a Atring1b and Atbmi1a Atbmi1b mutants as compared to Col-0. More important, the expression levels of these seed developmental genes were highest in double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 These data demonstrate that AtZRF1a/b, AtRING1a/b and AtBMI1a/b are involved in the repression of seed developmental genes during germination and early seedling growth. To investigate the mechanism of seed gene repression, we performed a ChIP analysis on H3K27me3, H3K4me3 or H2Aub levels during seed germination. ChIP fractions were analyzed using PCR primers covering the promoter, UTR and gene body regions of ABI3 (Figure III.35) and DOG1 (Figure III.35). In addition, we analyzed the deposition of H3K27me3, H3K4me3 or H2Aub1 marks at the gene body regions of CRC (Figure III.35) and AIL5 (Figure III.35). We found that H3K4me3 levels were slightly upregulated at all genes in Atbmi1a Atbmi1b mutants. Atring1a Atring1b, Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants showed a slight increase of H3K4me3 levels at CRC and AIL5. But Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants also showed a slight decrease of H3K4me3 levels at DOG1. Atbmi1a Atbmi1b mutants showed a drastic decrease of H3K27me3 levels at ABI3, DOG1, CRC and AIL5 (Figure III.35), indicating that these genes are specific targets of AtBMI1a/b. Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 and Atring1a Atring1b mutants also showed a drastic decrease of H3K27me3 levels at ABI3, CRC and AIL5 (Figure III.35). While

  seed development genes. Taken together, it indicates that AtZRF1a and AtZRF1b are required for maintaining H3K27me2 and H2Aub to repress the seed development genes ABI3, CRA1, CRC, PER and AIL5, to promote seed germination (Figure III.35).

Figure III. 34 .Figure

 34 Figure III.34. Expression of seed related genes during double mutants Atzrf1a-1 Atzrf1b-1, Atzrf1a-2 Atzrf1b-1 and Col germination Relative expression levels of ABI3, DOG1, CRA1, CYC, PER and AIL5 were compared by qRT-PCR between Col (blue bars) and double mutants Atzrf1a-1 Atzrf1b-1 (red bars), Atzrf1a-2 Atzrf1b-1 (green bars) at 5 DAS. Indicated values are meansSD from 3 technical replicates; three biological replicates gave similar results.

Figure

  Figure III.36. Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants mis-regulated genes.Venn diagrams showing the number and overlap of differentially expressed genes found in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 mutants. Microarray analyses were performed on Agilent Chip using total RNA extracted from 15 days old seedlings. The differentially expressed genes in the mutant compared to wild-type are validated by a change of at least 2fold and Bonferroni P value inferior to 0.05 from three replicates of hybridization. The corresponding overlaps are indicated by the percentage of common loci and the statistical significance is qualified by a representation factor (RF) and an associated p-value.

Figure III. 37 .

 37 Figure III.37. Comparison of mis-regulated loci identified by microarray analysis of gene expression in Atzrf1a Atzrf1b, Atring1a Atring1b and Atbmi1a Atbmi1b mutants seedlings.

  and GST-BMI1C. AtZRF1b protein comes from p35S::FLAG-ZRF1b transgenic plants in the wild-type background. Agarose beads coated with GST, GST-RING1A, GST-BMI1A, GST-BMI1B or GST-BMI1C were incubated with an equal aliquot of total nuclear protein extracts of Arabidopsis plants expressing FLAG-AtZRF1b. Then the pulldown fractions were analyzed by Western blot using antibodies against FLAG. We found that AtZRF1b can interact with AtBMI1A, AtBMI1B and AtBMI1C but not with AtRING1A (Figure III.38A). In order to confirm the observed interaction, we performed FLIM analysis to examine GFP-AtZRF1b interaction with RFP-AtRING1A, RFP-AtBMI1A, RFP-AtBMI1B or RFP-AtBMI1C, which are coexpressed in Nicotiana benthamiana leaves. We confirmed the interaction between AtZRF1b and AtBMI1A or AtBMI1B (Figure III.38B).

Figure III. 38 . 1 Atzrf1b- 1

 3811 Figure III.38. AtZRF1b physically interacts with AtBMI1 proteinsPulldown assay. Agarose beads coated with GST, GST-RING1a, GST-BMI1a, GST-BMI1b or GST-BMI1c were incubated with an equal aliquot of total protein extracts of Arabidopsis plants expressing FLAGAtZRF1b. The pulldown fractions and inputs were analyzed by Western blot using antibodies against FLAG (@FLAG, top panel). Coomassie staining is shown as loading control (bottom panel). The positions of GST, GST-RING1a, GST-BMI1a, GST-BMI1b and GST-BMI1c are indicated by red squares, respectively. FLIM detection of the GFP-AtZRF1b interaction with RFP-AtRING1a, RFP-AtBMI1a, RFP-AtBMI1b and RFP-AtBMI1c in planta. GFPand RFP-tagged proteins as indicated were transiently coexpressed in Nicotiana benthamiana leaves. The fluorescence lifetime of GFP fusion proteins was recorded two days post infiltration. Data represent the average GFP fluorescence lifetime decay 6 SD of three biological replicates, with over 30 nuclei for each recording. Values above 5% indicate positive protein-protein interactions.

Figure III. 39 .

 39 Figure III.39. Phenotype of quadruple mutants Left: Analysis of the expression of AtZRF1a, AtZRF1b, AtRING1a or AtRING1b in the quadruple mutant Atzrf1a-1 Atzrf1b-1 Atring1a Atring1b, and of the expression of AtZRF1a, AtZRF1b, AtBMI1a or AtBMI1b in the quadruple mutant Atzrf1a-1 Atzrf1b-1 Atbmi1a Atbmi1b. Full-length AtZRF1a, AtZRF1b, AtRING1a, AtRING1b, AtBMI1a and AtBMI1b sequences were amplified from wild-type (Col) and quadruple mutant cDNAs. ACTIN served as an internal control. Right: Phenotype of quadruple mutants.

  contains AtZRF1a and AtZRF1b exhibiting high sequence homology. The AtZRF1 function and the underlying molecular mechanisms remain largely unknown. So far, our study has revealed a redundant function of AtZRF1a and AtZRF1b in the regulation of plant development. The AtZRF1b protein is localized in both the cytoplasm and the nucleus. Moreover, both AtZRF1a and AtZRF1b genes are widely expressed in various plant organs including roots, stems, leaves, and inflorescences.The Atzrf1a Atzrf1b mutant plants are small in size. Plant size is intrinsically determined by cell division and cell expansion activities. In the Atzrf1a Atzrf1b mutant, the pavement cell size is smaller than wild-type, and the final leaf size is drastically reduced in Atzrf1a Atzrf1b compared with Col. The reduced leaf size is largely associated with a major reduction of cell expansion. Moreover, cell division and differentiation in Atzrf1a Atzrf1b double mutants are also affected; the G1 phase is relatively shorter, and polyploidy levels are slightly increased in the Atzrf1a Atzrf1b mutant leaves. And qPCR data showed a strong enrichment in M-phasespecific genes down-regulated in the Atzrf1a Atzrf1b mutant, namely, the A-type and B-type cyclins that are key regulators in the G2-to-M transition(Inzé and De Veylder, 2006).

AtZRF1a

  and AtZRF1b drastically delayed flowering time. To explore the molecular mechanisms responsible for the change in flowering time in Atzrf1a Atzrf1b double mutants, we tested flowering-related genes by quantitive PCR. In the Atzrf1a Atzrf1b double mutant, the expression of FLC was about 5 times lower than in wild type (Figure III.16A). And the expression of MAF1 (FLM), MAF2 and MAF4 was 2.5-5 times lower than that in wild type (Figure III.16A). As a result of the down-regulation of FLC and MAFs, the expression of FT in Atzrf1a Atzrf1b double mutant was about two times higher than in wild type (Figure III.16B). These results suggest that AtZRF1a and AtZRF1b are involved in flowering regulation.

AtZRF1a

  and AtZRF1b in flowering time regulation is opposite to that of the PRC1 complex, AtZRF1a and AtZRF1b promoting the expression of FLC and MAF4 through affecting their H3K27me3 levels to regulate the floral transition in Arabidopsis (FigureIII.18).The function of AtZRF1a and AtZRF1b in mediating H3K27me3 levels could be restricted to a limited number of target genes in a specific developmental context.As shown in this study, although elevated levels of H3K27me3 were found to be associated with down-regulation of FLC and MAF4 in Atzrf1a Atzrf1b, ,global H3K27 methylation levels are not altered in Atzrf1a Atzrf1b as compared to wild-type plants during the floral transition, indicating that AtZRF1a and AtZRF1b might only affect H3K27me3 levels at a few specific flowering regulators. We failed to detect an elevated level of H2Aub1, which would support the animal model where ZRF1 is involved in H2Aub1 removal for transcriptional activation. In this study, H2Aub1 levels at FLC and MAF4 are not altered and global H2Aub1 levels are slightly down-regulated in Atzrf1a Atzrf1b as compared to wildtype plants during the floral transition. This may be because the loss of AtZRF1a and AtZRF1b affects the expression levels of AtRING1a, AtRING 1b, AtBMI1a, AtBMI1b and AtBMI1c .However, the Atbmi1a Atbmi1b and Atring1a Atring1b mutants also did not show detectable changes of H2Aub1 at FLC and MAF4. Seed germination is crucial for next-generation plant growth and it is regulated by a very complicated signaling network and gene expression regulation. Different plants may share similar molecular mechanisms. Atzrf1a and Atzrf1b single mutants showed a normal germination ratio, while the Atzrf1a Atzrf1b double mutant exhibited a delayed germination under osmotic stress growth conditions (treatment with salt or mannitol). Similarly, the Atbmi1a Atbmi1b and Atring1a Atring1b double mutants also displayed a germination delay. The enhanced germination defects observed in the Atzrf1a Atzrf1b Atbmi1a Atbmi1b quadruple mutant indicate that AtZRF1a/b and AtBMI1a/b may also work in parallel pathways. AtZRF1a/b and AtBMI1a/b promote seed germination likely through repression of seed developmental genes. Consistently, the Atzrf1a Atzrf1b mutant and the Atbmi1a Atbmi1b mutant showed derepression of ABI3, CRU1/CRA1, CRU3/CRC, CHO1/AIL5

  ggatcc ATG CCG AGC CGG AGA AGT GAG TC Z1aR-EcoRI gaattc TCA TAC TCC GGT TTT CTT GTT TCT AAT GAT TTC GGC GGATCC GGC TGT GGG TTT CTT GGT TCT GAT G ZRF1bSANT-R GGC CTC GAG CTT CTC ATG AGT AGC AGT ATC C ZRF1bSANT-F GGC GAA TTC AAA GAG AAA CCT TGG AGC AAG ZRF1bSANT-R GGC CTC GAG GGC TGT GGG TTT CTT GG ZRF1bUBD-F GGC GAA TTC AGA AGA ATA TTT GAC TCT ACA GAT ZRF1bUBD-R GGC CTC GAG TTT CTG TAT TCT TAT GTC TTT TTT A ZRF1bUBD-R GGC GAA TTC TTT TGT TGA GTC CAT CAA AAC C Name Sequences 5'-3'

  R1 CAT TTT TAA CCA AGG TCT qChFT-F2 GAT CTA CAA TCT CGG CCT TCC qChFT-R2 ATC ATC ACC GTT CGT TAC TCG qChFLC-F1 ATT TAG CAA CGA AAG TGA AAA CTA AG qChFLC-R1 GCC ACG TGT ACC GCA TGA C qChFLC-F2 AGA AAT CAA GCG AAT TGA GAA CAA qChFLC-R2 CGT TGC GAC GTT TGG AGA A qChFLC-F3 AAT TGC ATG TCA TTC ACG ATT TG qChFLC-R3 TGA AAC TTC ACT CAA CAA CAT CGA qChFLC-F4 CAT CTC TCC AGC CTG GTC AAG qChFLC-R4 GGC TTT AAG ATC ATC AGC ATG CT qChFLC-F5 AGC CAG GTA ACG AAA GCT ACA TTT qChFLC-R5 ACA TGG ACA TTG GAC ACA CAA CA qChMAF4-F1 GAC CAA CGC GCC ACA AG qChMAF4-R1 CGG TGC GTT TTT AAT AGG AGT TTA G qChMAF4-F2 CCG AAT TGA GAC CTT GTA GAA GTA GA qChMAF4-R2 ATC AAG CAT TTG TGG TGT TAA GTA TGA qChMAF4-F3 GTT GTT TTC CTT TTC TGT TGT TTA TCT A qChMAF4-R3 ATA CTT ACA TTA TCG CTT TTC GCT TCT qChMAF4Mother plants are chosen at a stage when they have developed 5-6 inflorescences (they have the largest buds). A bud, at the correct stage, should contain short immature stamens with anthers that are greenish-yellow in color and should not have opened and potentially exposed its pistil to parental pollen. Father plants should have have started to form siliques (this indicates that the pollen is o.k.).

  ) and grow overnight. Measure the OD 600 of overnight culture. Pellet the bacteria (5000 x g, 15 min) and resuspend the pellet in Resuspension Solution. The final OD 600 should be adjusted to 0.4. Leave on the bench (room temperature) for 2-3 hours (or overnight) before infiltration. Perform the infiltration with 5 ml syringe. Simply press the syringe (no needle) on the underside of the leaf (avoid cotyledons!), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading "wetting" area in the leaf. 2-5 days after infiltration, observe the fluorescence labeled protein under a fluorescence microscope or confocal laser scanning microscope. Or harvest leaves for protein purification.

Then in 1 .

 1 5 ml tubes at room temperature add 40-100 fmol PCR product (a 1-kb PCR product is ~0.65 ng/fmol), ~150 ng donor vector (pDONR207) and TE or water to a final volume of 8 µl. Remove BP clonase TM II Enzyme Mix and thaw on ice (~2 min). Vortex briefly (2 s) twice and add 2 µl of BP clonase enzyme mix to BP reaction. Mix well by vortexing briefly twice. After that, reactions are incubated at 25°C for 60 min or overnight. Finally, add 1 µl of proteinase K solution and incubate for 10 min at 37°C to stop the reaction. LR reaction: In 1.5-ml tubes at room temperature add ~150 ng entry clone, ~150 ng destination vector and TE or water to a final volume of 8 µl. Remove LR clonase TM II Enzyme Mix and thaw on ice (~2 min). Vortex briefly (2 s) twice and add 2 µl of LR clonase enzyme mix to the LR reaction. Mix well by vortexing briefly

Day 4 :

 4 6.4 µl 0.5 M EDTA pH8.0, 12.8 µl Tris-HCl pH8.0 and 2 µl 20mg/ml proteinaes K were added and incubated at 45°C for 1 hour. Recover DNA was recovered by NucleoSpin Gel and PCR Clean-up kit (Macherey-nagel). At first, mix 1 volume of sample with 5 volumes of Buffer NTB to adjust DNA binding condition. Then place a NucleoSpin Gel and PCR Clean-up Column into a collection tube and load up to 700 µl sample, centrifuge for 30 s at 11000g. Discard flow-through and add 700 µl Buffer NT3 to the column. Centrifuge for 30 s at 11000 g, discard flowthrough and centrifuge for 1 min at 11000 g to remove Buffer NT3 completely. At last, place the column into 1.5 ml tube and add 150 µl Buffer NE, incubating at room temperature for 1 min. Centrifuge for 1 min at 11000g. Fold-enrichment of each fragment was determined by quantitative real-time PCR. Genomic fragments of ACTIN2 were amplified as internal controls for measurement of H3K27me3, H3K4me3 and H2AK119ub1 enrichment, respectively. Fold enrichment of each fragment was calculated first by normalizing the amount of a target DNA fragment against a genomic fragment of an internal control, and then by normalizing the value for immunoprecipitated samples against that for input. Fix buffer: 0.4 M sucrose; 10 mM Tris-HCl pH8.0; 1 mM EDTA pH8.0; 1.0% Formaldehyde add PMSF before use ChIP lysis buffer: 50 mM HEPES pH7.5; 1 mM EDTA pH8.0; 150 mM NaCl; 1% Triton X-100; 5 mM β-mercaptoethanol; 10% glycerol add cocktail before use Antibody binding buffer: 50 mM Tris-HCl pH8.0; 1 mM EDTA pH8.0; 150 mM NaCl; 0.1% Triton X-100; 5 mM β-mercaptoethanol add cocktail before use Low salt wash buffer: 50 mM HEPES pH7.5; 150 mM NaCl; 1 mM EDTA pH8.0 High salt wash buffer: 50 mM HEPES pH7.5; 500 mM NaCl; 1 mM EDTA pH8.0 LiCl wash buffer: 10 mM Tris-HCl pH8.0; 1 mM EDTA pH8.0; 0.5% NP-40; 0.25 M LiCl TE buffer: 10 mM Tris-HCl pH8.0; 1 mM EDTA pH8.0 Elution buffer: 1% SDS; 0.1 M NaHCO 3

  (1-2 mm side pieces or strips) with a razor blade in nuclear extraction buffer (CyStain UV precise P kit, Partec). All preparations were subsequently filtered through 50 µm (pore diameter) nylon mesh and stained with nuclear staining solution (CyStain UV-precise P) containing 4, 6-diamidino-2-phenylindole (DAPI). Flow cytometry was performed on a Ploid Analysis PA-1 (Partec). Ploidy levels of three individual plants were averaged and doublets were excluded from the analysis by gating on single nuclei in a DAPIwidth versus DAPI-area. A total of 5000 nuclei per sample were analyzed. Data were analyzed using Flowjo (TreeStar).V.2.10.1 Preparation of competent cellsSingle colonies were picked from a plate freshly grown for 16-20 hours at 37°C and transferred into 5 ml of LB broth medium and incubated by shaking at 37°C overnight. Overnight culture was used to inoculate new LB (1: 100) and incubated at 37°C with vigorous shaking (300 cycles per minute). Monitor growth until OD 600 reaches 0.4-0.6. Aseptically transfer the cells to sterile, disposable, ice-cold 50 mL Falcon tubes. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes. Spin cells at 4000 rpm for 10 minutes at 4°C. Pour off supernatant and stand tubes upside down for 1 minute to drain remaining media. Resuspend each pellet in 10 ml of icecold 0.1 M CaCl 2 and store on ice. Spin cells at 4000 rpm for 10 minutes at 4°C. Pour off supernatant and stand tubes upside down for 1 minute to drain remaining media.Resuspend each pellet in 2 ml of ice-cold 0.1 M CaCl 2 and store on ice. Dispense in 100 ul aliquots, freeze in liquid nitrogen, and store in at 80°C.In addition, we can prepare competent cells by electroporation. When the OD 600 equals 0.4-0.6 (log phase growth), remove the cells from the shaker and place on ice. Split the culture into four equal parts by pouring ~250 ml of culture into each chilled 250 ml Corning pointed bottle. Centrifuge at 4000 rpm, 25 min at 4°C. Place bottles on ice. Remove supernatant immediately as cell pellet begins to lift off quickly.

Add 1

 1 µl of DNA or 5-20 µl DNA ligation products to 100 µl of competent cells. Mix contents by swirling gently. Store the tubes on ice for 30 minutes. Transfer tubes to a rack placed in a circulating water bath that has been preheated to 42°C.Leave the tubes in the rack for exactly 45 seconds. Do not shake the tubes. Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 minutes. Add 400 ul LB to each tube. Incubate cultures for 45 minutes in a water bath set at 37°C or rotate cell at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded in the plasmid. Transfer the appropriate volume of transformed competent cells onto agar LB plate with appropriate antibiotic. Using a sterile bent glass rod gently spread the transformed cells over the surface of the agar plate. Leave the plate at room temperature until the liquid has been absorbed. Invert the plates and incubate at 37°C. Colonies should appear in 12-16 hours.V.2.10.3 Electroporation transformationLocate Electroporator power source and cuvette holder (Bio-Rad). Set the conditions for transformation according to strain. For DH5α cells, use 25 µFD, 200 Ω, and 2.5 kV. The time constant (tau value) should be 3-4 msec. Thaw required number of frozen cell aliquots (each tube 70 µl = two transformations) on ice. Thaw plasmid DNA in TE/H 2 0 on ice. Place 15 ml conical tube containing 10 ml of LB media without antibiotics on ice. Place 3 µl of DNA along wall of 0.2 cm cuvette. Pipet 35 µl of thawed electrocompetent cells onto DNA drop. Flick cuvette to settle DNA + cells mixture into bottom of cuvette. Have 1 ml pipette containing 1 ml of LB media ready. Dry off any moisture from cuvette outside and immediately place cuvette in white plastic holder. Slide holder into position and zap cells. If you hear a high constant tone, immediately add the 1ml of LB to cells! Transfer cells from cuvette into 1.5 ml eppendorf tube and store on ice. The tone indicates that you have successfully electroporated your cells. Record the time constant value. Repeat procedure for remaining samples. If you see or hear sparking coming from your cuvette of cells, then the cells are dead! Repeat that sample again. Things that can cause sparking: excess water on cuvette outside, human skin oil on cuvette outside, too high salt conc. in DNA sample (try diluting DNA 10-fold), and poorly made electrocompetent cells. Outgrow transformed cells in eppendorf tubes by incubating the tubes in 37°C water bath for 1-1.5 hrs. Place transformant plates in 37°C bacterial incubator for 16-24 hrs until colonies appear. V.2.10.4 Extraction of plasmid DNA Plasmid DNA was extracted by the alkaline lysis method from 1.5 ml bacterial culture. Overnight-grown bacterial cells were harvested and resuspended in ice-cold 100 µl of solution I by vortexing vigorously. Then freshly prepared 200 µl of solution II were added and gently mixed by inverting the tube 6-8 times. After chilling 5 min on ice, 150 µl of solution III were added and mixed by inverting the tube. The bacterial lysate was centrifuged at 13000 rpm for 5 min and the supernatant was transferred to a new tube. The solution was cleared with one volume of phenol: chloroform and plasmid DNA was precipitated with 2 volumes of ethanol and washed with 70% ethanol. Finally, the DNA pellet was dissolved in 50 µl of distilled water containing 10 ug/ml DNase-free RNase A (Fermentas). Solution I: 50 mM Glucose; 10 mM EDTA; 25 mM Tris-HCl pH8.

FIGURE 1 |

 1 FIGURE 1 | A proposed model for deposition and removal of histone H2B monoubiquitination in transcriptional activation of FLC and MAFs in flowering time regulation. In this model, HUB1 and HUB2 form a heterotetramer and recruit UBC1 or UBC2 to FLC/MAFs chromatin, leading to transfer of a ubiquitin (ub) monomer from UBC1 or UBC2 onto H2B. H2Bub1 formation enhances H3K4me3 deposition by methyltransferases, together promoting transcription initiation. UBP26 removes ubiquitin on H2B, favoring H3K36me3 deposition in promoting transcription elongation. Active transcription of FLC/MAFs represses Arabidopsis flowering, a transition from vegetative to reproductive plant development.

FIGURE 2 |

 2 FIGURE 2 | Proposed models for histone H2A monoubiquitination deposition in transcriptional repression of varied target genes. The Arabidopsis PRC1-like RING-finger proteins AtRING1a/b (RING1) and AtBMI1a/b/c (BMI1) have the E3 ligase activity in catalyzing H2A monoubiquitination (H2Aub1). Comparable to the classical model of sequential PRC2 then PRC1 action in Polycomb silencing in animal cells, the Arabidopsis PRC1-like protein LHP1 binds H3K27me3 pre-deposited by the evolutionarily conserved PRC2 complexes and recruits RING1, BMI1 and possibly also EMF1 through protein-protein interactions (A). This combinatorial action by PRC2 then PRC1 likely plays a broad role in

1 Atzrf1b- 1 Atzrf1b- 1 . 1 Atzrf1b- 1

 11111 2 (PRC2) et PRC1, responsables UHVSHFWLYHPHQW GH OD WULPpWK\ODWLRQ GH OD O\VLQH VXU O ¶KLVWRQH +.PH HW GH OD PRQRXELTXLWLQDWLRQ GH OD O\VLQH VXU O ¶KLVWRQH +$ (H2Aub1). Il est maintenant accepté TXH O ¶HQULFKLVVHPHQW HQ H3K27me3 par le PRC2 est un pré-requis essentiel à l'attachement du PRC1 à la chromatine, permettant la répression stable de la transcription chez divers organismes eucaryotes. Chez les végétaux, ces mécanismes de modification des histones par le PRC2 en lien avec la répression de la transcription sont bien connus. &HSHQGDQW FH Q ¶HVW TXH UpFHPPHQW TXH OHV KRPRORJXHV GHV 35& DQLPaux ont été identifiés chez Arabidopsis (AtRING1A/B et AtBMI1A/B/C) et que leur implication dans la monoubiquitination de H2A a été démontrée. En lien avec ce processus de répression, une étude intéressante a permis de démontrer le rôle joué par ZUOTIN-RELATED FACTOR 1 (ZRF1) dans la levé de la répression Polycomb-dépendante des gènes essentiels pour la différentiation des cellules humaines. Ainsi, ZRF1 se lie spécifiquement à H2Aub1 et dissocie le PRC1 de la chromatine, ce qui engendre ensuite la dissociation du PRC2 et par conséquence la levé de la répression. Deux homologues de ZRF1 ont été identifiés chez Arabidopsis et sont ci-après nommé AtZRF1a et AtZRF1b. Mon WUDYDLO GH GRFWRUDW V ¶HVW FRQFHQWUp VXU OD FDUDFWpULVDWLRQ IRQFWLRQQHOOH G$W=5)pull-GRZQ P ¶RQW SHUPLV GH GpPRntrer que la protéine de fusion His-AtZRF1b peut se lier à la protéine de fusion GST-ub1, mais pas à la GST seule. Par la suite, des analyses de mutagenèse dirigée ont révélé que le domaine conservé UBD est responsable de la liaison à ub1. De plus, le domaine UBD seul fusionné à la GST peut se lier à H2Aub1 et également à H2A. Ces résultats sont similaires aux observations faites avec la ZRF1 humaine.AtZRF1a et AtZRF1b ont des fonctions redondantes et jouent des rôles cruciaux pour la germination des graines 3DU XQH DSSURFKH JpQpWLTXH M ¶DL HQWUHSULV O ¶DQDO\VHV GHV U{OHV ELRORJLTXHV GH AtZRF1a et AtZRF1b chez Arabidopsis. A partir de différentes banques de mutant G ¶LQVHUWLRQ M ¶DL LVROp OHV VLPSOHV PXWDQWV Atzrf1a-1, Atzrf1a-2 et Atzrf1b-1, SXLV M ¶DL REWHQX SDU FURLVHPHQWV OHV GRXEOHV PXWDQWV Atzrf1aet Atzrf1a-2 Atzrf1b-1. Des graines de génotype sauvage (Col) ou provenant des simples et des doubles mutants ont été placées sur milieu MS dans des boites de pétri, puis stratifiées, afin d'analyser la cinétique de germination (l'émergence de la radicule étant ici l'indicateur de germination). 'ans des conditions standard, la germinatioQ Q ¶HVW SDV DIIHFWpH GH PDQLqUH significative chez les simples mutants Atzrf1a-1, Atzrf1a-2 et Atzrf1b-1. Cependant, dans les même conditions, le taux de germination des doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 est lui diminué. A un niveau moindre, nous avons également observé une diminution du taux de germination du double mutant Atbmi1a Atbmi1b. L'acide gibbérellique 3 (GA3) est connue pour favoriser la levée de dormance et la germination, pourtant malgré les différentes concentrations testées (0.5, 1.0 and 2.0µmol/L), ilQ ¶D SDV pWp SRVVLEOH GH UHVWDXUHU XQH JHUPLQDWLRQ QRUPDOH FKH] OHV doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1.Ensuite, nous avons soumis nos mutants à des stresses salin et osmotique, connus pour leurs effets négatifs sur la germination. En présence de 100 mM de NaCl ou de 200 mM de mannitol, le taux de germination des simples mutants Atzrf1a-1, Atzrf1a-2 et Atzrf1b-1 reste similaire à celui observé chez Col, alors que les doubles mutants Atbmi1a Atbmi1b, Atzrf1a-1 Atzrf1b-1et Atzrf1a-2 Atzrf1b-1 présentent une efficacité de germination significativement réduite (le taux de germination des doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 étant significativement inférieure à celui du double mutant Atbmi1a Atbmi1b). En effet, dans ces conditions, toutes les graines sauvages sont JHUPpHV MRXUV DSUqV VWUDWLILFDWLRQ DORUV TX ¶DX PrPH WHPSV OH WDX[ GH JHUPLQDWLRQ Q ¶HVW TXH G ¶HQYLURQ HQ SUpVHQFH GH P0 GH 1D&O HW G ¶HQYLURQ HQ SUpVHQFH GH P0 de mannitol pour les doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Le double mutant Atbmi1a Atbmi1b présente lui un taux de JHUPLQDWLRQ G ¶HQYLURQ HQ SUpVHQFH GH P0 GH 1D&O HW G ¶HQYLURQ HQ présence de 200 mM de mannitol. -DL HQWUHSULV O ¶pWXGH GHV PpFDQLVPHV PROpFXODLUHV j OD EDVH GHV GpIDXWV GH germination observés chez les doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 (Q SUHPLHU OLHX M ¶DL DQDO\Vp OH WDX[ G ¶H[SUHVVLRQ GH gènes impliqués dans le développement de la graine, tels que ABI3, DOG1, CRA1, CRC, PER and AIL5. Comme attendu, les différents gènes analysés présentent tous, jO ¶H[FHSWLRQ GH DOG1, une levé de répression de leur expression dans des plantules de Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1. $ILQ G ¶DOOHU SOXV HQ DYDQW GDQV O ¶pWXGH GH FHWWe dé-UpSUHVVLRQ M ¶DL UpDOLVp GHV DQDO\VHV G ¶LPPXQR-précipitation de la chromatine (ChIP) des gènes listés ci-GHVVXV j O ¶DLGH G ¶DQWLFRUSV GLULJpV VSpFLILTXHPHQW FRQWUH +.PH +.PH RX +$XE - ¶DL ainsi détecté chez Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 une légère augmentation GX QLYHDX G ¶+.PH VXU FHUWDLQV JqQHV DORUV TXH les niveaux en H3K27me3 HW +$XE VRQW G ¶XQH PDQLqUH JpQpUDOH GLPLQXpV VXU WRXV OHV gènes analysés. Ces résultats indiquent que AtZRF1a et AtZRF1b sont requis dans la maintenance du niveau de H3K27me3 et de H2Aub1 nécessaire à la répression des gènes du développement de la graine ABI3, CRA1, CRC, PER and AIL5 afin de permettre la germination. AtZRF1a et AtZRF1b sont impliqués dans la maintenance des cellules souches et la régulation des divers aspects développementaux des plantes En plus de son sévère défaut de germination, le double mutant Atzrf1a Atzrf1b SUpVHQWH G ¶DXWUHV GpIDXWV SKpQRW\SLTXHV $LQVL SDU UDSSRUW j GHV plantules sauvages, le phénotype des cotylédons de plantules Atzrf1a-1 Atzrf1b-1 peut présenter des degrés variables de sévérité (e.g. cotylédon unique, cotylédons asymétriques ou encore embryonnaires). Au stade végétatif, des mesures du poids frais de plantes de 4 semaines ont permis de confirmer le nanisme des doubles mutants Atzrf1a-1 Atzrf1b-1 (18.33 ± 6.87 mg, n = 10) et Atzrf1a-2 Atzrf1b-1 (18.59 ± 6.90 mg, n = 10) par rapport à Col (75.0 ± 11.18 mg, n = 10). En microscopie électronique à balayage, la taille des cellules chez les doubles mutants Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 apparaît réduite. La taille des cellules épidermique pavimenteuses est diminuée de 40% chez Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 par rapport à Col. Prises dans leur ensemblece qui pourrait en grande partie expliquer la taille réduite des feuille chez Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1. $ILQ G ¶H[DPLQHU OD SURJUHVVLRQ GX F\FOH FHOOXODLUH QRXV DYRQV FRPSDUp les niveaux de ploïdie entre des feuilles de Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 et des feuilles de Col en mesurant le contenu relatif en ADN nucléaire par cytométrie en flux. Le cycle cellulaire se divise en quatre phases : la phase G1 ou post-PLWRWLTXH DYHF VRQ QLYHDX & G ¶$'1 QXFOpDLUH OD SKDVH 6 RX SKDVH GH V\QWKqVH G ¶$'1 DYHF VRQ QLYHDX G ¶$'1 LQWHUPpGLDLUH HQWUH & HW & OD phase G2 ou post-UHSOLFDWLYH DYHF VRQ QLYHDX & G ¶$'1 HW ILQDOHPHQW OD SKDVH 0 RX PLWRWLTXH 0HV DQDO\VHV P ¶RQW SHUPLV G ¶REVHUYHU que la proportion de cellules 2C était sensiblement plus faible chez Atzrf1aet Atzrf1a-2 Atzrf1b-1 par rapport à Col, ce qui suggère une réduction de la durée de la phase G1 chez le double mutant. Les cellules ayant un niveau de ploïdie supérieur ou égale à 8C sont le résultat du de la cellule. La proportion de ces cellules est également légèrement augmentée chez Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 par rapport à Col. GFP, WOX5::GFP, SCR::GFP, CO2::GFP, J1092 and J2341 dans le double mutant Atzrf1a-1 Atzrf1b-1 M ¶DL FKHUFKp j FRPSUHQGUH OHV FDXVHV GH FH SKpQRW\SH 0HV REVHUYDWLRQV HQ PLFURVFRSLH FRQIRFDOH P ¶RQW SHUPLV GH FRQVWDWHU TXH OHV mutations perte de fonction de AtZRF1a et AtZRF1b conduisent à une importante désorganisation des différentes couches cellulaires au niveau de la racine et provoque la perte des cellules souches racinaires au niveau du centre TXLHVFHQW 'HV DQDO\VHV GH 3&5 TXDQWLWDWLYH P ¶RQW GH SOXV SHUPLV GH GpPRQWUHU TXH des gènes de régulation de la voie de l'auxine sont dérégulés chez les doubles mutants Atzrf1a-1 Atzrf1b-1et Atzrf1a-2 Atzrf1b-1 3RXUWDQW O ¶DSSOLFDWLRQ H[RJqQH GH différentes concentrations GH O ¶DX[LQH V\QWKpWLTXH $1$ DFLGH QDSKWDOqQH-acétique) ne semble pas restaurer le phénotype racinaire du double mutant. Le double mutant Atzrf1a Atzrf1b présente également un défaut de floraison. En comptant le nombre de feuilles formées dans la rosette avant que la KDPSH IORUDOH Q ¶DSSDUDLVVH M ¶DL SX constater que le double mutant Atzrf1a Atzrf1b SUpVHQWDLW XQ SKpQRW\SH GH IORUDLVRQ SUpFRFH - ¶DL HQVXLWH FRPELQp FHWWH DQDO\VH SKpQRW\SLTXH j O ¶DQDO\VH GX QLYHDX G ¶H[SUHVVLRQ GHV GLIIpUHQWV gènes de floraison que sont FLC, les gènes MAFs, FT, SOC1, AGL24 et SVP. Dans le GRXEOH PXWDQW OH QLYHDX G ¶H[SUHVVLRQ GH FLC et des gènes MAFs est fortement réduit. $LQVL FH SURILOH G ¶H[SUHVVLRQ HVW O ¶LQYHUVH GH FHOXL REVHUYp SRXU OHV JqQHV GX GpYHORSSHPHQW UDFLQDLUH GpFULWV SOXV KDXW G ¶R O ¶LQWpUrW G ¶pWXdier la structure de leur FKURPDWLQH 3DU GHV DQDO\VHV GH &K,3 M ¶DL SX REVHUYHU TXH OH QLYHDX GH +.PH HW G ¶+$XE UHVWH LQFKDQJp FKH] Atzrf1a-1 Atzrf1b-1 et Atzrf1a-2 Atzrf1b-1 par rapport j &RO DX QLYHDX GHV JqQHV GH IORUDLVRQ DORUV TXH OH QLYHDX G ¶+3K27me3 lui augmente. Ces résultats indiquent que AtZRF1a et AtZRF1b sont nécessaire au mDLQWLHQW G ¶XQ QLYHDX IDLEOH G ¶+.PH SHUPHWWDQW GH SURPRXYRLU O ¶H[SUHVVLRQ GHV JqQHV FLC et MAFs et donc de réprimer la floraison. AtZRF1a et AtZRF1b fonctionnent partiellement en relation avec PRC1 Certains défauts phénotypiques observés chez le double mutant Atzrf1a Atzrf1b sont similaires à ceux précédemment observés chez les mutants du PRC1 Atring1a Atring1b et Atbmi1a Atbmi1b. Mes donnés de microarray YRQW GDQV OH VHQV G ¶XQH LQWHUDFWLRQ JpQpWLTXH SXLVTX ¶HOOHV UpYqOHQW une superposition significative des gènes dérégulés chez Atzrf1a Atzrf1b et Atring1a Atring1b ou Atbmi1a Atbmi1b - ¶DL GH SOXV DQDO\Vp O ¶LQWHUDFWLRQ SK\VLTXe de AtZRF1b DYHF $W5,1* RX $W%0, 'HV ELOOHV G ¶DJDURVHV FRXSOpV j OD *67 RX DX[ SURWpLQHV de fusion GST-RING1A, GST-BMI1a, GST-BMI1B ou GST-$UDELGRSsis exprimant la protéine de fusion FLAG-AtZRF1b. Le pull-GRZQ GH OD *67 VXLYLH GH O ¶DQDO\VH SDU :HVWHUQ-EORW j O ¶DLGH G ¶DQWLFRUSV DQWL-)/$* P ¶D DORUV SHUPLV GH GpPRQWUHU O ¶LQWHUDFWLRQ HQWUH $W=5)% HW AtBMI1A, AtBMI1B ou AtBMI1C, mais pas AtR,1*$ - ¶DL SDU OD VXLWH FRQILUPp FHV UpVXOWDWV SDU GHV DQDO\VHV GH )/,0 HQ GpPRQWUDQW O ¶LQWHUDFWLRQ HQWUH OHV SURWpLQHV de fusion GFP-AtZRF1b et RFP-AtBMI1A ou RFP-AtBMI1B. Ensemble de mes résultats ont permis la première caractérisation fonctionnelle des gènes AtZRF1a et AtZRF1b. Mes données ont montrés que AtZRF1a et AtZRF1b jouent des rôles en partie en relation avec PRC1, mais également avec aspects spécifiques. Leur rôle dans l'enlèvement de H2Aub1, comme cela été proposé pour ZRF1 chez l'animal, n'a pas été observé chez les plantes. PROPOSITION DE MOTS-CLÉS : Chromatine regulateur; Épigénétique; Ubiquitine; H2Aub1; Régulation de la transcription; Développement de la plante; ZRF1; Germination des grains Polycomb group (PcG) proteins form two distinct complexes, polycomb repressive complex 2 (PRC2) and PRC1 that mediate trimethylation at histone 3 lysine 27 (H3K27me3) and monoubiquitination at histone H2A lysine 119 (H2AK119ub), respectively. H3k27me3 by PRC2 is believed to be a prerequisite for PRC1 binding, and such combination of PcG-mediated epigenetic modifications lead to transcriptional gene silencing in diverse eukaryotic organisms. PRC2-mediated histone modification and gene repression have also been intensively studied in plant. However, PRC1 in plants has only been more recently documented and the Arabidopsis PRC1-like RING-finger homologs (AtRNIG1A/B and AtBMI1A/B/C) have been characterized and shown to catalyze H2AK119ub. A recent study shows that ZUOTIN-RELATED FACTOR 1 (ZRF1) functions in the de-repression of polycomb-repressed genes in human cells. ZRF1 specifically binds to H2AK119ub and then displaces PRC1 from chromatin. The depletion of PRC1 subsequently causes the loss of PRC2 from the chromatin, consequently switching polycomb-repressed genes from repressive to active state. Two homologs of human ZRF1 have been identified in Arabidopsis, and are hereinafter named AtZRF1a and AtZRF1b. My PhD work focuses on the functional characterization of AtZRF1a and AtZRF1b.

AtZRF1a

  and AtZRF1b have redundant functions and play crucial roles for seed germination Using the powerful genetic tool in Arabidopsis, I investigated the biological roles of AtZRF1a and AtZRF1b. From the Arabidopsis seed store center, we got single mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1. Then by crossing, I obtained the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. Seeds of wild-type (Col), single mutants Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1 and double mutants Atzrf1a-1 Atzrfb-1, Atzrf1a-2

Atzrf1a- 1 1 Atzrf1b- 1 , 1 Atzrf1b- 1

 11111 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. It indicates that AtZRF1a and AtZRF1b are required for maintaining H3K27me3 and H2Aub to repress the seed development genes ABI3, CRA1, CRC, PER and AIL5, to promote seed germination.AtZRF1a and AtZRF1b are involved in stem cell maintenance and regulation of various developmental aspects of plantsIn addition to seed germination, the Atzrf1a Atzrf1b double mutants also exhibited other defective phenotypes. Compared to the wild-type plant, the Atzrf1a Atzrf1b double mutant seedlings showed varied degrees of phenotype severity on cotyledons, such as single cotyledon, asymmetrical cotyledon or embryonic cotyledon. Moreover, at the vegetative stage, fresh weight measurements of whole rosettes of 4-week-old plants confirmed the smaller size of Atzrf1a-1 Atzrf1b-1(18.33 ± 6.87 mg, n = 10) and Atzrf1a-2 Atzrf1b-1 (18.59 ± 6.90 mg, n = 10) compared with Col (75.0 ± 11.18 mg, n = 10). Scanning electron microscope revealed smaller cell size in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. The epidermal pavement cell surface is reduced to ~40% in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 as compared that in Col. Taken together, these data indicate that cell expansion is drastically constrained, which might largely account for the reduced leaf size in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1. To investigate cell cycle progression, we compared the ploidy levels of Atzrf1a-Atzrf1a-2 Atzrf1b-1 and Col leaves by measurement of the relative nuclear DNA content via flow cytometry analysis. The DNA was isolated from the first true leaf on three different plantlets. The cell cycle consists of four phases: postmitotic interphase (G1), with 2C nuclear DNA content; S phase, meaning DNA synthetic phase, nuclear DNA content intermediate 2C and 4C; postsynthetic interphase (G2), with a 4C nuclear DNA content; and finally the M phase, meaning mitosis. I observed that the proportion of 2C cells is slightly lower in Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 compared with Col, suggesting a relatively shorter duration of G1 in the mutant. Higher ploidy levels (ı8C) are the result of endoreduplication cycles in which nuclear DNA is replicated without a subsequent mitotic division. The relative proportion of cells with higher ploidy levels is slightly increased in Atzrf1aand Atzrf1a-2 Atzrf1b-1 compared with Col.Compared to wild-type, we found the primary root growth of the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 was strongly impaired. To further investigate the root phenotype, by crossing we introduced reporter genes DR5::GFP, WOX5::GFP, SCR::GFP, CO2::GFP, J1092 and J2341 into the double mutant Atzrf1a-1 Atzrf1b-1. Confocal observation results showed that loss-of-function of AtZRF1a and AtZRF1b drastically affects root cell layer organization and causes loss of root stem cells. RT-PCR analysis indicated that some auxin regulatory genes are mis-regulated in the double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1.

  It will be interesting to investigate H3K27me3, H3K4me3 and H2Aub at flowering genes for a comparison. I performed ChIP experiments using antibodies specific to H3K27me3, H3K4me3 or H2Aub. As results, we found that H3K4me3 and H2Aub levels were unchanged whereas the level of H3K27me3 up-regulated at the examined flowering time genes in Atzrf1a-1 Arzrf1b-1and Atzrf1a-2 Atzrf1b-1. It indicates that AtZRF1a and AtZRF1b are required for maintaining H3K27me3 to promote the flowering time genes FLC and MAFs, to repress flowering.

Table III .1. Segregation analysis of Atzrf1a Atzrf1b double mutant in progeny derived from self-pollination or reciprocal crosses

 III

  ± 3.6, n =10) and the Atzrf1a-2 Atzrf1b-1 double mutant plants grew approximately 46 days before bolting (46.1 ± 5.2, n =10) in LD. Moreover, in SD, the Atzrf1a-1Atzrf1b-1 double mutant plants grew approximately 115 days before bolting (115.1 ± 11.2, n =10) and the Atzrf1a-2 Atzrf1b-1 double mutant plants grew approximately 114 days before bolting (114.1 ± 10.5, n =10). These data indicate that

To examine whether loss of functions of AtZRF1a and AtZRF1b affect flowering time, we have grown Atzrf1a-1, Atzrf1a-2 and Atzrf1b-1 single mutants in long day (LD; 16 h light/8 h dark) and short day (SD; 8 h light/16 h dark) conditions, respectively., We counted the days the plants have grown until first flower emerged. As a result, in LD, the wild-type plants grew approximately 28 days before bolting (27.7 ± 2.2, n =10), the Atzrf1a-1 plants grew approximately 26 days before bolting (26.2 ± 0.6, n =10), the Atzrf1a-2 plants grew also approximately 26 days before bolting (26.0 ± 0.3, n =10) and the Atzrf1b-1 plants grew approximately 24 days before bolting (24.2 ± 0.6, n=10). This indicates that single mutants show slightly earlier flowering time than wild-type in LD condition. However, in SD, the wild-type plants grew approximately 78 days before bolting (78.8 ± 4.4, n =10), the Atzrf1a-1 plants grew approximately 66 days before bolting (66.6 ± 2.9, n =10), the Atzrf1a-2 plants grew approximately 69 days before bolting (69.7 ± 4.1, n =10) and the Atzrf1b-1 plants grew approximately 59 days before bolting (58.9 ± 1.0, n=10). Hence, single mutants show a more obvious earlier flowering time than wild-type in SD (Figure III.28C-D). Furthermore, we used double mutants Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 to study the flowering time in LD and SD conditions. We found that the heterozygous double mutant shows early flowering in LD (Figure III.29), however, the Atzrf1a-1Atzrf1b-1 double mutant plants grew approximately 47 days before bolting (47.3

  In Atring1a Atring1b double mutant, H3K27me3 levels at FLC and FT were not obviously affected, but reduced at MAF4. In Atzrf1a-1 Atzrf1b-1 and Atzrf1a-2 Atzrf1b-1 double mutants, the level of H3K27me3 was increased at both FLC and MAF4, but not obviously affected at FT (Figure III.32). ChIP analysis revealed that H2AK119ub1 levels at FLC, MAF4 and FT were not significantly changed in Atring1a Atring1b, Atbmi1a Atbmi1b, Atzrf1a Atzrf1b and clf (Figure III.32), indicating that H2AK119ub1 might not directly contribute to the modulation of FLC, MAF4 and FT expression by

To further understand the mechanism by which AtZRF1a and AtZRF1b regulate floral transition, we measured the H3K27me3, H3K4me3 and H2AK119ub1 levels at the FLC, MAF4 and FT loci by ChIP assays of 2-week-old wild-type and Atring1a Atring1b, Atbmi1a Atbmi1b, Atzrf1a Atzrf1b as well as clf seedlings.

H3K4me3 levels at FLC, MAF4 and FT were increased in clf seedlings. In the Atbmi1a Atbmi1b double mutant, H3K4me3 levels were increased at FLC and MAF4, but not affected at the FT locus. In Atring1a Atring1b and Atzrf1a Atzrf1b double mutants, H3K4me3 levels at FLC, MAF4 and FT were not obviously affected (Figure III.32).

Furthermore, as expected, we found that H3K27me3 levels at FLC, MAF4 and FT were strongly reduced in clf seedlings. In the Atbmi1a Atbmi1b double mutant, H3K27me3 levels at both FLC and MAF4 were also reduced, but at FT locus, the level of H3K27me3 was increased. Interestingly, although AtRING1a/b and AtBMI1a/b are RING finger proteins, and both atring1a atring1b and atbmi1a atbmi1b mutants are late flowering, we found that they have different methylation levels at FLC, MAF4 and FT.

  RF=3.73, P<1.32e-10) were down-regulated, which is 54.12% of commonly mis-regulated genes(Figure III.36). These results indicate that AtZRF1a and AtZRF1b are involved in establishing both an active transcriptional state and a repressive transcriptional state. The predominant function is involved in transcriptional activity.In seedlings, global gene expression was more strongly affected in the Atzrf1a Atzrf1b than in the Atring1a Atring1b and Atbmi1a Atbmi1b mutants. Indeed, a total of 8956 loci were affected in the Atzrf1a Atzrf1b mutant, while 678 were misregulated in the Atring1a Atring1b mutant(Molitor, unpublished results) and 432 in the Atbmi1a Atbmi1b mutant(Qin et al., 2008). Interestingly, in the Atzrf1a Atzrf1b mutant

	Atbmi1a Atbmi1b.
	The number of transcripts overlapping between Atzrf1a Atzrf1b-up and
	Atring1a Atring1b-up is 171 (25.22% of Atring1a Atring1b total; RF=2.42, P<1.47e-5)

10, P<1.2e-11) were up-regulated, which is 45.88% of commonly mis-regulated genes. And 4840 genes (the number of mis-regulated genes that were down-regulated (54.12%) was higher than those that were up-regulated (45.88%). In Atring1a Atring1b and Atbmi1a Atbmi1b mutants the up-regulated loci, were predominant, with 69.57% and 73.38%, respectively. Only 30.43% and 26.62% respectively of the genes were down-regulated in the two mutants. Furthermore, I analyzed the transcriptional overlaps of upregulated loci between Atzrf1a Atzrf1b and Atring1a Atring1b or Atzrf1a Atzrf1b and

  numbers of floral organs. Flower organ identity is determined by the interplay between homeotic transcription factor genes, including AG, PI, AP3, AP2, and AP1, which are subjected to chromatin-remodelling regulation (reviewed inShen and Xu, 2009). Consistent with its phenotype, downregulation of AG, PI and AP1 was observed in Atzrf1a Atzrf1b.The Atzrf1a Atzrf1b mutant plants are almost completely sterile. Several defects may contribute to Atzrf1a Atzrf1b sterility: first, abnormal floral organs, such as fewer stamens than wild-type or stamen filaments too short to allow effective pollination of the stigma; second, short siliques and seed abortion; and third, reduction of transmission efficiency of mutant alleles in heterozygous mutant plants (TableIII.1).

Mutant T-DNA line or Stock number

  mutant plants, but enhancer trap lines J2341 and J1092 are in C24 ecotype. The reporter lines WOX5::GFP, DR5::GFP, SCR::GFP, CO2::GFP and STM::GUS were provided by Dr. Donghong CHEN. Arabidopsis thaliana mutants were obtained from Vectors used during my thesis are listed below (Table V.2)

	V zrf1b.1-LP	AAAAGCTTTAGCTGAGTCGGG
	zrf1b.1-RP	GAAAAAGTTATCGCGATGCTG
	zrf1b.2-LP	TGGATATAACAAGGCCTGACG
	Vector zrf1b.2-RP pDONR207 zrf1b.3-LP zrf1b.3-RP	Resistance CTGGAGAATAGGAAACCTGCC Experiment Gateway cloning CGA AGC AAT CAA AAC CAA GAG Gentamycin ACC ATT CGA TAC TGT GCA AGG
	pENTR3C zrf1b.4-LP zrf1b.4-RP	Gateway cloning CGA AGC AAT CAA AAC CAA GAG Kanamycin ACC ATT CGA TAC TGT GCA AGG
	pGWB5 zrf1b.5-LP	Gateway cloning AAA AGC TTT AGC TGA GTC GGG Spectinomycin
	pGWB11 zrf1b.5-RP LBb1.3	Gateway cloning GAA AAA GTT ATC GCG ATG CTG Spectinomycin ATTTTGCCGATTTCGGAAC
	pB7WGF2 FLAG_LB1	Gateway cloning CGGCTATTGGTAATAGGACACTGG Spectinomycin
	pB7FWG2 SAIL LB	Gateway cloning GCC TTT TCA GAA ATG GAT AAA TAG CCT TGC TTC C Spectinomycin
	pH7WGR2 Name	Gateway cloning Sequences 5'-3' (Clone) Spectinomycin
	pH7RWG2 H2A.1-F	Gateway cloning GGC GGTACC ATGGCTGGTCGTGGAAAAAC Spectinomycin
	pGEX-4T-1	Protein expression Ampicillin
	pET30a	Protein expression Kanamycin
	pCAMBIA1300 Binary vector	Kanamycin
			Host	Company
	Table .1 Anti-trimethyl-Histone -H3-(K4) antibody (Cat. 07-473)	Rabbit Millipore
	Anti-trimethyl-Histone -H3-(K36) antibody (Cat. Ab9050) Rabbit Abcam
	Atzrf1a-1 Anti-trimethyl-Histone -H3-(K27) antibody (Cat. 07-449) Rabbit Millipore Sail_786_F09 (N876841)
	Atzrf1a-2 Anti-Histone -H3 antibody Salk_070956.55.25.X (N570956)	Rabbit Millipore
	Atzrf1a-3 anti-hH2Aub antibody (Cell Signaling Technology 8240) Salk_070965.50.20.X (N570965)	Rabbit
	Atzrf1b-1 Anti-FLAG (F1804) FLAG_110A05	Sigma-Aldrich
	Atzrf1b-2	Sail_716_D04 (N876215)
	Atzrf1b-3 Ⅴ	Sail_625_B03 (N826768)
	Atzrf1b-4 Name	Sail_629_F09 (N827014) Sequences 5'-3' (Genotying)
	Atzrf1b-5 zrf1a.1-LP zrf1a.1-RP	TTGTTGTTGTGCAGATTCTGC FLAG_099C10 CGTACTCTGAGGAGCTTGTGG
	zrf1a.2-LP	AGGCTAGAAAGGAGGAGCATG
	zrf1a.2-RP	TCATCGTTTTACCAGGGACAG
	zrf1a.3-LP	AGA AGA AGC AGG AAG AGG ACG
	zrf1a.3-RP	CTT GCT TCT CGC AAA GTA ACG

the Arabidopsis Biological Resource Center (ABRC, http://www.arabidopsis.org) and the European Arabidopsis Stock Center (NASC, http://arabidopsis.info). They were either grown on soil (16 h light and 8 h dark) in the greenhouse or in vitro on 0.8% MS medium. Seeds grown on plates were stratified in the dark at 4°C for 48 h to synchronize the germination time. Then the plants were transferred to a growth chamber (16 h light and 8 h dark, 22°C). To generate flowering plants, seedlings were transferred to soil 10 days after germination and cultivated under long day conditions (LD; 16 h light and 8 h dark).

Ⅴ

Antibodies used are listed below (Table

Ⅴ )

  Protein samples are loaded directly after heating in 1x SDS loading buffer at 95 for 5-10 min or after storage at -20 °C (in this latter case, samples are warmed up 5 min at 65 °C before loading because of SDS buffer). Gel is run in 1xSDS electrophoresis buffer at 100 V to allow the samples to separate. To visualize the proteins, the gel is incubated under shaking in a Coomassie solution for 20 minutes at RT. Remove Coomassie solution and incubate under shaking in destaining solution for 30 minutes (adapt to intensity of the staining). Finally, incubate the gel overnight in distilled water with glycerol (~500 µL for 150 mL H 2 O). Dry the gel if necessary.

	Staining solution: 10% acetic acid (v/v); 90% methanol (v/v); 0.05% Amidoblack
	(w/v)
	Washing solution: 10% acetic acid (v/v); 90% ethanol (v/v)
	V.2.5.3 SDS (Sodium dodecylsulfate) gel electrophoresis
	Scan.
	Resolving gel: 10-15% Acrylamide/Bis-acrylamide (29:1); 375 mM Tris-HCl pH8.8;
	0.1% SDS; 0.1% AP; 0.4 µl/ml TEMED
	Stacking gel: 5% Acrylamide/Bis-acrylamide (29:1); 125 mM Tris-HCl pH6.8; 0.1%
	SDS; 0.1% AP; 1 µl/ml TEMED
	1X SDS running buffer: 25 mM Tris; 250 mM glycine; 0.1% SDS
	1X SDS loading buffer: 50 mM Tris-HCl pH6.8; 100 mM DTT; 2% SDS; 0.1%
	bromophenol blue; 10% glycerol
	Coomassie blue solution: 40% methanol; 10% acetic acid 50% water; 0.1% (w/v)
	Coomassie brilliant blue R250

Dried pellets are resuspended in 250 µL NaOH 0.2 M and 200 µL of this solution are transferred to an ELISA plate for measurement of extinction at 595 nm (Microplatereader Model 680, BioRad).

  RESUMEDes études chez les animaux ont montré que ZRF1 a une fonction lectrice au niveau de H2AK119ub1 dans la dérépression de gènes réprimés par polycomb. Deux gènes homologues au gène humain ZRF1 ont été identifiés dans le génome d'Arabidopsis, et ont par la suite été appelés AtZRF1a et AtZRF1b. La caractérisation fonctionnelle de ces gènes n'a pas encore été rapportée.Ma premier objectif était d'obtenir des connaissances générales sur AtZRF1a et AtZRF1b. Tous les deux sont exprimés dans des plantes d'Arabidopsis et la protéine AtZRF1b est localisée dans le noyau et dans le cytoplasme. En plus, nous avons trouvé que la protéine AtZRF1b lie H2Aub1 avec les mêmes caractéristiques que la protéine ZRF1 humaine.J'ai utilisé les outils génétiques puissants disponibles pour Arabidopsis pour étudier la fonction d'AtZRF1a et AtZRF1b. Plusieurs lignées d'insertion de T-DNA indépendantes ont été identifiées. A cause d'une rédondance fonctionnelle, des mutants simples n'ont pas de défauts de développement évidents. C'est pourquoi j'ai étudié un mutant double qui montre une perte de fonction pour les deux gènes AtZRF1a et AtZRF1b. Ce double mutant révèle des rôles importants pour ces gènes dans la croissance et le développement, qui vont de la prolifération et la différenciation cellulaire jusqu'au contrôle du temps de floraison.J'ai ensuite étudié les rôles d'AtZRF1a et AtZRF1b dans la régulation de la transcription et j'ai constaté que AtZRF1a et AtZRF1b ont une fonction similaire a PRC1.

	OD 600	Optical Density, measured at 600 nm
	PAGE	Ammonium persulfate PolyAcrylamide Gel Electrophoresis
	ATP PBS	Adenosine Triphosphate Phosphate Buffer Saline
	Amp PCR	Ampicillin Polymerase Chain Reaction
	BLAST PI	Basic Local Alignment Search Tool Propidium Iodide
	bp PMSF	base pair Phenyl Methyl Sulphonyl Fluoride
	cm PRC1	centimeter Polycomb Repression Complex 1
	Col PVDF	Columbia PolyVinylidene DiFluoride
	ChIP qPCR	Chromatin Immunoprecipitation quantitative Polymerase Chain Reaction
	Da RNA	Dalton RiboNucleic Acid
	DAPI rpm	4',6-Diamidino-2-phenylindole rotations per minute
	DAS RT	Days After Stratification room temperature
	DNA RT PCR	DeoxyriboNucleic Acid Reverse Transcription Polymerase Chain Reaction
	DTT SANT	dithiothreitol Swi3, Ada2, NcoR1 and TFIIIB
	EDTA SD	Ethylene Diamine Tetraacetic Acid Short Day
	EGTA SDS	Ethylene Glycol Tetraacetic Acid Sodium Dodecyl Sulphate
	GFP SEM	Green Fluorescent Protein Scanning Electron Microscope
	GST TBS	Glutathione-S-Transferase Tris Buffer Saline
	g T-DNA	gram Transfer-DNA
	h TE	hour Tris-EDTA Buffer
	His TEMED	histidine N,N,N',N',-tetramethyl-ethylene.1,2-diamine
	Hyg Tris	Hygromycin Tris(hydroxymethyl) aminomethane
	Kan TTBS	Kanamycin Triton-Tris Buffer Saline
	kb UBD	kilo base pairs Ubiquitin Binding Domain
	LD Ub	Long Day ubiquitin
	µ YC	micro C-terminal half of yellow fluorescent protein
	m YFP	milli Yellow Fluorescent Protein
	M YN	Molar (mole/liter) N-terminal half of yellow fluorescent protein
	min ZRF1	minutes ZUO1-Related Factor 1
	MS	Murashige and Skoog
	MW	molecular weight
	NCBI	National Center for Bioinformation
	nm	nanometer

March 2014 | Volume 5 | Article 83 | 4

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ACKNOWLEDGMENTS

The work in Wen-Hui Shen laboratory was supported by the French "Centre National de la Recherche Scientifique" (CNRS) and in part by the French "Agence Nationale de la Recherche" (ANR-08-BLAN-0200-CSD7; ANR-12-BSV2-0013-02). Jing Feng received a fellowship from the China Scholarship Council of the Ministry of Education of P. R. China.

ACKNOWLEDGMENTS

The work in Wen-Hui Shen laboratory was supported by the French "Centre National de la Recherche Scientifique" (CNRS) and in part by the French "Agence Nationale de la Recherche" (ANR-08-BLAN-0200-CSD7; ANR-12-BSV2-0013-02). Jing Feng received a fellowship from the China Scholarship Council of the Ministry of Education of P. R. China.

for maintaining root development ---IV.4 AtZRF1a and AtZRF1b repressed flowering by promoting FLC and MAF gene expression - ------------------------------------------------------------------------------IV.5 AtZRF1a and AtZRF1b play crucial roles in seed germination ---------------IV.6 AtZRF1a and AtZRF1b functions are partially related to PRC1 -------------V. MATERIALS AND METHODS - -----------------------------------------------------------V.Protein quantification - ------------------------------V.2.5.3 SDS (sodium dodecylsulfate) gel electrophoresis -----------------V.2.5.4 Western blot -----------------------------------GST fusion protein purification - ------------------------------------V.2.5.7 GST pull-down assay ----------------------------V.2.8 Propidium iodide staining -------------------------

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

MOTS-CLÉS :

Chromatine regulateur; Épigénétique; Ubiquitine; H2Aub1; Régulation de la transcription; Développement de la plante; ZRF1; Germination des grains

Résumé en anglais

Studies in animals showed that ZRF1 can read the histone H2AK119ub1 modification in the derepression of polycomb-repressed genes. Two homologs of human ZRF1 have been identified in the Arabidopsis genome, and hereinafter are named AtZRF1a and AtZRF1b. So far, their functional characterization had not been reported yet.

My first objective was to acquire basic knowledge about AtZRF1a and AtZRF1b. Both genes are broadly expressed in Arabidopsis plants and the AtZRF1b protein is localized in the nucleus and the cytoplasm. Moreover, we found that AtZRF1b binds H2Aub1 with characteristics similar to those previously reported for the human ZRF1 protein.

I subsequently used the powerful genetic tools available in Arabidopsis to investigate the functions of AtZRF1a and AtZRF1b. Several independent T-DNA insertion Arabidopsis mutant lines were identified. Because of functional redundancy, single mutants have no obvious developmental defects. I therefore focused on double mutants displaying loss of function of both AtZRF1a and AtZRF1b. The study of a double mutant revealed important roles for these genes in plant growth and development ranging from cell proliferation and differentiation to flowering time control. I then investigated the roles of AtZRF1a and AtZRF1b in gene transcriptional regulation and found that AtZRF1a and AtZRF1b function in a way that is partially similar to PRC1 function. Lastly, I investigated H3K4me3, H3K27me3 and H2Aub1 levels in the chromatin regions of some expression-perturbed genes in double mutants.

The results show that ZRF1-mediated deubiquitination of H2Aub1 is not a major event in transcriptional regulation in Arabidopsis.

KEYWORDS :

Chromatin regulator ; Epigenetics ; Ubiquitin ; H2Aub1 ; Transcription regulation ; Plant development ; ZRF1 ; Seed germination