Changzheng Xu

Yun Shen

Fu He

Xiaokang Fu

Hong Yu

Wanxiang Lu

Yongli Li

Chaofeng Li

Di Fan

Hua Cassan Wang
email: huawang76@yahoo.com

Keming Luo
email: kemingl@swu.edu.cn

Auxin-mediated Aux/IAA-ARF-HB signaling cascade regulates secondary xylem development in Populus

Keywords: ARF5, Aux/IAA9, auxin, HD-ZIP III transcription factors, Populus, xylem development

Wood development is strictly regulated by various phytohormones and auxin plays a central regulatory role in this process. However, how the auxin signaling is transducted in developing secondary xylem during wood formation in tree species remains unclear.

Here, we identified an Aux/INDOLE-3-ACETIC ACID 9 (IAA9)-AUXIN RESPONSE FACTOR 5 (ARF5) module in Populus tomentosa as a key mediator of auxin signaling to control early developing xylem development.

PtoIAA9, a canonical Aux/IAA gene, is predominantly expressed in vascular cambium and developing secondary xylem and induced by exogenous auxin. Overexpression of PtoIAA9m encoding a stabilized IAA9 protein significantly represses secondary xylem development in transgenic poplar. We further showed that PtoIAA9 interacts with PtoARF5 homologs via the C-terminal III/IV domains. The truncated PtoARF5.1 protein without the III/IV domains rescued defective phenotypes caused by PtoIAA9m. Expression analysis showed that the PtoIAA9-PtoARF5 module regulated the expression of genes associated with secondary vascular development in PtoIAA9m-and PtoARF5.1-overexpressing plants. Furthermore, PtoARF5.1 could bind to the promoters of two Class III homeodomain-leucine zipper (HD-ZIP III) genes, PtoHB7 and PtoHB8, to modulate secondary xylem formation.

Taken together, our results suggest that the Aux/IAA9-ARF5 module is required for auxin signaling to regulate wood formation via orchestrating the expression of HD-ZIP III transcription factors in poplar.

Introduction

Meristems, specialized structures of reservoirs of stem cells undergoing proliferation, drive postembryonic developmental programs in plants [START_REF] Nakajima | Signaling in and out: control of cell division and differentiation in the shoot and root[END_REF]. In comparison with primary/longitudinal growth of shoots and roots mediated by apex-localized meristems, secondary/radial growth is dominated by vascular cambium [START_REF] Campbell | Regulation of vascular cell division[END_REF]. Cambial cells sequentially undergo proliferation and differentiation into secondary vascular tissues [START_REF] Matte Risopatron | The vascular cambium: molecular control of cellular structure[END_REF]. Different from herbaceous plants, trees exhibit perennial stem thickening during their life cycle due to continuous competence of the vascular cambium [START_REF] Sanchez | From thin to thick: major transitions during stem development[END_REF]. The secondary xylem, commonly called wood, overwhelmingly contributes to stem thickening of trees [START_REF] Sanchez | From thin to thick: major transitions during stem development[END_REF]. Wood production is a predominant proportion of biomass accumulation in terrestrial ecosystems and is also of outstanding economic value [START_REF] Ragauskas | The path forward for biofuels and biomaterials[END_REF][START_REF] Bonan | Forests and climate change: forcings, feedbacks, and the climate benefits of forests[END_REF].

Cambium-originated wood formation starts with specification of secondary xylem cell precursors, which in turn undergo a series of cellular events for maturation, including expansion, secondary cell-wall deposition and programmed cell death [START_REF] Ye | Molecular control of wood formation in trees[END_REF]. Cambial homeostasis between stem cell identity and xylem specification affects the deposition of xylem cell layers [START_REF] Campbell | Regulation of vascular cell division[END_REF]. Expansion of newly specified xylem cells contributes to the size of woody elements, while secondary wall deposition is important for functional maturation of xylem. Although vessels and fibers are lignified components of secondary xylem, they differ in cell size, secondary cell-wall deposition and cell viability [START_REF] Fukuda | Signals that control plant vascular cell differentiation[END_REF]). These differences result from the cellular changes in cytoskeletal arrangement, secondary wall patterning and autolysis that occur during differentiation of tracheary elements, the unit of xylem vessels [START_REF] Fukuda | Signals that control plant vascular cell differentiation[END_REF][START_REF] Turner | Tracheary element differentiation[END_REF]. These coordinated behaviors of cambial cells and their differentiating descendants are intricately and dynamically orchestrated by various genetic and environmental factors [START_REF] Dejardin | Wood formation in Angiosperms[END_REF][START_REF] Ye | Molecular control of wood formation in trees[END_REF]. Recent high-spatialresolution profiling analyses in Populus have revealed three transcriptome reprogramming events coinciding with transitions of distinct cellular behaviors in the context of wood formation [START_REF] Sundell | AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula[END_REF]. Although a comprehensive picture of the gene regulatory machinery involved in secondary growth has emerged in recent decades, precise regulation of spatially organized and temporally coordinated cellular events for wood differentiation remains largely unknown.

Auxin is a crucial phytohormone for cell-cell communication in meristems [START_REF] Leyser | Auxin distribution and plant pattern formation: how many angels can dance on the point of PIN?[END_REF]. Auxin signaling is able to direct cellular behaviors, including cell division, expansion and differentiation, for various developmental programs [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF][START_REF] Perrot-Rechenmann | Cellular responses to auxin: division versus expansion[END_REF]. Previous studies have shown that auxin accumulates in a radial gradient pattern across wood-forming tissues with a peak in concentration in cambium and bilateral decay towards differentiating secondary xylem and phloem [START_REF] Tuominen | A radial concentration gradient of indole-3-acetic acid is related to secondary xylem development in hybrid aspen[END_REF][START_REF] Uggla | Indole-3-acetic acid controls cambial growth in scots pine by positional signaling[END_REF][START_REF] Immanen | Cytokinin and auxin display distinct but interconnected distribution and signaling profiles to stimulate cambial activity[END_REF]. Auxin is thus considered as a primary regulator of secondary xylem formation in a morphogen-like manner [START_REF] Uggla | Auxin as a positional signal in pattern formation in plants[END_REF][START_REF] Sundberg | Cambial growth and auxin gradients[END_REF]. In Pinus, a lack of auxin supply from shoot apex leads to a loss of fusiform shape of cambial derivatives [START_REF] Savidge | The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta[END_REF]. Introduction of a stabilized version of PttIAA3 (homolog of IAA20s in Arabidopsis and Populus; Supporting Information Fig. S1) that represses auxin response in hybrid aspen attenuates periclinal division of cambial cells, but enhances stem cell characteristics as indicated by an enlarged zone of anticlinal cell division in cambium [START_REF] Nilsson | Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen[END_REF]. These findings reveal a dual role of auxin in coordinating cambial identity and proliferation activity. Recently, detection of auxin signaling via a high-affinity sensor indicates its moderate level in cambial stem cells, while increased level in differentiating cambial descendants [START_REF] Brackmann | Spatial specificity of auxin responses coordinates wood formation[END_REF]. These results implicate the key role of auxin signaling in cambial differentiation. Consistently, exogenous auxin is able to induce differentiation of intact and functionally normal secondary xylem cells [START_REF] Bj€ Orklund | Cross-talk between gibberellin and auxin in development of Populus wood: gibberellin stimulates polar auxin transport and has a common transcriptome with auxin[END_REF]. Local enhancement of auxin signaling is required for wood formation, also supporting the action of auxin in recruiting cells for differentiation [START_REF] Bargmann | A map of cell type-specific auxin responses[END_REF][START_REF] Muller | PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis[END_REF]. However, the precise regulation of auxin signaling on these coordinated cellular events for wood differentiation remains obscure in trees.

Auxin perception starts with auxin binding to TIR1 (TRANSPORT INHIBITOR RESPONSE1)/AFB (AUXIN SIGNALING F-BOX) receptors, and leads to subsequent degradation of the Aux/IAA proteins that repress auxin signaling via physical interactions with auxin response factor (ARF) proteins [START_REF] Mockaitis | Auxin receptors and plant development: a new signaling paradigm[END_REF]. The auxin-stimulated protein turnover of Aux/IAAs releases the transcriptional activity of their partner ARFs to activate downstream auxin responsive gene expression [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF]. Different Aux/IAA-ARF modules are known to regulate corresponding auxinresponsive genes and developmental processes [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF]. In Arabidopsis, the IAA12/BODENLOS (BDL)-ARF5/MONOPTEROS (MP) module was identified to control provascular specification and patterning during embryogenesis [START_REF] Hardtke | The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development[END_REF][START_REF] Hamann | The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning[END_REF]. ARF5/ MP is also crucial for leaf (pre)procambial specification during leaf vein patterning [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF]. In hybrid aspen, eight Aux/IAA members were assayed for expression of auxin responsive genes in wood tissues and during transitions of cambial activity [START_REF] Moyle | Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen[END_REF], and PttIAA3 was identified as an important mediator of auxin-dependent regulation of cambial proliferation activity [START_REF] Nilsson | Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen[END_REF]. When a Eucalyptus Aux/IAA member IAA4 (EgrIAA4) was ectopically expressed in Arabidopsis, secondary xylem formation of the transgenic plants was dramatically reduced [START_REF] Yu | Comprehensive genomewide analysis of the Aux/IAA gene family in Eucalyptus: evidence for the role of EgrIAA4 in wood formation[END_REF].

Various plant model systems have been used to study the mechanisms governing plant vascular development. The construction of genetic networks underlying vascular tissue specification has been mainly achieved based on research on four organs/ systems: provascular specification during embryogenesis, procambial cell specification during root development (primary growth), (pre)procambial cell specification during leaf vein patterning (primary growth), and cambium specification during secondary growth in stem for wood formation [START_REF] Campbell | Regulation of vascular cell division[END_REF]. For embryogenesis and procambial specification during primary growth in roots and leaf veins, the key components of auxin signaling have been identified: the IAA12/BDL-ARF5/MP module for embryogenesis [START_REF] Hardtke | The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development[END_REF][START_REF] Hamann | The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning[END_REF], the ARF5/MP-mediated pathway for leaf vein patterning [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF][START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF], and the IAA20/ IAA30-ARF5/MP combinations for root development [START_REF] Muller | PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis[END_REF]. To date, however, it remains unknown which Aux/ IAA-ARF combination mediates auxin-dependent cambial specification of secondary growth for wood formation.

Here, we identified PtoIAA9 as a novel key component in modulating wood formation in Populus tomentosa. Functional characterization of the PtoIAA9-PtoARF5 module revealed auxin-dependent differentiation of secondary xylem derived from the cambium, and demonstrated their roles in orchestrating xylem cell specification, woody cell size and vessel density. Moreover, we provided biochemical and genetic evidence that PtoHB7 and PtoHB8, encoding HD-ZIP III transcription factors, are direct targets of the PtoIAA9-PtoARF5 module to coordinate cellular behaviors associated with woody cell differentiation. Our results provide novel insights into auxin-dependent regulation on the dynamic and coordinated cellular events for secondary xylem differentiation in woody species.

Materials and Methods

Gene cloning and plasmid construction

The full-length coding sequence of PtoIAA9, PtoARF5.1 and PtoARF5.2 was amplified from cDNA of P. tomentosa using gene-specific primer pairs (Table S1), and constructed into the pCXSN vector [START_REF] Chen | A versatile zero background Tvector system for gene cloning and functional genomics[END_REF], respectively. To substitute the first proline (P) with serine (S) within the Degron motif, overlap PCR was performed using pCXSN-PtoIAA9 as templates. The resulting PCR product was ligated into pCXSN under control of the CaMV 35S promoter to generate the plasmid for overexpressing PtoIAA9m. The full-length sequence of PtoARF5.1 was subcloned into the modified pCAMBIA1305 with a kanamycin resistant gene. The sequence of PtoARF5.1D harboring the Nterminal 1995 nucleotides of PtoARF5.1 was amplified from pCXSN-PtoARF5.1 and constructed into the modified pCAMBIA1305 vector. A 2-kb promoter fragment upstream of PtoIAA9 was amplified from genomic DNA of P. tomentosa using the primer pair Pro-PtoIAA9-fw/rv (Table S1), and inserted into pXGUS-P to drive the GUS reporter gene [START_REF] Chen | A versatile zero background Tvector system for gene cloning and functional genomics[END_REF].

Genetic transformation and growth conditions

P. tomentosa was stably transformed using the method of Agrobacterium-mediated infiltration of leaf disks as described previously [START_REF] Jia | The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr[END_REF]. PCR genotyping with the primers of hygromycin/kanamycin-resistant genes was performed for the identification of positive transgenic plants. Transgenic and WT poplar plants were propagated via in vitro microcutting. For clonal propagation, shoot segments of 3-4 cm with two or three young leaves were cut from sterilized seedlings and cultivated on woody plant medium solid medium at 25°C with 16 h light of 5000 lux and 8 h dark. For phenotype analyses, 4-wk-old microcutting-propagated seedlings were transferred to soil in pots, and cultivated in a glasshouse at 23-25°C with the light of 10 000 lux under a 16 h: 8 h, day : night cycle.

Cross-sectioning and histological staining

The 7 th internodes were sectioned with a razor blade, and then stained with 0.05% (w/v) toluidine blue for 5 min. The crosssections were observed and captured using a microscope (Zeiss). The images were analyzed using IMAGEJ (https://imagej.nih.gov/ ij/) for quantifying morphological parameters of xylem cells.

RNA in situ hybridization

For probe preparation, 276-and 223-bp gene-specific cDNA fragments were amplified for PtoIAA9 and PtoARF5.1, respectively (Table S1). The two probes were labeled using a DIG RNA Labeling Kit (Roche). Section pretreatment, hybridization and immunological detection were performed as previously described [START_REF] Sang | CHIMERIC FLORAL ORGANS1, encoding a monocotspecific MADS box protein, regulates floral organ identity in rice[END_REF].

GUS staining

For GUS staining [START_REF] Jefferson | Assaying chimeric genes in plants: the GUS gene fusion system[END_REF], cross-sections of the 7 th internodes of 2-month-old plants were fixed in acetone for 1 h at À20°C, and then washed twice in double distilled H 2 O (ddH 2 O). The cross-sections were soaked in GUS staining solution (0.5 M Tris, pH 7.0, 10% Triton X-100 with 1 mM X-Gluc (5-bromo-4chloro-3-indolyl-D-glucuronide)) for 15 min at 37°C in the dark. After reaction, Chl was removed by use of 75% ethanol three times at 65°C. The Chl-free stained stems were observed under an Olympus 566 SZX16 microscope (Tokyo, Japan) and documented using an Olympus DP73 camera.

qRT-PCR

Total RNA was extracted from tissues of 2-month-old P. tomentosa plants using a Plant RNeasy Mini Kit (Qiagen). cDNA was synthesized using a PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative real time polymerase chain reaction (qRT-PCR) was performed using SYBR Premix ExTaqTM (Takara) in a TP800 Real-Time PCR machine (Takara). The poplar 18S rRNA gene was used as the reference gene as an internal standard. The primers used for qRT-PCR are listed in Table S1.

Transactivation test in yeast

The recombinant plasmids were introduced into the yeast strain Saccharomyces cerevisiae Gold2 using the PEG/LiAC method. The transformed strains were screened on synthetic dropout (SD medium) lacking tryptophan (Trp; SD/-T) for selection of positive clones. Subsequently, positive clones were transferred to SD medium lacking Trp, histidine (His) and adenine (Ade; SD/-ATH) and cultivated at 28°C for 2 d. Positive clones were used for transactivation analysis on X-a-gal indicator plates. A digital camera (EOS 550D, Canon, Tokyo, Japan) was used to photograph yeast cells.

Yeast two-hybrid (Y2H) assays Y2H assays were performed based on the manufacturer's instructions (Clontech, Palo Alto, CA, USA). The AD and BD fusion constructs were co-transformed into yeast strain Gold2 as described above. The transformants were screened on SD medium lacking tryptophan (Trp) and leucine (Leu; SD/-TL) at 28°C. After 3 d, positive clones were cultured in YPD liquid medium for 1 d to an OD 600 of 1.0, and the yeast solution was diluted with ddH 2 O to 1-10 3 x, and then transferred to SD medium lacking Trp, Leu, histidine (His) and adenine (Ade; SD/-AHTL) and cultured for 2 d. A EOS 550D digital camera was used to photograph yeast cells.

Bimolecular fluorescence complementation (BiFC)

For BiFC assays, full-length coding sequences of PtoIAA9 and PtoARF5.1 were cloned from pCXSN-PtoIAA9 and pCXSN-PtoARF5.1, and ligated into pXY104 and pXY106 vectors, respectively. The vectors pXY104 and pXY106 carrying the Nand C-terminal halves of yellow fluorescent protein (YFP) were used. The open reading frame (ORF) of PtoIAA9 was cloned into pXY106, while that of PtoARF5.1 was constructed into pXY104. The constructs were co-transformed into tobacco as described above, and observed by confocal laser microscopy (Olympus 589 FV1200).

Chromatin immunoprecipitation (ChIP) assay

ChIP analysis was performed as previously described [START_REF] Yang | A companion cell-dominant and developmentally regulated H3K4 demethylase controls flowering time in Arabidopsis via the repression of FLC expression[END_REF]. One-month-old PtoARF5.1 transgenic poplar plants harboring the HA epitope were used as samples; the HA antibody and normal mouse IgG were used for immunoreaction. All primers used in ChIP assays are listed in Table S1.

Effector-reporter test

Promoters of the PtoHB7 and PtoHB8 genes were amplified via specific primers (Table S1) and constructed into pCXGUS-P. The 35S:PtoARF5.1 and 35S:PtoARF5.1D vectors were used as effectors. Tobacco leaves were transformed by Agrobacteria containing the effectors and reporters as described above. After 3 d of infiltration, GUS activity was measured by monitoring the cleavage of 4-methyl umbelliferyl b-D-glucuronide (MUG), the substrate of b-glucuronidase, which produces the fluorescent 4-methyl umbelliferone (4MU) upon hydrolysis. Protein concentrations were determined via the Bradford method.

Gene accessions

GeneBank accession numbers of the P. tomentosa genes are: PtoIAA9 (MH345700), PtoARF5.1 (MH352401), PtoARF5.2 (MH352402) and PtoHB7 (MH345699).

Results

IAA9 is highly expressed across wood-forming tissues in poplar

A total of 35 Aux/IAA genes were previously predicted in the poplar genome [START_REF] Kalluri | Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa[END_REF]. These Aux/IAA genes were identified in the more recent release of the Populus trichocarpa genome (v3.0; https://phytozome.jgi.doe.gov) and designated according to phylogenetic relationships with their Arabidopsis orthologues (Fig. S1a). To determine the members that play a key role during wood formation, expression levels of all Aux/IAA genes were comprehensively evaluated using the high-spatialresolution RNA sequencing data of the wood-forming tissues in poplar [START_REF] Sundell | AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula[END_REF]. Among them, IAA9, -11, -16.1, -16.2 and -29.1 showed high expression (Fig. S1). Continuous assays across secondary xylem tissues in poplar stems [START_REF] Sundell | AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula[END_REF] allowed us to evaluate spatial expression patterns of these highly expressed Aux/IAAs and some other members, such as IAA12.1, -12.2, -20.1 and -20.2 (Fig. S2b). A highly similar expression pattern was shared by IAA9, -16.1 and -16.2, extending from the cambium to differentiated xylem (Fig. S2b), which was validated by correlation tests (Pearson correlation coefficient R 2 = 0.82 or 0.51, P < 0.01; Fig. S2c). For this pattern, the transcripts of these three Aux/IAAs accumulated in the cambial zone and developing xylem, and then decreased towards differentiated xylem (Fig. S2d), consistent with the previously reported auxin distribution across wood-forming tissues of poplar stems [START_REF] Uggla | Indole-3-acetic acid controls cambial growth in scots pine by positional signaling[END_REF][START_REF] Immanen | Cytokinin and auxin display distinct but interconnected distribution and signaling profiles to stimulate cambial activity[END_REF]. By contrast, IAA11 and -29.1 displayed totally different expression patterns (Fig. S2b). Thus, IAA9, -16.1 and -16.2 were considered key Aux/IAA genes of auxin signaling involved in wood formation in poplar. Tissuespecific expression assays by qRT-PCR in P. tomentosa further confirmed the enriched transcript abundance of IAA9, -16.1 and -16.2 in stems (Fig. S2d). We selected PtoIAA9 from P. tomentosa as a candidate gene to further investigate auxin-dependent regulation of wood formation.

To determine the exact expression pattern of PtoIAA9 in secondary vascular tissues, RNA in situ hybridization was performed using the fifth internode of 1.5-month-old poplar (Fig. 1a,b). Transcripts of PtoIAA9 were preferentially accumulated in the cambial zone and neighboring cells (Fig. 1a). Furthermore, the GUS reporter driven by the PtoIAA9 promoter in transgenic poplar confirmed its expression in the cambium zone and closely neighboring cell layers of the wood-developing stem (Fig. 1c,d). qRT-PCR assays revealed a more than 3-fold induced transcript abundance of PtoIAA9 in stems within 3 h of exogenous auxin (Fig. 1e). The auxin-inducible expression was also examined by quantification of GUS activity driven by the PtoIAA9 promoter in wood-forming tissues (Fig. 1f). These results indicated that PtoIAA9 is an auxin-inducible gene predominantly expressed in cambium and adjacent cells towards xylem differentiation.

PtoIAA9 encodes a canonical Aux/IAA protein belonging to a distinct clade PtoIAA9 protein harbors the characteristic domains (Domain I, II, III and IV; [START_REF] Reed | Roles and activities of Aux/IAA proteins in Arabidopsis[END_REF] of Aux/IAAs in high sequence similarity to its close homologs in other plant species (Fig. S3). Remarkably, PtoIAA9 belongs to a distinct clade among all Aux/IAA members due to its 50% longer amino acid sequence (c. 300 aa) compared with the average length (c. 200 aa) of other Aux/IAA members [START_REF] Wang | The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF]. Transient expression of a PtoIAA9-GFP fusion gene revealed that PtoIAA9 is a nucleus-localized protein (Fig. S4a; Methods S1), consistent with the prediction of both bipartite and SV40-type nuclear localization signals (NLS) present within its sequences (Fig. S3). Aux/IAA proteins usually contain a short amino acid stretch (VGWPP) called Degron that confers auxin-stimulated protein turnover [START_REF] Worley | Degradation of Aux/IAA proteins is essential for normal auxin signalling[END_REF]. Due to the presence of a typical Degron motif in its Domain II, auxin-responsive protein stability of PtoIAA9 fused with a green fluorescent protein (GFP) tag was monitored in transiently expressed epidermal cells of tobacco leaves (Fig. S4b; Methods S1). The fluorescent signals of PtoIAA9-GFP were significantly reduced in the leaf epidermal cells subject to IAA (Fig. S4b), as validated by quantifying the percentage of fluorescent nuclei and fluorescence intensity per nucleus (Fig. S4c,d). By contrast, this auxin-induced protein instability disappeared when the first proline (P) within the Degron motif of PtoIAA9 was replaced by serine (S) (ns, not significant; P > 0.05; Fig. S4be). Moreover, stronger fluorescence was detected in these leaf epidermal cells transiently transformed by the PtoIAA9 mutant gene with impaired Degron motif, compared to the wild-type control (Fig. S4b-d), indicating that the auxin-inducible rapid turnover of PtoIAA9 depends on its Degron motif. Self-activation assays in yeast showed that PtoIAA9 has no transcriptional activating activity (Fig. S4f). Additionally, PtoIAA9 compromised the activating capability of the VP16 domain, demonstrating that it is a transcriptional repressor. The truncated PtoIAA9 protein without New Phytologist (2019)

Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com Domain I at the C-terminal region (BD-VP16-PtoIAA9t) showed repressing activity (Fig. S4f ). Taking this together, PtoIAA9 exhibits molecular features typical of canonical Aux/ IAA repressors.

Overexpression of an auxin-resistant PtoIAA9m represses wood formation

To identify its role in regulating secondary xylem development during wood formation, the auxin-resistant PtoIAA9 containing the impaired Degron sequence (Fig. S4e; PtoIAA9m) was constitutively expressed in poplar (PtoIAA9m-OE; Fig. S5a). Representative overexpressing lines displayed significantly reduced plant growth compared to the wildtype (WT; Fig. 2a). Quantitative measurement revealed that overexpression of PtoIAA9m led to a 40-50% reduction in plant height and 35% reduction in stem diameter (Fig. 2b,c). Similar changes were indicated by a timecourse analysis of these two parameters (Fig. S5b,c). The internode number of WT and transgenic plants was not changed (ns; P > 0.05; Fig. S5d). Secondary vascular tissues were examined in PtoIAA9m-OE plants stained with toluidine O (Fig. 2d).

Secondary xylem development was significantly repressed by overexpressing PtoIAA9m (Fig. 2d), with a 40% decrease in the number of xylem cell layers, compared with WT (Fig. 2e). The percentage of secondary xylem occupying the whole stem was 46.8% in WT, but was attenuated to 39.1% in the PtoIAA9m-OE lines, whereas that of phloem was not affected (Fig. 2f). These results indicated that PtoIAA9m might inhibit wood formation.

PtoIAA9-attenuated auxin signaling impairs secondary xylem formation

To determine PtoIAA9-dependent regulation of wood formation, we first examined phenotypes of wood-associated cell types, including cambium, xylem fibers and vessels in transgenic poplar. The results showed that cambial cells were arrayed more tightly in PtoIAA9m-OE plants than WT (Fig. 2g), but the number of cambial cell layers was not significantly affected by overexpression of PtoIAA9m (ns; P > 0.05; Fig. S5e). Cross-sections of different internodes in WT revealed the presence of one or two particular cell layers on the side of the cambium zone towards the xylem S5f). These cells were not stained by toluidine blue, suggesting that lignin deposition was not initiated for secondary cell wall formation. These cells were distinctly larger than cambial cells but smaller than mature xylem cells (Figs 2g, S5f). These features indicated that these cells were newly differentiated from cambium zone towards xylem and undergoing cell expansion, and thereby considered as early developing xylem (EDX).

However, these EDX cell layers were greatly reduced and almost absent in cross-sections of PtoIAA9m-OE stems (Fig. 2g), resembling EDX cells just before dormancy due to reduced rate of cambium cell periclinal division. This greatly reduced EDX cell layer suggests that overexpression of stabilized PtoIAA9 inhibited the periclinal division of cambium, thus leading to reduced wood formation. Quantitative measurement showed that the cell size of xylem fibers and vessels was reduced by 36% and 28% in PtoIAA9m-OE plants relative to WT (Fig. 2g-i). By contrast, vessel density was significantly increased by 42% in PtoIAA9m-OE plants (Fig. 2g,j). Cell wall thickness was not affected by overexpressing PtoIAA9m (ns; P > 0.05; Fig. S5g). Together, these data suggested that PtoIAA9m inhibited developing xylem cell (vessel and fiber) differentiation and expansion.

(a) (d) (b) (c) (e) (f) (g) (h) (i) (j)

PtoIAA9 interacts with PtoARF5s

PtoIAA9 contains intact Domains III and IV (also called PB1 domain) that mediate physical interaction with ARFs, a family of plant-specific B3-type transcription factors directly driving the expression of auxin responsive genes [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF]. We speculated that PtoIAA9 might interact with ARF proteins to regulate wood formation in poplar. A previous study has shown that ARF5/MP is a key regulator of auxin-dependent vascular patterning during embryogenesis and leaf vein patterning in Arabidopsis [START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF][START_REF] Brackmann | Spatial specificity of auxin responses coordinates wood formation[END_REF]. The poplar genome harbors duplicated ARF5 members, designated ARF5.1 and ARF5.2, which are expressed in secondary vascular tissues of stems [START_REF] Johnson | Populus trichocarpa MONOPTEROS/AUXIN RESPONSE FACTOR5 (ARF5) genes: comparative structure, subfunctionalization, and Populus Arabidopsis microsynteny[END_REF]. Poplar ARF5s share conserved modular structure with their Arabidopsis homolog containing intact Domains III and IV (Fig. S6a), implying that they may interact with PtoIAA9. To determine the interactions between PtoIAA9 and PtoARF5s, we performed Y2H assay (Fig. 3a). Owing to the presence of the B3 domain with DNAbinding activity, the coding region of PtoARF5s was fused to AD, and the resulting fused protein was subsequently coexpressed in yeast cells with PtoIAA9-BD. Both PtoARF5 paralogs were shown to interact with PtoIAA9, but PtoARF5.1 displayed stronger interaction ability than PtoARF5.2 (Fig. 3a). The PtoIAA9-PtoARF5 interaction was subsequently confirmed by BiFC analyses (Fig. 3b).

Despite the physical interactions, the PtoIAA9-PtoARF5dependent regulation on wood formation requires their coexpression in secondary vascular tissues. Analysis of expression patterns based on RNAseq datasets [START_REF] Sundell | AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula[END_REF] revealed the highly correlated transcript accumulation between IAA9 and ARF5s in poplar wood-forming tissues (Fig. S6b,c). Similar to the expression patterns of PtoIAA9, RNA in situ hybridization results showed that PtoARF5.1 was highly expressed in developing wood-associated tissues of poplar (Fig. 3c,d).

PtoARF5.1 rescues PtoIAA9m-affected secondary xylem differentiation

Physical interactions and overlapping expression of PtoIAA9 and PtoARF5s suggested their cooperative involvement in the regulation of wood formation. To test this hypothesis, functional complementation of PtoARF5.1 was performed on PtoIAA9m-OE-resulting phenotypes of secondary xylem development (Fig. 4). To avoid the undesirable effects of PtoIAA9 overexpression and other endogenous Aux/IAA genes on its function, a fulllength PtoARF5.1 and a truncated form without C-terminal Domains III and IV responsible for Aux/IAA interactions (PtoARF5.1D) were introduced into the PtoIAA9m-OE transgenic lines, respectively (Figs 4, S6). Y2H tests showed that the C-terminal deletion abolished the interactions of PtoARF5.1 with the PtoIAA9 protein (Fig. S7a), but did not affect transcriptional activation (Fig. S7b).

Phenotypic analysis of transgenic plants showed that reduced growth of the PtoIAA9m-OE lines could be partially rescued by PtoARF5.1, whereas the truncated PtoARF5.1 displayed stronger recovery than the full-length form (Fig. 4a). Quantitative measurement of plant height and stem diameter confirmed these changes in different transgenic lines (Fig. 4b,c). By contrast with the partial recovery (c. 50%) by the full-length PtoARF5.1, the PtoARF5.1D almost completely rescued the decreased stem diameter of the PtoIAA9m-OE plants relative to that of WT (Figs 4c, S7d). Stem cross-sectioning was conducted to establish if rescue of secondary xylem growth by constitutive expression of PtoARF5.1 occurred in the PtoIAA9m-OE lines (Fig. 4d-f). Both the full-length and the truncated PtoARF5.1 were able to rescue the inhibition of overexpressing PtoIAA9m on secondary xylem development, as evidenced by the increased number of xylem cell layers and xylem percentage (40% by PtoARF5.1 and 94% by PtoARF5.1D) in stems (Fig. 4d-f). Noticeably, PtoARF5.1D conferred stronger recovery of the PtoIAA9m-OE-resulting defective wood formation than the full-length PtoARF5.1 (Fig. 4d-f), leading to even more xylem cell layers than WT.

Subsequently, the cambium zone and its adjacent cell layers were characterized in detail to reveal the possible modulation of cell differentiation by the PtoIAA9-PtoARF5 module (Fig. 5). By contrast with the absence of EDX in PtoIAA9m-OE plants, these cell layers reoccurred in both PtoARF5.1 and PtoARF5.1Dcomplementing lines (Fig. 5a). Similar recovery of defective xylem cell expansion resulting from overexpression of PtoIAA9m was detected for PtoARF5.1 and PtoARF5.1D, as indicated by measurement of fiber and vessel cell size (Fig. 5b-d). Moreover, elevated vessel density was also rescued by introduction of PtoARF5.1 and PtoARF5.1D into the PtoIAA9m-OE lines (Fig. 5b,e). The stronger recovery of PtoARF5.1D than its fulllength form indicated that the PB1 domain mediating AUX/ IAA-ARF protein interactions compromises the rescue of PtoIAA9m-resulting phenotypes of secondary xylem differentiation. Therefore, PtoARF5 is able to drive the PtoIAA9dependent cellular behaviors for secondary xylem differentiation in poplar.

The PtoIAA9-PtoARF5 module directly regulates expression of PtoHB7/8

Diverse auxin-triggered developmental phenotypes depend on expression of specific batteries of auxin responsive genes targeted by Aux/IAA-ARF pairs [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF]. To identify auxin responsive genes targeted by the PtoIAA9-PtoARF5 module during wood formation in poplar, comparative transcriptomic profiling via RNAseq was performed with the PtoIAA9m-OE lines and WT, revealing 3873 differentially expressed genes (fold change ≥ 2 and false discovery rate ≤ 1%; Fig. S8a; Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust New Phytologist (2019) www.newphytologist.com Methods S1; Dataset S1). A number of Aux/IAAs, SAURs and GH3s, which fall into classic families of early auxin responsive genes in plants [START_REF] Guilfoyle | Auxin-regulated genes and promoters[END_REF], displayed significantly reduced transcript abundance (2.1-to 15.2-fold) in the PtoIAA9m-OE lines (Fig. S7b), suggesting that auxin signaling was weakened by PtoIAA9m overexpression in poplar. Gene ontology (GO) analysis of differentially expressed genes showed enrichment of the term of nucleic acid binding transcription factor activity (Fig. S7c). We thus investigated the differentially expressed genes encoding transcription factors that are known to regulate wood formation in poplar (Fig. S7d). All of these genes, including WNDs, ANT and HBs, displayed decreased expression in the PtoIAA9m-OE lines (Fig. S8d). It has previously been established that class III HD-ZIP transcription factors are critical regulators of vascular formation and patterning in plants [START_REF] Ramachandran | Class III HD-ZIPs govern vascular cell fate: an HD view on patterning and differentiation[END_REF]. This family, designated HB transcription factors, comprises eight members in poplar (Fig. S8e). RNAseq and qRT-PCR showed that five of them displayed PtoIAA9minhibited expression (Fig. S8d,f). Among them, PtoHB7 and PtoHB8 were identified as critical regulators of vascular cambium differentiation to secondary xylem in poplar [START_REF] Zhu | PtrHB7, a class III HD-Zip gene, plays a critical role in regulation of vascular cambium differentiation in Populus[END_REF]. Therefore, we investigated whether the PtoHB7/8 genes are downstream targets of PtoIAA9-PtoARF5 during wood formation.

To test this hypothesis, we determined the regulation of PtoIAA9-PtoARF5 on PtoHB7 in the PtoIAA9m-OE lines. As shown in Fig. 6(a), the expression of both PtoHBs was significantly decreased by 71% and 74% in the PtoIAA9m-OE lines, respectively, and could be rescued by the introduction of the truncated PtoARF5.1 to even higher levels than that of WT. We also found that expression of PtoHB7 and PtoHB8 was increased 3.9-and 1.9-fold, respectively, in response to auxin treatment (Fig. 6b). Noticeably, overexpression of PtoIAA9 almost completely blocked the auxin-induced expression of both PtoHB7 and PtoHB8 in the wood-forming stem (Fig. 6b), indicating that the auxin responsive expression of PtoHB7 and PtoHB8 is dependent on PtoIAA9. Promoter sequence analysis revealed that a series of core and canonical auxin response elements (AuxREs), potential DNA binding sites for ARFs, are predicted in the 1.5 kb upstream regions of the start codons of PtoHB7 and PtoHB8 (Fig. 6c,d). ChIP was performed using PtoARF5.1-HA transgenic plants and the enrichment of AuxREs harbored by the PtoHB7 and PtoHB8 promoters was quantified by qPCR (Fig. 6e,f). In comparison with negative controls, some AuxRE-containing promoter regions of PtoHB7 (Regions II/III) and PtoHB8 (Regions III/IV) were significantly enriched after immunoprecipitation (Fig. 6e,f), indicating the direct binding of PtoARF5.1 to their promoters. The PtoIAA9/PtoARF5-dependent regulation of PtoHB7/8 was subsequently confirmed by effector-reporter assays in transiently expressed tobacco leaves. The reporter constructs carrying the GUS reporter gene driven by the 1.5 kb promoter regions of PtoHB7 and PtoHB8, respectively, were co-transfected with different combinations of the effectors harboring PtoIAA9 and PtoARF5.1 (Fig. S9). Determination of GUS activity revealed that PtoARF5.1 was able to independently activate expression of PtoHB7, whereas the activation was completely abolished by the addition of PtoIAA9 (P < 0.05; Fig. S9a). By contrast, PtoIAA9 did not compromise the activation induced by PtoARF5.1D without the C-terminal III/IV domains, indicating that PtoIAA9-mediated repression of PtoHB7 expression relies on its interactions with PtoARF5.1 via the III/V domains. Similar results were obtained from the promoter of PtoHB8 (Fig. S9b).

To further validate direct regulation of PtoARF5.1 on PtoHBs via binding to AuxREs, short promoter fragments harboring New Phytologist (2019) www.newphytologist.com canonical AuxREs (À447 to À581 bp upstream of the start codon of PtoHB7, and À344 to À434 bp upstream of the start codon of PtoHB8), which were strongly enriched in ChIP assays, were constructed to drive the firefly luciferase reporter. Consistently, PtoARF5.1 significantly activated the expression of luciferase reporter driven by the AuxRE-harbored promoter fragments of PtoHB7/8 (P < 0.05; Fig. 6g). By contrast, PtoARF5.1-driven activation disappeared when the AuxREs harbored in the promoter fragments of PtoHB7 and PtoHB8 were disrupted by sitedirected mutagenesis (Fig. 6g). Therefore, the PtoIAA9-PtoARF5 module directly regulates the expression of PtoHB7 and PtoHB8 via binding of PtoARF5 to the AuxREs within their promoters.

PtoHB7 partially rescues PtoIAA9m-resulting phenotypes of wood formation

To establish the link of biological functions between auxin signaling and the HB transcription factors during wood formation, we conducted complementation of the PtoIAA9m-resulting impaired secondary xylem phenotypes by PtoHB7 via stable transformation (Figs 7, S9). Phenotypic characterization revealed partial recovery of the PtoIAA9m-repressing plant growth and stem development by PtoHB7 (Fig. S10b-d). Stem cross-sections showed that introduction of PtoHB7 into the PtoIAA9m-OE background could partially rescue the impaired secondary xylem development, as validated by the quantification of xylem cell layers (Fig. 7a,b). Constitutive expression of PtoHB7 led to the reoccurrence of the EDX cell layers, which were repressed in the PtoIAA9m-OE lines (Fig. 7c). Similarly, the decreased cell size of xylem fibers and vessels as well as the enhanced vessel density were partially rescued by the introduction of PtoHB7 into the PtoIAA9m-OE plants (Fig. 7d-h).

Moreover, we determined the expression of several genes, including ACL5, CesA7A, PAL4, GT43B and WND1B, which were previously reported HB7/8-regulated genes involved in wood formation of poplar [START_REF] Milhinhos | Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem[END_REF][START_REF] Zhu | PtrHB7, a class III HD-Zip gene, plays a critical role in regulation of vascular cambium differentiation in Populus[END_REF]. The down-regulated expression of these genes in PtoIAA9m-OE plants was at least partially rescued in PtoARF5.1and PtoHB7-complementing transgenic lines (Fig. S11). A similar result was obtained in EXPA1, a marker gene for cell expansion during wood formation [START_REF] Sundell | AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula[END_REF]. We also

New Phytologist

found that expression of WND6A and B, which are the closest homologs of Arabidopsis VND6 and VND7 [START_REF] Zhong | Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar[END_REF], was enhanced by PtoIAA9m overexpression, but was rescued by PtoARF5.1 and PtoHB7 (Fig. S11), in accordance with elevated vessel formation by PtoIAA9m-OE and complemented by PtoARF5 and PtoHB7. These results strongly suggest that auxininduced secondary xylem development depends on the activation of the PtoHB genes by the PtoIAA9/PtoARF5 complex in poplar.

Discussion

Auxin is considered a positional signal that drives cambiumderived wood formation [START_REF] Uggla | Auxin as a positional signal in pattern formation in plants[END_REF]. Previous studies have demonstrated auxin-mediated regulation of cambial cell division in trees [START_REF] Sundberg | Cambial growth and auxin gradients[END_REF][START_REF] Nilsson | Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen[END_REF]. However, a highly sensitive analysis of auxin signaling in Arabidopsis revealed its maximal levels in differentiating cambial descendants, spatially divergent from the maximum auxin concentration in cambial initials [START_REF] Brackmann | Spatial specificity of auxin responses coordinates wood formation[END_REF]. In this study, we report that auxin coordinates multiple aspects of the cellular changes occurring for cambium specification into secondary xylem through the IAA9-ARF5 pathway in Populus. We further demonstrate that the PtoIAA9-PtoARF5 module directly targets PtoHB7 and PtoHB8 to mediate auxin-triggered early developing xylem (EDX) cell differentiation during wood formation. Aux/IAA proteins function as molecular switches of auxin signaling conserved among flowering plants [START_REF] Paponov | The evolution of nuclear auxin signalling[END_REF]. Comprehensive analyses of Populus Aux/IAAs revealed variable levels and spatial patterns of expression across wood-forming tissues (Fig. S2), implying functional diversification in wood formation [START_REF] Moyle | Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen[END_REF][START_REF] Kalluri | Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa[END_REF]. The transcripts of IAA9, -16.1 and -16.2 were enriched in cambium-neighboring cells undergoing differentiation into xylem but declined with xylem maturation (Fig. S2c). Highly correlated expression patterns among these Aux/IAA genes (Fig. S2c,d) and common protein interactions with ARF5 (Fig. S6d) suggested their functional redundancy in wood formation. Although compromised, wood differentiation could not be completely suspended by overexpressing PtoIAA9, implying regulatory complexity depending on some other pathways besides auxin signaling.

Compromised auxin signaling by expressing stable Aux/IAAs allows auxin-mediated coordination of cellular behaviors during wood formation to be explored. Overexpression of a hybrid aspen IAA3 (PttIAA3) attenuates periclinal cell division in cambium but enlarges cell files harboring anticlinal cell division, suggesting dual regulation of auxin signaling on cambial proliferation [START_REF] Nilsson | Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen[END_REF]. Despite more tightly arranged cambial cells (Fig. 2g), however, overexpressing PtoIAA9 did not lead to significant changes in cambial cell layers (Fig. S5e), implicating its weak role in cambial activity. IAA20.1 and -20.2, the PttIAA3 homologs in our nomenclature, displayed much lower expression levels than IAA9, and different expression patterns from IAA9 during wood formation (Fig. S2). Aux/IAAs usually require functional cooperation of ARFs via protein interactions, and harbor variable interacting affinities with different ARFs [START_REF] Vanneste | Auxin: a trigger for change in plant development[END_REF]. Thus, specific ARF partners of PttIAA3 and PtoIAA9 may cause their differential regulation on cambial activity.

Compared to WT, the proportion of woody tissues of transgenic plants overexpressing PtoIAA9m was significantly reduced (Fig. 2e, f), implying inhibition of secondary xylem development by PtoIAA9. Both reduced xylem cell layers and restricted xylem cell size led to PtoIAA9-inhibited wood formation that was rescued by overexpressing PtoARF5 (Figs 2, 4, 5). Cambial proliferation and cambium specification to xylem cooperatively determine the number of xylem cell layers [START_REF] Sanchez | From thin to thick: major transitions during stem development[END_REF]. Given the excluded PtoIAA9-mediated effects on cambial proliferation, we propose that the reduced periclinal cell division rate may be responsible for PtoIAA9-repressed wood formation. This is supported by the disappearance and reappearance of the EDX cell layers in PtoIAA9overexpressing and PtoARF5.1-complementing lines, respectively (Figs 2,5). These cell layers are initially specified from cambial cells and at early stage of wood formation. Active cambium periclinal cell division leads to rapid xylem mother cell accumulation, while a low rate of cambium periclinal cell division slowly produces xylem mother cells, resulting in an absence of the EDX cell layers in the PtoIAA9m-OE lines. Similar phenotypes were also found in trees during dormancy [START_REF] Gri Car | Plastic and locally adapted phenology in cambial seasonality and production of xylem and phloem cells in Picea abies from temperate environments[END_REF]. Therefore, specification and expansion of xylem cells during wood differentiation are coordinated by IAA9-ARF5-mediated auxin signaling in poplar.

Recent studies have shown that Arabidopsis MP/ARF5 directly represses WOX4 expression for restrained stem cell quantity [START_REF] Brackmann | Spatial specificity of auxin responses coordinates wood formation[END_REF], and switches of MP/ARF5 phosphorylation mediated by BIL1 kinase integrate peptide and cytokinin signaling for cambial activity [START_REF] Han | BIL1-mediated MP phosphorylation integrates PXY and cytokinin signalling in secondary growth[END_REF]. Here we provide evidence in Populus that ARF5 coordinates multiple behaviors for woody cell differentiation via directly targeting HB7/8 paralogs. BDL/IAA12 is a canonical Aux/IAA partner of ARF5/MP in Arabidopsis during embryogenesis [START_REF] Hamann | The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning[END_REF]. Due to promiscuous protein interactions between Aux/IAAs and ARFs, cooperative functions of IAA12-ARF5 could not be excluded in poplar. The significantly lower transcript abundance of IAA12 paralogs than IAA9 and their different expression patterns in wood-forming stems (Fig. S2) suggest possible variable roles in wood differentiation. We expected that IAA9 also regulates procambial specification during root development and leaf vein pattering. Indeed, the knockdown of IAA9 in tomato led to abnormal leaf formation with different vascular tissue patterning [START_REF] Wang | The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF].

Arabidopsis HD-ZIP III transcription factors are key regulators in orchestrating vascular patterning in developmental contexts of root, leaf and stem [START_REF] Ramachandran | Class III HD-ZIPs govern vascular cell fate: an HD view on patterning and differentiation[END_REF]. AtHB8 displays specific expression in procambial cells and is involved in vascular differentiation [START_REF] Baima | The Arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems[END_REF]. HB7, the poplar ortholog of AtHB8, has been revealed to be a dosedependent regulator that balances cambium activity and xylem differentiation during secondary growth [START_REF] Zhu | PtrHB7, a class III HD-Zip gene, plays a critical role in regulation of vascular cambium differentiation in Populus[END_REF]. We found that a series of core and canonical auxin response elements (AuxREs), the potential DNA binding sites for ARFs, are present in the promoters of poplar HB7 and HB8. ChIP and effector-reporter tests revealed transcriptional activation of PtoARF5.1 via direct binding to the AuxRE elements harbored in the promoters of HB genes (Fig. 6). Importantly, PtoHB7 is able to functionally rescue phenotypes of xylem cell specification, expansion and vessel density (Fig. 7). Therefore, our data suggested that the PtoIAA9-PtoARF5 module directly targets PtoHB7 and PtoHB8 for regulating secondary cell differentiation. In Arabidopsis, ARF5/MP has been identified to control leaf vein patterning by directly targeting AtHB8 [START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF], demonstrating ARF5-HB7/8 as a conserved pathway for vascular patterning of leaves and stems in herbaceous and woody species. Moreover, the HB8 orthologs can directly regulate the expression of ACL5 encoding thermospermine synthase in both Arabidopsis and poplar for xylem differentiation, and also form a feedback regulation on the auxin pathway [START_REF] Milhinhos | Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem[END_REF][START_REF] Baima | Negative feedback regulation of auxin signaling by ATHB8/ ACL5-BUD2 transcription module[END_REF]. We also found that the expression of ACL5 was significantly decreased by PtoIAA9 overexpression and rescued by PtoARF5 complementation (Fig. S11).

Collectively, our data allow us to propose a model for IAA9-ARF5-mediated coordination for wood differentiation (Fig. 8). Auxin releases the repression of PtoIAA9, by triggering its protein degradation, on PtoARF5-activated auxin responsive gene expression during cambium-derived wood formation. In parallel, auxin-inducible transcript abundance of PtoIAA9 switches-off auxin signaling in a self-controlled manner. The PtoIAA9-PtoARF5 module directly targets PtoHB7 and PtoHB8 to mediate auxin regulation on multiple cellular events, including xylem specification, expansion and vessel formation for secondary xylem development. In conclusion, our findings show that the auxin-Aux/IAA-ARF pathway plays a key role in controlling wood formation via HB-driven cambium differentiation in Populus. Table S1 Primer sequences used in this study.

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Fig. 1

 1 Fig. 1 Expression pattern and auxin induction of PtoIAA9 transcripts in wood-forming tissues of Populus tomentosa stem. (a, b) RNA in situ hybridization of PtoIAA9 in secondary vascular tissues of poplar. The 5 th internodes of 1.5-month-old poplar plants cultivated in soil were cross-sectioned for hybridization with antisense (a) and sense (b) probes of PtoIAA9. Red triangles indicate in situ hybridization signals for PtoIAA9 transcripts. Ca, cambium; Ph, phloem; Pi, pith; Xy, xylem. (c, d) Histological staining of the GUS reporter driven by the promoter of PtoIAA9 in poplar stems. The 7 th internodes of 1.5-month-old poplar plants cultivated in soil were cross-sectioned for GUS staining. Ca, cambium; Ph, phloem; Xy, xylem.. (e) Time-course assays of auxin-induced transcript abundance of PtoIAA9 in poplar stems. The microcutting-propagated poplar seedlings cultivated in vitro for 4 wk were subjected to 5 lM IAA for 0, 1, 2 and 3 h, and stem tissues were collected for RNA extraction followed by qRT-PCR assays. 18S rRNA was used as a reference gene. Expression levels are indicated relative to values for 0 h (with 0 h set arbitrarily to 1). Error bars represent AE SD. Asterisks indicate significant differences between mock and auxin treatment at each time point (Student's t test): *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3. (f) Quantification of auxin-induced GUS activity driven by the promoter of PtoIAA9 in poplar stem. IAA treatment (5 lM) was performed for 6 h as indicated in (d). The values under mock treatment were normalized to 1. Error bars represent SD. Asterisks indicate significant differences with respect to mock (Student's t test): **, P < 0.01; n = 4. Bars: (a, b) 200 lm; (c) 500 lm; (d) 100 lm.

Fig. 2

 2 Fig. 2 Wood phenotypes resulting from PtoIAA9m overexpression in Populus tomentosa. (a) Dwarf phenotypes of 2-month-old plants of independent PtoIAA9m-overexpressing (PtoIAA9m-OE) transgenic poplar lines (L1 and L2). (b, c) Measurement of plant height (b) and stem diameter (c) of PtoIAA9m-OE transgenic poplar lines corresponding to (a). (d) Cross-sectioning and staining with toluidine blue of the 7 th internode of 2-month-old wild-type (WT) and PtoIAA9m-OE transgenic plants (L1). Ph, phloem; Pf, phloem fibers; Xy, xylem. (e) Quantification of secondary xylem cell layers in WT and PtoIAA9m-OE transgenic plants (L1). The number of secondary xylem cell layers was counted in toluidine blue-stained anatomical sections of the 7 th internode of WT and PtoIAA9m-OE transgenic plants. (f) Percentage of secondary xylem and bark in the stem of WT and PtoIAA9m-OE transgenic plants (L1). The area of secondary xylem, bark and total stem was measured via IMAGEJ in toluidine blue-stained anatomical sections of the 7 th internode of WT and PtoIAA9m-OE transgenic plants. (g) Detailed observation of the cambial zone and woody cells of secondary xylem in WT and PtoIAA9m-OE plants. The images were captured on toluidine blue-stained anatomical sections of the 7 th internode of the corresponding lines. White lines indicate cambium (Ca), and red stars represent the cells of early developing xylem (EDX). V, vessel. (h, i) Quantification of the size of a single fiber (h) and vessel (i) cell in stem of WT and PtoIAA9m-OE plants. The area of fiber and vessel cells was measured and calculated via IMAGEJ based on the images of toluidine blue-stained anatomical sections as described in the Materials and Methods section. (j) Density of vessels in stem of WT and PtoIAA9m-OE plants. The number of vessels was counted based on the images of toluidine blue-stained anatomical sections. Error bars represent SD. Asterisks indicate significant differences with respect to values of WT (Student t-test): *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4. Bars: (a) 5 cm; (d) upper, 250 lm, lower, 100 lm; (g) 50 lm.

Fig. 3

 3 Fig. 3 Protein interactions of PtoIAA9 with PtoARF5s and expression of PtoARF5 in secondary vascular tissues of Populus tomentosa stem. (a) Yeast-twohybrid analysis of protein interactions between PtoIAA9 and PtoARF5.1/5.2. PtoIAA9 and PtoARF5.1/5.2 were fused with a Gal4 DNA-binding domain (BD) and a GAL4 activation domain (AD), respectively. The interaction between BD-p53 and AD-RecT (SV40 large T-antigen) was used as a positive control, while those between blank constructs (BD or AD) with BD-PtoIAA9 or AD-PtoARF5.1/5.2 were used as negative controls. Yeast cells were inoculated on selective medium in a 10-fold gradient dilution. SD/-TL, double dropout medium lacking tryptophan and leucine; SD/-AHTL, quadruple dropout medium lacking adenine, histidine, tryptophan and leucine. (b) Bimolecular fluorescence complementation (BiFC) assays validating physical interactions between PtoIAA9 and PtoARF5 in nuclei. nYFP and cYFP represent the N-and C-terminal part of yellow fluorescent protein (YFP), respectively. The PtoIAA9-nYFP and PtoARF5.1-cYFP constructs were cotransfected into tobacco epidermal leaf cells via Agrobacterium-mediated infiltration. The blank constructs of cYFP or nYFP were cotransfected with PtoIAA9-nYFP or PtoARF5.1-cYFP, respectively, as negative controls. The fluorescence emitted by YFP was examined with a confocal microscope. Nuclei were identified by DAPI staining. (c, d) RNA in situ hybridization of PtoARF5 in secondary vascular tissues of poplar stem. The 7 th internodes of 6-wk-old poplar plants cultivated in soil were cross-sectioned for hybridization with sense (c) and anti-sense (d) probes. The probes were designed within the identical region of PtoARF5.1 and PtoARF5.2 for detection of both ARF5 paralogs in poplar. Red triangles indicate the in situ hybridization signals. Ca, cambium; Ph, phloem; Pi, pith; Xy, xylem. Bars: (b) 50 lm; (c, d) 200 lm.

Fig. 4

 4 Fig. 4 Rescue of PtoIAA9m-resulting wood-associated phenotypes in Populus tomentosa by PtoARF5. (a) Recovered morphological phenotypes of 1.5month-old PtoIAA9m-overexpressing (OE) transgenic poplar plants by PtoARF5. The constructs of 35S:PtoARF5.1 and 35S:PtoARF5.1D (encoding for 1-665 amino acids of PtoARF5.1 without C-terminal III/IV domains) were transformed into poplar in the background of PtoIAA9m-OE. (b, c) Measurement of plant height and stem diameter of wild-type (WT), PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE transgenic poplar lines. (d) Anatomical sections stained with toluidine blue of the 7 th internode of 1.5-month-old WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. Red lines indicate xylem. Xy, xylem. (e) Quantification of secondary xylem cell layers of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE lines. The number of xylem cell layers was counted in toluidine blue-stained anatomical sections of the 7 th internode of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. (f) Percentage of secondary xylem and bark in the stem of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The area of secondary xylem, bark and total stem was quantified via IMAGEJ in toluidine blue-stained cross-sections of the 7 th internode of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. Error bars represent SD. The letters above error bars indicate statistically significant differences (one-way ANOVA followed by Dunnett's test for pairwise comparisons; n = 4). Bars: (a) 5 cm; (d) 100 lm.

Fig. 5 Fig. 6

 56 Fig. 5 PtoARF5 restores PtoIAA9-affected phenotypes of secondary xylem cells in Populus tomentosa during wood formation. (a, b) Detailed observation of cambial zone and woody cells of secondary xylem in wild-type (WT), PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The images were captured on toluidine blue-stained anatomical sections of the 7 th internode of the corresponding lines. White lines indicate cambium (Ca), and red stars represent the cells of early developing xylem (EDX). V, vessel. Bars, 50 lm. (c, d) Quantification of size of a single fiber and vessel cell in stems of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The area of fiber and vessel cells was measured and calculated via IMAGEJ based on images of toluidine blue-stained anatomical sections as described in the Materials and Methods. (e) Density of vessels in stems of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The number of xylem vessels was counted based on images of toluidine blue-stained anatomical sections. Error bars represent SD. The letters above error bars indicate significant differences (one-way ANOVA followed by Dunnett's test for pairwise comparisons; n = 10-12).

Fig. 7

 7 Fig. 7 PtoHB7 partially rescues deficient phenotypes of wood formation resulting from constitutive expression of PtoIAA9 in Populus tomentosa. (a) Crosssections stained with toluidine blue of the 7 th internode of 1-month-old WT, PtoIAA9m-OE and PtoHB7-OE/PtoIAA9m-OE lines. Red lines indicate xylem. Xy, xylem. (b) Number of xylem cell layers of wild-type (WT), PtoIAA9m-OE and PtoHB7-OE/PtoIAA9m-OE lines. (c, d) Detailed phenotypes of cambial zone and woody cells of secondary xylem in wood-forming stem of WT, PtoIAA9m-OE and PtoHB7/PtoIAA9m-OE plants. The images were captured on toluidine blue-stained anatomical sections of the 7 th internode of the corresponding lines. White lines indicate cambium (Ca), and red stars represent the cells of early developing xylem (EDX). V, vessel. (e, f) Quantification of size of a single fiber and vessel cell in stems of WT, PtoIAA9m-OE, and PtoHB7/ PtoIAA9m-OE plants. The area of fiber and vessel cells was measured and calculated via IMAGEJ based on images of toluidine blue-stained anatomical sections as described in the Materials and Methods. (g) Density of vessels in stem of WT, PtoIAA9m-OE and PtoHB7/PtoIAA9m-OE plants. The number of xylem vessels was counted based on the images of toluidine blue-stained anatomical sections. Error bars represent SD. The letters above error bars indicate significant differences (one-way ANOVA followed by Dunnett's test for pairwise comparisons; n = 10-12). Bars: (a) 100 lm; (c, d) 50 lm.

Fig 8

 8 Fig 8 A model for Aux/IAA-ARF-HB-dependent wood formation in Populus tomentosa. Auxin displays complex regulation of various steps during wood formation in poplar. The auxin-dependent cambial cell division was demonstrated by Nilsson et al. (2008). The role of auxin in xylem maturation, mainly including secondary wall deposition, remained unclear. In this study, PtoIAA9-ARF5-HB7/8-mediated wood differentiation is proposed. The auxin-dependent PtoIAA9-ARF5 module directly regulates the expression of PtoHB7/8, which drives multiple cellular events including xylem cell specification, expansion and vessel formation during secondary xylem development. Arrows represent a positive regulatory action of one component on another. Lines ending with a trait represent a negative regulatory action. Dotted lines indicate indirect regulation.

Fig. S9

 S9 Fig.S9Regulation of the PtoIAA9/ARF5 module on PtoHB7/8 promoter activities via effector-reporter tests using GUS as a reporter gene.

Fig. S10

 S10 Fig. S10 Constitutive expression of PtoHB7 in the PtoIAA9m-OE lines.

Fig. S11

 S11 Fig. S11 Expression of some secondary xylem developmentrelated genes in PtoIAA9m-OE, PtoARF5.1-and PtoHB7-complementing transgenic lines.

Acknowledgements

We thank Drs Guoqing Niu, Xinqiang He and Jianquan Liu for helpful comments. This work was supported by the National Natural Science Foundation of China (31500544, 31670669, 31870657, 31800505 and 31870175), National Key Project for Research on Transgenic Plant (2016ZX08010-003) and Fundamental Research Funds for the Central Universities (XDJK2018AA005, XDJK2014a005). The authors declare no conflicts of interest.

Author contributions

KL, CX and HCW designed the work; CX, FH, YS, XF, WL, CL, HY, YL and DF performed experiments and data analyses; CX drafted the manuscript; KL and HCW revised the manuscript. All authors approved the final version of the manuscript for publication. CX, YS, FH, XF and HY contributed equally to this work.

Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

Dataset S1 Differentially expressed genes in the RNAseq-based transcriptomic analysis of PtoIAA9m-OE vs WT.