Islam El-Sharkawy

Sherif M Sherif

Brian Jones

Isabelle Mila

Prakash P Kumar

Mondher Bouzayen

Subramanian Jayasankar

TIR1-like auxin-receptors are involved in the regulation of plum fruit development

Keywords: Auxin-receptors, plum fruit development, Prunus salicina, subcellular localization, protein-protein interaction, auxin/ ethylene crosstalk

Ethylene has long been considered the key regulator of ripening in climacteric fruit. Recent evidence showed that auxin also plays an important role during fruit ripening, but the nature of the interaction between the two hormones has remained unclear. To understand the differences in ethylene-and auxin-related behaviours that might reveal how the two hormones interact, we compared two plum (Prunus salicina L.) cultivars with widely varying fruit development and ripening ontogeny. The early-ripening cultivar, Early Golden (EG), exhibited high endogenous auxin levels and auxin hypersensitivity during fruit development, while the late-ripening cultivar, V98041 (V9), displayed reduced auxin content and sensitivity. We show that exogenous auxin is capable of dramatically accelerating fruit development and ripening in plum, indicating that this hormone is actively involved in the ripening process. Further, we demonstrate that the variations in auxin sensitivity between plum cultivars could be partially due to PslAFB5, which encodes a TIR1-like auxin receptor. Two different PslAFB5 alleles were identified, one (Pslafb5) inactive due to substitution of the conserved F-box amino acid residue Pro 61 to Ser. The early-ripening cultivar, EG, exhibited homozygosity for the inactive allele; however, the late cultivar, V9, displayed a PslAFB5/afb5 heterozygous genotype. Our results highlight the impact of auxin in stimulating fruit development, especially the ripening process and the potential for differential auxin sensitivity to alter important fruit developmental processes.

Introduction

Fruit and seeds are essential for human nutrition. Postfertilization, fruits develop rapidly through a coordinated programme of molecular, biochemical and structural changes that optimize the potential for the development and dissemination of the seeds within. The fundamental importance of these processes has prompted considerable research into how they are governed. Research into fruit developmental processes has been greatly aided by analysing the outcome of both naturally occurring and induced genetic diversity [START_REF] Kelly | Insensitivity of the diageotropica tomato mutant to auxin[END_REF][START_REF] Lau | The evolving complexity of the auxin pathway[END_REF]. One outcome of this research has been the identification of phytohormones as master regulators of the many processes involved [START_REF] Mcatee | A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening[END_REF]. Ethylene, auxin, GA, ABA, and cytokinin have all been shown to play important integrative roles [START_REF] Crane | The role of hormones in fruit set and development[END_REF][START_REF] Srivastava | Hormonal regulation of tomato fruit development: A molecular perspective[END_REF]. Given its almost ubiquitous importance, it was not unexpected that auxin would play a prominent role in reproductive growth [START_REF] Friml | Auxin transport-shaping the plant[END_REF]. Collectively, data from auxin application, quantification, and molecular genetic studies have shown that auxin is integral to most reproductive processes [START_REF] Sedgley | Sexual reproduction of tree crops[END_REF][START_REF] Sundberg | Distinct and dynamic auxin activities during reproductive development[END_REF]. For example, auxin is required for floral meristem formation and acts with homeotic genes in determining floral organogenesis [START_REF] Coen | Flower development[END_REF]. Auxin is critical for the cell divisions that occur in response to fertilization (fruit-set), to the subsequent expansion and development of these cells [START_REF] Mapelli | Relationship between set, development and activities of growth regulators in tomato fruits[END_REF], and in regulating the onset and coordination of ripening processes [START_REF] Gillaspy | Fruits: a developmental perspective[END_REF][START_REF] Trainotti | The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches[END_REF][START_REF] El-Sharkawy | Regulation of two germin-like protein genes during plum fruit development[END_REF]. Recent studies have shown that exogenous auxin accelerates fruit development and ethylene production, acting at least partially by triggering the expression of several genes, such as ACS and ERF, encoding ethylene-synthesis and -response components [START_REF] Trainotti | The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches[END_REF][START_REF] El-Sharkawy | Differential regulation of four members of ACC synthase gene family in plum[END_REF], 2009).

Auxin responses are regulated at multiple levels, but they basically fall into two categories: responses that affect the hormone content and responses that regulate the response capacity [START_REF] Lau | The evolving complexity of the auxin pathway[END_REF]. Previous studies in tomato and Arabidopsis have identified auxin-signalling components, such as a member of the Aux/IAA family (IAA9) and two members of the Auxin Response Factor protein family, ARF7 and ARF8, that are clearly important for early fruit development [START_REF] Wang | The tomato Aux/ IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF][START_REF] Goetz | AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis[END_REF][START_REF] Goetz | Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato[END_REF]de Jong et al., 2009b). Downregulation of these transcriptional regulators results in pleiotropic phenotypes, including fertilization-independent fruit-set.

The discovery that certain F-box proteins are auxinreceptors considerably improved the understanding of how auxin mediates cellular responses (Dharmasiri et al., 2005a;[START_REF] Kepinski | The Arabidopsis F-box protein TIR1 is an auxin receptor[END_REF]. Basically, Aux/IAAs and ARF-transcriptional regulators interact in homo-and heterodimers, forming complexes that repress auxin-dependent changes in gene expression and therefore auxin action. Auxin binding to an F-box receptor promotes the binding of Aux/IAAs to the SCF TIR1/AFB complex, leading to the ubiquitin-dependent proteolysis of the Aux/IAA [START_REF] Dharmasiri | Auxin signaling and regulated protein degradation[END_REF][START_REF] Leiser | Dynamic integration of auxin transport and signalling[END_REF]. Loss of Aux/IAA repressors allows ARF-mediated auxin-responsive changes in gene transcription. Although this archetypal response mechanism is widespread, genetic studies have demonstrated alternative mechanisms. For example, TIR1-and AFB2-type auxinreceptors have been shown to be positive regulators of the auxin-response; however, loss-of-function studies revealed that members of the distinct AFB4 group act as negative regulators (Dharmasiri et al., 2005b;[START_REF] Parry | Complex regulation of the TIR1/AFB family of auxin receptors[END_REF][START_REF] Greenham | The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings[END_REF]. Given that auxin is a key regulator of early fruit development, it might be expected that TIR1like receptors also regulate the process [START_REF] Ren | The auxin receptor homologue in Solanum lycopersicum stimulates tomato fruit-set and leaf morphogenesis[END_REF].

In this study, we evaluated the role of auxin in flower and fruit development of two plum cultivars that vary in their flowering and ripening dates. We showed that the diversity in fruit development behaviours and auxin-responsiveness between the early-ripening/auxin-hypersensitive EG cultivar, and the late-ripening/reduced auxin-sensitivity V9 cultivar, could be partially due to a mutation occurred within the essential F-box domain of the auxin-receptor PslAFB5. This mutation resulted in a substitution in the highly conserved residue Pro 61 -to-Ser. Using yeast twohybrid (Y2H) and bimolecular fluorescence complementation (BiFC) strategies we provided evidence that the allele with Pro 61 (PslAFB5) encodes a functional auxin-receptor and the allele with the substitution Ser 61 (Pslafb5) is inactive. Interestingly, genetic analysis of the two plum cultivars revealed that the early cultivar, EG, exhibited homozygosity for the inactive allele gPslafb5; however, the late cultivar, V9, displayed a gPslAFB5/afb5 heterozygous genotype. The data presented here suggest that the auxin-hypersensitivity in the EG cultivar is at least partially due to the loss of PslAFB5 activity. Collectively, our results highlight the importance of auxin in coordinating the progression of fruit development, particularly in terms of the developmental phase transition that leads to the onset of ripening.

Materials and methods

Plant materials and post-harvest treatments

Reproductive structures from flower buds to ripening fruit were collected from two Japanese plum (Prunus salicina L.) cultivars, Early Golden (EG) and V98041 (V9). These were frozen in liquid-N 2 , and stored at -80°C until analysis. These two varieties were chosen according to the diversity of their responses to auxin application. Fruit were picked at stages S1 (first exponential growth phase), 16-34 and 32-50 days after bloom (DAB); S2 (pit hardening), 36-54 and 52-72 DAB; and S3 (second exponential growth phase), 57-77 and 74-108 DAB for EG and V9, respectively. Fruits from S4 (climacteric ripening) were collected from EG between 78 and 83 DAB, and from V9 between 110 and 128 DAB. To evaluate the effects of exogenous auxin in flower and fruit development, EG and V9 trees were sprayed during the reproductive cycle (flower bud and immature expanded fruit, stage 3) with two different concentrations of the synthetic auxin naphthalene acetic acid (NAA) (10 and 100 µM). Mature fruits of EG (76 DAB) and V9 (108 DAB) were harvested, surface sterilized, and subjected to various treatments, including: propylene (1000 µl l -1 ); the ethylene-inhibitor 1-MCP (1 µl l -1 ); NAA (10 and 100 µM for EG and V9, respectively); and the auxin transport-inhibitor TIBA (10 µM). Non-treated fruit were used as controls. See supplementary material for other details concerning hormone quantification, PslTIR1/AFBs sequence isolation and analysis, nucleic acid extraction, qPCR assays, and subcellular localization.

Generation of truncated and mutated versions of PslTIR1-like proteins

Fragments containing N-terminal (∆N) and C-terminal (∆C) deletions of PslTIR1, PslAFB2, and PslAFB5 were obtained by PCR using gene-specific primer pairs (primers 11-22, Supplementary Table S1 available at JXB online). Internal deletions of the F-box domain (∆F) from each sequence were created using two different strategies depending on the variation in N-terminal length (see supplementary material for further details). Mutants of PslTIR1 (Pro 9 to Ser) and PslAFB5 (Pro 61 to Ser) were created using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA).

Y2H assays

Yeast two-hybrid (Y2H) assays were performed using the Matchmaker Gold Yeast two-hybrid System (Clontech, Palo Alto, CA, USA). Full-length and modified PslTIR1, PslAFB2, and PslAFB5 ORFs were cloned into the BamHI-SalI site of the pGBKT7 bait vector (GAL4 binding-domain; DBD). Tomato and Arabidopsis cDNAs were fused into either the NdeI-BamHI site (AtIAA7) or the BglII-BamHI site (ASK1, SlIAA3, and SlIAA9) of the pGADT7 prey vector (GAL4 activation-domain; AD). Bait and prey vectors (100 ng) were then introduced into Y2HGold and Y187 yeast strains, respectively, using Yeastmaker yeast transformation system 2. Interactions between the proteins were assayed by the mating method, according to the manufacturer's instructions, in 96-well plates containing medium with or without 100 µM IAA. All assays were repeated at least three independent times.

BiFC assays

Full-length and mutated PslTIR1 and PslAFB5 ORFs were fused into the SalI-BamHI site of the pSAT1-N vector, containing N-terminal YFP (NY). ASK1 and AtIAA7 sequences were cloned into the BglII-BamHI and XhoI-BamHI sites, respectively, of the pSAT1-C vector containing C-terminal YFP (CY). The different combinations of constructs encoding NY and CY at similar concentrations were mixed and then co-transfected into protoplasts obtained from suspension-cultured tobacco BY-2 cells in the presence of 100 µM IAA, as previously described [START_REF] El-Sharkawy | Characterization of gibberellin-signalling elements during plum fruit ontogeny defines the essentiality of gibberellin in fruit development[END_REF]. Empty BiFC vectors (NY/CY), (NY:gene /CY) and (NY/ CY:gene) were used as negative controls. All assays were repeated at least three times and visualized using confocal microscopy.

Results

Auxin enhances fruit development

To assess the role of auxin in fruit development, a range of plum genotypes that vary in their flowering and ripening dates were selected and treated with NAA (Supplementary Table S2 available at JXB online). Two cultivars, early-ripening, EG, and late-ripening, V9, were selected for further analysis based on the diversity of their responses to the auxin treatment. Application of auxin to floral buds considerably enhanced flower development in both cultivars as determined by flower sizes at different developmental stages (Supplementary Table S3 available at JXB online). In EG, the maximum response to auxin in terms of flowering occurred at an NAA concentration of 10 µM. However, higher concentrations of NAA diminished responses in EG flowering (Fig. 1A, B; Supplementary Table S3 available at JXB online). In contrast, a flowering response to auxin application occurred only at a concentration of 100 µM in V9. In both cultivars, auxin treatment accelerated the fruit-set by 5 ± 1 days compared to the untreated controls. Moreover, treatment of mature fruits from both cultivars with auxin resulted in significant increases in fruit size (~28%) and weight (~35%) over the untreated controls (Fig. 1C,D,E), indicating the potential for auxin to affect fruit development.

Ripening is the final stage of fruit development. Many of the processes integral to the ripening of plums and other climacteric fruit are known to be triggered by ethylene [START_REF] Lelièvre | Ethylene and fruit ripening[END_REF]. To examine the contribution of auxin to the coordination of fruit development, the onset of ethylene production, and ripening, we monitored the ethylene production and endogenous IAA levels during EG and V9 fruit development. In stone fruit, including plum, the fruit growth followed a typical double-sigmoidal curve (Fig. 2A,B), in which four developmental stages (S1-S4) can clearly be recognized [START_REF] El-Sharkawy | Isolation and characterization of four ethylene signal transduction elements in plums (Prunus salicina L.)[END_REF]. V9 exhibited significant delays in fruit developmental progression compared to EG, due to a delay in flowering (16 ± 2 days later than in EG) and extended S3 duration (14 d± 2 days longer than in EG), resulting in prolonged fruiting period (Supplementary Table S2 available at JXB online). Ethylene was undetectable in immature fruit, but both cultivars produced significant amounts of ethylene during ripening (Fig. 2C,D). EG fruit, in particular exhibited a sharp increase in ethylene production during ripening. In contrast, the onset of ripening was delayed in V9 (22 ± 2 days later than in EG) with lower ethylene levels, resulting in slower ripening process (13 ± 2 days longer than in EG).

The IAA accumulation patterns were similar in the two cultivars during the early stages of fruit development (Fig. 2E,F). Endogenous IAA levels were low at anthesis, but rose rapidly in flowers post-fertilization. IAA levels gradually declined thereafter in both cultivars to low or undetectable levels by S2. A clear divergence in IAA accumulation profiles between the two cultivars occurred subsequent to this minimum. In EG, the concentration of endogenous IAA increased rapidly as the fruit matured, reaching maximal levels during climacteric ripening. In stark contrast, the levels of IAA increased only slightly in V9, and remained low through to the end of the ripening phase.

Auxin application accelerated the onset of ripening; thus, to ascertain the relationship between the auxin treatment and ethylene production and ripening, ethylene was assessed during the ripening of EG and V9 fruits pre-exposed to different treatments designed to alter auxin-or ethylene-responses. EG and V9 fruit displayed a climacteric peak of ethylene production at ~5 days and ~13 days after the onset of ethylene production, respectively. Ethylene emission at the peak of the climacteric was 48.2 ± 4.2 and 5.7 ± 0.9 nl g -1 h -1 for EG and V9, respectively. Propylene-treatment accelerated ripening and increased ethylene production in both cultivars. Maximal levels of ethylene were observed at ~2 days (EG) and ~5 days (V9) post-treatment with a correspondingly increased ethylene production of 80.5 ± 5.1 and 58.6 ± 3.2 nl g -1 h -1 for EG and V9, respectively (Supplementary Figure S1A, B available at JXB online). As expected, all fruit treated with the ethylene response inhibitor, 1-MCP, were unable to ripen autonomously and their ethylene production remained low (Supplementary Figure S1C, D available at JXB online).

Auxin dramatically accelerated the ripening of both plum cultivars, but in a slightly different manner for each one. Ethylene production in the auxin-treated EG fruit peaked at a high level (69.5 ± 5.5 nl g -1 h -1 ) ~3 days after treatment. Relative to NAA-treated EG fruit, a lower climacteric ethylene peak of 30.6 ± 2.2 nl g -1 h -1 was observed in NAA-treated V9 fruit and this peak occurred much later, ~15 days after the treatment (Supplementary Figure S1E, F available at JXB online). Treatment with the auxin transport inhibitor TIBA had almost no effect on ethylene production in EG, but had more pronounced effects in V9 (Supplementary Figure S1G, H available at JXB online), as determined by the time necessary to have the climacteric peak after the treatment and the levels of ethylene at the peak.

Identification and characterization of PslTIR1/ AFB genes

To investigate the molecular basis of auxin action in fruit development, genes encoding three novel proteins closely related to the F-box TIR1-like gene family of auxin-receptors were isolated from plum. The isolated sequences were designated as PslTIR1, PslAFB2, and PslAFB5 to comply with the names retained for the homologues from other plant species. The relationships between the predicted plum and Arabidopsis amino acid sequences (Dharmasiri et al., 2005b;[START_REF] Walsh | Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis[END_REF], as indicated by percentage similarity over the whole sequence, are presented in Supplementary Table S4 available at JXB online. Despite strong sequence diversity among the different gene members, there were also highly homologous sequences putatively coding for closely related proteins. Strong sequence similarity between specific plum and Arabidopsis sequences (80-89%) indicates that these sequences are likely to be orthologous. The derived PslTIR1/AFB sequences comprise three typical domains that are associated with the F-box subunit protein of SCF (E3) ubiquitin ligase (Supplementary Figure S2 available at JXB online). The deduced amino acid sequences of PslTIR1 and PslAFBs start by an N-terminal domain with unknown function that exhibits high sequence divergence in length [3-58 amino acids (aa)] and homology (12-66% similarity), followed by the characteristic F-box domain (42-48 aa), which shows higher sequence similarity, ranging from 65-81%. Finally, the C-terminal domain, which is composed of several LRRs, occupies the major part of the predicted protein (521-535 aa; 64-90% similarity). The number of LRRs varied among the different sequences [predicted to be 5-(PslAFB5), 6-(PslTIR1), and 7-LRRs (PslAFB2)]. See supplementary material for gene structure and organization.

PslTIR1/AFBs are components of an SCF complex that interacts with Aux/IAAs F-box subunits are anchored into SCF complexes via binding of the F-box domain to Skp1 [START_REF] Zheng | Structure of the Cul1-Rbx1-Skp1-F box Skp2 SCF ubiquitin ligase complex[END_REF]. If PslTIR1/AFB proteins are part of a functional SCF PslTIR1/AFBs complex, they should interact with one or more Skp1 homologues [START_REF] Gray | Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana[END_REF]. We tested for the capacity of PslTIR1/ AFBs to interact with the Arabidopsis Skp1-related protein (ASK1) using Y2H. Consistent with the Arabidopsis TIR1-like proteins, all PslTIR1/AFBs interacted strongly with ASK1 in a manner independent of auxin action (Fig. 3). We next sought to determine if Aux/IAA proteins are potential substrates of the SCF PslTIR1/AFBs E3 ubiquitin-ligase by assessing interactions between PslTIR1/AFBs and several Aux/IAAs using the Y2H system in the presence or absence of IAA (Fig. 3). The well-characterized Aux/IAA proteins chosen for this study were the Arabidopsis IAA7 (AtIAA7), and the tomato IAA3 and IAA9 (SlIAA3 and SlIAA9), which represent three different sub-clades of Aux/IAAs and play distinct roles in mediating auxin responses in planta [START_REF] Nagpal | AXR2 encodes a member of the Aux/IAA protein family[END_REF][START_REF] Wang | The tomato Aux/ IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF][START_REF] Chaabouni | Sl-IAA3, a tomato Aux/IAA at the crossroads of auxin and ethylene signalling involved in differential growth[END_REF]. The analysis revealed a clear difference in the binding specificity and auxin-dependence of the PslTIR1/AFB proteins for the various Aux/IAAs. The binding results confirmed the auxin-induced assembly of stable IAA::PslTIR1/AFBs::Aux/IAAs co-receptors in yeast.

Characterization of PslTIR1/AFBs domains

All TIR1/AFB gene family members are structurally conserved across land plant lineages. Their predicted proteins comprise three typical domains-N-terminal, F-box, and C-terminal-that are commonly associated with the F-box subunit protein of SCF (E3) ubiquitin-ligase (Supplementary Figure S2 available at JXB online). To explore the structural contribution of each domain in mediating auxin signalling, we performed a series of experiments, including subcellular localization and Y2H assays of full-length ORFs and ORFs containing independent deletions of the three PslTIR1, PslAFB2, and PslAFB5 domains. The full-length proteins are referred to Fig. 3. Differences in PslTIR1/AFBs-ASK1 and -Aux/IAAs interactions in yeast. Y2H assays of the interaction (indicated by formation of blue colour) were performed using PslTIR1/AFBs (as bait) in Y2HGold strain yeast and ASK1 or Aux/IAAs (as prey) in Y187 strain yeast. The mated yeast (DBD-PslTIR1/AFBs and AD-ASK1 or -Aux/IAAs) were grown in 96-well plates containing DDO/X/A selective medium in the presence or absence of 100 µM IAA. All experiments were repeated at least three times.

as wild-type (WT). Proteins lacking the N-terminal, F-box, or C-terminal domains are referred to as ∆N, ∆F, or ∆C, respectively. Analysis of TIR1/AFB sequences from different plant species revealed the absence of any feasible targeting sequence that can signify the localization of such protein in the plant cell. Fluorescence microscopy revealed, however, that as expected the full-length PslTIR1/AFBs::GFP fusions were localized exclusively in the nucleus (Fig. 4A). Treatment of cells with IAA before transfection of the GFP fusion construct did not alter the protein localization (data not shown). Deletion of the N-terminal (∆N) domain did not visually affect nuclear localization. By contrast, removal of the C-terminal domain (∆C) noticeably altered the protein localization. Without the C-terminal domain, the GFP signal was uniformly spread throughout the cytoplasm and nucleus. An even more dramatic disruption of protein localization occurred when the F-box domain was eliminated (∆F). Small amounts of the protein localized in the nucleus or cytoplasm; however, most of the GFP signal was observed on the outer surface of the nuclear envelope. To further assess the contribution of each domain in forming SCF-like complexes and activating auxin signalling, the different ORFs were challenged to interact with ASK1 and AtIAA7 in the presence of auxin using a Y2H approach (Fig. 4B). Compared with full-length ORFs, the lack of an N-terminal domain did not noticeably affect the interaction of ∆N with ASK1. Interestingly, although PslTIR1∆N and PslAFB2∆N were able to assemble a co-receptor complex with AtIAA7, the interaction activity of PslAFB5∆N was visibly reduced. The ∆C-derivative remained able to interact with ASK1, albeit at a significantly lower efficiency. In contrast, lack of a C-terminal domain resulted in a complete failure to interact with AtIAA7. Deletion of the F-box domain completely abolished the ability of the protein to interact with ASK1 and AtIAA7. Therefore, although the F-box domain appears to be the key factor in successful Skp1-like protein complex formation, the loss of the C-terminal domain is similarly critical for proper function.

PslTIR1/AFB expression during fruit development

To investigate the contribution of the plum auxin receptors in fruit development, the accumulation profiles of the three genes were determined at different developmental stages in EG and V9 fruit. During the immature stages (flowering to S3), both cultivars exhibited similar transcript accumulation profiles for all three genes, with slightly, but consistently higher levels, in EG compared with V9 (data not shown).

All transcripts were initially present at low levels in flower buds, but accumulated during flower development, peaking soon after fertilization at ~7 DAB (Fig. 5A). The levels detected at this stage were the highest detected over the experiment, with the transcript levels for all three genes declining to very low levels by the S2 stage (~52 DAB). PslTIR1 and PslAFB2 transcript levels, however, increased markedly during S3, with the progression to maturity. By contrast, those of PslAFB5 were barely detectable during this phase.

Transcript levels for the three genes were closely assessed during the S4 stage, where most ripening-related metabolic changes take place. Clear differences in transcript accumulation profiles were observed during this stage (Fig. 5B). In EG, PslTIR1 and PslAFB2 levels were low before the onset

Identification of PslTIR1/AFB alleles among plum cultivars

In an attempt to further unravel the effects of auxin during fruit development and links between auxin receptors and the variation in auxin sensitivity among cultivars, the fulllength genomic sequences of the three genes were isolated and sequenced from EG and V9. All three gPslTIR1/AFB genes in both cultivars have open reading frames comprising three exons and two introns (Supplementary Figure S4 available at JXB online) with the exon/intron boundaries occurring in the same positions in both cultivars (Supplementary Figure S2 available at JXB online). The two cultivars were homozygous for gPslTIR1 and gPslAFB2; however, two different alleles were identified for gPslAFB5, with the alterations in nucleotide composition leading to four predicted changes in amino acid residues (Supplementary Figure S5 available at JXB online). One alteration in particular occurred within the F-box domain, causing a predicted substitution in the highly conserved residue Pro 61 (Pro to Ser). Pro 61 has been shown to be essential for TIR1-like function [START_REF] Gray | Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana[END_REF][START_REF] Kepinski | The Arabidopsis F-box protein TIR1 is an auxin receptor[END_REF]. Given the nature of the substitution, we predicted that the allelic form with Pro 61 (designated gPslAFB5) would be functional and the allele with the substitution Ser 61 (designated gPslafb5) would be inactive, or at best differentially functional. Strikingly, the auxin-hypersensitive cultivar, EG, was homozygous for gPslafb5; and the reduced auxinsensitivity cultivar, V9, had the gPslAFB5/afb5 heterozygous genotype (Supplementary Figure S6 available at JXB online). To confirm the distribution of gPslAFB5 alleles among cultivars, we performed a cleaved-amplified polymorphic sequence (CAPS) analysis (Fig. 6). Sequence analysis revealed that the exon-1 fragment of PslAFB5 (632 bp) is interrupted by a unique AccIII-restriction site that divides it into two smaller fragments (121 and 511 bp). Due to a nucleotide substitution, the AccIII-site is not present in the gPslafb5 allele. Direct digestion of the amplified gPslAFB5 exon-1 from the two cultivars confirmed that gPslAFB5 was represented by a single allele (gPslafb5) in EG and by two different alleles in V9.

The Pslafb5 protein is an inactive auxin-receptor

To determine whether the observed Pro 61 -to-Ser mutation in Pslafb5 influences SCF assembly and/or subsequent coreceptor formation with Aux/IAAs, the corresponding proline residues in PslTIR1 (Pro 9 ) and PslAFB5 (Pro 61 ) were mutated into serine. The consequences of these mutations were evaluated by assessing the interaction capacity of the full-length WT ORFs (as a positive control) and ORFs carrying mutations, using both Y2H and BiFC approaches (Fig. 7). Relative to WT ORFs, all mutated proteins were unable to interact with ASK1 or AtIAA7 in yeast or tobacco protoplasts, providing strong evidence that the Pslafb5 allele encodes an inactive TIR1-like protein.

Discussion

Fruit growth is a complex process where cell division, expansion and differentiation are coordinated by a diverse range of hormones. Auxin is a regulator of patterning and tissue specification in many processes in plants, including the development of reproductive and fruiting tissues [START_REF] Sundberg | Distinct and dynamic auxin activities during reproductive development[END_REF]. In flowers, auxin specifies the site of floral primordium initiation and regulates floral organogenesis and patterning [START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Cheng | A role for auxin in flower development[END_REF]. Post-fertilization, an interaction between auxin and gibberellin triggers fruit-set, the term given to the onset of rapid cell division necessary for early embryo development and fruiting structure enlargement (de Jong et al., 2009a;[START_REF] El-Sharkawy | Characterization of gibberellin-signalling elements during plum fruit ontogeny defines the essentiality of gibberellin in fruit development[END_REF]. In both berry-and stone-fruit, auxin has also been shown to be important during the immature fruit development and ripening processes [START_REF] Miller | Measurement of indole-3acetic acid in peach fruits (Prunus persica L. Batsch cv. Redhaven) during development[END_REF][START_REF] Agustí | Synthetic auxin 3,5,6-TPA promotes fruit development and climacteric in Prunus persica L. Batsch[END_REF][START_REF] Jones | Down-regulation of DR12, an auxin-responsefactor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit[END_REF][START_REF] El-Sharkawy | Regulation of two germin-like protein genes during plum fruit development[END_REF], 2012;[START_REF] Seymour | Fruit development and ripening[END_REF]. Our results show that the IAA content in plums is high during flowering and in most phases of fruit development. The major exception is the S2 phase of fruit growth where the fruit appears to fall into a quiescent phase. Whereas auxin was once thought to be exclusively involved in the early stages of fruit development (S1 and S3), it is now understood also to be important during the S4 phase, where autocatalytic ethylene production is critical for the suite of changes that comprise the ripening process [START_REF] Miller | Measurement of indole-3acetic acid in peach fruits (Prunus persica L. Batsch cv. Redhaven) during development[END_REF][START_REF] Trainotti | The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches[END_REF]. We have previously shown the effect of auxin in enhancing ethylene production and ripening in Shiro, a late plum cultivar [START_REF] El-Sharkawy | Differential regulation of four members of ACC synthase gene family in plum[END_REF], 2009[START_REF] El-Sharkawy | Regulation of two germin-like protein genes during plum fruit development[END_REF], 2012). This finding led us to hypothesize that the differences in ripening-associated ethylene production observed in different plum cultivars are at least partially due to variations in the auxin-content and/ or -responsiveness in fruiting tissues. The early ripening cultivar, EG, exhibited a sharp increase in auxin levels during fruit ripening and a strong auxin-sensitivity. Treatment with auxin elicited an enhanced ethylene climacteric comparable to that observed in fruit treated with propylene, a known ethylene inducer [START_REF] Mcmurchie | Treatment of fruit with propylene gives information about the biogenesis of ethylene[END_REF]. Together, the data indicate that auxin plays a key role in progressing fruit development towards the transition phase that leads to the initiation of autocatalytic ethylene production. It also indicates that auxin regulates ethylene production and the progression of ripening. A capacity to advance and enhance ripening in EG suggests that the relative lack of auxin and/or reduced auxin-sensitivity in V9 is, at least partially, responsible for the suppressed ethylene production and prolonged ripening in this cultivar.

To investigate the molecular basis of auxin action in fruit development, three auxin-receptors, belonging to the F-box TIR1like gene family, were isolated and characterized. TIR1-like proteins are components of an SCF-ubiquitin ligase complex that targets the Aux/IAA auxin-repressors for ubiquitination and subsequent degradation [START_REF] Gray | Auxin regulates SCF TIR1 -dependent degradation of AUX/IAA proteins[END_REF][START_REF] Dharmasiri | Auxin action in a cell-free system[END_REF]. Consistent with published data on TIR1-like proteins, we demonstrated that the isolated PslTIR1/AFBs were able to interact with the receptor complex component, ASK1 and that they were differentially responsive to various Aux/IAA proteins. Interestingly, the absence of auxin did not prevent the assembly of PslTIR1/PslAFB2::AtIAA7; however, the tomato Aux/IAA (SlIAA3) interacts poorly with PslAFB5, even in the presence of auxin. Auxin orchestrates a broad range of processes and many levels of regulation are known to be involved in determining responses [START_REF] Ljung | Auxin metabolism and homeostasis during plant development[END_REF], including biosynthesis/ metabolism, transport, and response. Our data and those of others [START_REF] Parry | Complex regulation of the TIR1/AFB family of auxin receptors[END_REF][START_REF] Calderón-Villalobos | A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin[END_REF] show that another level of regulation exists at the level of receptor:: auxin:: Aux/IAA-binding capacity.

Assessing the role of different PslTIR1/AFBs domains in protein function confirmed the essential contribution of the F-box and C-terminal domains in ensuring appropriate receptor activity. Loss of the N-terminal domain in PsITIR1 and PslAFB2 had little effect on ligand-receptor complex formation and function; however, the long N-terminal region of PslAFB5 was required for full protein function. Similarly, other studies have also shown the importance of sequences outside the F-box for Skp1 association [START_REF] Li | Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: Coupling glucose sensing to gene expression and the cell cycle[END_REF][START_REF] Gray | Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana[END_REF].

An analysis of transcripts for three PslTIR1/AFB mRNAs during EG and V9 fruit development did not correlate with the observed cultivar differences in terms of auxin-sensitivity and fruit development. Sequence data indicated, however, that while only one type of allele was present for two of the receptor genes, gPslTIR1 and gPslAFB2, two different allele types were identified for gPslAFB5. The auxin-hypersensitive cultivar, EG, is homozygous for the gPslafb5 allele, whereas V9 is gPslAFB5/afb5 heterozygous. In the gPslafb5 allele, a serine residue is substituted for the highly conserved Pro 61 located within the F-box domain. A targeted mutation in the corresponding proline (Pro 10 to Ala) of the Arabidopsis TIR1 abolished auxin responses by inhibiting its capacity to interact with Skp1-like and Aux/IAA proteins [START_REF] Gray | Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana[END_REF][START_REF] Kepinski | The Arabidopsis F-box protein TIR1 is an auxin receptor[END_REF]. This suggests that the Pslafb5 allele encodes an inactive auxin-receptor. In Arabidopsis, the loss of the closest homologues of PslAFB5, AFB4, and/or AFB5 resulted in a range of growth defects consistent with auxin hypersensitivity, suggesting that this class of auxin receptors act as negative regulators of auxin responses [START_REF] Greenham | The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings[END_REF]. EG is therefore associated with the homozygous presence of the inactive Pslafb5 allele, and conversely, V9 is associated with the presence of a functional PslAFB5 allele. Further, we were able to show that a substitution of a highly conserved F-box proline residue (PslTIR1-Pro 9 and PslAFB5-Pro 61 ) into serine, simulating the naturally occurring sequence in the Pslafb5 allele, completely abolished auxin responses by inhibiting interactions with ASK1 and AtIAA7. This provides strong evidence that this proline residue is essential for protein function and therefore that the Pslafb5 allele in the EG cultivar is non-functional.

In the auxin-hypersensitive Arabidopsis afb4-2 mutant, IAA levels are not significantly altered compared to the wild type [START_REF] Greenham | The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings[END_REF]. Intriguingly, EG also exhibited auxin hypersensitivity. Taken together, the results suggest that the active PslAFB5 is involved in the auxin-signalling network that negatively regulates fruit development processes, particularly from flowering until the end of the S1 stage. Thus, the loss of PslAFB5 function may increase auxin responses and consequently accelerate early fruit development processes in EG. Auxin can also be a part of the mechanisms that control the capacity of the fruit to ripen. However, the absence of PslAFB5 transcripts during late fruit growth and ripening phases (S3 and S4) in both plum cultivars suggested that the auxin content character should be more important than the auxin hypersensitivity feature for determining the auxindependence of fruit development and ripening. Auxin acts upstream of ethylene to control the initiation of autocatalytic ethylene production and the transition of a fruit from the end of growth into the onset of ripening. So the high auxin levels in EG should accelerate fruit growth and force the fruit to early transit into ripening; however, the lack of sufficient auxin in V9 delays both processes. Once autocatalytic ethylene production is initiated during ripening, the process will progress in both auxin-and ethylene-dependent manners [START_REF] El-Sharkawy | Differential regulation of four members of ACC synthase gene family in plum[END_REF],2009;[START_REF] Ziliotto | Transcriptome profiling of ripening nectarine (Prunus persica L. Batsch) fruit treated with 1-MCP[END_REF].

The data presented here provide further evidence of the integral role played by auxin in fruit development, and provide a basis for continuing research to determine the precise nature of the many complex regulatory processes involved.

Fig. 1 .

 1 Fig. 1. Variation in response to NAA application due to diversity in auxin sensitivity between early (EG) and late (V9) plum cultivars. In (A) and (B), the two plum cultivars were treated with two NAA concentrations (10 and 100 µM). Image (A) illustrates the effects of NAA concentration on flowering of EG and V9. Image (B) demonstrates the changes in flower developmental process due to auxin application. Floral stages were selected when ~70% of the flowers in the shoot were in the same developmental stage. I, II, III, and IV represent flower stages at 2, 3, 4, and 6 days after treatment (DAT). (C) Close-up views of plum fruits before and after auxin application, in which EG and V9 fruits were treated with 10 and 100 µM NAA, respectively. Images (D) and (E) show the alterations in EG and V9 fruit size and weight due to NAA treatment.

Fig. 2 .

 2 Fig. 2. Representative fruit growth curves (A, B), changes in ethylene production (C, D), and IAA levels (E, F) during plum fruit development for the earlyand late-ripening cultivars EG and V9, respectively. Values represent the mean ±SD, as derived from nine independent replicates (5 fruit per replicate). The x axis represents the developmental stages indicated by DAB. The developmental stages include flower (FL), and stages 1-4 (S1-S4) of fruit development. The y axis refers to the changes in fruit fresh mass (A, B), ethylene levels (C, D) and IAA content (E, F) during development, respectively. The grey arrow (C-F) represents the fruit at climacteric stage.

Fig. 4 .

 4 Fig. 4. Functional characterization of PslTIR1, PslAFB2, and PslAFB5 domains. Constructs containing full-length ORFs are referred to as WT. Truncated derivatives, including independent deletion of the N-terminal, F-box, and C-terminal domains, are referred to as ∆N, ∆F, and ∆C, respectively. (A) Subcellular localization of WT and truncated derivatives fused to the GFP tag. All constructs were transiently transformed for the assay into Nicotiana tabacum protoplasts. NLS-mCherry was included in each transfection to indicate the location of the nucleus. GFP fluorescence is shown as green; the merged image is a digital merge of bright field and fluorescent images to illustrate the protein compartments. All experiments were repeated a minimum of three independent times. Bars, 10 µm. (B) Y2H assays were performed using previously indicated derivatives with ASK1 and AtIAA7 in the presence of IAA. Other details as described in Fig. 3.

  of the climacteric phase (~78 DAB), but steadily increased with the progression in ripening. In contrast, PslTIR1 and PslAFB2 transcript levels during ripening of V9 (~110-128 DAB) remained constitutively low throughout the process. PslAFB5 transcripts were undetectable during ripening in both cultivars.

Fig. 5 .

 5 Fig. 5. Steady-state transcript levels of PslTIR1, PslAFB2, and PslAFB5 mRNAs assessed by qPCR during (A) EG flower and fruit development and (B) throughout ripening of early -EG and late -V9 fruit. The expression was determined from fruit pulp. Results represent data from three biological and three technical replicates. Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to PslAct expression. Error bars represent SD. The y axis refers to the mean molecules of the target gene per reaction/mean molecules of PslAct. The x axis in each figure represents the developmental stage as indicated by the number of days after bloom (DAB).

Fig. 7 .

 7 Fig. 7. Pslafb5 encodes an inactive auxin receptor. Constructs containing full-length ORFs of PslTIR1 and PslAFB5 were used as a positive control and referred to as WT. Mutations were created by a sitedirected mutagenesis approach through substitution of Pro9 and Pro61 of PslTIR1 and PslAFB5, respectively, into serine to simulate Pslafb5. (A) Y2H interaction assays of PslTIR1 and PslAFB5 (WT) and mutated derivatives with ASK1 and AtIAA7 (other details as in Fig. 4). (B) BiFC interaction visualization of PslTIR1 and PslAFB5 (WT and mutated ORFs) with ASK1 and AtIAA7. PslTIR1-and PslAFB5-related proteins were fused with the N-terminus (NY) of YFP; ASK1 and AtIAA7 were fused with the C-terminus (CY) of YFP. Different combinations of NY and CY constructs were transiently co-expressed in tobacco protoplasts. NLS-mCherry was included in each transfection to highlight the location of the nucleus. YFP fluorescence is yellow; the merged image is a digital merge of bright field and fluorescent images to illustrate the interaction location. Bars, 10 µm.

Fig. 6 .

 6 Fig.6. CAPS analysis of PslAFB5 alleles. The PCR products of PslAFB5 exon-1 were amplified using EG and V9 gDNA, digested with AccIII and then electrophoresed on 2.5% agarose gel. Lane L refers to 100-bp ladder; lanes indicated by AFB5 and afb5 are the digested products from subcloned exon-1 fragments. Lanes EG and V9 refer to digested PCR products using the gDNA of the plum cultivars indicated as a template.

Acknowledgements

We thank Dr Corinne Audran-Delalande, University of Toulouse INP/ ENSAT, France for providing the tomato IAA3 and IAA9 clones; and Pratibha Ravindran and Pavithra Amruthur Jyothi Prakash, National University of Singapore for help during auxin quantification.

Supplementary material

Supplementary data can be found at JXB online.

Supplementary Table S1. Oligonucleotide primers used in this study.

Supplementary Table S2. Flowering, maturation dates and fruiting duration in different plum genotypes.

Supplementary Table S3. Changes in plum flower developmental characteristics due to auxin treatment. Supplementary Table S4. Amino acid sequence comparison between the predicted full-length plum and Arabidopsis auxin receptors.

Supplementary Figure S1. Alteration in ethylene production during the ripening of fruits from early and late cultivars pre-exposed to different treatments designed to alter auxinor ethylene-responses.

Supplementary Figure S2. Amino acid sequence alignment of plum and Arabidopsis TIR1/AFB proteins. Supplementary Figure S3. Phylogenetic relationships between TIR1/AFB genes from plum and other plant species.

Supplementary Figure S4. Genomic structure of PslTIR1/ AFB genes.

Supplementary Figure S5. Amino acid sequence comparison of the two plum AFB5 alleles (PslAFB5 and Pslafb5).

Supplementary Figure S6. Sequencing traces of PslAFB5 allelic genotypes in EG and V9 plum cultivars.

Funding

Partial funding for this work was provided by Ontario Tender Fruit Marketing Board and Ontario Ministry of Agriculture, Food and Rural Affairs to SJ.


Franck Petitdemange
email: franck.petitdemange@irisa.fr

Isabelle Borne
email: isabelle.borne@irisa.fr

Jeremy Buisson
email: jeremy.buisson@irisa.fr

Approach Based Patterns for System-of-Systems Reconfiguration

Systems-of-systems (SoS) are a particular class of systems that recruit dynamically their constituents to achieve a global goal. To accomodate this approach, the architecture of SoS is usually described by architectural patterns to be instantiated at runtime. Based on the study of an example, we introduce reconfiguration patterns to help reasoning on reconfiguration and maintaining the architectural patterns of the SoS.

I. INTRODUCTION

System-of-systems (SoS) are a particular class of systems. An SoS is a set of Constituent Systems(CSs) which collaborate to fulfill a mission. Each CS has its own managerial independance, meaning that it has its own teams of managers, developers, administrators, maintainers who control its lifecycle (e.g., deciding when the CS is started, stopped, updated) independently of the SoS. Each CS has also operational independance, meaning that the CS can fulfilled valid missions if disassembled from overall system. In this context, the architect who designs an SoS has autority on interactions between CSs but has no control over the CSs themselves. Due to the lack of coercitive power on the environnement of the SoS some architectural details are delayed until runtime. For instance a CS can leave or join the SoS on its own right, therefore the SoS must reconfigure its architecture at runtime to accomodate such changes.

The architecture of the SoS defines the role of each constituent system and the interactions between them. In order to be assisted with regard to this point, the SoS architect can use patterns. A pattern is a generic building block which defines a structure and behaviors as a solution to solve a well-identified design problem in a specific context with wellknown properties.

One challenge of SoS engineering is that the constituent systems, because of their own managerial and operational independence, can decide to engage or disengage at their will. But regardless of the decisions of the constituent systems, the SoS must self-reconfigure to continuously achieve its global mission. In the case of SoS, we identify two cases for reconfiguration: either maintaining the SoS pattern or switching to a new coalition.

A difficulty arises because the role a constituent system plays after reconfiguration depends not only on its role before reconfiguration, but also on its involvement in other SoS as well as its own missions. In this paper we consider the example of a fire emergency system. Clearly, if an ambulance system is transporting injured persons at the time of the reconfiguration, this ambulance will not play the same role as if it were idle. To solve this issue, we propose to assist reconfiguration with patterns. Our idea is that the architect defines configurations and reconfigurations with patterns. Then reconfiguration patterns can be used as patches on the architecture in order to change it at runtime while maintaining the consistency of SoS and preserving the other patterns (i.e., the other SoS).

Section II presents the main concepts of the SoSADL architecture description language we are relying on, as well as an illustrative example expressed in terms of these SoSADL concepts. Section III presents how we propose to use patterns to assist reconfiguration. Section IV provides an overview of related works. Section V concludes the paper with future directions.

II. BACKGROUND A. SoSADL Concepts

This paper focuses on the reconfiguration concern in SoS. To make the examples more concrete, we rely on SoSADL, a language under construction in our team targeted the SoS description architectures. SoSADL is an evolution of π-ADL [START_REF] Oquendo | pi-ADL: An Architecture Description Language Based on the Higher-order Typed pi-calculus for Specifying Dynamic and Mobile Software Architectures[END_REF], a language used to describe static and dynamic architectural specifications. π-ADL is based on the π-calculus, a formal basis to verify specification of architectural structures and behaviors. SoSADL extends π-ADL with new architectural artifacts for SoS. In comparison to other ADL, SoSADL allows to declare sets of architectural constraints solved at runtime to generate the configuration. This language relies on three main concepts.

• Systems are the constituents of an SoS. Following the definition of SoS, each system has its own mission and is operationally and managerially independent, while contributing to the global mission of the SoS. Systems are described in terms of their interaction points, called gates, and a model of their interaction behavior. The gates are the means given to the SoS to interact with the system such that the system achieves some tasks for the SoS. • Mediators are communicating elements that specify the interactions among systems. Mediators belong to the SoS, i.e., the SoS has full control over them. They are described in terms of their interaction points, which we call duties, and their internal behavior. • Each SoS is described by a coalition, which identifies systems and controls mediators. Coalition are described by-intention. It means that each coalition is defined by 1) sets of to-be-discovered systems and sets of to-beinstantiated mediators; and 2) a constraint that describes how the system gates and mediator duties are bound to one another to establish communications. With SoSADL, we consider that SoS can themselves be systems involved in other SoS. Hence a coalition may expose some gates to its environment, like any other system. The resolution of the coalition constraint determines the instances of mediators that are required as well as the bindings between system gates and mediator duties. When systems are discovered or disengage from the SoS, the resolution of the constraint yields to a new architecture and triggers a reconfiguration.

B. Illustrative Example

To illustrate our proposal we study the case of an emergency organization. It is a reference case study commonly used in the SoS area [START_REF] Forcolin | Accident response use case engineering analysis report using current methods & tools[END_REF], [START_REF] Lochow | Concept alignment example description[END_REF]. In this paper we focus the example on a single fire emergency service, independently of the other services that may be involed. This service is composed of several ambulances, fire cars and officers that are dispatched to emergency scenes by a strategic command center (SCC) which fulfills the global mission by controling the allocation of these elements.

When an incident scene requires the coordination of multiple resources (ambulances, fire cars and/or officers), the SCC creates a temporary tactical command (TC). An officer is in charge of the command and control of the resources allocated to the TC. Following a hierarchical command scheme, the TC issues operational orders to its allocated ressources based on strategic decisions received from the SCC. In this case, resources are subordinates of the TC, rather than the SCC.

The fire emergency service can be considered an SoS because:

• ambulances and fire cars are distributed over several districts in order to minimise intervention time (geographical distribution); • an ambulance can operate standalone to rescue one wounded (operational independence); • in such situation, the ambulance has its own strategy to operate and control its own cycle life (managerial independence); and • the number of ambulances and fire cars changes over the time (evolution).

The constituent systems (CSs) of the fire emergency service are SCC, ambulances, fire cars and officers. Because we consider a hierarchical command chain (coercive power on CSs), the SoS belongs to the directed category [START_REF] Maier | Architecting principles for systems-of-systems[END_REF].

C. Modelization of the example in terms of SoSADL concepts

In our example, we identify two coalitions: the global architecture of the emergency service; and the adaptation of the service to specific incident scenes.

1) Fire emergency service: The fire emergency service contains four sets of constituent systems: one SCC, some officers, some ambulances and some fire cars. It contains a set of mediators named transmitter which model communication equipment. The coalition defines the SCC as the central authority that is responsible for the control and command of the other CSs. The coalition constraint states that a transmitter handles the communications between SCC and the CSs of a district: it broadcasts orders from SCC to CSs and transmits reports from CSs to SCC. As consequence, the SoS must instantiate a set of transmitters in order to comply to this rule. As shown in Figure 1, to resolve the coalition constraint, the SoS instantiates one transmitter when all the CSs are located in the same district. But in the case the ambulances, officers and fire cars are dispacthed over several districts the SoS instantiates several transmitters.

2) Tactic command: When an incident occurs in the environment of the SoS, a tactical command coalition is temporarily instantiated by the SCC, resulting in a reconfiguration of the global architecture. The SCC reshapes the coalition, introducing a new intermediate authority (an officer) and allocates some resources to it. The new constraint of the reconfigured coalition states that communications must pass through the officer, involving a second set of transmitters between officers and SCC. SCC maintains direct authority on officers. The reconfiguration brings for instance the SoS from Figure 1 to Figure 2: transmitter 2 is instantiated and ambulance and fire car are connected to it. Once again, depending on geographical distribution of the CSs, several transmitter 1 and several transmitter 2 may be instantiated to accomodate hardware constraints of the equipment modeled by transmitters.

III. PATTERNS FOR DYNAMIC RECONFIGURATION

Architectural patterns provide a high level view of a system architecture and define rules to support software construction with well-defined properties. According to [START_REF] Coplien | Software patterns[END_REF]:

Architectural patterns capture important structure, practice, and technique that are key competencies in a given field. Due to the increased complexity of SoS (compared to regular systems), the use of architectural patterns is mandatory to help architects understanding and documenting the behavior and structure of the SoS [START_REF] Kalawsky | Using architecture patterns to architect and analyze systems of systems[END_REF]. Existing projects such as COM-PASS [START_REF] Ingram | Modelling patterns for systems of systems architectures[END_REF] and DANSE [START_REF] Kalawsky | Report on danse architectural approaches[END_REF] study how existing patterns in the literature may be applied in a SoS architecture.

Constant evolution is a key feature of a SoS, but this topic is not fully addressed in classical architectural patterns. While, e.g., [START_REF] Léger | Reliable dynamic reconfigurations in a reflective component model[END_REF], [START_REF] Buisson | Coqcots & pycots: Non-stopping components for safe dynamic reconfiguration[END_REF] verify that primitive reconfiguration actions preserve the architectural invariants of their respective component model, the challenge for the SoS field arises from The objective of this work is to explore a new approach in order to reason about reconfiguration with specific patterns.

The architect could apply a set of reconfigurations described by reconfiguration patterns. Reconfiguration patterns should preserve the architectural decisions defined through architectural patterns. In our work we focus on the relation between architectural patterns and reconfigurations.

We identify two cases for SoS reconfiguration:

• On the one hand, reconfigurations should maintain consistency in the hierarchy of architectural patterns defined in the SoS. The architectural patterns will be instantiated by coalitions, and re-instantiated when CSs leave or join. Based on our example (figure 1), if a CS loses its connection with SCC by moving to another district (out of range of its transmitter), a new transmitter must be instantiated for that district (if it does not exist) and then the CS reconnected to this transmitter. • On the other hand, reconfigurations must allow the evolution of the hierarchy of patterns with composition and fusion operations of other patterns. At runtime when the SoS creates a new coalition, the process which selects and composes CSs must preserve the consistency in the SoS. For instance, when an incident occurs, the SCC may want to create a new tactical command coalition. When multiple incidents occur at the same time, several tactical commands could be instantiated. Consider that a tactical command assigns an ambulance to transport an injured person to the hospital: in this case the SCC cannot disengage this ambulance to provision a new tactical command. According to the domain knowledge and current state of SoS, dynamic reconfigurations should determine a set of reconfiguration operations that preserve consistency, and select and compose a new coalition.

The former clearly concerns the repeated resolution of the constraint in order to instantiate the pattern depending on the joining and leaving CSs. The latter shows that the role of the CSs depends not only on the target pattern, but also on their role before reconfiguration in regard to both the SoS and the environment.

The reconfiguration patterns will be designed by identifying the steps and constraints of a reconfiguration case. For example starting from the main coalition (figure 1) to get a tactical command coalition (figure 2) constitutes a reconfiguration pattern composed of smaller patterns that explain the necessary steps.

The main coalition will be described with SoSADL and the reconfiguration patterns will include some SoSADL templates to guide the reconfiguration process.

Several advantages could be excepted. Relating architectural and reconfiguration patterns, we will describe what can be reconfigured in a SoS and how to achieve these kinds of reconfiguration. The combination of multiple concurrent reconfigurations should also be better controlled if the effects of well-designed reconfigration patterns are bounded by the related architectural pattern. Reconfiguration patterns will be a means to reason about reconfiguration in SoS. Using an adequate language, such as SoSADL, will allow to check and validate the reconfiguration operations. This approach should allow to have the traceability of the reconfiguration process.

IV. RELATED WORK Some major European projects gave some ideas to deal with reconfiguration of SoS (COMPASS and DANSE european project). COMPASS addresses the problem of emergent property dependences against dynamicity (changes in SoS configuration) and evolution (changes in the behaviours of SoS). This dependability relies on a contract approach [START_REF] Huang | Contract support for evolving sos[END_REF], to specify CSs behaviors, and a framework to model and reason about fault tolerance. This project also proposes a collection of architectural patterns [START_REF] Perry | Final report on sos architectural models[END_REF] extracted from literature (software system and engineering) which adress particular needs of a SoS.

Another European project named DANSE [START_REF] Kalawsky | Report on danse architectural approaches[END_REF] proposes an approach of development process for architectures based on architectural patterns. It demonstrates the usefulness of architectural patterns in SoS enginering (modeling, analysis, dealing with complexity). Architectural patterns are used to generate optimized architectures. From informations extracted from architectual patterns (e.g connections between CS, constraints on their behaviors and capacities) a solver generates an optimized architecture which satisfies goals of the SoS. The approach does not address issuing reconfiguration operations to switch to the target architecture from current one. To deal with dynamicity and evolution, DANSE has abstracted models from SoS characteristics and used a contract approach to define semantics on behaviors, evolutions and structural parts of SoS [START_REF] Etzien | Danse modelling formalism, including domain metamodel & semantics: Focused on support for analysis and optimization[END_REF]. Our approach mainly addresses evolution of SoS with a dynamic reconfiguration process based on reconfiguration patterns. As such it is a complementary approach to those projects.

Regarding dynamic reconfiguration, [START_REF] Léger | Reliable dynamic reconfigurations in a reflective component model[END_REF], [START_REF] Buisson | Coqcots & pycots: Non-stopping components for safe dynamic reconfiguration[END_REF] address the preservation of invariants solely at the primitive operation level. Both focus on invariants coming from the component model, while considering that the approach generalizes well to invariants coming from patterns or from the application. In our work we aim to propose reconfiguration patterns similar to architectural patterns, i.e., templates to be instantiated for each application. Our approach is complementary to those previous work.

V. FUTURE WORK

In this paper we proposed an approach to describe a particular kind of SoS and how we intend to manage reconfigurations in a SoS architecture. We propose to assist reconfiguration with a pattern-based approach. Relying on patterns implemented by an SoS, our approach suggests a set of reconfiguration patterns which could be applied to maintain consistency in the SoS. As mentioned reconfiguration implementation should follow the reconfiguration pattern. Another case study in our team based on a real situation : a flood monitoring system developed at the University of São Paulo, will allow us to collect more reconfiguration examples. We are starting by decomposing the reconfiguration steps and defining the elementary patterns. We will study how to compose the elementary patterns [START_REF] That | Compositioncentered architectural pattern description language[END_REF] to form reconfiguration patterns. Finally we will design a catalog of architectural patterns devoted to SoS and will build a system of patterns, where each pattern will describe an architectural solution to a SoS reconfiguration problem.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Pattern of the fire emergency service coalition


ЧТО МЫ МОЖЕМ УЗНАТЬ ИЗ ГЛУБИННОГО АНАЛИЗА ЗАПРОСА?

Keywords: information retrieval, query expansion, analysis of query terms, relevance feedback, global analysis, co-occurrence

Россия

Одна из основных задач информационного поиска-извлечение документов, релевантных информационной потребности пользователя, выраженной запросом. Зачастую пользовательские запросы не превосходят 3 слов, что усложняет задачу. Многочисленные исследования показали, что автоматическое расширение запроса в среднем повышает точность, несмотря на то, что для некоторых запросов результаты ухудшаются. В статье предлагается новый метод автоматического расширения запроса, основанный на оценки важности словкандидатов, определяемой силой их связи со словами из запроса

Introduction

Information Retrieval (IR) systems aim at retrieving information that answers a user's needs/he expresses through a query. Retrieving relevant information to a query implies a two-step process: off line, the system indexes documents, generally using a bag of words representation; online, the system computes the similarity between the user's query and the document representations (indexing terms) to retrieve the most similar documents.

IR systems have to face the problem of query term ambiguity inherent in natural language; it is even more a challenging problem since users' queries are very short [START_REF] Chifu | Word Sense Disambiguation to Improve Precision for Ambiguous Queries[END_REF]. Indeed, more than 90% of the queries are 3 words or less long. Considering such a small number of terms, it is a challenge for the systems to "understand" the query or to disambiguate it.

To face these challenges, IR systems consider several strategies. One of them is to diversify the results by providing document related to the various senses of query terms, the system optimizes the chance of providing relevant information [START_REF] Vargas | Selecting Effective Expansion Terms for Diversity[END_REF]). Another strategy is to expand the query [START_REF] Gauch | Search Improvement via Automatic Query Reformulation[END_REF].

The principle of query expansion (QE) is to add new query terms to the initial query in order to enhance its formulation. Candidate terms for expansion are either extracted from external resources such as WordNet or from the documents themselves; based on their links with the initial query terms. In the latter types of methods, the most popular one is the pseudo-relevance feedback [START_REF] Buckley | Automatic Query Expansion Using SMART : TREC 3[END_REF].

Pseudo-relevance feedback has been shown as an effective method in average; however, it can lower results for some queries. For example, it is most probable that for poor performing queries query expansion is helpless since it will be based on the first retrieved documents that are probably non-relevant documents. It is thus important to know in advance if QE will be helpful or on the contrary if it will degrade the results. Selective query expansion aims at making this decision. However, current methods use blind methodologies and uses learning methods as black boxes. On the contrary, we think that a deep analysis of queries and query expansion terms could help understanding when QE would be useful and if there are some sort of typology of QE usefulness.

This paper pursues two objectives; first of all, we suggest a new automatic query expansion method. This new method estimates the importance of expansion candidate terms by the strength of their relation to the query terms. The second objective is to deeply analyze the results: both the initial and expanded queries and the terms they are composed of, and the cases when the expansion lowers the results and when it improves them.

The remaining of the paper is organized as follows: Section 2 reports related works. In Section 3, we present the new QE method we propose. Section 4 presents the evaluation framework. Section 5 discusses the results and presents a deep analysis of query terms, on a linguistic point of view.

Literature Review

QE and Pseudo Relevance Feedback

QE is either based on the analysis of a document collection (Carpineto and Romano 2012) or they imply dictionary or ontology-based methods [START_REF] Bhogal | A Review of Ontology Based Query Expansion[END_REF]. This study focuses on document analysis methods. This analysis may be either [START_REF] Amati | Probability Models for Information Retrieval Based on Divergence from Randomness[END_REF] global (corpus analysis to detect word relationships) (Carpineto and Romano 2012) or (2) local feedback (analysis of documents retrieved by the initial query) [START_REF] Rocchio | Relevance Feedback in Information Retrieval[END_REF][START_REF] Xu | Query Expansion Using Local and Global Document Analysis[END_REF]. Local analysis or local feedback methods rely on the hypothesis that relevant documents contain terms that could be useful to enhance query formulation. Rocchio defined a method in which term weights are re-computed so that the terms that occur in relevant documents contribute positively to the new query whereas the weight of the terms from non-relevant documents are lowered [START_REF] Rocchio | Relevance Feedback in Information Retrieval[END_REF]). Rocchio's method implies to know document relevance. Buckley suggested pseudo-relevance feedback (PRF) in which the top retrieved document are automatically considered as relevant [START_REF] Buckley | Automatic Query Expansion Using SMART : TREC 3[END_REF]. PRF is now common practice and used in many expansion methods [START_REF] Carpineto | A Survey of Automatic Query Expansion in Information Retrieval[END_REF]. Global methods work alike but in that case candidate terms come from the entire document collection rather than just (pseudo-) relevant documents.

Divergence from Randomness (DFR) models were developed by the School of Computing Science, University of Glasgow [START_REF] Ounis | Terrier: A High Performance and Scalable Information Retrieval Platform[END_REF]). These models are based on the assumption that informative words are relatively more frequent in relevant documents than in others. During QE the best-scored terms from the top-ranked documents are extracted. Terms are ranked using one of the DFR weighting model. DFR models include Kullback-Leibler divergence, Chi-square divergence, Bose-Einstein 1 (Bo1) and 2 (Bo2) models. In the DFR models QE is performed by ordering the candidate terms by their information content given the query Q. DFR models are presented in (G. [START_REF] Amati | Probability Models for Information Retrieval Based on Divergence from Randomness[END_REF]. In this paper, we compare our results with Bo2.

Analysis of Queries and Results

A few studies have reported analysis of results. The deeper analysis has been conducted in the RIA Workshop that took place in 2004. One of the objectives of the workshop was to analyze the variability in systems: some systems answering well on some queries and badly on others; some other systems behaving oppositely. One of the conclusion of the workshop was that the comprehension of variability is complex because of various parameters: query formulation, the relation between the query and the documents as well as the characteristics of the system (Harman and Buckley 2009). Moreover, they conducted failure analysis for 45 of the TREC topics. After using various systems on "hard" topics, the workshop participants analyzed why the system failed. For 39 topics out of 45 the systems failed for the same reason. Moreover, even if they did not retrieve the same documents, they were missing the same aspect in the top documents. Predicting query difficulty remains a challenge [START_REF] Mothe | Linguistic Features to Predict Query Difficulty-A Case Study on Previous TREC Campaign[END_REF].

The work presented in this paper combines local analysis, namely relevance feedback, and global analysis.

Method Description

The key idea of our new QE method is to estimate the importance of candidate terms by the strength of their relation to the query terms. In contrast to DFR models we do not compare the term frequency in PRF and the entire collection. In our approach, documents from PRF provide term candidates that are analyzed in two aspects: their frequency in PRF and their co-occurrence with query terms in the whole collection. Indeed, DFR models are based on two metrics: term frequency in PRF and the frequency of the term in the collection. Particularly, Bo2 uses the extrapolation of term frequency in PRF on the whole collection.

In our method candidate terms are selected from the PRF and are based on the underlain hypotheses: the strength of their relation to the query terms is proportional to the fraction of the number of the documents containing both candidate terms and query terms and the product of the number of documents containing at least one of these sets.

A query is first preprocessed. It is cleared from stop-words, punctuation; duplicate terms are removed. However if a query contains only stop-words, this could mean that a user is interested, for example, in grammar. For instance, the query "a and the" may imply that a user wants to find how to use English articles. Thus, if a query contains only stop-words, we keep all of them.

The importance of term combinations is estimated by the formula:

where is the term combination, is the -th term from , is the number of documents containing the -th term.

is similar to IDF. For widelyspread terms with low the importance of their combination is approximately equal to their number.

The importance of candidate terms is computed as follows:

where is the set of all possible term combinations, and is the analogue of non-negative point-wise mutual information calculated by the formula:

where is the number of documents containing the candidate term , is the number of documents containing all terms from the term combination , is the length of their intersection, and n is the total number of documents in the collection.

All weights are normalized. The best-scored term candidates are selected for query expansion.

Evaluation

Data Collections and Evaluation Measures

The evaluation was performed on two kinds of datasets: TREC (Text Retrieval Conference) Ad Hoc Track data sets for three years (1997-1999) ( In our evaluation, we considered the following evaluation measures:\begin {itemize} Precision at 10; Mean Average. Precision (P) is the fraction of retrieved documents that are relevant. P at 10 (P@10) is the fraction of the top 10 retrieved documents that are relevant. Average precision (AP) is the average of precision computed each time a relevant document is retrieved. Mean average precision (MAP) is calculated as the mean of average precision over queries.

System Details

We compared our system (Co) with the Bo2 DFR model implemented in Terrier platform.

Both systems used InL2c1.0 model for PRF, 3 documents from which 10 best scored terms were extracted. InL2c1.0 is a DFR (divergence from randomness) model based on TF-IDF measure with L2 term frequency normalization (Gianni Amati and Van Rijsbergen 2002; He and Ounis 2005)Heidelberg","page":"200-214", "event-place": "Berlin, Heidelberg","URL":"http://dx.doi.org/10.1007/978-3-540-31865-1_15","DOI": "10.1007/978-3-540-31865-1_15","ISBN":"3-540-25295-9, 978-3-540-25295-5","author": [{"family":"He","given":"Ben"},{"family":"Ounis","given":"Iadh"}],"issued":{"date-parts": [["2005"]]}}}],"schema":"https://github.com/citation-style-language/schema/raw/ master/csl-citation.json"}.

Performance Results

Table 1 provides information about the number of relevant retrieved documents (RRD), MAP, and P@10. On adhoc data set by all metrics our systems showed the best results, which are much higher than the baseline. The Student's test confirmed that the differences between MAP values of Co and Bo2 is not significant at the level $p < 0.05$. Significant difference is marked by *, the best results are marked-up in bold. Our system showed lower results than Bo2 according to P@10. In case of web data, Bo2 obtained the same MAP score as Co. Co remains the best according to P@10.

Table 1. General results

Co

Discussion and Further Analysis

Detailed Results

In the previous section, we reported the results when averaged over the set of topics. In this section, we aim at analyzing the results deeper. Table 2 reports the number of topics in each collection for which the method (column) got the best results according to AP. We also report these numbers when the 25 hardest and 25 easiest topics are considered. The hardest and easiest topics are defined as the ones that got the highest and lowest AP using the initial query. For example, in the Adhoc collection, from 150 topics, 33 are best treated without QE, 52 best when using Bo2 and 63 when using our method. The percentage of best treated topic per method is slightly different when considering the easiest topics: 7 are best without QE, 10 are best treated using Bo2 and 7 using our method.

Two examples are provided below. Each topic is composed of the title part which was used as a submitted query to the system, as well as descriptive and narrative parts that helps in understanding the user's need.

Example 1 <num>530 <title>do pheromone scents work? <desc>What is the scientific evidence that suggests pheromones stimulate the opposite sex? <narr>A relevant document will discuss how pheromones act as an attractor or repellent among humans, other animals, or plants. Example 2. <num>494 <title>nirvana <desc>Find information on members of the rock group Nirvana. <narr>Descriptions of members' behavior at various concerts and their performing style is relevant. Information on who wrote certain songs or a band member's role in producing a song is relevant. Biographical information on members is also relevant.

Table 3 provides the results in terms of AP and the various query formulations the system really used as well as the term weights. For example, with regard to topic 530, "do" was removed as a stop word and the other words have been stemmed. In topic 530 both reformulation went to the "sex" concept which leaded to some noise in the answers.

In topic 494, it is clear that adding "kurt" and "cobain", the lead singer, guitarist, and primary songwriter of the band Nirvana help in retrieving relevant documents. Either the weight of the terms (which are stronger in our reformulation than in Bo2) or some additional terms such as "band" made our reformulation better than Bo2.

Types of Initial Queries

Types of initial queries play an essential role in the prediction of successful information retrieval. As usual initial queries include, besides articles and other grammar words, nouns and entities, sometimes attributes and verbs. Grammatical structure of a title does not influence on the information retrieval process, because every title while processing the query is ruined into words and even word chunks. So types of queries are limited by a number of words and topic. Types of the query terms are restricted to words grammatical classes, such as parts of speech, and words semantic classes, such as terminology, entities, peculiarities, etc.

Potentially a document matches the initial query thanks to one term, or one term with its attribute, or two (or more) different terms. The last possibility is the best one, since a number of documents with two (or more) unconnected terms from the initial query is less, than a number of documents with the term and its attribute (noun phrase). In other words, co-occurrence of two (or more) semantically unconnected query terms in a document guarantees more accurate matching the initial query, while occurrence of one term just presupposes matching in a topic. Thus, one term query is less informative, than two and more terms queries. Hence for a one-word initial query QE is a productive way to increase the relevance of results, however, it depends on semantics of the one-word query. Our results for QE for one-word queries are slightly better regardless of the QE methods.

As a consequence of the diffusive character of the category, there are a lot of different factors which influence on the document frequency of the words associated with the topic. Thus, the more texts we use for the QE in the global analysis, the more unpredictable candidates we get for the QE. That is why the query generated on the basis of the title What is a Bengals Cat? (Animals) provides slightly better results with Bo2 QE system, while, with our system, we get a lot of useless extensions.

The structure of scientific categories is more compact and hierarchical. We assume that the initial query in the field of scientific categories evokes texts with less associative and more logical connections. The QE allows directing the IR process in a narrow relevant field. The title Unsolicited Faxes (Ad Hoc) refers to a multi-topic document, which simultaneously belongs to at least two topics in our set ("crimes" and "technology"). The results of QE performed by both systems are very good, but for our system it is significantly better: 0.6638 against 0.6015.

Therefore, the topic of the initial query is a strong factor, which influences on the necessity of the QE. Within homogenous text collection, every QE system works good, producing better results, than an initial query. Within naive topics categories the simple QE system is appropriate, while our QE generates complicated associative queries. So for the IR on the topic from naive category within heterogeneous text collection our QE system is overcomplicated, and that is why it works worse.

Conclusion

In this paper, we first suggest a new method for QE we call Co. The key idea of the proposed method is to estimate the importance of candidate terms by the strength of their relation to the query terms.

In our experiments, we show that the Co method has similar results as the Bo2 method from the literature. However, our finer analysis shows that the type of initial query can have an influence on the success of QE.

In our future work, we will work on the relationship between the types of queries and the field associated to the query in order to detect correlation with these features and the best method to treat the query. We think that using more linguistic features can help in selective approaches in IR.

  Voorhees and Harman 2000) containing 150 topics in total and composed of news articles, and WT10g from TREC Web track 2000-2001 (Hawking and Craswell 2002) which is a 10GB subset of the web snapshot of 1997 from Internet Archive. There are 100 topics in this second collection from track 2000 and 2001.

Table 2 .

 2 Detailed statistics

		# topics # Baseline best	# Bo2 best # Co best
	Adhoc	150 33	(22%) 52 (34.6%) 63	(42%)
	Adhoc 25 hardest	25 8	(32%) 7	(28%) 10	(40%)
	Adhoc 25 easiest	25 7	(28%) 10	(40%) 7	(28%)
	Web	98 34	(34.7%) 30 (30.6%) 31 (31.6%)
	Web 25 hardest	25 11	(44%) 5	(20%) 7	(28%)
	Web 25 easiest	25 10	(40%) 10	(40%) 4	(16%)

Table 3 .

 3 Query reformulation examples

		AP / Initial
	# Topic	query	AP / Bo2 reformulation	AP / Co reformulation
	530	0.3838 /	0.1551/	0.1765 /
		pheromon^	pheromon^1.60 scent^	pheromon^
		1.0 scent^	1.30 work^1.00 fragranc^	2.0 scent^ 1.68 work^
		1.0 work^	1.00 perfum^0.96 men^	1.42 perfum^ 1.0 fragranc^
		1.0	0.45 attract^	0.97 aphrodisiac^
			0.41 design^ 0.41 sex^	0.36 sex^
			0.38 natur^ 0.34 sexual^	0.22 men^ 0.22 attract^
			0.34	0.21 sexual^ 0.17 world^
				0.16 cologn^ 0.15 natur^
				0.15 nerd^ 0.13
	494	0.1706 /	0.3508 /	0.5703 /
		nirvana^	nirvana^	nirvana^2.0 cobain^
		1.0	2.00 kurt^0.29 cobain^	1.0 kurt^0.82 bootleg^
			0.25 bootleg^0.17 world^	0.42 song^0.30 nerd^
			0.17 list^0.15 song^	0.26 music^
			0.13 new^0.11 contain^	0.18 unplug^0.18 band^
			0.11 dedic^0.11	0.17 world^0.16 sound^
				0.15 stuff^0.14