An NLR integrated domain toolkit to identify plant pathogen effector targets.

Plant immune receptors, known as NOD-like receptors (NLRs), possess unique integrated decoy domains that enable plants to attract pathogen effectors and initiate a specific immune response. The present study aimed to create a library of these integrated domains (IDs) and screen them with pathogen effectors to identify targets for effector virulence and NLR-effector interactions. This works compiles IDs found in NLRs from seven different plant species and produced a library of 78 plasmid clones containing a total of 104 IDs, representing 43 distinct InterPro domains. A yeast two-hybrid assay was conducted, followed by an in planta interaction test, using 32 conserved effectors from Ralstonia pseudosolanacearum type III. Through these screenings, three interactions involving different IDs (kinase, DUF3542, WRKY) were discovered interacting with two unrelated type III effectors (RipAE and PopP2). Of particular interest was the interaction between PopP2 and ID#85, an atypical WRKY domain integrated into a soybean NLR gene (GmNLR-ID#85). Using a Förster resonance energy transfer-fluorescence lifetime imaging microscopy technique to detect protein-protein interactions in living plant cells, PopP2 was demonstrated to physically associate with ID#85 in the nucleus. However, unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, GmNLR-ID#85 could not be acetylated by PopP2 and failed to activate RPS4-dependent immunity when introduced into the RRS1-R immune receptor. The generated library of 78 plasmid clones, encompassing these screenable IDs, is publicly available through Addgene. This resource is expected to be valuable for the scientific community with respect to discovering targets for effectors and potentially engineering plant immune receptors.

The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

The RIN-regulated Small Auxin-Up RNA SAUR69 is involved in the unripe-to-ripe phase transition of tomato fruit via enhancement of the sensitivity to ethylene.

Ethylene is the main hormone controlling climacteric fruit ripening; however, the mechanisms underlying the developmental transition leading to the initiation of the ripening process remain elusive, although the presumed role of active hormone interplay has often been postulated. To unravel the putative role of auxin in the unripe-to-ripe transition, we investigated the dynamics of auxin activity in tomato fruit and addressed the physiological significance of Sl-SAUR69, previously identified as a RIN target gene, using reverse genetics approaches. Auxin signalling undergoes dramatic decline at the onset of ripening in wild-type fruit, but not in the nonripening rin mutant. Sl-SAUR69 exhibits reduced expression in rin and its up-regulation results in premature initiation of ripening, whereas its down-regulation extends the time to ripening. Overexpression of Sl-SAUR69 reduces proton pump activity and polar auxin transport, and ectopic expression in Arabidopsis alters auxin transporter abundance, further arguing for its active role in the regulation of auxin transport. The data support a model in which Sl-SAUR69 represses auxin transport, thus generating auxin minima, which results in enhanced ethylene sensitivity. This defines a regulation loop, fed by ethylene and auxin as the main hormonal signals and by RIN and Sl-SAUR69 as modulators of the balance between the two hormones.

Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants.

Plants are sessile organisms, hence to face environmental constrains they developed strategies that rely on the activation of stress-response genes under the control of specific transcription factors. The plant hormone ethylene mediates physiological, developmental and stress responses through the activation of Ethylene Response Factors (ERFs) which belong to a large multigene family of transcription factors. While an increasing number of studies supports the involvement of ERFs in abiotic stress responses, so far the specific role of ERF family members in different abiotic stress conditions remains unexplored. The present work investigates the expression profile of a set of ERFs, representative of different ERF types, in tomato plants subjected to cold, heat, salt, drought and flooding conditions. The study revealed that a group of ERFs is preferentially associated with cold and heat stress responses while another set is expressed in response to salt, water and flooding stresses. Transactivation assays indicated that ERFs can regulate the expression of abiotic stress genes regardless of whether or not they harbor conserved GCC or DRE cis-elements in their promoter region. The outcome of the study provides clue on which ERFs should be targeted when aiming to improve adaptation to a particular stress type.

Molecular cloning and characterization of novel WIN1/SHN1 ethylene responsive transcription factor HvSHN1 in barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is the fourth major cereal crop and shows high adaptive capabilities to diverse environments. Thus, it might represent a potential reservoir of novel genes to improve abiotic stress tolerance. In this study, a novel AP2/ERF transcription factor gene designated as HvSHN1 was isolated from barley. Protein sequence analysis showed that the HvSHN1 protein contained a nuclear localization signal and the conserved AP2/ERF domain. Phylogenetic analysis showed that HvSHN1 belongs to the group Va protein in the ERF subfamily which contains the Arabidopsis genes (SHN1, 2 and 3) and the wheat gene TdSHN1 with which it has 94.7% protein sequence identity. Expression profile analysis revealed that HvSHN1 is strongly induced by heat, cold, salt and drought. Transient expression using tobacco BY-2 protoplast coupled to confocal microscopy analysis revealed that HvSHN1 is exclusively targeted to the nucleus. Interestingly, when constitutively expressed in transgenic tobacco, HvSHN1 up-regulated stress responsive genes known to harbor GCC or DRE motif in their promoter regions. Therefore, HvSHN1 might represent a potential candidate for improvement of abiotic stress tolerance in economically important crops.

The tomato Ethylene Response Factor Sl-ERF.B3 integrates ethylene and auxin signaling via direct regulation of Sl-Aux/IAA27.

Plant growth and development is coordinated by complex networks of interacting hormones, and cross-talk between ethylene and auxin signaling is essential for a wide range of plant developmental processes. Nevertheless, the molecular links underlying the interaction between the two hormones remain poorly understood. In order to decipher the cross-talk between the Ethylene Response Factor Sl-ERF.B3 and Sl-IAA27, mediating ethylene and auxin signaling, respectively, we combined reverse genetic approaches, physiological methods, transactivation experiments and electrophoretic mobility shift assays. Sl-ERF.B3 is responsive to both ethylene and auxin and ectopic expression of its dominant repressor version (ERF.B3-SRDX) results in impaired sensitivity to auxin with phenotypes recalling those previously reported for Sl-IAA27 downregulated tomato lines. The expression of Sl-IAA27 is dramatically reduced in the ERF.B3-SRDX lines and Sl-ERF.B3 is shown to regulate the expression of Sl-IAA27 via direct binding to its promoter. The data support a model in which the ethylene-responsive Sl-ERF.B3 integrates ethylene and auxin signaling via regulation of the expression of the auxin signaling component Sl-IAA27. The study uncovers a molecular mechanism that links ethylene and auxin signaling in tomato.

Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum).

Survival biomass production and crop yield are heavily constrained by a wide range of environmental stresses. Several phytohormones among which abscisic acid (ABA), ethylene and salicylic acid (SA) are known to mediate plant responses to these stresses. By contrast, the role of the plant hormone auxin in stress responses remains so far poorly studied. Auxin controls many aspects of plant growth and development, and Auxin Response Factors play a key role in the transcriptional activation or repression of auxin-responsive genes through direct binding to their promoters. As a mean to gain more insight on auxin involvement in a set of biotic and abiotic stress responses in tomato, the present study uncovers the expression pattern of SlARF genes in tomato plants subjected to biotic and abiotic stresses. In silico mining of the RNAseq data available through the public TomExpress web platform, identified several SlARFs as responsive to various pathogen infections induced by bacteria and viruses. Accordingly, sequence analysis revealed that 5' regulatory regions of these SlARFs are enriched in biotic and abiotic stress-responsive cis-elements. Moreover, quantitative qPCR expression analysis revealed that many SlARFs were differentially expressed in tomato leaves and roots under salt, drought and flooding stress conditions. Further pointing to the putative role of SlARFs in stress responses, quantitative qPCR expression studies identified some miRNA precursors as potentially involved in the regulation of their SlARF target genes in roots exposed to salt and drought stresses. These data suggest an active regulation of SlARFs at the post-transcriptional level under stress conditions. Based on the substantial change in the transcript accumulation of several SlARF genes, the data presented in this work strongly support the involvement of auxin in stress responses thus enabling to identify a set of candidate SlARFs as potential mediators of biotic and abiotic stress responses.

Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits.

MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3, both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling highlighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.

Comprehensive Profiling of Ethylene Response Factor Expression Identifies Ripening-Associated ERF Genes and Their Link to Key Regulators of Fruit Ripening in Tomato.

Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripening-associated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening.

Auxin Response Factor SlARF2 Is an Essential Component of the Regulatory Mechanism Controlling Fruit Ripening in Tomato.

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

Comprehensive genome-wide analysis of the Aux/IAA gene family in Eucalyptus: evidence for the role of EgrIAA4 in wood formation.

Auxin plays a pivotal role in various plant growth and development processes, including vascular differentiation. The modulation of auxin responsiveness through the auxin perception and signaling machinery is believed to be a major regulatory mechanism controlling cambium activity and wood formation. To gain more insights into the roles of key Aux/IAA gene regulators of the auxin response in these processes, we identified and characterized members of the Aux/IAA family in the genome of Eucalyptus grandis, a tree of worldwide economic importance. We found that the gene family in Eucalyptus is slightly smaller than that in Populus and Arabidopsis, but all phylogenetic groups are represented. High-throughput expression profiling of different organs and tissues highlighted several Aux/IAA genes expressed in vascular cambium and/or developing xylem, some showing differential expression in response to developmental (juvenile vs. mature) and/or to environmental (tension stress) cues. Based on the expression profiles, we selected a promising candidate gene, EgrIAA4, for functional characterization. We showed that EgrIAA4 protein is localized in the nucleus and functions as an auxin-responsive repressor. Overexpressing a stabilized version of EgrIAA4 in Arabidopsis dramatically impeded plant growth and fertility and induced auxin-insensitive phenotypes such as inhibition of primary root elongation, lateral root emergence and agravitropism. Interestingly, the lignified secondary walls of the interfascicular fibers appeared very late, whereas those of the xylary fibers were virtually undetectable, suggesting that EgrIAA4 may play crucial roles in fiber development and secondary cell wall deposition.

Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF) gene family in Eucalyptus grandis.

Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF) are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation.

The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

Isolation and molecular characterization of ERF1, an ethylene response factor gene from durum wheat (Triticum turgidum L. subsp. durum), potentially involved in salt-stress responses.

As food crop, wheat is of prime importance for human society. Nevertheless, our understanding of the genetic and molecular mechanisms controlling wheat productivity conditions has been, so far, hampered by the lack of sufficient genomic resources. The present work describes the isolation and characterization of TdERF1, an ERF gene from durum wheat (Triticum turgidum L. subsp. durum). The structural features of TdERF1 supported the hypothesis that it is a novel member of the ERF family in durum wheat and, considering its close similarity to TaERF1 of Triticum aestivum, it probably plays a similar role in mediating responses to environmental stresses. TdERF1 displayed an expression pattern that discriminated between two durum wheat genotypes contrasted with regard to salt-stress tolerance. The high number of cis-regulatory elements related to stress responses present in the TdERF1 promoter and the ability of TdERF1 to regulate the transcription of ethylene and drought-responsive promoters clearly indicated its potential role in mediating plant responses to a wide variety of environmental constrains. TdERF1 was also regulated by abscisic acid, ethylene, auxin, and salicylic acid, suggesting that it may be at the crossroads of multiple hormone signalling pathways. Four TdERF1 allelic variants have been identified in durum wheat genome, all shown to be transcriptionally active. Interestingly, the expression of one allelic form is specific to the tolerant genotype, further supporting the hypothesis that this gene is probably associated with the susceptibility/tolerance mechanism to salt stress. In this regard, the TdERF1 gene may provide a discriminating marker between tolerant and sensitive wheat varieties.

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

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 Pro61 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.

Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing.

BACKGROUND: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. RESULTS: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5'-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. CONCLUSION: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.

Genome-wide identification, phylogenetic analysis, expression profiling, and protein-protein interaction properties of TOPLESS gene family members in tomato.

Members of the TOPLESS gene family emerged recently as key players in gene repression in several mechanisms, especially in auxin perception. The TOPLESS genes constitute, in 'higher-plant' genomes, a small multigenic family comprising four to 11 members. In this study, this family was investigated in tomato, a model plant for Solanaceae species and fleshy fruits. Six open reading frames predicted to encode topless-like proteins (SlTPLs) containing the canonical domains (LisH, CTLH, and two WD40 repeats) were identified in the tomato genome. Nuclear localization was confirmed for all members of the SlTPL family with the exception SlTPL6, which localized at the cytoplasm and was excluded from the nucleus. SlTPL genes displayed distinctive expression patterns in different tomato organs, with SlTPL1 showing the highest levels of transcript accumulation in all tissues tested except in ripening fruit where SlTPL3 and SlTPL4 were the most prominently expressed. To gain insight into the specificity of the different TOPLESS paralogues, a protein-protein interaction map between TOPLESS and auxin/indole-3-acetic acid (Aux/IAA) proteins was built using a yeast two-hybrid approach. The PPI map enabled the distinction of two patterns: TOPLESS isoforms interacting with the majority of Aux/IAA, and isoforms with limited capacity for interaction with these protein partners. Interestingly, evolutionary analyses of the TOPLESS gene family revealed that the highly expressed isoforms (SlTPL1, SlTPL3, and SlTPL4) corresponded to the three TPL-related genes undergoing the strongest purifying selection, while the selection was much weaker for SlTPL6, which was expressed at a low level and encoded a protein lacking the capacity to interact with Aux/IAAs.

A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components.

Ethylene Response Factors (ERFs) are downstream components of the ethylene signal transduction pathway, although their role in ethylene-dependent developmental processes remains poorly understood. As the ethylene-inducible tomato Sl-ERF.B3 has been shown previously to display a strong binding affinity to GCC-box-containing promoters, its physiological significance was addressed here by a reverse genetics approach. However, classical up- and down-regulation strategies failed to give clear clues to its roles in planta, probably due to functional redundancy among ERF family members. Expression of a dominant repressor ERF.B3-SRDX version of Sl-ERF.B3 in the tomato resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The dominant repressor etiolated seedlings displayed partial constitutive ethylene response in the absence of ethylene and adult plants exhibited typical ethylene-related alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlated with the altered expression of ethylene biosynthesis and signaling genes and suggested the involvement of Sl-ERF.B3 in a feedback mechanism that regulates components of ethylene production and response. Moreover, Sl-ERF.B3 was shown to modulate the transcription of a set of ERFs and revealed the existence of a complex network interconnecting different ERF genes. Overall, the study indicated that Sl-ERF.B3 had a critical role in the regulation of multiple genes and identified a number of ERFs among its primary targets, consistent with the pleiotropic phenotypes displayed by the dominant repression lines.

SlARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development.

Successful completion of fruit developmental programs depends on the interplay between multiple phytohormones. However, besides ethylene, the impact of other hormones on fruit quality traits remains elusive. A previous study has shown that down-regulation of SlARF4, a member of the tomato (Solanum lycopersicum) auxin response factor (ARF) gene family, results in a dark-green fruit phenotype with increased chloroplasts (Jones et al., 2002). This study further examines the role of this auxin transcriptional regulator during tomato fruit development at the level of transcripts, enzyme activities, and metabolites. It is noteworthy that the dark-green phenotype of antisense SlARF4-suppressed lines is restricted to fruit, suggesting that SlARF4 controls chlorophyll accumulation specifically in this organ. The SlARF4 underexpressing lines accumulate more starch at early stages of fruit development and display enhanced chlorophyll content and photochemical efficiency, which is consistent with the idea that fruit photosynthetic activity accounts for the elevated starch levels. SlARF4 expression is high in pericarp tissues of immature fruit and then undergoes a dramatic decline at the onset of ripening concomitant with the increase in sugar content. The higher starch content in developing fruits of SlARF4 down-regulated lines correlates with the up-regulation of genes and enzyme activities involved in starch biosynthesis, suggesting their negative regulation by SlARF4. Altogether, the data uncover the involvement of ARFs in the control of sugar content, an essential feature of fruit quality, and provide insight into the link between auxin signaling, chloroplastic activity, and sugar metabolism in developing fruit.

Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene.

BACKGROUND: The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. RESULTS: A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. CONCLUSIONS: The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.