AUXIN RESPONSIVE FACTOR8 regulates development of the feeding site induced by root-knot nematodes in tomato.

Root-knot nematodes (RKN) from the genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression, and auxin is known to be a key player in their development. However, little is known about how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their miRNA167 regulators, were identified as robust gene-miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-ß-glucuronidase (GUS) fusions showed the up-regulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR (clustered regularly interspaced palindromic repeats) mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Genetic diversity of tomato response to heat stress at the QTL and transcriptome levels.

Tomato is a widely cultivated crop, which can grow in many environments. However, temperature above 30°C impairs its reproduction, subsequently impacting fruit yield. We assessed the impact of high-temperature stress (HS) in two tomato experimental populations, a multi-parental advanced generation intercross (MAGIC) population and a core-collection (CC) of small-fruited tomato accessions. Both populations were evaluated for 11 traits related to yield components, phenology and fruit quality in optimal and HS conditions. HS significantly impacted all traits in both populations, but a few genotypes with stable yield under HS were identified. A plasticity index was computed for each individual to measure the extent of the heat impact for each trait. Quantitative trait loci (QTL) were detected in control and HS conditions as well as for plasticity index. Linkage and genome-wide association analyses in the MAGIC and CC populations identified a total of 98 and 166 QTLs, respectively. Taking the two populations together, 69 plasticity QTLs (pQTLs) were involved in tomato heat response for 11 traits. The transcriptome changes in the ovary of six genotypes with contrasted responses to HS were studied, and 837 genes differentially expressed according to the conditions were detected. Combined with previous transcriptome studies, these results were used to propose candidate genes for HS response QTLs.

De Novo Transcriptome Assembly, Gene Annotations, and Characterization of Functional Profiling Reveal Key Genes for Lead Alleviation in the Pb Hyperaccumulator Greek Mustard (Hirschfeldia incana L.).

Lead (Pb) contamination is a widespread environmental problem due to its toxicity to living organisms. Hirschfeldia incana L., a member of the Brassicaceae family, commonly found in the Mediterranean regions, is characterized by its ability to tolerate and accumulate Pb in soils and hydroponic cultures. This plant has been reported as an excellent model to assess the response of plants to Pb. However, the lack of genomic data for H. incana hinders research at the molecular level. In the present study, we carried out RNA deep transcriptome sequencing (RNA-seq) of H. incana under two conditions, control without Pb(NO3)2 and treatment with 100 µM of Pb(NO3)2 for 15 days. A total of 797.83 million reads were generated using Illumina sequencing technology. We assembled 77,491 transcript sequences with an average length of 959 bp and N50 of 1330 bp. Sequence similarity analyses and annotation of these transcripts were performed against the Arabidopsis thaliana nr protein database, Gene Ontology (GO), and KEGG databases. As a result, 13,046 GO terms and 138 KEGG maps were created. Under Pb stress, 577 and 270 genes were differentially expressed in roots and aboveground parts, respectively. Detailed elucidation of regulation of metal transporters, transcription factors (TFs), and plant hormone genes described the role of actors that allow the plant to fine-tune Pb stress responses. Our study revealed that several genes related to jasmonic acid biosynthesis and alpha-linoleic acid were upregulated, suggesting these components' implication in Hirschfeldia incana L responses to Pb stress. This study provides data for further genomic analyses of the biological and molecular mechanisms leading to Pb tolerance and accumulation in Hirschfeldia incana L.

Histone posttranslational modifications rather than DNA methylation underlie gene reprogramming in pollination-dependent and pollination-independent fruit set in tomato.

Fruit formation comprises a series of developmental transitions among which the fruit set process is essential in determining crop yield. Yet, our understanding of the epigenetic landscape remodelling associated with the flower-to-fruit transition remains poor. We investigated the epigenetic and transcriptomic reprogramming underlying pollination-dependent and auxin-induced flower-to-fruit transitions in the tomato (Solanum lycopersicum) using combined genomewide transcriptomic profiling, global ChIP-sequencing and whole genomic DNA bisulfite sequencing (WGBS). Variation in the expression of the overwhelming majority of genes was associated with change in histone mark distribution, whereas changes in DNA methylation concerned a minor fraction of differentially expressed genes. Reprogramming of genes involved in processes instrumental to fruit set correlated with their H3K9ac or H3K4me3 marking status but not with changes in cytosine methylation, indicating that histone posttranslational modifications rather than DNA methylation are associated with the remodelling of the epigenetic landscape underpinning the flower-to-fruit transition. Given the prominent role previously assigned to DNA methylation in reprogramming key genes of the transition to ripening, the outcome of the present study supports the idea that the two main developmental transitions in fleshy fruit and the underlying transcriptomic reprogramming are associated with different modes of epigenetic regulations.

Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation.

As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.

Multilevel comparative bioinformatics to investigate evolutionary relationships and specificities in gene annotations: an example for tomato and grapevine.

BACKGROUND: "Omics" approaches may provide useful information for a deeper understanding of speciation events, diversification and function innovation. This can be achieved by investigating the molecular similarities at sequence level between species, allowing the definition of ortholog and paralog genes. However, the spreading of sequenced genome, often endowed with still preliminary annotations, requires suitable bioinformatics to be appropriately exploited in this framework. RESULTS: We presented here a multilevel comparative approach to investigate on genome evolutionary relationships and peculiarities of two fleshy fruit species of relevant agronomic interest, Solanum lycopersicum (tomato) and Vitis vinifera (grapevine). We defined 17,823 orthology relationships between tomato and grapevine reference gene annotations. The resulting orthologs are associated with the detected paralogs in each species, permitting the definition of gene networks, useful to investigate the different relationships. The reconciliation of the compared collections in terms of an updating of the functional descriptions was also exploited. All the results were made accessible in ComParaLogs, a dedicated bioinformatics platform available at http://biosrv.cab.unina.it/comparalogs/gene/search . CONCLUSIONS: The aim of the work was to suggest a reliable approach to detect all similarities of gene loci between two species based on the integration of results from different levels of information, such as the gene, the transcript and the protein sequences, overcoming possible limits due to exclusive protein versus protein comparisons. This to define reliable ortholog and paralog genes, as well as species specific gene loci in the two species, overcoming limits due to the possible draft nature of preliminary gene annotations. Moreover, reconciled functional descriptions, as well as common or peculiar enzymatic classes and protein domains from tomato and grapevine, together with the definition of species-specific gene sets after the pairwise comparisons, contributed a comprehensive set of information useful to comparatively exploit the two species gene annotations and investigate on differences between species with climacteric and non-climacteric fruits. In addition, the definition of networks of ortholog genes and of associated paralogs, and the organization of web-based interfaces for the exploration of the results, defined a friendly computational bench-work in support of comparative analyses between two species.

TomExpress, a unified tomato RNA-Seq platform for visualization of expression data, clustering and correlation networks.

The TomExpress platform was developed to provide the tomato research community with a browser and integrated web tools for public RNA-Seq data visualization and data mining. To avoid major biases that can result from the use of different mapping and statistical processing methods, RNA-Seq raw sequence data available in public databases were mapped de novo on a unique tomato reference genome sequence and post-processed using the same pipeline with accurate parameters. Following the calculation of the number of counts per gene in each RNA-Seq sample, a communal global normalization method was applied to all expression values. This unifies the whole set of expression data and makes them comparable. A database was designed where each expression value is associated with corresponding experimental annotations. Sample details were manually curated to be easily understandable by biologists. To make the data easily searchable, a user-friendly web interface was developed that provides versatile data mining web tools via on-the-fly generation of output graphics, such as expression bar plots, comprehensive in planta representations and heatmaps of hierarchically clustered expression data. In addition, it allows for the identification of co-expressed genes and the visualization of correlation networks of co-regulated gene groups. TomExpress provides one of the most complete free resources of publicly available tomato RNA-Seq data, and allows for the immediate interrogation of transcriptional programs that regulate vegetative and reproductive development in tomato under diverse conditions. The design of the pipeline developed in this project enables easy updating of the database with newly published RNA-Seq data, thereby allowing for continuous enrichment of the resource.

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 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 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.

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.

Carotenoid accumulation during tomato fruit ripening is modulated by the auxin-ethylene balance.

BACKGROUND: Tomato fruit ripening is controlled by ethylene and is characterized by a shift in color from green to red, a strong accumulation of lycopene, and a decrease in ß-xanthophylls and chlorophylls. The role of other hormones, such as auxin, has been less studied. Auxin is retarding the fruit ripening. In tomato, there is no study of the carotenoid content and related transcript after treatment with auxin. RESULTS: We followed the effects of application of various hormone-like substances to "Mature-Green" fruits. Application of an ethylene precursor (ACC) or of an auxin antagonist (PCIB) to tomato fruits accelerated the color shift, the accumulation of lycopene, α-, ß-, and δ-carotenes and the disappearance of ß-xanthophylls and chlorophyll b. By contrast, application of auxin (IAA) delayed the color shift, the lycopene accumulation and the decrease of chlorophyll a. Combined application of IAA + ACC led to an intermediate phenotype. The levels of transcripts coding for carotenoid biosynthesis enzymes, for the ripening regulator Rin, for chlorophyllase, and the levels of ethylene and abscisic acid (ABA) were monitored in the treated fruits. Correlation network analyses suggest that ABA, may also be a key regulator of several responses to auxin and ethylene treatments. CONCLUSIONS: The results suggest that IAA retards tomato ripening by affecting a set of (i) key regulators, such as Rin, ethylene and ABA, and (ii) key effectors, such as genes for lycopene and ß-xanthophyll biosynthesis and for chlorophyll degradation.

Auxin perception is required for arbuscule development in arbuscular mycorrhizal symbiosis.

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells.

A natural system of chromosome transfer in Yersinia pseudotuberculosis.

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4-borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches.

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.

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.

Proteomic analysis of chloroplast-to-chromoplast transition in tomato reveals metabolic shifts coupled with disrupted thylakoid biogenesis machinery and elevated energy-production components.

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

The Knockdown of AUXIN RESPONSE FACTOR 2 Confers Enhanced Tolerance to Salt and Drought Stresses in Tomato (Solanum lycopersicum L.).

Auxin response factors (ARFs) act as key elements of the auxin-signaling pathway and play important roles in the process of a plant's growth, development, and response to environmental conditions. We studied the implication of the SlARF2 gene in the tomato response to salt (150 mM of NaCl) and drought (15% PEG 20000) stresses. The functional characterization of SlARF2 knockdown tomato mutants revealed that the downregulation of this gene enhanced primary root length and root branching and reduced plant wilting. At the physiological level, the arf2 mutant line displayed higher chlorophyll, soluble sugars, proline, and relative water contents as well as lower stomatal conductance and a decreased malondialdehyde content. Moreover, SlARF2 knockdown tomato mutants demonstrated higher activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) under salt and drought stresses than the wild type. Indeed, the stress tolerance of the arf2 mutant was also reflected by the upregulation of stress-related genes involved in ROS scavenging and plant defense, including SOD, CAT, dehydration-responsive element-binding protein, and early responsive to dehydration, which can ultimately result in a better resistance to salt and drought stresses. Furthermore, the transcriptional levels of the Δ1-pyrroline-5-carboxylate synthase (P5CS) gene were upregulated in the arf2 mutant after stress, in correlation with the higher levels of proline. Taken together, our findings reveal that SlARF2 is implicated in salt and drought tolerance in tomato and provides some considerable elements for improving the abiotic stress tolerance and increasing the crop yields of tomato.

Genome-wide identification, functional analysis and expression profiling of the Aux/IAA gene family in tomato.

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk.