The Impact of Elevated Atmospheric Carbon Dioxide Exposure on Magic Tomatoes' Nutrition-Health Properties.

The release of carbon dioxide (CO2) into the atmosphere has accelerated during the last two decades. Elevated atmospheric CO2 concentration (eCO2) is known as an agent that improves plant photosynthesis. However, eCO2 was also correlated with alterations in the macronutrient and micronutrient compositions of various dietary crops. In order to explore the effect of eCO2 on the nutritional and health properties of tomatoes, three parental lines of the Magic population, which includes a large part of the genetic diversity present in large fruit varieties, were used as models. The plants were grown in growth chambers under ambient (400 ppm) or eCO2 (900 ppm) conditions. The macronutrient and micronutrient contents were measured. The anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. These analyses highlighted that the carbohydrate content was not affected by the eCO2, whereas the protein, carotenoid, lycopene, and mineral contents decreased. Regarding the anti-oxidant properties, no influence of eCO2 exposure was observed. Similarly, the anti-inflammatory properties were not affected by the eCO2. These data are in contrast with previous studies conducted on different plant species or accessions, indicating that the effect of eCO2 on crops' nutrition and health properties is based on complex mechanisms in which growth conditions and genetic backgrounds play a central role.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.

Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3.

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

Identification of client iron-sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana.

Iron-sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins.

Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes.

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

Deciphering the Molecular Mechanisms Underpinning the Transcriptional Control of Gene Expression by Master Transcriptional Regulators in Arabidopsis Seed.

In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.

Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast.

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

MYB118 represses endosperm maturation in seeds of Arabidopsis.

In the exalbuminous species Arabidopsis thaliana, seed maturation is accompanied by the deposition of oil and storage proteins and the reduction of the endosperm to one cell layer. Here, we consider reserve partitioning between embryo and endosperm compartments. The pattern of deposition, final amount, and composition of these reserves differ between the two compartments, with the embryo representing the principal storage tissue in mature seeds. Complex regulatory mechanisms are known to prevent activation of maturation-related programs during embryo morphogenesis and, later, during vegetative growth. Here, we describe a regulator that represses the expression of maturation-related genes during maturation within the endosperm. MYB118 is transcriptionally induced in the maturing endosperm, and seeds of myb118 mutants exhibit an endosperm-specific derepression of maturation-related genes associated with a partial relocation of storage compounds from the embryo to the endosperm. Moreover, MYB118 activates endosperm-induced genes through the recognition of TAACGG elements. These results demonstrate that the differential partitioning of reserves between the embryo and endosperm in exalbuminous Arabidopsis seeds does not only result from developmental programs that establish the embryo as the preponderant tissue within seeds. This differential partitioning is also regulated by MYB118, which regulates the biosynthesis of reserves at the spatial level during maturation.

Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis.

TT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYB-bHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated. Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches. Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA- and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized. Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.

Evolution goes GAGA: GAGA binding proteins across kingdoms.

Chromatin-associated proteins (CAP) play a crucial role in the regulation of gene expression and development in higher organisms. They are involved in the control of chromatin structure and dynamics. CAP have been extensively studied over the past years and are classified into two major groups: enzymes that modify histone stability and organization by post-translational modification of histone N-Terminal tails; and proteins that use ATP hydrolysis to modify chromatin structure. All of these proteins show a relatively high degree of sequence conservation across the animal and plant kingdoms. The essential Drosophila melanogaster GAGA factor (dGAF) interacts with these two types of CAP to regulate homeobox genes and thus contributes to a wide range of developmental events. Surprisingly, however, it is not conserved in plants. In this review, following an overview of fly GAF functions, we discuss the role of plant BBR/BPC proteins. These appear to functionally converge with dGAF despite a completely divergent amino acid sequence. Some suggestions are given for further investigation into the function of BPC proteins in plants.

Transcriptional regulation of Arabidopsis LEAFY COTYLEDON2 involves RLE, a cis-element that regulates trimethylation of histone H3 at lysine-27.

LEAFY COTYLEDON2 (LEC2) is a master regulator of seed development in Arabidopsis thaliana. In vegetative organs, LEC2 expression is negatively regulated by Polycomb Repressive Complex2 (PRC2) that catalyzes histone H3 Lys 27 trimethylation (H3K27me3) and plays a crucial role in developmental phase transitions. To characterize the cis-regulatory elements involved in the transcriptional regulation of LEC2, molecular dissections and functional analyses of the promoter region were performed in vitro, both in yeast and in planta. Two cis-activating elements and a cis-repressing element (RLE) that is required for H3K27me3 marking were characterized. Remarkably, insertion of the RLE cis-element into pF3H, an unrelated promoter, is sufficient for repressing its transcriptional activity in different tissues. Besides improving our understanding of LEC2 regulation, this study provides important new insights into the mechanisms underlying H3K27me3 deposition and PRC2 recruitment at a specific locus in plants.

Regulation of HSD1 in seeds of Arabidopsis thaliana.

The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.