Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

The effect of low ascorbic acid content on tomato fruit ripening.

MAIN CONCLUSION: The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

MAIN CONCLUSION: Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

Genome-wide analysis of intraspecific DNA polymorphism in 'Micro-Tom', a model cultivar of tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. The genome sequencing of the tomato cultivar 'Heinz 1706' was recently completed. To accelerate the progress of tomato genomics studies, systematic bioresources, such as mutagenized lines and full-length cDNA libraries, have been established for the cultivar 'Micro-Tom'. However, these resources cannot be utilized to their full potential without the completion of the genome sequencing of 'Micro-Tom'. We undertook the genome sequencing of 'Micro-Tom' and here report the identification of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) between 'Micro-Tom' and 'Heinz 1706'. The analysis demonstrated the presence of 1.23 million SNPs and 0.19 million indels between the two cultivars. The density of SNPs and indels was high in chromosomes 2, 5 and 11, but was low in chromosomes 6, 8 and 10. Three known mutations of 'Micro-Tom' were localized on chromosomal regions where the density of SNPs and indels was low, which was consistent with the fact that these mutations were relatively new and introgressed into 'Micro-Tom' during the breeding of this cultivar. We also report SNP analysis for two 'Micro-Tom' varieties that have been maintained independently in Japan and France, both of which have served as standard lines for 'Micro-Tom' mutant collections. Approximately 28,000 SNPs were identified between these two 'Micro-Tom' lines. These results provide high-resolution DNA polymorphic information on 'Micro-Tom' and represent a valuable contribution to the 'Micro-Tom'-based genomics resources.

High light stress induces H2O2 production and accelerates fruit ripening in tomato.

Increased synthesis of H2O2 is observed during the initiation of fruit ripening. However, its association with plant cell processes triggering the maturation of fruit has not yet been demonstrated. The aim of this work is to investigate whether H2O2 participates in the tomato ripening process and particularly through its association with the ethylene signaling pathway. The experiments were carried out with two ethyl methanesulfonate mutant lines of Micro-Tom tomato deficient in GDP-L-galactose phosphorylase activity and displaying lower ascorbic acid content than the corresponding parental genotype (i.e. wild type). Plants were subjected to a high irradiance (HI) treatment to stimulate H2O2 synthesis. HI treatment enhanced H2O2 production and reduced the timing of fruit ripening in both mutants and wild-type fruits. These results could be linked to an increase of the expression of H2O2-related genes and changes in the expression of ethylene-related genes. The fruit H2O2 production increased or decreased after applying the treatments that induced ethylene synthesis or blocked its action, respectively. The results presented in this work give an evidence of the association of redox and hormonal components during fruit ripening in which H2O2 participates downstream in the events regulated by ethylene.

A quick protocol for the identification and characterization of early growth mutants in tomato.

Root system architecture (RSA) manipulation may improve water and nutrient capture by plants under normal and extreme climate conditions. With the aim of initiating the genetic dissection of RSA in tomato, we established a defined ontology that allowed the curated annotation of the observed phenotypes on 12 traits at four consecutive growth stages. In addition, we established a quick approach for the molecular identification of the mutations associated with the trait-of-interest by using a whole-genome sequencing approach that does not require the building of an additional mapping population. As a proof-of-concept, we screened 4543 seedlings from 300 tomato M3 lines (Solanum lycopersicum L. cv. Micro-Tom) generated by chemical mutagenesis with ethyl methanesulfonate. We studied the growth and early development of both the root system (primary and lateral roots) and the aerial part of the seedlings as well as the wound-induced adventitious roots emerging from the hypocotyl. We identified 659 individuals (belonging to 203 M3 lines) whose early seedling and RSA phenotypes differed from those of their reference background. We confirmed the genetic segregation of the mutant phenotypes affecting primary root length, seedling viability and early RSA in 31 M4 families derived from 15 M3 lines selected in our screen. Finally, we identified a missense mutation in the SlCESA3 gene causing a seedling-lethal phenotype with short roots. Our results validated the experimental approach used for the identification of tomato mutants during early growth, which will allow the molecular identification of the genes involved.

Identification of Two New Mechanisms That Regulate Fruit Growth by Cell Expansion in Tomato.

Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection. Their thorough characterization at organ, tissue and cellular levels revealed two major clusters controlling fruit growth and tissue morphogenesis either through (i) the growth of all fruit tissues through isotropic cell expansion or (ii) only the growth of the pericarp through anisotropic cell expansion. These likely correspond to new cell expansion modules controlling fruit growth and tissue morphogenesis in tomato. Our study therefore opens the way for the identification of new gene regulatory networks controlling tomato fruit growth and morphology.

Culture of the Tomato Micro-Tom Cultivar in Greenhouse.

Micro-Tom tomato cultivar is particularly adapted to the development of genomic approaches in tomato. Here, we describe the culture of this plant in greenhouse, including climate regulation, seed sowing and watering, vegetative development, plant maintenance, including treatment of phytosanitary problems, and reproductive development.

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Regulation of the fruit-specific PEP carboxylase SlPPC2 promoter at early stages of tomato fruit development.

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the ß-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5' SlPPC2 promoter deletions fused to LUC in fruits at the 8(th) day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5' UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

SNP discovery and linkage map construction in cultivated tomato.

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between 'Micro-Tom' and either 'Ailsa Craig' or 'M82'. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/.

Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.

L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P(35S):Slgalldh(RNAi) silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P(35S):Slgalldh(RNAi) plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P(35S):Slgalldh(RNAi) plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.

The expression of cell proliferation-related genes in early developing flowers is affected by a fruit load reduction in tomato plants.

Changes in photoassimilate partitioning between source and sink organs significantly affect fruit development and size. In this study, a comparison was made of tomato plants (Solanum lycopersicum L.) grown under a low fruit load (one fruit per truss, L1 plants) and under a standard fruit load (five fruits per truss, L5 plants), at morphological, biochemical, and molecular levels. Fruit load reduction resulted in increased photoassimilate availability in the plant and in increased growth rates in all plant organs analysed (root, stem, leaf, flower, and fruit). Larger flower and fruit size in L1 plants were correlated with higher cell number in the pre-anthesis ovary. This was probably due to the acceleration of the flower growth rate since other flower developmental parameters (schedule and time-course) remained otherwise unaffected. Using RT-PCR, it was shown that the transcript levels of CYCB2;1 (cyclin) and CDKB2;1 (cyclin-dependent kinase), two mitosis-specific genes, strongly increased early in developing flower buds. Remarkably, the transcript abundance of CYCD3;1, a D-type cyclin potentially involved in cell cycle regulation in response to mitogenic signals, also increased by more than 5-fold at very early stages of L1 flower development. By contrast, transcripts from fw2.2, a putative negative regulator of cell division in tomato fruit, strongly decreased in developing flower bud, as confirmed by in situ hybridization studies. Taken together, these results suggest that changes in carbohydrate partitioning could control fruit size through the regulation of cell proliferation-related genes at very early stages of flower development.

Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato.

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

Physiological, biochemical and molecular analysis of sugar-starvation responses in tomato roots.

Two-month-old tomato plants were submitted to day/night cycles and to prolonged darkness in order to investigate the physiological and biochemical response to sugar starvation in sink organs. Roots appeared particularly sensitive to the cessation of photosynthesis, as revealed by the reduction of the growth rate and the decline of the carbohydrate and protein content. Therefore, excised tomato roots were used as a model to deepen the characterization of sugar starvation symptoms. In excised roots, the endogenous sugars were rapidly exhausted and significant degradation of protein was observed. Glutamine and asparagine accounted for most of the nitrogen released by protein breakdown. Respiration declined and proliferation- and growth-associated genes were repressed soon after the beginning of the sugar depletion. Among the genes studied, only the gene encoding asparagine synthetase was strongly induced. All the starvation symptoms were reversible when the roots were resupplied with sugar. When the culture conditions deteriorated, the metabolic and molecular changes led to the triggering of apoptosis of the root cells.

A fruit-specific phospho enolpyruvate carboxylase is related to rapid growth of tomato fruit.

Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.