Chlorophyll (chl) is essential for light capture and is the starting point that provides the energy for photosynthesis and thus plant growth. Obviously, for this reason, retention of the green chlorophyll pigment is considered a desirable crop trait. However, the presence of chlorophyll in mature seeds can be an undesirable trait that can affect seed maturation, seed oil quality, and meal quality. Occurrence of mature green seeds in oil crops such as canola and soybean due to unfavorable weather conditions during seed maturity is known to cause severe losses in revenue. One recently identified candidate that controls the chlorophyll degradation machinery is the stay-green gene, SGR1 that was mapped to Mendel's I locus responsible for cotyledon color (yellow versus green) in peas. A defect in SGR1 leads to leaf stay-green phenotypes in Arabidopsis and rice, but the role of SGR1 in seed degreening and the signaling machinery that converges on SGR1 have remained elusive. To decipher the gene regulatory network that controls degreening in Arabidopsis, we have used an embryo staygreen mutant to demonstrate that embryo degreening is achieved by the SGR family and that this whole process is regulated by the phytohormone abscisic acid (ABA) through ABSCISIC ACID INSEN-SITIVE 3 (ABI3); a B3 domain transcription factor that has a highly conserved and essential role in seed maturation, conferring desiccation tolerance. Misexpression of ABI3 was sufficient to rescue cold-induced green seed phenotype in Arabidopsis. This finding reveals a mechanistic role for ABI3 during seed degreening and thus targeting of this pathway could provide a solution to the green seed problem in various oil-seed crops.freezing tolerance | nondormant T he success of angiosperms impinges on their ability to desiccate and protect their embryos in a dormant state until favorable conditions are perceived. In many angiosperms and oilseed plants such as Arabidopsis and canola, this desiccation process during seed maturation is intricately coupled to loss of chlorophyll (chl) from photosynthetically active embryos (1). During the embryo maturation phase, as the embryos begin to lose their chlorophyll, they concomitantly initiate the process of acquisition of desiccation tolerance and dormancy, thereby producing mature, brown (degreened) and dormant seeds. The persistence of chlorophyll in mature seeds has negative impacts on seed storability in many commercial plant species such as canola, cabbage, carrot, geranium, and soybean (2-4). Apart from contributing to reduced storability, prevalence of green seeds in mature oil seeds (canola and soybean) is also associated with reduced shelf life of oil and production of unfavorable odors and flavors. Particularly, in canola, which is one of the major global cash crops, the frost-induced green seed problem has been estimated to result in an annual loss of $150 million in revenue in North America alone (5, 6).During seed development, abscisic acid (ABA) is known to control mid to late stages of embryo maturation...

HighlightThe two tomato GDP-D-mannose epimerase isoforms play specific roles in cell wall biosynthesis and plant development but participate similarly in ascorbate biosynthesis.

Tomato is currently the model plant for fleshy fruit development and for Solanaceae species. Recent genomic approaches including transcriptome, proteome and metabolome analyses and genetic mapping have produced a wealth of candidate genes whose function needs to be assessed. The recent development in model and crop plants of TILLING (Targeting Induced Local Lesions IN Genomes), which reveals allelic series corresponding to several independent point mutations, and the current availability of deep sequencing tools further increase the interest of generating artificiallyinduced genetic diversity in tomato. We describe here the generation and use of EMS (ethyl methanesulfonate) tomato mutants in the miniature cultivar Micro-Tom and provide as example the identification of new fruit size and morphology mutants. We further propose new deep sequencing-based strategies for the discovery of mutations underlying phenotypic variations observed in mutant collections that will considerably increase the interest of exploiting Micro-Tom mutant collections for gene discovery in tomato.

Ascorbate is a major antioxidant buffer in plants, so several approaches have been developed to increase the ascorbate contents of fruits and vegetables. In this study, we combined forward genetics with mapping-by-sequencing approaches using an EMSMicro-Tom population to identify putative regulators underlying a high ascorbate phenotype in fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5 line, up to 5 times the WT) strongly impaired flower development and produced seedless fruit. Without progeny, genetic characterization was performed by outcrossing the P17C5 line with S. Lycopersicum cv. M82. We successfully identified the mutation responsible for the high ascorbate trait in a cis-acting upstream open reading frame (uORF) that is involved in the downstream regulation of GDP-L-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enrichment trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5xM82 plants, and the CRISPR-mutated line. These studies revealed that a high ascorbate content is linked to impaired floral organ architecture, particularly anthers and pollen development, thus leading to male sterility. RNAseq analysis suggests that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

Besides being a model for fleshy fruits and Solanaceae species, tomato represents one of the main sources of ascorbate (vitamin C) in human diet in many parts of the world. Ascorbate fulfills various roles in plants due to its antioxidant potential and to its connection with other metabolic pathways e.g. cell wall biosynthesis. Among the functional genomic tools recently developed in tomato, EMS (ethyl methanesulfonate) mutant collections provide an opportunity for identifying allelic series of mutations in target genes by TILLING (Targeting Induced Local Lesions IN Genomes). We describe here the use of tomato EMS mutant collections in the miniature cv. Micro-Tom for the discovery of allelic variants in three ascorbate biosynthetic genes encoding the GDP-d-mannose pyrophosphorylase (GMP), the GDP-dmannose epimerase (GME) and the GDP-l-galactose phosphorylase (GGP) respectively. We report on the discovery of several missense, truncation and splice junction mutations in these genes affecting plant ascorbate content to various levels, and show that several tomato mutant lines with strongly reduced ascorbate content undergo severe bleaching upon exposure to high light intensity.

Ascorbate (vitamin C) is one of the most essential antioxidants in fresh fruits and vegetables. To get insights into the regulation of ascorbate metabolism in plants, a mutant producing ascorbate-enriched fruits was studied. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a new class of photoreceptor named PAS/LOV protein (PLP, Solyc05g07020), which acts as a negative regulator of ascorbate biosynthesis in tomato. This trait was confirmed by CRISPR/Cas9 gene editing, and further found in all plant organs, including fruit that accumulated 2-3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the two 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 mutated. These results were confirmed by an optogenetic 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 non-competitive inhibitor of GGP that is inactivated after blue light exposure. This discovery sheds light on the light-dependent regulation of ascorbate metabolism in plants.

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 2-3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the two 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 non-competitive 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.