Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit.

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato. In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1 In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes. These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes. Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes.

Critical function of DNA methyltransferase 1 in tomato development and regulation of the DNA methylome and transcriptome.

DNA methylation confers epigenetic regulation on gene expression and thereby on various biological processes. Tomato has emerged as an excellent system to study the function of DNA methylation in plant development. To date, regulation and function of DNA methylation maintenance remains unclear in tomato plants. Here, we report the critical function of tomato (Solanum lycopersicum) Methyltransferase 1 (SlMET1) in plant development and DNA methylome and transcriptome regulation. Using CRISPR-Cas9 gene editing, we generated slmet1 mutants and observed severe developmental defects with a frame-shift mutation, including small and curly leaves, defective inflorescence, and parthenocarpy. In leaf tissues, mutations in SlMET1 caused CG hypomethylation and CHH hypermethylation on a whole-genome scale, leading to a disturbed transcriptome including ectopic expression of many RIN target genes such as ACC2 in leaf tissues, which are normally expressed in fruits. Neither the CG hypomethylation nor CHH hypermethylation in the slmet1 mutants is related to tissue culture. Meanwhile, tissue culture induces non-CG hypomethylation, which occurs more frequently at gene regions than at TE regions. Our results depict SlMET1- and tissue culture-dependent tomato DNA methylomes, and that SlMET1 is required for maintaining a normal transcriptome and normal development of tomato.

Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato.

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit-ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8-ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild-type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation-related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd-mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S-ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole-plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post-harvest senescence and decay.

Functional analysis of a tomato (Solanum lycopersicum L.) rhamnogalacturonan lyase promoter.

The enzyme rhamnogalacturonan lyase (RGL) cleaves α-1,4 glycosidic bonds located between rhamnose and galacturonic acid residues in the main chain of rhamnogalacturonan-I (RG-I), a component of the plant cell wall polymer pectin. Although the mode of action of RGL is well known, its physiological functions associated with fruit biology are less understood. Here, we generated transgenic tomato plants expressing the ß-glucuronidase (GUS) reporter gene under the control of a -504 bp or a -776 bp fragment of the promoter of a tomato RGL gene, Solyc11g011300. GUS enzymatic activity and the expression levels of GUS and Solyc11g011300 were measured in a range of organs and fruit developmental stages. GUS staining was undetectable in leaves and roots, but high GUS enzymatic activity was detected in flowers and red ripe (RR) fruits. Maximal expression levels of Solyc11g011300 were detected at the RR developmental stage. GUS activity was 5-fold higher in flowers expressing GUS driven by the -504 bp RGL promoter fragment (RGFL3::GUS) than in the isogenic line, and 1.7-fold higher when GUS gene was driven by the -776 bp RGL promoter fragment (RGLF2::GUS) or the constitutive CaMV35S promoter. Quantitative real-time polymerase chain reaction analysis showed that the highest expression of GUS was in fruits at 40 days after anthesis, for both promoter fragments. The promoter of Solyc11g011300 is predicted to contain cis-acting elements, and to be active in pollen grains, pollen tubes, flowers and during tomato fruit ripening, suggesting that the Solyc11g011300 promoter is transcriptionally active and organ-specific.

Functional analysis of tomato rhamnogalacturonan lyase gene Solyc11g011300 during fruit development and ripening.

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life.

Maturity and ripening-stage specific modulation of tomato (Solanum lycopersicum) fruit transcriptome.

Tomato (Solanum lycopersicum) fruit is a model to study molecular basis of fleshy fruit development and ripening. We profiled gene expression during fruit development (immature green and mature green fruit) and ripening (breaker stage onwards) program to obtain a global perspective of genes whose expression is modulated at each stage of fruit development and ripening. A custom made cDNA macroarray containing cDNAs representing various metabolic pathways, defense, signaling, transcription, transport, cell structure and cell wall related functions was developed and used to quantify changes in the abundance of different transcripts. About 34 % of 1066 unique expressed sequence tags (ESTs) printed on the macroarray were differentially expressed during tomato fruit ripening. Out of these, 25 % genes classify under metabolism and protein biosynthesis/degradation related processes, while a significant proportion represented stress-responsive genes and about 44 % represented genes with unknown functions. RNA gel blot analysis validated changes in a few representative genes. Although the mature green fruit was found transcriptionally quiescent, the K-means cluster analysis highlighted coordinated up or down regulation of genes during progressive ripening; emphasizing that ripening is a transcriptionally active process. Many stress-related genes were found up-regulated, suggesting their role in the fruit ripening program.