Elevated methylglyoxal levels inhibit tomato fruit ripening by preventing ethylene biosynthesis.

Methylglyoxal (MG), a toxic compound produced as a by-product of several cellular processes, such as respiration and photosynthesis, is well known for its deleterious effects, mainly through glycation of proteins during plant stress responses. However, very little is known about its impact on fruit ripening. Here, we found that MG levels are maintained at high levels in green tomato (Solanum lycopersicum L.) fruits and decline during fruit ripening despite a respiratory burst during this transition. We demonstrate that this decline is mainly mediated through a glutathione-dependent MG detoxification pathway and primarily catalyzed by a Glyoxalase I enzyme encoded by the SlGLYI4 gene. SlGLYI4 is a direct target of the MADS-box transcription factor RIPENING INHIBITOR (RIN), and its expression is induced during fruit ripening. Silencing of SlGLYI4 leads to drastic MG overaccumulation at ripening stages of transgenic fruits and interferes with the ripening process. MG most likely glycates and inhibits key enzymes such as methionine synthase and S-adenosyl methionine synthase in the ethylene biosynthesis pathway, thereby indirectly affecting fruit pigmentation and cell wall metabolism. MG overaccumulation in fruits of several nonripening or ripening-inhibited tomato mutants suggests that the tightly regulated MG detoxification process is crucial for normal ripening progression. Our results underpin a SlGLYI4-mediated regulatory mechanism by which MG detoxification controls fruit ripening in tomato.

A glutathione-independent DJ-1/PfpI domain-containing tomato glyoxalaseIII2, SlGLYIII2, confers enhanced tolerance under salt and osmotic stresses.

In plants, glyoxalase enzymes are activated under stress conditions to mitigate the toxic effects of hyperaccumulated methylglyoxal (MG), a highly reactive carbonyl compound. Until recently, a glutathione-dependent bi-enzymatic pathway involving glyoxalase I (GLYI) and glyoxalase II (GLYII) was considered the primary MG-detoxification system. Recently, a new glutathione-independent glyoxalase III (GLYIII) mediated direct route was also reported in plants. However, the physiological significance of this new pathway remains to be elucidated across plant species. This study identified the full complement of 22 glyoxalases in tomato. Based on their strong induction under multiple abiotic stresses, SlGLYI4, SlGLYII2 and SlGLYIII2 were selected candidates for further functional characterisation. Stress-inducible overexpression of both glutathione-dependent (SlGLYI4 + SlGLYII2) and independent (SlGLYIII2) pathways led to enhanced tolerance in both sets of transgenic plants under abiotic stresses. However, SlGLYIII2 overexpression (OE) plants outperformed the SlGLYI4 + SlGLYII2 OE counterparts for their stress tolerance under abiotic stresses. Further, knockdown of SlGLYIII2 resulted in plants with exacerbated stress responses than those silenced for both SlGLYI4 and SlGLYII2. The superior performance of SlGLYIII2 OE tomato plants for better growth and yield under salt and osmotic treatments could be attributed to better GSH/GSSG ratio, lower reactive oxygen species levels, and enhanced antioxidant potential, indicating a prominent role of GLYIII MG-detoxification pathway in abiotic stress mitigation in this species.

Genome-wide analysis of genes encoding MBD domain-containing proteins from tomato suggest their role in fruit development and abiotic stress responses.

In tomato, DNA methylation has an inhibitory effect on fruit ripening. The inhibition of DNA methyltransferase by 5-azacytidine results in premature fruit ripening. Methyl CpG binding domain (MBD) proteins are the readers of DNA methylation marks and help in the recruitment of chromatin-modifying enzymes which affect gene expression. Therefore, we investigate their contribution during fruit development. In this study, we identified and analyzed 18 putative genes of Solanum lycopersicum and Solanum pimpinellifolium encoding MBD proteins. We also identified tomato MBD syntelogs in Capsicum annum and Solanum tuberosum. Sixty-three MBD genes identified from four different species of solanaceae were classified into three groups. An analysis of the conserved domains in these proteins identified additional domains along with MBD motif. The transcript profiling of tomato MBDs in wild-type and two non-ripening mutants, rin and Nr, indicated constructive information regarding their involvement during fruit development. When we performed a stage-specific expression analysis during fruit ripening, a gradual decrease in transcript accumulation in the wild-type fruit was detected. However, a very low expression was observed in the ripening mutants. Furthermore, many ethylene-responsive cis-elements were found in SlMBD gene promoters, and some of them were induced in the presence of exogenous ethylene. Further, we detected the possible role of these MBDs in abiotic stresses. We found that few genes were differentially expressed under various abiotic stress conditions. Our results provide an evidence of the involvement of the tomato MBDs in fruit ripening and abiotic stress responses, which would be helpful in further studies on these genes in tomato fruit ripening.

Silencing of SlSPX1 and SlSPX2 promote growth and root mycorrhization in tomato (Solanum lycopersicum L.) seedlings.

Owing to the essential requirement of phosphorus (P) for growth and development, plants tightly control inorganic phosphate (Pi) homeostasis. SPX-PHR regulatory circuit not only control phosphate homeostasis responses but also root mycorrhization by arbuscular mycorrhiza (AM) fungi. Besides sensing Pi deficiency, SPX (SYG1/Pho81/XPR1) proteins also control the transcription of P starvation inducible (PSI) genes by blocking the activity of PHR1 (PHOSPHATE STARVATION RESPONSE1) homologs in plants under Pi-sufficient conditions. However, the roles of SPX members in Pi homeostasis and AM fungi colonization remain to be fully recognized in tomato. In this study, we identified 17 SPX-domain containing members in the tomato genome. Transcript profiling revealed the high Pi-specific nature of their activation. Four SlSPX members have also induced in AM colonized roots. Interestingly, we found that SlSPX1 and SlSPX2 are induced by P starvation and AM fungi colonization. Further, SlSPX1 and SlSPX2 exhibited varying degrees of interaction with the PHR homologs in this study. Virus-induced gene silencing-based (VIGS) transcript inhibition of these genes alone or together promoted the accumulation of higher total soluble Pi in tomato seedlings and improved their growth. It also enhanced AM fungi colonization in the roots of SlSPX1 and SlSPX2 silenced seedlings. Overall, the present study provides evidence in support of SlSPX members being good candidates for improving AM fungi colonization potential in tomato.

Identification, evolutionary profiling, and expression analysis of F-box superfamily genes under phosphate deficiency in tomato.

F-box genes are an integral component of the Skp1-cullin-F-box (SCF) complex in eukaryotes. These genes are primarily involved in determining substrate specificities during cellular proteolysis. Here we report that 410 members constitute the F-box superfamily in tomato. Based on the incidence of C-terminal domains, these genes fell into ten subfamilies, leucine-rich repeat domain-containing F-box members constituting the largest subfamily. The F-box genes are present on all 12 chromosomes with varying gene densities. Both segmental and tandem duplication events contribute significantly to their expansion in the tomato genome. The syntenic analysis revealed close relationships among F-box homologs within Solanaceae species genomes. Transcript profiling of F-box members identified several ripening-associated genes with altered expression in the ripening mutants. RNA-sequencing data analysis showed that phosphate (Pi) deficiency affected 55 F-box transcripts in the Pi-deficient seedlings compared to their control seedlings. The persistent up-regulation of eight members, including two phloem protein 2B (PP2-B) genes, PP2-B15, and MATERNAL EFFECT EMBRYO ARREST 66 (MEE66) homologs, at multiple time-points in the roots, shoot, and seedling, point towards their pivotal roles in Pi starvation response in tomato. The attenuation of such upregulation in sucrose absence revealed the necessity of this metabolite for robust activation of these genes in the Pi-deficient seedlings. Altogether, this study identifies novel F-box genes with potential roles in fruit ripening and Pi starvation response and unlocks new avenues for functional characterization of candidate genes in tomato and other related species.

Identification, structure analysis, and transcript profiling of purple acid phosphatases under Pi deficiency in tomato (Solanum lycopersicum L.) and its wild relatives.

Purple acid phosphatases (PAPs), a family of metallo-phosphoesterase enzymes, are involved in phosphorus nutrition in plants. In this study, we report that the tomato genome encodes 25 PAP members. Physio-biochemical analyses revealed relatively lower total root-associated acid phosphatase activity in the seedlings of Solanum pimpinellifolium than their cultivated tomato seedlings under Pi deficiency. Scrutiny of their transcript abundance shows that most of PAPs are activated, although to varying levels, under Pi deficiency in tomato. Further investigation demonstrates that the magnitude of induction of phosphate starvation inducible root-associated PAP homologs remains lower in the Pi-starved S. pimpinellifolium seedlings, hence, accounting for the lower acid phosphatase activity in this wild relative. Examination of their amino acid sequences revealed significant variation in their substrate-specificity defining residues. Among all members, only SlPAP15 possesses the critical lysine residue (R337) and atypical REKA motif in its C-terminal region. Homology modeling and docking studies revealed that ADP and ATP are preferred substrates of SlPAP15. We also identified other amino acid residues present in the vicinity of the active site, possibly facilitating such physical interactions. Altogether, the results presented here will help in the functional characterization of these genes in the tomato in the future.

AtMBD4: A methylated DNA binding protein negatively regulates a subset of phosphate starvation genes.

DNA methylation is an important epigenetic modification that governs transcriptional regulation. The methylation mark is read by a special class of proteins called methyl-CpG-binding domain proteins. The role of DNA methylation has been found in X-chromosome inactivation, genomic imprinting, transposon silencing, and self-incompatibility. Recently, remodeling of global DNA methylation was demonstrated in Arabidopsis during low phosphate availability. The present study reports that AtMBD4 gene of Arabidopsis negatively regulates phosphate starvation. The T-DNA insertion mutation at the AtMBD4 locus exhibited altered root architecture as compared to wild-type plants. Using microarray hybridization and analysis, an increased transcript accumulation of 242 genes was observed in the mutant. Many of these genes were related to phosphate transporters and transcription factors, involved in phosphate starvation response. Comparison of data of atmbd4 mutant with publicly available microarray data of phosphate starvation response indicated the role of AtMBD4 protein in phosphate starvation response. Further, promoter analysis of up-regulated genes suggested that cis-regulatory elements like MBS, W-box, and B1BS are more prominent in the promoters of up-regulated genes. Upon performing a methylation-specific PCR, a decreased DNA methylation in the promoter regions of up-regulated genes was observed. The accumulation of anthocyanin and inorganic phosphate in the atmbd4 mutant was found to be higher than the wild-type plant. Altered root morphology, up-regulation of phosphate starvation-induced genes in atmbd4 mutant suggests that AtMBD4 negatively regulates the phosphate starvation response.

AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins.

DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.