The pattern of DNA methylation alteration, and its association with the changes of gene expression and alternative splicing during phosphate starvation in tomato.

DNA methylation is changed and associates with gene expression alterations in plant response to phosphate starvation (Pi-), a common stress that impacts plant growth and productivity. However, in the horticultural model species Solanum lycopersicum (tomato), the dynamics of DNA methylation and its relationship with changes in gene transcription and alternative splicing (AS) under Pi- are unknown. Here, we performed integrative methylome and transcriptome analyses of tomato seedlings under Pi-deficient and -sufficient conditions. We found Pi- caused a slight increase in the overall methylation level, with millions of differentially methylated cytosines (DmCs) and a few hundred differentially methylated regions (DMRs). We also identified thousands of differentially expressed (DE) and differential AS (DAS) genes induced by Pi-, and found that DmCs were more abundant in non-expressed genes than in DE or DAS genes. Moreover, DNA methylation alterations weakly correlated with transcription changes but not with DAS events, and hyper-CHH-DMRs overlapping with transposable elements (TEs) were enriched in a subset of Pi starvation response (PSR) genes. We propose that changes in DNA methylation may be associated with the differential expression of some PSR genes, but that most of these changes probably control the expression of nearby TEs, rather than directly affecting the transcription or AS of PSR genes. Besides, the pattern of methylation changes upon Pi- may largely be shaped by TE distributions. Together, our study provides comprehensive insights into the association of DNA methylation with gene transcription and AS under Pi- in tomato and may contribute to unveiling novel roles of epigenetic mechanisms in plant stress response.

SlMIR164A regulates fruit ripening and quality by controlling SlNAM2 and SlNAM3 in tomato.

MiRNAs are important posttranscriptional regulators of plant development. Many miRNAs, such as the conserved miR164 species, are encoded by families of MIRNA genes, but the specific roles of individual MIRNA genes are largely undefined. Here, we characterize the functions and regulatory mechanisms of SlMIR164A, one of the primary genes of Sly-miR164, in tomato. We show that SlMIR164A is preferentially expressed at late stages of fruit development and plays a vital role in controlling fruit ripening and quality. Loss of function of SlMIR164A by CRISPR/Cas9-mediated mutagenesis results in accelerated fruit ripening and enhanced chloroplast development, which leads to altered sugar and organic acid contents and affects the nutritional quality of fruits. We also show that SlMIR164A modulates fruit ripening and quality through specific target genes, SlNAM2 and SlNAM3, which control key regulators of chloroplast function and fruit ripening processes. MIR164 genes have been shown to play conserved roles in regulating organ ageing, such as leaf senescence and fruit ripening, in a variety of plants, but whether and how their family members in tomato exert the same function remain to be elucidated. Our results reveal a previously undiscovered role of SlMIR164A in ripening control, which will further our understanding of the actions of MIR164 family, as well as the mechanisms of fruit ripening and quality control in tomato. Moreover, as loss of SlMIR164A exhibits minor impacts on organ morphology, our results can be leveraged in tomato breeding for specific manipulation of fruit ripening and quality to facilitate tomato improvement in agriculture.

Sly-miR159 regulates fruit morphology by modulating GA biosynthesis in tomato.

Fruit morphology is an important agronomical trait of many crops. Here, we identify Sly-miR159 as an important regulator of fruit morphology in tomato, a model species of fleshy-fruit development. We show that Sly-miR159 functions through its target SlGAMYB2 to control fruit growth. Suppression of Sly-miR159 and overexpression of SlGAMYB2 result in larger fruits with a reduced length/width ratio, while loss of function of SlGAMYB2 leads to the formation of smaller and more elongated fruits. Gibberellin (GA) is a major phytohormone that regulates fruit development in tomato. We show the Sly-miR159-SlGAMYB2 pathway controls fruit morphology by modulating GA biosynthesis. In particular, we demonstrate that Sly-miR159 promotes GA biosynthesis largely through the direct repression of the GA biosynthetic gene SlGA3ox2 by SlGAMYB2. Together, our findings reveal the action of Sly-miR159 on GA biosynthesis as a previously unidentified mechanism that controls fruit morphology in tomato. Modulating this pathway may have potential applications in tomato breeding for manipulating fruit growth and facilitating the process of fruit improvement.

Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis.

In Arabidopsis, the miR171-GRAS module has been clarified as key player in meristem maintenance. However, the knowledge about its role in fruit crops like tomato (Solanum lycopersicum) remains scarce. We previously identified tomato SlGRAS24 as a target gene of Sly-miR171. To study the role of this probable transcription factor, we generated transgenic tomato plants underexpressing SlGRAS24, overexpressing SlGRAS24, overexpressing Sly-miR171 and expressing ß-glucuronidase (GUS) under the SlGRAS24 promoter (proSlGRAS24-GUS). Plants overexpressing SlGRAS24 (SlGRAS24-OE) had pleiotropic phenotypes associated with multiple agronomical traits including plant height, flowering time, leaf architecture, lateral branch number, root length, fruit set and development. Many GA/auxin-related genes were down-regulated and altered responsiveness to exogenous IAA/NAA or GA3 application was observed in SlGRAS24-OE seedlings. Moreover, compromised fruit set and development in SlGRAS24-OE was also observed. These newly identified phenotypes for SlGRAS24 homologs in tomato were later proved to be caused by impaired pollen sacs and fewer viable pollen grains. At anthesis, the comparative transcriptome results showed altered expression of genes involved in pollen development and hormone signalling. Taken together, our data demonstrate that SlGRAS24 participates in a series of developmental processes through modulating gibberellin and auxin signalling, which sheds new light on the involvement of hormone crosstalk in tomato development.

Ectopic expression of SlAGO7 alters leaf pattern and inflorescence architecture and increases fruit yield in tomato.

ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential.

Integration of multi-omics analyses highlights the secondary metabolism response of tomato fruit to low temperature storage.

Inappropriate low temperature storage usually leads to quality deterioration of harvested tomato fruits. In this study, we performed comparative metabolome, transcriptome, and proteome analyses to comprehensively understand the effects of low temperature on metabolic changes in tomato fruit (fresh fruit, C0d; 4 °C 8 days, C8d; 4 °C 7 days and then 25 °C 1 day, C7dS1). Large amounts of secondary metabolites (including flavonoids and phenolic acids) increased after low temperature treatment. The overlap differentially accumulated metabolites in three comparative groups (C0d vs. C8d, C0d vs. C7dS1, C8d vs. C7dS1) were mainly flavonoid metabolites. A total of 1438 differentially expressed genes identified in these three comparative groups were primarily enriched in metabolic pathways and secondary metabolites biosynthesis pathways. Similarly, proteomic analysis showed that the differentially expressed proteins were enriched in the secondary metabolites biosynthesis and phenylpropanoid biosynthesis pathways. There was a strong correlation between changes in flavonoid metabolites and the expression of chalcone synthase (SlCHS), chalcone isomerase-like (SlCHIL), and coumarate 3-hydroxylase (SlC3H), which are involved in the phenylpropanoid and flavonoid biosynthesis. Additionally, seven differentially expressed MYB transcription factors were identified; SlMYB91, SlMYB106, and SlMYB70 strongly correlated with flavonoid biosynthesis structural genes after low temperature treatment. Other genes involved in fruit ripening and quality were also affected by low temperature. The data generated in this study may unravel the transcriptional regulatory network of secondary metabolism associated with low-temperature storage and provide a solid foundation for future studies.

Metabolite alteration in response to low phosphorus stress in developing tomato fruits.

Alteration of fruit quality caused by environmental stress is a common but largely unresolved issue for plant cultivation and breeding practices. Phosphorus (P) deficiency may interfere with a variety of metabolic processes whose intermediate products are correlated with important fruit quality traits. However, how low P stress affects fruit quality has not been investigated in detail. In this study, we assessed the contents of major metabolites associated with tomato fruit quality under two low P treatments that started at the seedling or flowering stage. The major pigments and the key organic acids related to fruit sourness were differentially over-accumulated as fruit ripened under two low P treatments compared to those under the control treatment, while the total content of soluble sugars contributing to fruit sweetness was substantially reduced under both treatments. These changes were largely attributed to the alteration of enzyme activities in the relevant metabolic pathways. In particular, we found that low P stress from different developmental stages had differential effects on the activation of γ-aminobutyric acid shunt that were likely responsible for the preferential accumulation of different organic acids in tomato fruits. Our study suggested that low P stress strongly affected tomato fruit quality and the effects appeared to be variable under different regimes of low P conditions.

Overexpression of SlGRAS40 in Tomato Enhances Tolerance to Abiotic Stresses and Influences Auxin and Gibberellin Signaling.

Abiotic stresses are major environmental factors that inhibit plant growth and development impacting crop productivity. GRAS transcription factors play critical and diverse roles in plant development and abiotic stress. In this study, SlGRAS40, a member of the tomato (Solanum lycopersicum) GRAS family, was functionally characterized. In wild-type (WT) tomato, SlGRAS40 was upregulated by abiotic stress induced by treatment with D-mannitol, NaCl, or H2O2. Transgenic tomato plants overexpressing SlGRAS40 (SlGRAS40-OE) were more tolerant of drought and salt stress than WT. SlGRAS40-OE plants displayed pleiotropic phenotypes reminiscent of those resulting from altered auxin and/or gibberellin signaling. A comparison of WT and SlGRAS40-OE transcriptomes showed that the expression of a large number of genes involved in hormone signaling and stress responses were modified. Our study of SlGRAS40 protein provides evidence of how another GRAS plays roles in resisting abiotic stress and regulating auxin and gibberellin signaling during vegetative and reproductive growth in tomato.

Overexpression of SlREV alters the development of the flower pedicel abscission zone and fruit formation in tomato.

Versatile roles of REVOLUTA (REV), a Class III homeodomain-leucine zipper (HD-ZIP III) transcription factor, have been depicted mainly in Arabidopsis and Populus. In this study, we investigated the functions of its tomato homolog, namely SlREV. Overexpression of a microRNA166-resistant version of SlREV (35S::REV(Ris)) not only resulted in vegetative abnormalities such as curly leaves and fasciated stems, but also caused dramatic reproductive alterations including continuous production of flowers at the pedicel abscission zone (AZ) and ectopic fruit formation on receptacles. Microscopic analysis showed that meristem-like structures continuously emerged from the exodermises of the pedicel AZs and that ectopic carpels formed between the first and second whorl of floral buds in 35S::REV(Ris) plants. Transcriptional data suggest that SlREV may regulate genes related to meristem maintenance and cell differentiation in the development of the flower pedicel abscission zone, and modulate genes in homeodomain and MADS-box families and hormone pathways during fruit formation. Altogether, these results reveal novel roles of SlREV in tomato flower development and fruit formation.