Genome-Wide Analysis of MBF1 Family Genes in Five Solanaceous Plants and Functional Analysis of SlER24 in Salt Stress.

Multiprotein bridging factor 1 (MBF1) is an ancient family of transcription coactivators that play a crucial role in the response of plants to abiotic stress. In this study, we analyzed the genomic data of five Solanaceae plants and identified a total of 21 MBF1 genes. The expansion of MBF1a and MBF1b subfamilies was attributed to whole-genome duplication (WGD), and the expansion of the MBF1c subfamily occurred through transposed duplication (TRD). Collinearity analysis within Solanaceae species revealed collinearity between members of the MBF1a and MBF1b subfamilies, whereas the MBF1c subfamily showed relative independence. The gene expression of SlER24 was induced by sodium chloride (NaCl), polyethylene glycol (PEG), ABA (abscisic acid), and ethrel treatments, with the highest expression observed under NaCl treatment. The overexpression of SlER24 significantly enhanced the salt tolerance of tomato, and the functional deficiency of SlER24 decreased the tolerance of tomato to salt stress. SlER24 enhanced antioxidant enzyme activity to reduce the accumulation of reactive oxygen species (ROS) and alleviated plasma membrane damage under salt stress. SlER24 upregulated the expression levels of salt stress-related genes to enhance salt tolerance in tomato. In conclusion, this study provides basic information for the study of the MBF1 family of Solanaceae under abiotic stress, as well as a reference for the study of other plants.

Genome-wide identification and characterization of tomato 14-3-3 (SlTFT) genes and functional analysis of SlTFT6 under heat stress.

The plant 14-3-3 proteins are essential for many biological processes and responses to abiotic stress. We performed genome-wide identification and analysis of the 14-3-3 family genes in tomato. To explore the properties of the thirteen Sl14-3-3 found in the tomato genome, their chromosomal location, phylogenetic, and syntenic relationships were analyzed. The Sl14-3-3 promoters were found to have a number of growth-, hormone-, and stress-responsive cis-regulatory elements. Moreover, the qRT-PCR assay revealed that Sl14-3-3 genes are responsive to heat and osmotic stress. Subcellular localization experiments evidenced that the SlTFT3/6/10 proteins occur in the nucleus and cytoplasm Additional analysis on Sl14-3-3 putative interactor proteins revealed a number of prospective clients that potentially participate in stress reactions and developmental processes. Furthermore, overexpression of an Sl14-3-3 family gene, SlTFT6, improved tomato plants thermotolerance. Taken together, the study provides basic information on tomato 14-3-3 family genes in plant growth and abiotic stress response (high temperature stress), which can be helpful to further study the underlying molecular mechanisms.

A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato.

Drought stress caused by water deficit reduces plant productivity in many regions of the world. In plants, basic helix-loop-helix (bHLH) transcription factors regulate a wide range of cellular activities related to growth, development and stress response; however, the role of tomato SlbHLHs in drought stress responses remains elusive. Here, we used reverse genetics approaches to reveal the function of SlbHLH96, which is induced by drought and abscisic acid (ABA) treatment. We found that SlbHLH96 functions as a positive regulator of drought tolerance in tomato. Overexpression of SlbHLH96 in tomato improves drought tolerance by stimulating the expression of genes encoding antioxidants, ABA signaling molecules and stress-related proteins. In contrast, silencing of SlbHLH96 in tomato reduces drought tolerance. SlbHLH96 physically interacts with an ethylene-responsive factor, SlERF4, and silencing of SlERF4 in tomato also decreases drought tolerance. Furthermore, SlbHLH96 can repress the expression of the ABA catabolic gene, SlCYP707A2, through direct binding to its promoter. Our results uncover a novel mechanism of SlbHLH96-mediated drought tolerance in tomato plants, which can be exploited for breeding drought-resilient crops.

Overexpression of SlBBX17 affects plant growth and enhances heat tolerance in tomato.

Heat stress is one of the major limiting factors that affect plant growth and production. In this study, we identified SlBBX17, which encodes a B-Box (BBX) protein and functions as a negative regulator of plant growth and a positive regulator of heat tolerance in tomato (Solanum lycopersicum). The expression of SlBBX17 is induced by hormones and heat stress. Overexpression of SlBBX17 (SlBBX17-OE) in tomato led to less chlorophyll content and lower net photosynthetic rate relative to the wild type. The growth retardation in the SlBBX17-OE plants may be attributed to the change of endogenous gibberellin (GA) metabolism and the decrease of photosynthetic capacity. SlBBX17-OE plants exhibited increased tolerance to heat stress, as reflected by the better membrane stability, higher antioxidant enzyme activities, and less reactive oxygen species (ROS) accumulation. Transcriptome analysis revealed that overexpression of SlBBX17 affected the expression of genes involved in GA biosynthetic process, photosynthesis, heat stress, ROS, and other cellular processes. The qRT-PCR analysis indicated that many SlHsf and SlHSP genes are up-regulated by SlBBX17 under heat stress. These results demonstrate that SlBBX17 plays important roles in regulating tomato growth and resistance to heat stress.

Identification, Classification, and Expression Analysis of the Triacylglycerol Lipase (TGL) Gene Family Related to Abiotic Stresses in Tomato.

Triacylglycerol Lipases (TGLs) are the major enzymes involved in triacylglycerol catabolism. TGLs hydrolyze long-chain fatty acid triglycerides, which are involved in plant development and abiotic stress responses. Whereas most studies of TGLs have focused on seed oil metabolism and biofuel in plants, limited information is available regarding the genome-wide identification and characterization of the TGL gene family in tomato (Solanum lycopersicum L.). Based on the latest published tomato genome annotation ITAG4.0, 129 SlTGL genes were identified and classified into 5 categories according to their structural characteristics. Most SlTGL genes were distributed on 3 of 12 chromosomes. Segment duplication appeared to be the driving force underlying expansion of the TGL gene family in tomato. The promoter analysis revealed that the promoters of SlTGLs contained many stress responsiveness cis-elements, such as ARE, LTR, MBS, WRE3, and WUN-motifs. Expression of the majority of SlTGL genes was suppressed following exposure to chilling and heat, while it was induced under drought stress, such as SlTGLa9, SlTGLa6, SlTGLa25, SlTGLa26, and SlTGLa13. These results provide valuable insights into the roles of the SlTGL genes family and lay a foundation for further functional studies on the linkage between triacylglycerol catabolism and abiotic stress responses in tomato.

A tomato dynein light chain gene SlLC6D is a negative regulator of chilling stress.

Dynein light chain (DLC) proteins are an important component of dynein complexes, which are widely distributed in plants and animals and involved in a variety of cellular processes. The functions of DLC genes in plant chilling stress remain unclear. In this study, we isolated a DLC gene from tomato, designated SlLC6D. Promoter analysis revealed many cis-elements involved in abiotic stress in the SlLC6D promoter. Expression of SlLC6D was induced by heat and salt stress, and inhibited by polyethylene glycol and chilling stress. Knockdown of SlLC6D in tomato exhibited low relative electrolyte leakage, malondialdehyde content, and reactive oxygen species (ROS) accumulation under chilling stress. The content of proline and activities of superoxide dismutase and peroxidase in knockdown lines were higher than in the wild type and overexpression lines during chilling stress. The high transcript abundances of three cold-responsive genes were detected in knockdown lines in response to chilling stress. Seedling growth of knockdown lines was significantly higher than that of the wild type and overexpression lines under chilling stress. These results suggest that SlLC6D is a negative regulator of chilling stress tolerance, possibly by regulating ROS contents and the ICE1-CBF-COR pathway.

A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato.

Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Specifically, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identified a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of SlGME1, which encodes GDP-Man-3',5'-epimerase, a pivotal enzyme in the D-mannose/L-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the D-mannose/L-galactose pathway. Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.

Modification of Serine 1040 of SIBRI1 Increases Fruit Yield by Enhancing Tolerance to Heat Stress in Tomato.

High temperature is a major environmental factor that adversely affects plant growth and production. SlBRI1 is a critical receptor in brassinosteroid signalling, and its phosphorylation sites have differential functions in plant growth and development. However, the roles of the phosphorylation sites of SIBRI1 in stress tolerance are unknown. In this study, we investigated the biological functions of the phosphorylation site serine 1040 (Ser-1040) of SlBRI1 in tomato. Phenotype analysis indicated that transgenic tomato harbouring SlBRI1 dephosphorylated at Ser-1040 showed increased tolerance to heat stress, exhibiting better plant growth and plant yield under high temperature than transgenic lines expressing SlBRI1 or SlBRI1 phosphorylated at Ser-1040. Biochemical and physiological analyses further showed that antioxidant activity, cell membrane integrity, osmo-protectant accumulation, photosynthesis and transcript levels of heat stress defence genes were all elevated in tomato plants harbouring SlBRI1 dephosphorylated at Ser-1040, and the autophosphorylation level of SlBRI1 was inhibited when SlBRI1 dephosphorylated at Ser-1040. Taken together, our results demonstrate that the phosphorylation site Ser-1040 of SlBRI1 affects heat tolerance, leading to improved plant growth and yield under high-temperature conditions. Our results also indicate the promise of phosphorylation site modification as an approach for protecting crop yields from high-temperature stress.

The tomato 2-oxoglutarate-dependent dioxygenase gene SlF3HL is critical for chilling stress tolerance.

Low temperature is a major stress that severely affects plant development, growth, distribution, and productivity. Here, we examined the function of a 2-oxoglutarate-dependent dioxygenase-encoding gene, SlF3HL, in chilling stress responses in tomato (Solanum lycopersicum cv. Alisa Craig [AC]). Knockdown (KD) of SlF3HL (through RNA interference) in tomato led to increased sensitivity to chilling stress as indicated by elevated levels of electrolyte leakage, malondialdehyde (MDA) and reactive oxygen species (ROS). In addition, the KD plants had decreased levels of proline and decreased activities of peroxisome and superoxide dismutase. The expression of four cold-responsive genes was substantially reduced in the KD plants. Furthermore, seedling growth was significantly greater in AC or SlF3HL-overexpression plants than in the KD plants under either normal growth conditions with methyl jasmonate (MeJA) or chilling stress conditions. SlF3HL appears to positively regulate JA accumulation and the expression of JA biosynthetic and signaling genes under chilling stress. Together, these results suggest that SlF3HL is a positive regulator of chilling stress tolerance and functions in the chilling stress tolerance pathways, possibly by regulating JA biosynthesis, JA signaling, and ROS levels.

An InDel in the Promoter of Al-ACTIVATED MALATE TRANSPORTER9 Selected during Tomato Domestication Determines Fruit Malate Contents and Aluminum Tolerance.

Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in crops such as tomato (Solanum lycopersicum). Here, we integrated a metabolite-based genome-wide association study with linkage mapping and gene functional studies to characterize the genetics of malate accumulation in a global collection of tomato accessions with broad genetic diversity. We report that TFM6 (tomato fruit malate 6), which corresponds to Al-ACTIVATED MALATE TRANSPORTER9 (Sl-ALMT9 in tomato), is the major quantitative trait locus responsible for variation in fruit malate accumulation among tomato genotypes. A 3-bp indel in the promoter region of Sl-ALMT9 was linked to high fruit malate content. Further analysis indicated that this indel disrupts a W-box binding site in the Sl-ALMT9 promoter, which prevents binding of the WRKY transcription repressor Sl-WRKY42, thereby alleviating the repression of Sl-ALMT9 expression and promoting high fruit malate accumulation. Evolutionary analysis revealed that this highly expressed Sl-ALMT9 allele was selected for during tomato domestication. Furthermore, vacuole membrane-localized Sl-ALMT9 increases in abundance following Al treatment, thereby elevating malate transport and enhancing Al resistance.

The tomato HD-Zip I transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of the D-mannose/L-galactose pathway.

Ascorbate (AsA) is an antioxidant that can scavenge the reactive oxygen species (ROS) produced when plants encounter stressful conditions. Here, it was revealed by a yeast one-hybrid assay that a tomato (Solanum lycopersicum) HD-Zip I family transcription factor, SlHZ24, binds to the promoter of an AsA biosynthetic gene encoding GDP-D-mannose pyrophosphorylase 3 (SlGMP3). Both the transient expression system and electrophoretic mobility shift assay (EMSA) showed that SlHZ24 binds to a regulatory cis-element in the SlGMP3 promoter, and further overexpression of SlHZ24 in transgenic tomato lines resulted in increased AsA levels. In contrast, suppressing expression of the gene using RNA interference (RNAi) had the opposite effect. These data suggest that SlHZ24 can positively regulate the accumulation of AsA, and in support of this it was shown that SlGMP3 expression increased in the SlHZ24-overexpressing lines and declined in SlHZ24-RNAi lines. SlHZ24 also affected the expression of other genes in the D-mannose/L-galactose pathway, such as genes encoding GDP-mannose-3',5'-epimerase 2 (SlGME2), GDP-L-galactose phosphorylase (SlGGP) and SlGMP4. The EMSA indicated that SlHZ24 bound to the promoters of SlGME2 and SlGGP, suggesting multi-targeted regulation of AsA biosynthesis. Finally, SlHZ24-overexpressing plants showed less sensitivity to oxidative stress; we therefore conclude that SlHZ24 promotes AsA biosynthesis, which in turn enhances oxidative stress tolerance.

Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis.

Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

Genome-wide analysis of plant-specific Dof transcription factor family in tomato.

The Dof (DNA binding with One Finger) family encoding single zinc finger proteins has been known as a family of plant-specific transcription factors. These transcription factors are involved in a variety of functions of importance for different biological processes in plants. In the current study, we identified 34 Dof family genes in tomato, distributed on 11 chromosomes. A complete overview of SlDof genes in tomato is presented, including the gene structures, chromosome locations, phylogeny, protein motifs and evolution pattern. Phylogenetic analysis of 34 SlDof proteins resulted in four classes constituting six clusters. In addition, a comparative analysis between these genes in tomato, Arabidopsis and rice was also performed. The tomato Dof family expansion has been dated to recent duplication events, and segmental duplication is predominant for the SlDof genes. Furthermore, the SlDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth conditions. This is the first step towards genome-wide analyses of the Dof genes in tomato. Our study provides a very useful reference for cloning and functional analysis of the members of this gene family in tomato and other species.

Over-expression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant.

Plant microRNAs (miRNAs) are vital components of the translation control system that regulates plant development and reproduction. The biological function of sly-miR156 was investigated by over-expression in tomato plants. Transgenic tomato plants exhibited a drastically altered phenotype, with reduced height, smaller but more numerous leaves, and smaller fruit. The inflorescence structure of sly-miR156 over-expressing plants phenocopied the sft mutant. The putative targets of sly-miR156 were identified by data base search and included six SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box transcription factor genes. Their expression patterns were then determined in 35S-miR156a and wild type tomato plants. These target genes, as well as the tomato FLOWERING LOCUS T (FT) ortholog SFT, were significantly down-regulated in sly-miR156 over-expressing plants. These studies reveal novel phenotypes regulated by miR156.