Changxian Yang

Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato

To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (β-amylase), methionine biosynthesis (cystathionine β-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato.

A regulatory gene induces trichome formation and embryo lethality in tomato

Trichomes are universal biological structures originating from the aerial epidermis, which serve as an excellent model to study plant differentiation at the cell level. Although the pathway regulating trichome formation in the Rosids has been well characterized, only very recently a few genes were identified for trichome initiation in the Asterids. In this study, we cloned Woolly (Wo), essential for trichome formation in tomato. Transgenic experiments revealed that the woolly phenotype is caused by the mutation in Wo which encodes a homeodomain protein containing a bZIP motif and a START domain. We identified three alleles of Wo and found that each allele contains a missense mutation, which respectively results in an amino acid substitution at the C terminus. Microarray and expression analysis showed that the expression of a B-type cyclin gene, SlCycB2, is possibly regulated by Wo, which also participates in trichome formation. Suppression of Wo or SlCycB2 expression by RNAi decreased the number of type I trichomes, and direct protein–protein interaction was detected between them, implying that both proteins may work together in the regulation of this type of trichome formation. Cytological observation and Wo transcript analysis in the developing seeds showed that embryo development was also correlated with Wo.

A STAY-GREEN protein SlSGR1 regulates lycopene and β-carotene accumulation by interacting directly with SlPSY1 during ripening processes in tomato

As a primary source of lycopene in the human diet, fleshy fruits synthesize this compound both de novo and via chlorophyll metabolism during ripening. SlSGR1 encodes a STAY-GREEN protein that plays a critical role in the regulation of chlorophyll degradation in tomato leaves and fruits. We report that SlSGR1 can regulate tomato (Solanum lycopersicum) lycopene accumulation through direct interaction with a key carotenoid synthetic enzyme SlPSY1, and can inhibit its activity. This interaction with SlSGR1 mediates lycopene accumulation during tomato fruit maturation. We confirmed this inhibitory activity in bacteria engineered to produce lycopene, where the introduction of SlSGR1 reduced dramatically lycopene biosynthesis. The repression of SlSGR1 in transgenic tomato fruits resulted in altered accumulation patterns of phytoene and lycopene, whilst simultaneously elevating SlPSY1 mRNA accumulation and plastid conversion at the early stages of fruit ripening, resulting in increased lycopene and β-carotene (four- and nine-fold, respectively) in red ripe fruits. SlSGR1 influences ethylene signal transduction via the altered expression of ethylene receptor genes and ethylene-induced genes. Fruit shelf-life is extended significantly in SlSGR1-repressed tomatoes. Our results indicate that SlSGR1 plays a pivotal regulatory role in color formation and fruit ripening regulation in tomato, and further suggest that SlSGR1 activity is mediated through direct interaction with PSY1.

Reducing AsA leads to leaf lesion and defence response in knock-down of the AsA biosynthetic enzyme GDP-D-mannose pyrophosphorylase gene in tomato plant.

As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

Identification and Expression Pattern of a ZPR1 Gene in Wild Tomato ( Solanum Pennellii )

The mammalian zinc finger protein ZPR1 is known to be essential in cell cycle progression, but its homolog in plants remains to be identified. Here, we isolated SpZPR1 from the wild tomato species Solanum pennellii LA0716 and found that its protein sequence shares homology with ZPR1 genes from other organisms. SpZPR1 mRNA was found to accumulate in tomato apical meristem in response to ABA (abscisic acid) treatment and heat stress. A transactivation activity assay in yeast revealed that SpZPR1 functions as a transcriptional activator, while the results of our gel electrophoresis mobility shift assay showed that SpZPR1 was able to bind the ABRE (ABA-responsive element) sequence in vitro. Our study is the first to provide evidence that SpZPR1 is involved in the ABA signaling network and plays a potential role in plant cell development and abiotic stress response.

Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato

Homeodomain leucine zipper IV (HD-ZIP IV) proteins are plant-specific transcription factors that play important roles in development of epidermal cell layers and cuticle formation. The functions of two HD-ZIP IV family genes, CD2 and Wo, have been well characterized in tomato (Solanum lycopersicum). CD2 and Wo are involved in cuticle biosynthesis and trichome formation, respectively. In this study, we identified 13 novel tomato HD-ZIP IV (SlHDZIV) genes. We analyzed the structures, chromosome locations, phylogeny, protein motifs, and expression profiles of these SlHDZIV genes. Gene structure analysis revealed that a module of 11 exons and 10 introns existed in the SlHDZIV genes. These genes were asymmetrically distributed on chromosomes, except on chromosome 4 and 5. Segmental duplication possibly contributed to the expansion of tomato HD-ZIP IV genes. The expression profiles of these genes revealed their broad expression pattern and high expression in young leaves and flowers. Each gene responded to more than one of different phytohormones [abscisic acid, ethephon, 4-(indolyl)-butyric acid, jasmonic acid, salicylic acid, gibberellic acid, and 6-benzylaminopurine] and four abiotic stress treatments (cold, heat, salt, and drought). This study provided significant insights into the diverse roles of SlHDZIV genes in tomato growth and development.

The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense

Cyclins exist extensively in various plant species. Among them, B-type cyclins play important roles in the transition of G2-to-M. However, few B-type cyclins have been reported to participate in reproductive organ development and trichome formation. In this study, transgene analysis showed that SlCycB2 overexpression caused abnormal flower with the unclosed stamen, shortened style and aberrant pollen. In addition, nearly all non-glandular trichomes, as well as the glandular ones were disappeared. On the contrary, suppression of SlCycB2 could promote type III and type V trichomes formation. Detection of secondary metabolites indicated that the production of monoterpene and sesquiterpene were significantly decreased in SlCycB2-OE plants, which thus resulted in the reduction of the defense against Prodenia litura. Transcriptome profile demonstrated that the differentially expressed genes mainly participate in the biosynthesis of terpenes, cutin, suberine and wax. Furthermore, we identified several homologs of SlCycB2, SlCycB3, NtCycB2, AtCycB2, which have similar regulatory functions in trichome formation. These results indicate that SlCycB2 plays a critical role in reproductive organ development, multicellular trichome initiation, secondary metabolite biosynthesis and Prodenia litura defense in tomato. The similar roles of its homologs in multicellular trichome formation suggest that Solanaceous species may share common regulatory pathway.

HyPRP1 Gene Suppressed by Multiple Stresses Plays a Negative Role in Abiotic Stress Tolerance in Tomato

Many hybrid proline-rich protein (HyPRP) genes respond to biotic and abiotic stresses in plants, but little is known about their roles other than as putative cell-wall structural proteins. A HyPRP1 gene encodes a protein with proline-rich domain, and an eight-cysteine motif was identified from our previous microarray experiments on drought-tolerant tomato. In this study, the expression of the HyPRP1 gene in tomato was suppressed under various abiotic stresses, such as drought, high salinity, cold, heat, and oxidative stress. Transgenic functional analysis showed no obvious changes in phenotypes, but enhanced tolerance to various abiotic stresses (e.g., oxidative stress, dehydration, and salinity) was observed in RNAi transgenic plants. Interestingly, several SO2 detoxification-related enzymes, including sulfite oxidase, ferredoxins (Fds), and methionine sulfoxide reductase A (Msr A), were revealed in HyPRP1-interacting proteins identified by Yeast Two-Hybrid screening. More sulfates and transcripts of Msr A and Fds were accumulated in HyPRP1 knockdown lines when wild-type plants were exposed to SO2 gas. Our findings illustrate that the tomato HyPRP1 is a negative regulator of salt and oxidative stresses and is probably involved in sulfite metabolism.

Overexpression of SlRBZ Results in Chlorosis and Dwarfism through Impairing Chlorophyll, Carotenoid, and Gibberellin Biosynthesis in Tomato

ZFPs play important roles in many biological processes, including plant development, stress response, and phytohormone response. RanBP2-type zinc finger transcription factors have been characterized in animals and humans. However, their functions remain largely unknown in plants. In this study, we identified a RanBP2-type zinc finger protein gene (SlRBZ) in tomato. SlRBZ was constitutively expressed in roots, stems, leaves, flowers, and fruits. The SlRBZ-GFP fused protein was localized in the nucleus. Overexpression of SlRBZ resulted in chlorosis and dwarf phenotypes in tomato. Determination of physiological index showed that chlorophyll, carotenoid, and GAs contents were evidently decreased in transgenic plants. Furthermore, the qRT-PCR and RNA-Seq analyses demonstrated that the transcription of the genes involved in these biosynthesis pathways obviously decreased in SlRBZ-OE plants. In addition, ultrastructural observation by transmission electron microscopy indicated that plastids could not develop into mature chloroplasts with normal chloroplast membrane and thylakoid membrane system in SlRBZ-OE plants. The results suggest that overexpression of SlRBZ may impair the biosynthesis of chlorophyll, carotenoid, and gibberellin through blocking chloroplast development, resulting in chlorosis and dwarfism in tomato.

Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome-wide association study (GWAS) on type-I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map-based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc-finger protein. Transgenic experiments showed that hair-absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type-I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein-protein interaction between them was further detected through pull-down and yeast two-hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.