Project description:Herein, we identified the tomato <i>SlMYB102</i> gene as a MYB family transcription factor of the R2R3-MYB subfamily. We additionally determined that the <i>SlMYB102</i> promoter region contains photoresponsive, abiotic stress-responsive, and hormone-responsive regulatory elements, and we detected higher <i>SlMYB102</i> expression in the reproductive organs of tomato than that in vegetative organs, with the expression being highest in ripe fruits and in roots. <i>SlMYB102</i> expression was also shown to be cold-inducible. The protein encoded by <i>SlMYB102</i> localized to the nucleus wherein it was found to mediate the transcriptional activation of target genes through its C-terminal domain. Overexpression of <i>SlMYB102</i> in tomato plants conferred enhanced tolerance to cold stress. Under such cold stress conditions, we found that proline levels in the leaves of <i>SlMYB102</i> overexpressing transgenic plants were higher than those in WT plants. In addition, <i>S1MYB102</i> overexpression was associated with the enhanced expression of cold response genes including <i>SlCBF1, SlCBF3, SlDREB1, SlDEB2,</i> and <i>SlICE1</i>. We also found that the overexpression of <i>SlMYB102</i> further enhanced the cold-induced upregulation of <i>SlP5CS</i> and <i>SlAPX2.</i> Taken together, these results suggest that <i>SlMYB102</i> may be involved in the C-repeat binding transcription factor (CBF) and proline synthesis pathways, thereby improving tomato plant cold resistance.
Project description:Our containment trials have established cold tolerance in Nicotiana tabacum osmotin (Nt Osm) transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby). Though, the stress tolerance mechanisms have been studied at physio-biochemical levels, molecular mechanisms underlying the tolerant response are still not well studied. Therefore, quantitative transcript expression of Osmotin and other stress responsive genes (CBF1, P5CS and APX) was studied in response to cold (4°C; 2 and 24 h) treatment in the transgenic and wild type tomato plants. The expression analysis revealed differential transcript regulation in the transgenic and wild type plants on the cold exposure. In general, the genes were either earlier induced or the extent of fold change in transcript expression over the respective untreated controls was higher in transgenic than in the wild type plants on cold exposure. The transcript expression data also supported the metabolite analysis on free Proline and ascorbate content. The results thus suggest that constitutive over expression of the Osmotin modulate transcript abundance and functional expression products of the other stress responsive genes thereby, imparting cold tolerance in the transgenic tomato plants.
Project description:To understand the gene network that controls plant tolerance to cold stress, we carried out a near full genome transcript expression profiling in Arabidopsis using Affymetrix GeneChips that contain approximately 24,000 genes. For microarray analysis, Arabidopsis seedlings were cold treated at 0 C for 0 h, 3 h, 6 h, and 24 h. A total of 939 genes were statistically determined to be cold-regulated with 655 being up-regulated and 284 down-regulated. A large number of the early cold-responsive genes encode transcription factors that likely control late-responsive genes, which implies a multitude of transcriptional cascades. In addition, many genes involved in post-transcriptional and chromatin level regulation were also cold regulated suggesting their involvement in cold responsive gene regulation. A number of genes important for the biosynthesis or signaling of plant hormones, such as abscisic acid, gibberellic acid and auxin, are regulated by cold stress, which is of potential importance in coordinating cold tolerance with growth and development. We compared the cold-responsive transcriptomes of wild type and ice1, a mutant defective in an upstream transcription factor required for chilling and freezing tolerance. The transcript levels of many cold-responsive genes were altered in the ice1 mutant not only during cold stress conditions, but also before cold treatments. Our study provides a global picture of the Arabidopsis cold-responsive transcriptome and its control by ICE1, and thus will be valuable for understanding gene regulation under cold stress and the molecular mechanisms of cold tolerance. Experiment Overall Design: Two replicates for each time point of 0 hours, 3 hours, 6 hours and 24 hours of cold treatment for the wildtype (control) and ice1 (mutant).
Project description:ICE1 transcription factor plays an important role in plant cold stress via regulating the expression of stress-responsive genes. In this study, a PuICE1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. The expression levels of the PuICE1 were induced by cold, dehydration and salt, with the greatest induction under cold conditions. PuICE1 was localized in the nucleus and could bind specifically to the MYC element in the PuDREBa promoter. The PuICE1 fused to the GAL4 DNA-binding domain to have transcriptional activation activity. Ectopic expression of the PuICE1 in tomato conferred enhanced tolerance to cold stress at cold temperatures, less electrolyte leakage, less MDA content, higher chlorophyll content, higher survival rate, higher proline content, higher activities of enzymes. In additon, steady-state mRNA levels of six stress-responsive genes coding for either functional or regulatory genes were induced to higher levels in the transgenic lines by cold stress. Yeast two-hybrid, transient assay, split luciferase complementation and BiFC assays all revealed that PuHHP1 protein can physically interact with PuICE1. Taken together, these results demonstrated that PuICE1 plays a positive role in cold tolerance, which may be due to enhancement of PuDREBa transcriptional levels through interacting with the PuHHP1.
Project description:<h4>Background</h4>Saussurea involucrata survives in extreme arctic conditions and is very cold-resistant. This species grows in rocky, mountainous areas with elevations of 2400-4100?m, which are snow-covered year-round and are subject to freezing temperatures. S. involucrata's ability to survive in an extreme low-temperature environment suggests that it has particularly high photosynthetic efficiency, providing a magnificent model, and rich gene pool, for the analysis of plant cold stress response. Fructose-1, 6-bisphosphate aldolase (FBA) is a key enzyme in the photosynthesis process and also mediates the conversion of fructose 1, 6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and glycerol triphosphate (GAP) during glycolysis and gluconeogenesis. The molecular mechanisms underlying S. involucrata's cold tolerance are still unclear; therefore, our work aims to investigate the role of FBA in plant cold-stress response.<h4>Results</h4>In this study, we identified a cold-responsive gene, SiFBA5, based on a preliminary low-temperature, genome-wide transcriptional profiling of S. involucrata. Expression analysis indicated that cold temperatures rapidly induced transcriptional expression of SiFBA5, suggesting that SiFBA5 participates in the initial stress response. Subcellular localization analysis revealed that SiFBA5 is localized to the chloroplast. Transgenic tomato plants that overexpressed SiFBA5 were generated using a CaMV 35S promoter. Phenotypic observation suggested that the transgenic plants displayed increased cold tolerance and photosynthetic efficiency in comparison with wild-type plants.<h4>Conclusion</h4>Cold stress has a detrimental impact on crop yield. Our results demonstrated that SiFBA5 positively regulates plant response to cold stress, which is of great significance for increasing crop yield under cold stress conditions.
Project description:BACKGROUND: Since its discovery more than 100 years ago, potato (Solanum tuberosum) tuber cold-induced sweetening (CIS) has been extensively investigated. Several carbohydrate-associated genes would seem to be involved in the process. However, many uncertainties still exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated with CIS, such as beta-amylases and invertases, have still to be identified at a sequence level. In addition, little is known about the early events that trigger CIS and on the involvement/association with CIS of genes different from carbohydrate-associated genes. Many of these uncertainties could be resolved by profiling experiments, but no GeneChip is available for the potato, and the production of the potato cDNA spotted array (TIGR) has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated with CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to identify which potato cold-responsive gene family members should be further studied in detail by Real-Time (RT)-PCR (qPCR). RESULTS: A tomato-potato Global Match File was generated for the interpretation of various aspects of the heterologous dataset, including the retrieval of best matching potato counterparts and annotation, and the establishment of a core set of highly homologous genes. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over 26 days. We detected biphasic behaviour of mRNA accumulation for carbohydrate-associated genes and our combined GeneChip-qPCR data identified, at a sequence level, enzymatic activities such as beta-amylases and invertases previously reported as being involved in CIS. The GeneChip data also unveiled important processes accompanying CIS, such as the induction of redox- and ethylene-associated genes. CONCLUSION: Our Global Match File strategy proved critical for accurately interpretating heterologous datasets, and suggests that similar approaches may be fruitful for other species. Transcript profiling of early events associated with CIS revealed a complex network of events involving sugars, redox and hormone signalling which may be either linked serially or act in parallel. The identification, at a sequence level, of various enzymes long known as having a role in CIS provides molecular tools for further understanding the phenomenon.
Project description:BACKGROUND: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. RESULTS: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5'-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. CONCLUSION: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.
Project description:The wild species Solanum habrochaites is more cold tolerant than the cultivated tomato (S. lycopersicum). To explore the mechanisms underlying cold tolerance of S. habrochaites, seedlings of S. habrochaites LA1777 introgression lines (ILs), as well as the two parents, were evaluated under low temperature (4°C). The IL LA3969 and its donor parent LA1777 were found to be more cold tolerant than the recurrent parent S. lycopersicum LA4024. The differences in physiology and global gene expression between cold-tolerant (LA1777 and LA3969) and -sensitive (LA4024) genotypes under cold stress were further investigated. Comparative transcriptome analysis identified 1613, 1456, and 1523 cold-responsive genes in LA1777, LA3969, and LA4024, respectively. Gene ontology (GO) term enrichment analysis revealed that more GO biological process terms were significantly enriched among the up-regulated genes in the two tolerant genotypes, whereas more biological processes were significantly repressed by cold stress in the sensitive one. A total of 92 genes with significant differential expression between tolerant and sensitive genotypes under cold stress were identified. Among these, many stress-related GO terms were significantly enriched, such as 'response to stimulus' and 'response to stress'. Moreover, GO terms 'response to hormone stimulus', 'response to reactive oxygen species (ROS)', and 'calcium-mediated signaling' were also overrepresented. Several transcripts involved in hormone or ROS homeostasis were also differentially expressed. ROS, hormones, and calcium as signaling molecules may play important roles in regulating gene expression in response to cold stress. Moreover, the expression of various transcription factors, post-translational proteins, metabolic enzymes, and photosynthesis-related genes was also specifically modulated. These specific modifications may play pivotal roles in conferring cold tolerance in tomato. These results not only provide new insights into the molecular mechanisms of cold tolerance in tomato, but also provide potential candidate genes for genetic improvement.
Project description:Cold stress is considered as one of the major environmental factors that adversely affects the plant growth and distribution. Therefore, there arises an immediate need to cultivate effective strategies aimed at developing stress-tolerant crops that would boost the production and minimise the risks associated with cold stress. In this study, a novel cold-responsive protein1 (BoCRP1) isolated from Brassica oleracea was ectopically expressed in a cold susceptible tomato genotype Shalimar 1 and its function was investigated in response to chilling stress. BoCRP1 was constitutively expressed in all the tissues of B. oleracea including leaf, root and stem. However, its expression was found to be significantly increased in response to cold stress. Moreover, transgenic tomato plants expressing BoCRP1 exhibited increased tolerance to chilling stress (4 °C) with an overall improved rate of seed germination, increased root length, reduced membrane damage and increased accumulation of osmoprotectants. Furthermore, we observed increased transcript levels of stress responsive genes and enhanced accumulation of reactive oxygen species scavenging enzymes in transgenic plants on exposure to chilling stress. Taken together, these results strongly suggest that BoCRP1 is a promising candidate gene to improve the cold stress tolerance in tomato.
Project description:Perceiving incoming environmental information is critical for optimizing plant growth and development. Multiple B-box proteins (BBXs) play essential roles in light-dependent developmental processes in plants. However, whether BBXs function as a signal integrator between light and temperature in tomato plants remains elusive. In this study, 31 <i>SlBBX</i> genes were identified from the newly released tomato (<i>Solanum lycopersicum</i>) genome sequences and were clustered into five subgroups. Gene structure and protein motif analyses showed relatively high conservation of closely clustered <i>SlBBX</i> genes within each subgroup; however, genome mapping analysis indicated the uneven distribution of the <i>SlBBX</i> genes on tomato chromosomes. Promoter <i>cis</i>-regulatory elements prediction and gene expression indicated that <i>SlBBX</i> genes were highly responsive to light, hormones, and stress conditions. Reverse genetic approaches revealed that disruption of <i>SlBBX7, SlBBX9</i>, and <i>SlBBX20</i> largely suppressed the cold tolerance of tomato plants. Furthermore, the impairment of <i>SlBBX7, SlBBX9</i>, and <i>SlBBX20</i> suppressed the photosynthetic response immediately after cold stress. Due to the impairment of non-photochemical quenching (NPQ), the excess photon energy and electron flow excited by low temperature were not consumed in <i>SlBBX7-, SlBBX9-</i>, and <i>SlBBX20-</i> silenced plants, leading to the over reduction of electron carriers and damage of the photosystem. Our study emphasized the positive roles of light signaling transcription factors SlBBXs in cold tolerance in tomato plants, which may improve the current understanding of how plants integrate light and temperature signals to adapt to adverse environments.