Transcriptome Analysis Of Cold-Tolerant Tomato Germplasm Under Cold Stress And Functional Characterization Of Cold Responsive Genes

The cultivated tomato(Solanum lycopersicum) is unable to cold acclimate, and easily suffers cold injury at all stages of plant growth and development. Several practical benefits of increased cold tolerance in cultivated tomato would be:prevention of plant and fruit damage from cold stress, extension of the growing season, and reduction of input of facility cultivation. Thus, screening of cold tolerant tomato resources, and breeding new varieties that can tolerate cold stress have important practical value.The C-repeat binding factor (CBF) cold response pathway is currently the best documented system that plays a pivotal role in gene regulation during cold acclimation. Tomato also has the CBF cold response pathway, but its function appears to be considerably smaller. The molecular basis of cold adaptation in non-cold-acclimated tomato may differ from that of the cold-acclimated plants.The wild tomato S. habrochaites is more tolerant to low temperature than cultivated tomato. Significant progress has been made in the past decade in elucidating the differences in physiological responses under cold stress between S. habrochaites and S. lycopersicum. However, the differences in global gene expression under cold stress between the two genotypes and the molecular mechanisms responsible for cold tolerance in S. habrochaites are largely unknown.In this study, seedlings of S. habrochaites LA1777, S. lycopersicum LA4024, and introgression lines (ILs) of them, were evaluated for their tolerance to low temperature. To explore the molecular mechanisms of cold tolerance in tomato, the TOM2array was used to compare the transcriptome differences between the tolerant genotypes (the donor parent LA1777and the sleeted IL LA3969) and the sensitive one (the recurrent parent LA4024) under cold stress. Based on the microarray results, three cold-responsive genes were cloned from S. habrochaites and functionally analyzed. The main results are as follows:1. Identification of chromosomal regions conferring cold tolerance in S. habrochaites. A system for cold tolerance evaluation of the tomato seedlings was developed by comparing phenotypic and physiological responses of seedlings of S. habrochaites LAI777and S. lycopersicum LA4024under cold stress. Seedlings of LAI777ILs were evaluated under low temperature (4℃) using this system. Twenty-two ILs with S. habrochaites introgressions on chromosomes1,2,3,4,5,6,7,9,11, and12exhibited less severe wilting than LA4024after3d of cold treatment. Among these, the phenotypic performance of LA3969was quite close to that of LA1777during cold stress and recovery, and showed stronger cold tolerance than LA4024and other ILs. LA3969and LA1777suffered less membrane damage during cold stress and showed significantly higher survival rates than LA4024after10d of cold stress and recovery for one week. LA3969contains a large introgressed segment from S. habrochaites on chromosome12. This indicates that at least one major QTL/gene responsible for cold tolerance is located on S. habrochaites chromosome12.2. The molecular differences between tolerant and sensitive tomato in reponse to cold stress. Transcriptome analysis of LA1777, LA3969, and LA4024seedlings under cold stress were performed using TOM2array. After3d of cold stress (4℃), a total of1613,1456, and1523cold-responsive genes were identified in LA1777, LA3969, and LA4024, respectively. Among these,103cold-responsive genes were exclusively identified in both LA1777and LA3969, whereas196cold-responsive genes were uniquely observed in LA4024. These genes may play important roles in conferring tolerance or sensitivity to chilling in tomato. Functional classification of cold responsive genes showed that more genes involved in ’response to stress’,’response to endogenous stimulus’,’signal transduction’,’transcription’,’biosynthetic process’,’secondary metabolic process’, and ’protein metabolic process’ were up-regulated in the two tolerant genotypes, whereas more genes involved in photosynthesis were down-regulated in the sensitive genotype. 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 of7biochemical pathways varied significantly between tolerant and sensitive genotypes under cold stress, including jasmonic acid biosynthesis, brassinosteroid metabolic process, phenylpropanoid biosynthesis, starch degradation, leucine biosynthesis, Calvin cycle, and removal of superoxide radicals.3. The molecular mechanisms of cold tolerance in tomato. A total of92cold-responsive genes with statistically significant differences in expression between the two tolerant and sensitive genotypes were identified. In addition, the expression of126 genes in L1777showed significantly different from that in LA3969and LA4024under cold stress. These genes may play important roles in conferring cold tolerance in LA3969and/or LA1777. Among these,80genes were located on the introgressed chromosomal segments of the22selected cold-tolerant ILs and/or cold tolerance QTLs identified previously in S. habrochaites. Of these,11genes were localized to the introgressed chromosomal segment of LA3969. These genes may play critical roles in conferring cold tolerance in LA3969. GO term enrichment analysis showed that many genes involved in calcium-mediated signaling, ROS and hormone homeostasis were differentially expressed. Calcium, ROS, and hormones as signaling molecules may play important roles in regulating gene expression in response to cold stress in tomato. The modulation of these signaling pathways caused differential expression of many transcripts between tolerant and sensitive genotypes under cold stress, including transcription factors (e.g., MYBs, HSFs, and NACs), post-translational modification proteins (e.g., SKP2A, LAP-A1, and XERICOs), functional proteins (e.g., HSPs, PRs, and dehydrin), and metabolic enzymes (e.g., GSTs, LOXs, and BAM), and etc. These specific modifications make LA1777and LA3969more cold tolerant than LA4024.4. Functional characterization of differentially expressed gene ShDNN. Based on the microarray results, a dehydrin gene was isolated form S. habrochaites and designated ShDHN. Both microarray and quantitative real-time RT-PCR analysis indicated that the expression of this gene was more strongly induced by cold stress in the tolerant genotype than the sensitive one. In addition, ShDHN expression was also induced by drought, salt, osmotic stress, ABA, and MeJA. Overexpression of ShDHN in LA4024increased tolerance to cold and drought stress, and improved the early seedling development under salt and osmotic stress. Compared with the wild type, the transgenic lines accumulated more proline, maintained higher activities of SOD and CAT, and suffered less membrane damage under cold and drought stress. Under cold stress, the accumulation of H2O2and O2-was less in the transgenic plants than in the wild type. The expression of SOD1, GST, and PR1were increased in the ShDHN overexpression lines, while the transcripts of POD, LOX, and PR2were inhibited. These results indicate ShDHN confers abiotic stress tolerance by enhancing ROS scavenging capacity, accumulating higher amounts of compatible solutes, and regulating other signaling pathways and genes expression.5. Functional characterization of differentially expressed gene ShCHL P. Based on the microarray results, a geranylgeranyl reductase gene, was isolated from LA1777and designated ShCHL P. ShCHL P is highly expressed in the leaf and stem, and nearly no expression in root. Its expression was suppressed by drought, salt, low or high temperature, and oxidative stress. The overexpression vector of ShCHL P was constructed and translated into the cultivated tomato LA4024. Interestingly, we got both overexpression and co-suppression of CHL P in transgenic tomato plants. Overexpression of ShCHL P increased the leaf chlorophyll content, improved the early seedling development under normal, slat and osmotic stress conditions. Whereas the leaves of the co-suppression lines were yellow, the contents of chlorophyll in leaf, stem, and even fruit were decreased, and the early seedling development was significantly inhibited in co-suppression lines under slat and osmotic stress conditions. Both overexpression and co-suppression of CHL P transgenic plants enhanced oxidative stress tolerance. The molecular machines lead to this need to be further analysis. The results indicate that CHL P is required for chlorophyll biosynthesis, and it plays an important role in growth and development, and abiotic stress response in plants.6. Functional analysis of differentially expressed transcription factor ShNAC. Based on the microarray results, a NAC transcription factor (ShNAC) was isolated from LA1777. ShNAC encodes a protein of405amino acids, which has a conserved NAM domain in N-terminal. ShNAC is constitutively expressed in various tissues of LA1777, and the expression in fruit and flower is the highest, whereas the transcript abundance in stem is the lowest. Under normal growth condition, the plant height of ShNAC overexpression lines was significant lower than that of the wild type. The bottom of stem of the transgenic plant was flexible, and the plant was unable to stand erect. The ShNAC transgenic plants were hypersensitive to drought and cold stress compared with wild type. The results indicate ShNAC may act a negative regulator of growth and development, and abiotic stress responses in tomato.