The Study On Solanum Lycopersicum, Solanum Habrochaites, And Solanum Lycopersicoides Under Low Temperature Base On Transcriptomes

Many plants increase their freezing tolerance in response to low temperatures. This phenomenon is known as cold acclimation. Plants differ greatly in their abilities to cope with freezing temperatures. Those plants that have adapted to temperate environments generally increase in freezing tolerance in response to low nonfreezing temperatures. In contrast, plants that have adapted to tropical and subtropical climates, such as tomato, generally have little chilling tolerance and are unable to acclimate to cold temperatures. The roles of cold regulated genes in plants cold acclimation show that different expression of genes are related to different abilities of plants cold adaption. Moreover, some studies have demonstrated that different expression of cold-responsive gene due to differences in cold tolerance in plants. Alternative splicing（AS） has been confirmed widely at the functional level in A. thaliana, rice, and maize. AS can be regulated spatially and developmentally under environmental stress, so AS could play an important role under cold stress or other abiotic stress. Many studies have indicated that the key role of mi RNAs in regulating organ development and biological processes. mi RNAs are also related with abiotic stress responses. Solanum lycopersicum, Solanum habrochaites, and Solanum lycopersicoides are closely related plant species that differ in their abilities to cold acclimate; whereas the wild species Solanum habrochaites, and Solanum lycopersicoides is more freeze-tolerant than the cultivated tomato S. lycopersicum.The low temperature transcriptomes of S. lycopersicum, S. habrochaites, and S. lycopersicoides leaf tissue was studied using an Illumina platform for ultra-high-throughput RNA sequencing（RNA-seq）. More than 200 million reads were mapped to define genes, Micro RNAs（mi RNAs） and alternative splicing events（AS） and to quantify transcript abundance in response to cold stress. De novo assembly was performed using the Trinity method with default parameters. The sequence reads were finally assembled into 68,051 non-redundant unigenes from S. habrochaites c DNA libraries. The sequence reads were finally assembled into 59,286 non-redundant unigenes from S. lycopersicoides c DNA libraries. All unigenes were longer than 200 bp.The results indicated that three species alter gene expression in response to cold stress. In S. lycopersicum, transcripts for 1,256 and 3,350 ESTs increased at 1 and 12 h, respectively, and 804 ESTs increased at both time points tested; transcripts for 856 and 3,022 ESTs decreased at 1 and 12 h, respectively, and 339 ESTs decreased at both time points tested. In S. habrochaites, transcripts IV for 1,725 and 2,940 ESTs increased at 1 and 12 h, respectively, and 722 ESTs increased at both time points tested; transcripts for 1,967 and 3,126 ESTs decreased at 1 and 12 h, respectively, and 1,000 ESTs decreased at both time points tested. In S. lycopersicoides, transcripts for 617 and 1,928 ESTs increased at 1 and 12 h, respectively, and 136 ESTs increased at both time points tested; transcripts for 1,066 and 1,171 ESTs decreased at 1 and 12 h, respectively, and 185 ESTs decreased at both time points tested. In sum, the expression profiling experiments indicated that in S. lycopersicum, S. habrochaites, and S. lycopersicoides, gene expression was altered in response to low temperature to dissimilar degrees. To verify the correctly of the RNA-Seq data, some increased expressed genes, decreased expressed genes, and non-differentially expressed genes were choose for Real-time PCR（q RT-PCR） under cold stress. The results indicated that the expression patterns of these genes in q RT-PCR were similar with that in RNA-Seq.To understand the molecular mechanism why S. habrochaites can survive freezing temperatures, we report the results of an GO analysis by DAVID tool. We analyzed the genes that we determined to be responsive to cold at 1 h. The GO terms enriched in each species were comparable and were generally related to “response to abiotic stimulus”. From the heat-map, it was also obvious that S. lycopersicum was less affected by cold than S. habrochaites at 1 h. Genes which enriched in GO categories corresponding to “cell wall metabolism” were increase expressed under cold stress in S. lycopersicum, but the opposite result was observed in S. habrochaites. We observed a similar contrast in the GO category “response to organic substance”. In the GO categories “response to chitin”, “response to carbohydrate stimulus” and “DNA-binding WRKY”, there was a significant enrichment in S. lycopersicum, but S. habrochaites showed no enrichment. For the categories “chloroplast”, “transit peptide”, “pentatricopeptide repeat”, “phenylpropanoid metabolic process”, “flavonoid metabolic process”, and “amino acid derivative biosynthetic process”, no significant enrichment was observed in S. lycopersicum, but enrichment was observed in S. habrochaites. We next compared responses to cold at 12 h. Analysis of GO terms for cold-regulated gene, suggested that the categories “response to organic substance” and “response to hormone stimulus” were enriched in both S. lycopersicum and S. habrochaites（Additional file 3）. In the case of the GO category “UDP-glucuronosyl/ UDP-glucosyltransferase”, there was significant enrichment for S. lycopersicum, but S. habrochaites showed no enrichment.An AS analysis identified 75,885 novel splice junctions of 172,910 total splice junctions and suggested that the relative abundance of isoforms of S. lycopersicum and S. habrochaites significantly shift under cold stress. Using RNA-seq data, we identified 105,663, 109,251, 102,316, 106,690, 104,440, and 105,323 splicing junctions in samples C0, C1, C12, Tsh0, Tsh1, and Tsh12 with 21,548, 25,492, 22,870, 20,909, 19,957, and 23,179 novel junctions, respectively. We categorized each AS events using the primary known types of AS and the sequencing data. We found that intron retention was the primary type of AS. We tried to identify difference in altered AS events between the two species at 0, 1, and 12 h of cold treatment at 4°C. 131（sample C1 vs. C0）, 575（sample C12 vs. C0）, 114（sample Tsh1 vs. Tsh0）, and 606（sample Tsh12 vs. Tsh0） of the AS events were increased under cold stress; 119（sample C1 vs. C0）, 152（sample C12 vs. C0）, 122（sample Tsh1 vs. Tsh0）, and 130（sample Tsh12 vs. Tsh0） events were decreased. 121（sample C1 vs. C0）, 522（sample C12 vs. C0）, 112（sample Tsh1 vs. Tsh0）, and 553（sample Tsh12 vs. Tsh0） of the AS genes were increased under cold stress; 110（sample C1 vs. C0）, 140（sample C12 vs. C0）, 111（sample Tsh1 vs. Tsh0）, and 122（sample Tsh12 vs. Tsh0） of the genes were decreased. We compared the functions of the AS genes that were regulated in response to cold at 1 h and 12 h were compared with the DEG.In addition, we identified a total of 1041 mi RNA sequences, which may regulate relevant target genes. In S. lycopersicum, transcripts for 14 and 8 mi RNAs increased at 1 and 12 h, respectively; transcripts for 7 and 6 mi RNAs decreased at 1 and 12 h, respectively. In S. habrochaites, transcripts for 2 and 4 mi RNAs increased at 1 and 12 h, respectively; transcripts for 5 and 8 mi RNAs decreased at 1 and 12 h, respectively. In S. lycopersicoides, transcripts for 151 and 59 mi RNAs increased at 1 and 12 h, respectively; transcripts for 151 and 20 mi RNAs decreased at 1 and 12 h. To further characterize the role of the mi RNAs in the cold response, we examined the target list for genes that could be related to the cold response and that were either induced or repressed by cold based on our Illumina results. Our data indicated that e.g., sly-mi R396a- Solyc07g041640.2（S. lycopersicum under cold 1h）, sly-mi R5303bSolyc09g009550.2（S. lycopersicum under cold 12h）, sly-mi R162- Solyc01g080500.2（S. habrochaites under cold 1h）, sly-mi R172a-Solyc03g044300.2（S. habrochaites under cold 12h）, unconservative_SL2.50ch00₃860- c50426.graph_c0（S. lycopersicoides under cold 1h）, sly-mi R156a- c49736.graph_c0（S. lycopersicoides under cold 12h） play roles in response to cold stress. Thus, differences in cold regulatory programs may contribute to the differences in the freezing tolerance of these three species.