Preliminary Analysis On Molecular Mechanism Of Chilling In Upland Cotton Seedling Stage

Cotton originates from tropical and subtropical regions.During natural selection,domestication and farming,cotton gradually adapt to relative high-latitude areas,but cotton still prefers warmth.Xinjiang is the main cotton producing area in China,which is prone to reverse spring cold due to its unique geographical location.At present,researches on chilling of cotton mainly focus on the characterization of chilling level based on the evaluation of physiological and biochemical indexes over each developmental stage.Another research panel employed the cotton homologuus genes from the chilling-tolerance network in model plants.There are only few original studies on the molecular mechanism using cotton accessions with different tolerance to chilling.Lacking of the knowledges on molecular regulation on chilling tolerance for cotton is one of the barrier to prevent the breeding on cotton improvement.In this study,the chilling resistant upland cotton accession Junmian1 and chilling sensitive accession C1470 were selected via chilling resistance evaluation.Then physiological and biochemical indexes,photosynthetic fluorescence and cell submicrostructure were examined to compare the effects of chilling on different grades of chilling tolerance of cotton accessions and upland cotton genetic standard line TM-1.Based on the transcriptome analysis of cotton seedling under chilling,a molecular regulatory network in response to low-temperature was constructed by WGCNA analysis.A subset of protein-coding RNAs and lnc RNAs specifically responsive to chilling tolerance were characterized.Further integrated analysis of transcriptome and metabolome using cotton seedling with low-temperature treatment revealed a genetic mechanism of chilling tolerance difference between Junmian1 and C1470.The main results of this study are as follows:1.The screening of chilling tolerant cotton accessions.During the seed germination stage,12 cotton accessions were treated with different low-temperature treatment time.The integrated indexes suggested that Junmian1,TM-1 and Simian 3 were relative tolerant to chilling,and C1470 and KK1543 were chilling sensitive during the germination.The results showed that Zhongmiansuo16,Junmian1,TM-1 and Xinluzao42 have strong chilling tolerance,while Simian 3,C1470 and KK1543 were sensitive to chilling during the cotyledon period.The chilling tolerance of Junmian1 at germination and cotyledon stage and chilling sensitive C1470 at seedling stage also showed the same trend.There were significant differences in chilling resistance between Junmian 1 and C1470 at germination,cotyledon and seedling stages.2.Physicochemical and cytological properties of cotton seedling under chilling.Physicochemical indicators showed that chilling-tolerant materials could remove malondialdehyde and increase catalase activity as well as proline content in vivo more spontenously than chilling-sensitive materials.The increased level of superoxide anion in leaves could decrease superoxide dismutase activity.As a result,the photoprotection of leaves were prevented which led to the leaf cell damages with ruined chloroplast.Meanwhile,the actual light energy conversion efficiency was enhanced.3.Cotton has a concensus of molecular response system in response to chilling.The difference on chilling tolerance may due to the unique expression pattern of chilling response gene cluster from each cotton species.By comparing the differentially expressed genes of three cotton seeds before and after lowtemperature treatment,it was found that the common differential genes were mainly enriched in plant hormone signal transduction,plant MAPK signal pathway,photosynthesis,fatty acid elongation,lipid metabolism and other metabolic pathways.The DEGs of chilling-resistant accessions were mainly enriched in protein kinase,α-linolenic acid metabolism,peroxysome,degradation and metabolism of various amino acids,etc.The DEGs of chilling-sensitive materials were mainly enriched in metabolic pathways such as ribosome synthesis,mitochondrial biosynthesis and circadian rhythm of plants.The DEGs of TM-1 under low-temperature treatment was enriched in flavonoid biosynthesis,plant-pathogen interaction and GTPbinding proteins.4.Brown module,which is related to the genetic background of three cotton accessions,has a positive correlation with chilling tolerance.The gene expression networks of three cotton accessions before and after low-temperature treatment were established by WGCNA analysis,and a positive correlation module with chilling tolerance Brown was identified.Brown was also one of differential modules among the three accessions.Three functional clusters were identified in this module.One was dominanted by lnc RNAs enriched with functional annotation of photosynthesis.The other was the gene network enriched in heat response,and the third was the expression network containing six known genes associated with chilling.Candidate lnc RNAs and genes in these three networks can be used as resources for further research.5.Differential metabolites of cotton seedlings under chilling were related to gene transcriptional regulation.A total of 499 metabolites were identified in this study,including 21 differential metabolites under chilling.The common differential metabolite enrichment pathway was α-linolenic acid metabolism.All the differential metabolites identified in three cotton accessions were associated with WGCNA functional module by association analysis.It was found that octadecenoic acid,hydroxylinoleic acid,phenylalanine,proline,isoleucine and methiopurine might be related to the difference of chilling tolerance of different cotton accessions.6.Jasmonic acid(JA)take a role in the low-temperature tolerance regulation of cotton.α-linolenic acid metabolism was a common metabolic pathway associated with Junmian1’s specific genes and common differential metabolites under chilling.The contents of JA were abundently increased in all three cotton varieties after low-temperature treatment.The expression of the LOX and AOC4 in Junmian1 was significantly increased.The expression of COI1 in Junmian1 was specifically induced by chilling,while JAZ4 was ssuppressed.The JA signal could be transmitted more efficiently in respond to chilling in Junmian1.In this study,we screened and obtained the upland cotton accessions with different low-temperature tolerance at seedling stage,according to the chilling stress tolerance of cotton.It was found that the lowtemperature tolerance of Junmian1 was significantly correlated with the improvement of jasmonic acid metabolism and the activity of jasmonic acid signaling system,integrated with analysis of cytology,transcriptome and metabonomics.Jasmonic acid metabolism and signal transduction can be used as an effective approach for genetic improvement of low-temperature tolerance in cotton seedling stage.