Transcriptome Analysis Of Sugarcane Response To Cold Stress And Mining Of Cold Related Genes

Sugarcane(Saccharum spp.),being the most important sugar crop,accounts for about 80%of the gross product of sugar in the world,and over 90%in China.Sugarcane originates from the tropical region,and now mainly cultivates in the sub-tropical region after a long-term domestication.For sugarcane,the new-planting and germination of ratoon cane usually begin from the winter and continue until early spring.The weather being cold or together with rainy during this period which has a seriouly impact on the sprout bud,bud seedling and plantlets of sugarcane due to their sensitivity to cold.Thus,it has important scientific significance to elucidate the mechanism of cold tolerance and practical significance to develop new germplasm of sugarcane by mining important cold responsive genes.Until now,research on the sugarcane response to cold stress mainly focused on the phenotypic,morphological,physiological and biochemical changes,while only a little involved in the molecular mechanism.In this study,we explored the mechanism of sugarcane response to cold stress at the transcriptome level using the high-throughput sequencing.We also try to identify the function of several cold responsive miRNAs and mRNA genes using many methods,including the bioinformatics analysis,physiological and biochemical analysis,gene cloning,gene expression detection,transient expression,DAB staining and so on.This study helps to demonstrate the biomolecular regulatory network of sugarcane response to cold stress,and provides new gene resources for the cold tolerance breeding.The major results and conclusions are as followings.1.Evaluation and selection of sugarcane internal reference genes for microRNA detection.For identifying the most suitable reference genes for normalizing miRNAs expression in sugarcane under cold stress,13 candidates among 17 were investigated using four algorithms:geNorm,NormFinder,deltaCt and Bestkeeper,and four candidates were excluded because of unsatisfactory efficiency and specificity.Verification was carried out using cold-related genes miR319 and miR393 in cold-tolerant and sensitive cultivars.The results suggested that miR171/18S rRNA and miR171/miR5059 were the best reference gene sets for normalization for miRNA qRT-PCR,followed by the single miR171 and 18S rRNA.This study is the first report concerning the reference gene selection of miRNA qRT-PCR in sugarcane.These results can aid research on miRNA responses during sugarcane stress,and the development of sugarcane tolerant to cold stress.2.The miRNA transcriptome analysis in sugarcane bud response to low temperature.In spout buds,412 sugarcane miRNAs were obtained from 4 small RNA libraries through the Illumina sequencing method.Among them,62 exhibited significant differential expression under cold stress,with 34 being upregulated and 28 being downregulated.A total of 2,805 target genes were predicted for 202 known miRNA.GO and KEGG analysis indicated that these miRNAs were involved in several stressrelated biological pathways.To further investigate the involvement of these miRNAs in tolerance to abiotic stresses,sugarcane miR156 was selected for functional analysis.qRT-PCR revealed that miR156 levels increased in sugarcane during cold,salt and drought stress treatments.Nicotiana benthamiana plants transiently overexpressing miR156 exhibited better growth status,lower ROS levels,higher anthocyanin contents as well as the induction of some cold-responsive genes,suggesting its positive role in the plant cold stress response.We also deduced that miR156,by regulating target gene SPL,promotes the accumulation of anthocyanin,and eventually enhances the plant cold tolerance.3.The miRNA transcriptome analysis in sugarcane plantlet response to low temperature.Under cold treatment for 0 d,2 d,and 4 d,withered and yellow was observed on the leaves in cold relatively sensitive cultivar ROC22 after cold treatment for 2 d.And the cold relatively tolerant cultivar FN39 appeared the similar phenotypic change under cold treatment for 4 d.At this time,the chlorophyll content decreased,ion conductivity and MDA content increased,proline content and antioxidase activities increased,and some cold resistant genes were induced in their leaves in both cultivars.It indicated that low temperature will result in multi-level serious impact on the sugarcane.In this study,a total of 144 miRNAs were identified in four sugarcane small RNA libraries using high-throughput sequencing.Among them,32 miRNAs showed differential expression in two relative cold tolerant and sensitive sugarcane cultivars.Compared with the transcriptome analysis result of spout buds with a short-time(3 h)cold treatment,fewer number of miRNAs were identified in plantlets treated for 2 d,suggesting that the expressions of some miRNAs in plant may have space-time speciality.In addition,1,138 target genes for the 117 miRNAs were predicted,and their functional annotation analysis showed that they were involved in several pathways.According to results of the miRNA transcriptome analysis and qRT-PCR validation,the expression of miR393 was induced in cold-tolerant cultivar FN39 and inhibited in cold-sensitive cultivar ROC22,suggesting it may be one critical cold responsive factor.To further investigate the miRNA role,the miR393 in plants was selected and overexpressed in Arabidopsis.After a relative long-time cold treatment,the over-miR393 Arabidopsis showed better growth than the wild type,and this phenomenon was supported by the higher anthocyanin and proline content,lower MDA and H2O2 contents,and higher SOD,POD and CAT activities,as well as the induction of some cold resistant genes including CBFs.Besides,the transcriptome analysis reveals that the overexpression of miR393 inhibits the auxin signal pathway by targeting TIR1,and this also leads to the ethylene signaling inhibition.The inhibited ethylene signaling may induce the cold resistant genes and promote the anthocyanin accumulation and bolting,and eventually the cold tolerance of Arabidopsis is improved.4.The mRNA transcriptome analysis in sugarcane plantlet response to low temperature.The high-throughput sequencing was used for the mRNA transcriptome analysis of sugarcane leaves after 2 days of cold treatment.270,451 unigenes were identified,which significantly contributed to the resources of sugarcane genes.There were 11,574 and 13,340 differential expressed genes in FN39 and ROC22 respectively.Some of the differential expressed genes exhibited the cultivar specificity.The functional analysis showed that these differential expressed genes were involved in many metabolic pathways.And some difference exited in the two sugarcane cultivars,such as the carbon metabolism,which may be the reasons why these two cultivars had different cold tolerances.Glucose6-phosphate dehydrogenase is one of the key enzymes in oxidative pentose phosphate pathway.The subcellular localization experiment showed that it located in the cytoplasm in sugarcane and was a member of the cytosolic G6PDH family.Based on a real-time quantitative RT-PCR performed under salt,drought,heavy metal(CdCl2)and low temperature(4?)treatments,the transcription levels of the ScG6PDH gene were higher compared with transcription levels where these treatments were not imposed,suggesting a positive response of this gene to these environmental stresses.Furthermore,G6PDH activity was stimulated under 4?,CdCl2,NaCl and PEG treatments,but the increments varied with treatment and sampling time,implying positive response to abiotic stresses,similar to the transcript of the ScG6PDH gene.Ion conductivity measurement and a histochemical assay provided indirect evidence of the involvement of the ScG6PDH gene in defense reaction to the above-mentioned abiotic stresses.