Analysis Of The Molecular Mechanism Underlying The Pollen In Response To Low-temperature Stress Based On Transcriptome Profiling In Brassica Rapa

In recent years,extreme weather,such as low temperature,is occurred frequently due to the global climate change.For cruciferous vegetables such as Chinese cabbage,low temperature in early spring often leads to pollen abortion,which seriously affects the yield and quality of seeds.Thus,it is critical to understand the effect of cold stress on pollen development and the underlying molecular mechanism of how pollen response to cold stress and it will have certain theoretical guidance meaning for vegetable breeding and enhancement of crop resistance to low temperature.By treating Brasscia rapa L.ssp.chinensis with 0 ? for 120 h during the pollen development processes,our previous study has demonstrated that the tetrad stage is the most sensitive stage to low temperature stress.Low temperature mainly caused the abnormal deposition of callose wall in this period.in vitro germination assay found reduced germination in the mature pollen grains developed from the tetrads after low temperature treatment.In order to explore the molecular mechanism of the effect of low temperature on pollen development,transcriptome sequencing(RNA-seq)was performed on the flower buds at the tetrad stage before and after0 ? treatments.In this paper,the results of RNA-seq were analyzed by bioinformatics,and the long non-coding RNAs(lncRNAs)response to low temperature stress were identified.Quantitative real time PCR(qRT-PCR)was performed to verify the co-expression correlation between lncRNAs and their target genes.BrlncCBF4 and BrlncCPL3 were selected for gene function verification to explore the role of lncRNA in B.rapa pollen in response to low temperature stress.At the same time,RNA-seq and bioinformatics analysis were carried out on the flower buds at the tetrad stage and seeding leaves of B.rapa treated at 0 ? for 0 h,2 h,4 h,6 h,12 h and 24 h,respectively,to further explore the similarities and differences in response to low temperature stress between the two organs.The main results are as follows:(1)The expression the callose synthase encoding gene,GSL2/CalS5,which is required for the formation of callose wall was significantly decreased in the flower buds at the tetrad stage after 0 ? treatment for 120 h.Most of the genes encoding proteins related to the transport and deposition of the callose synthase,such as Rho GTPase,UDP-glucose transporter,annexin,actin and microtubule related protein were also down-regulated.Most of the genes related to pollen germination,such as GRP17,AGP11,CMLs,ADFs and genes encoding autophagy related proteins,were up-regulated.In addition,the genes critical for pollen tube growth,such as PME/PMEI,RALFL,CNGC18,FIM and ROPGEFs,and most ofthe genes related to lipid and carbohydrate metabolism,photosynthesis and other important metabolic pathways were also differentially expressed after low temperature treatment.The differential expression of genes involved in pollen germination and pollen tube growth may contribute to the reduced germination of pollen grains after the transient low temperature stress during the tetrad stage.(2)152 differently expressed lncRNAs(DELs)were selected out in the results of RNA-seq of flower buds at the tetrad stage treated at 0 ? for 120 h and corresponding controls and the expression of seven lncRNAs were significantly correlated with their target genes.LncRNA BrlncCBF4 and BrlncCPL3 were selected for gene function verification.Primary phenotypic observation of BrlncCBF4 and BrlncCPL3 overexpressing and repress expressing plants manifest that BrlncCBF4 and BrlncCPL3 may negatively regulate the expression of BrCBF4 and BrCPL3,respectively.However,the specific functions and regulatory mechanisms of these two lncRNAs in pollen development and the response to low temperature stress need to be further clarified.(3)Further analysis of lncRNAs in the results of RNA-seq of flower buds at the tetrad stage treated at 0 ? for 120 h and corresponding controls showed that there were significant differences in the expression of some lncRNAs and target genes between the flower buds and seedling leaves.While comparative analysis of RNA-seq of flower buds and seedling leaves under normal temperature,revealed apparent differences in the expression of protein-coding genes in flower buds between seedling leaves.A total of 14666 differentially expressed genes were detected.These differentially expressed genes mainly focused on transcription,translation,carbon metabolism,starch and sucrose metabolism and plant hormone signal transduction.(4)Comparative analysis of RNA-seq between flower buds and seedling leaves treated at 0 ? for 0 h,2 h,4 h,6 h,12 h,and 24 h revealed apparent differences of protein-coding genes between these two organs in the response to low temperature stress.In total,4178 differential expression genes(DEGs)were detected in seedling leaves,and the number of DEGs increased slowly at first,then rapidly increased to the peak at the treatment of 24 h.Compared with the flower buds in the tetrad stage,the leaves in the seedling stage have more DEGs in transcription,protein synthesis and processing,plant hormone signal transduction and plant-pathogen interaction.While,the number of DEGs in the flower buds in the tetrad stage reaches the peak at 2 h under treatment,and then keeps the trend of slow decline,and reaches the peak at 12 h of treatment.Compared with the leaves at seedling stage,the flowerbuds have more DEGs in carbohydrate transport and metabolism,lipid transport and metabolism and defense mechanism.There are more specific differential expression genes in transcription factors,plant hormone signal transduction related genes and pollen development and stress response related protein families,especially in MYB family,bHLH family,CCAAT family,NF-Y family,genes related to auxin and cytokinin signal transduction pathway.In addition,the expression patterns of some lncRNAs and their target genes responding to low temperature stress in the flower buds and seedling leaves are quite different.Some lncRNAs and target genes responding to low temperature stress in the seedling leaves are not induced by low temperature stress in the flower buds or only have extremely low expression.