Explanation Of Molecular Mechanisms To Low Phosphorus Stress Through RNA-Seq Transcriptome Analysis In Maize

Maize(Zea mays L.)is an important cereal and energy crop throughout the world.The low phosphate concentration in the soil extremely restricts the growth of maize.Therefore it is utmost significant to understand the molecular mechanism of resistance to low phosphorous in maize.Although microarray and RNA-seq technologies have identified plenty of phosphorous-stress related genes and greatly increased our general knowledge about molecular mechanisms of maize responses to P stress,but microarray can only detect known genes,while traditional RNA-seq couldn't distinguish the sequencing data from the first-and second-strand cDNA.Meanwhile a great understanding of the diversity of responses in maize genotypes is still needed.In this study,we first evaluated the tolerance to low phosphate of maize inbred lines under hydroponic conditions and selected two differential inbred lines responses to P stress for the transcriptome sequencing.Besides related physiological indexes;our study illustrates the different molecular mechanisms involved in the diversity of responses to P stress among maize accessions.We further excavated the lncRNAs from the RNA-seq data,and analysed the potential role of these lncRNA to adapt to low phosphorous stress in maize.The specific results are as follows:1.15 low-P tolerance accessions and 15 low-P sensitive accessions were selected from field experiment.They were further verified and screened under hydroponic conditions.According to the seedling biomass ratio of P-deficient to P-sufficient conditions,anthocyanin content and P concentrations,the low phosphorus tolerant line CCM454 and the low phosphorus sensitive line 31778 were selected for the further research.2.The plant samples were obtained from hydroponic grown seedlings that had been provided with sufficient P for 2 days or low P for 2 days or 8 days.These samples were constructed to strand-specific cDNA libraries and sequenced by Illumina Hiseq 2500 sequencing technology with PE125 strategy.Analyzing the sequencing datas,we found that the P deficiency-responsive genes common to CCM454 and 31778 were involved in various metabolic processes,including acid phosphatase(APase)activity.Determination of root-secretory APase activities between the two accessions showed that the root-secretory APase activity in CCM454 was significantly induced after 2 days of P deficiency,whereas the root-secretory APase activity in 31778 was significantly induced only after 6 days of P deficiency.The results agreed with the RNA-seq datas,and indicated that the low P-tolerant line responded more rapidly than the low P-sensitive line to P deficiency.Gene Ontology analysis of differentially expressed genes,SOD,POD and CAT activities between 31778 and CCM454 under P-sufficient and P-deficient conditions demonstrated that CCM454 has a greater ability to eliminate reactive oxygen species.3.We also obtained 65099 lncRNA transcripts after assembling and filtering with the sequencing datas.The features of these lncRNA were consistant with other reports and confirmed by real-time RT-PCR.The results agree with the RNA-seq data,indicating the reliability of these lncRNAs.4.A total of 16 miRNAs in roots and 12 miRNAs in shoots were found to be differently expressed in CCM454 versus 31778,including the well known miR399 related to P stress.Small RNA Northern blot analysis showed that miR399 was much higher in 31778 than in CCM454 after 8 days of P deficiency stress.We also obtained 6787 lncRNAs as potential targets of miR399 predicted by psRobot software,and these target lncRNAs also showed differently expression between the two lines,indicating that miR399 could be involved in maize adaptation to low phosphorus stress by interacting with lncRNAs.5.Through co-expression of lncRNAs and mRNAs network analysis by WGCNA method,we get several hub lncRNAs that are potentially related to low P stress based on the genes connectivity.