Molecular Mechanism Of Phosphate Deficiency Response In Maize Seedling Based On Multi-omics

Phosphorus(P)is one of essential mineral-nutrient involved in plant developmental process.In soil,total phosphate(Pi)is quite abundant but available Pi is low for plants results in “Pi deficiency in heredity”,so continuous inputs of P fertilizer are necessary to sustain crop yields.However,sources of P rock will be used up in the future,and applying P fertilizer in a large amount brings environmental contamination.Recently,most studies focus on model plants in Pi-responsive mechanism,but limited in maize(Zea mays L.).Based on high-throughput sequencing technology,the strategy of multi-omics help to resolve key metabolic pathways and identify important candidate genes.In this study,multi-omics strategy of small RNA sequencing combined with RNA-seq sequencing and proteome sequencing(i TRAQ)was used to carry out the Pi homeostasis mechanism in maize seedling.The specific results as follows:1.Firstly,we recorded the phenotype under Pi starvation in seedling of maize Pi-tolerance inbred line 178.Phenotype of inbred line 178 started to change after 6d low-Pi treatment,and dwarf plants,elongating of roots,increased number of lateral roots and purple leaves showed at 12 d under Pi deficiency.The concentration of soluble Pi showed significantly decreased at 6 d under Pi limited conditions.The index of root morphology in total root length,total root surface and total root volumes increased significantly after 6 d low-Pi treatment.These results suggested that the seedling stage of maize Pi-tolerance inbred line 178 was suitable for research of low-Pi tolerant response mechanism,and provided basis for the selection of tissues and time points in further omics study.2.159 known mi RNAs were identified by small RNA sequencing in early stage(12h and 24 h)of maize seedling under Pi deficiency,of these,mi R396 is the most abundant mi RNA family.In addition,28 mi RNAs showed differentially expressed under Pi deprivation,and 10 root-specific and 8 leaf-specific mi RNAs were included.Functional annotation results showed that mi RNA396 and mi RNA169 and related target genes are mainly involved in plant root and leaf growth and development biological pathways,indicating that low P stress response in early maize seedling stage is a result of multiple and complex regulatory pathway.3.Eight c DNA libraries of 12 d roots and leaves treated with low-Pi in maize seedlings were constructed by RNA-seq sequencing,and a total of 1944 differentially expressed transcripts(DET)were identified.These DETs are involved in numerous signaling pathways such as glucose metabolism,energy metabolism,and secondary metabolism.The GO-enrichment analysis of DET revealed that cellular response to Pi starvation process and acid phosphatase activity GO entries enriched significantly,indicating that the DET enriched in these GO entries are involved in the low-Pi stage of maize seedlings at the transcriptional level.Notabley,a large number of transcription factors are differentially expressed at the transcriptional level.4.340 differentially expressed proteins(DEP)were identificed by i TRAQ,and 167 and 163 DEPs were leaf-and root-specific expression protein,respectively.In addition,10 proteins showed differential expression in both leaf and root.The significance GO entries included acid phosphatase activity,catalyze activity,transcription activity,Pi starvation response process,acid metabolic process and photosynthesis process,and the genes in these significance GO entries were involved in Pi homeostasis meachanism in maize.5.Combined with trancripteome and proteome data,ZmSPX3 and ZmSPX5 were screened as Pi responsive gene for preliminary functional study.Firstly,the gene sequences of ZmSPX3 and ZmSPX5 were cloned by using the c DNA of inbred line178,and they were located on nucleus and cell membrane.Under Pi starvation,both ZmSPX3 and ZmSPX5 were up-regulated by low-Pi stress.ZmSPX3 was strongly up-regulated in the roots of low-Pi treatment for 12 d,while ZmSPX5 was found in roots and leaves at 6 d under limited Pi conditions.Further analysis of protein interaction between ZmSPX3,ZmSPX5 and ZmPHR1 indicated ZmSPX3 and ZmSPX5 interacted with ZmPHR1 to participate in Pi homeostasis mechanism in maize seedling.Combined with small RNA,trancriptome and proteome sequencing,we found regulation of growth and development is the main mechanism in response to Pi deficiency in early stage of maize seedling,and mi R169/mi R396 and related target genes participated in the response process.In the late stage of low-Pi treatment,genes involved in the absorption and reutilization of Pi,such as acid phosphatase(PAP)and Pi transporter(PHT),play an important role in promoting the absorption of Pi from the soil to meet the plant growth and development.The genes involved in anthocyanin synthesis,represented by DFR,ANS and 3GT,which were up-regulated to promote the accumulation of anthocyanins in low-Pi stress responses.The response of hormone-regulated genes such as auxin-related may play an important role in the changes of root architecture of maize seedlings.Down-regulated expression of ribosomal proteins associated with protein synthesis may be systematically involved in Pi homeostasis mechanism in leaf.The transcription factors represented by SPX(ZmSPX3 and ZmSPX5)regulate the downstream target genes at the transcriptional level to participate in the molecular mechanism of low-Pi stress response in maize seedlings.