The Molecular Mechanism Of MiR156 And SPL9 Regulate MiR399f Under Low Phosphorus Stress

Plants have evolved many strategies to cope with phosphorus(Pi)limitation such as the alteration of root system architecture,increased the activity of Pi absorption,the secretion of organic acids and phospholipase.Many factors which are involved in low Pi regulatory network have been identified.However,how plants perceive and response to the signal of low Pi is not clear.MicroRNA(miRNAs)are endogenous non-coding small RNAs and have been reported to modulate plant stress responses.According to Chiou article,in addition to miR399-PHO2 specifically response to low Pi,miR156 can also be induced under low Pi.Whether there is a relationship between miR156 and miR399 under low Pi,it is still unclear.Here,we found that the phenotypes of 35S:MIM156 and 35S:rSPL9 mutants under low Pi: increased Pi content in leaf,loss function of rhizosphere acidification,reduced anthocyanin content and lateral root number and length.The miR156-SPL9 is involved in the regulation of low Pi.Simultaneously,we detected some marker gene expression in these mutants.The results show that the expression of PLDZ2,PHO2 in the mutants is significantly inhibited,while other genes are up-regulated.By observing the precursor gene miR156 a and miR399 f and SPL9 tissue staining,we found that miR399,miR156 and SPL9 showed similar expression patterns.Meanwhile,miR399 precursor genes expression level in mutants under low Pi significantly increased.Using one-yeast hybrid and transient assay,we found that SPL9 could combine with miR399 f in the promoter of GTAC motif.The above results show that by regulating the expression level of miR399 f,miR156 and its target gene SPL9 participate in low Pi.To sum up,our results suggest that the miR156 and SPL9 participate in the process of low Pi response,and participate in the Pi signaling pathway by modulating the downstream genes of miR399 f.The functional analysis of miR156 and SPL9 reinforces our understanding of transcriptional regulation in higher plants under phosphorus deficiency.The miR156-SPLs module may play a broad and vital role in plant development and stress responses.