Molecular Mechanism Of ZmmiR399 To Low Phosphate Stress In Maize

Plant miRNAs are a group of small non-coding RNAs,transcripted by the endogenous MIR genes and processed into 21-24 nucleotides,regulate its target mRNAs through transcript cleavage or translational inhibition and involve in several growth,development and metabolism processes.The AtmiR399 was the first miRNA demonstrated to be involved in Pi-deprivation responses in plants.The miR399 is specifically induced by Pi-deficiency,controls Pi homeostasis by suppressing PHO2(a ubiquitin-conjugating E2 enzyme),which mediates the ubiquitination of endomembrane-localized PHT1;1 and PHO1 to maintain Pi homeostasis in plants.As the predicted targets of ZmmiR399 in maize mainly include Pi transporters(PHTs)but not AtPHO2 homologs,which implies that the biological roles of miR399 in maize might be different in Arabidopsis and rice.Therefore the present study aims to characterize the molecular mechanism of ZmmiR399 responses to phosphate deficiency in maize.The main results are as follows:1.ZmmiR399 and targets are induced by Pi stress in maize.Under Pi stress,the level of ZmmiR399 increased substantially in both roots and shoots of maize.Four out of five predicted targets of ZmmiR399 in maize including ZmPHT1;1,Zm PHT1;3,ZmPHT1;7 and ZmPHT1;13 were strongly induced by Pi deficiency in both roots and shoots.While ZmPHT1;8 was induced by low-Pi in shoots but not in roots.We tested the expression of the ZmPHTs in the transgenic maize and found that only ZmPHT1;3 and ZmPHT1;8 were abruptly decreased.We then carried out a 5?-RACE assay and transient co-expression assays in N.benthamianato,all the results showed that ZmPHT1;3 and ZmPHT1;8 were the targets of ZmmiR399.2.Zm MIR399 b over-expressing transgenic maize accumulated excessive Pi in old leaves.Transgenic maize showed typical Pi-toxicity phenotypes with chlorosis or necrosis at the tips and margins of old leaves under normal Pi concentration.The total Pi concentration was significantly higher in the old leaves of transgenic maize as compared to WT,while significantly lower as compared to the roots of WT.When seedlings were transferred to Pi-deficient medium,Pi-toxicity phenotypes were not observed in the transgenic maize.Total Pi concentration was found higher in shoots while lower in roots of transgenic maize as compared to the WT.These observations indicated that more Pi was translocated from roots to shoots in ZmMIR399b-overexpressing transgenic maize as compared to WT.3.ZmPHO2 is also a target of ZmmiR399 in maize.Real-time RT-PCR showed that ZmPHO2 mRNA decreased significantly in transgenic maize.We reannotated the ZmPHO2 by 5?-RACE and found that six cleavage sites existed in the newly annotated ZmPHO2.We confirmed four out of six predicted cleavage sites.To confirm our results we also carried out transient co-expression assays in N.benthamianato.The results showed that ZmPHO2 was also a target of ZmmiR399 in maize.4.ZmPHO2 interacts with ZmPHT1;3 and ZmPHT1;8 at the plasma membrane.We performed transient co-expression assays in N.benthamiana and maize protoplast and found that ZmPHT1;3,ZmPHT1;8 and ZmPHO2 were localized on the plastma membrance.The BiFC results showed that ZmPHO2 and ZmPHT1;3 or ZmPHT1;8 were physically interacting.5.ZmPHO2 might mediate the degradation of ZmPHT1;3 and ZmPHT1;8.Co-expressed Zm PHO2 with ZmPHT1;3 or ZmPHT1;8 in tobacco leaves revealed that the protein concentration of ZmPHT1;3 or ZmPHT1;8 was significantly decreased with no significant difference at the mRNA level.These results implied that ZmPHO2 might mediate the degradation of ZmPHT1;3 and ZmPHT1;8.6.The differential expression of ZmmiR399 and ZmPHO2 in low Pi-sensitive and low Pi-tolerant maize genotypes.The transcriptome sequences showed that the level of ZmmiR399 was significantly higher in 31778 than CCM454 under Pi-deficient conditions.To further verify the results,we selected three low Pi-sensitive lines(FR19/1538/JI419)and three low Pi-tolerant lines(XI14/HAI9-21/Q1261)and cultured them under low-Pi and normal-Pi medium respectively.The reaults indicated that the expression of ZmmiR399 under low Pi concentration was significantly higher in the low Pi-sensitive lines as compared to the low Pi-tolerant lines.The transcripts of Zm PHO2 were up-regulated in the four low Pi-sensitive lines and down-regulated in the low Pi-torlerant lines after Pi-deficiency treatment except Q1261.These results suggested that ZmmiR399 guided postranscriptional repression of ZmPHO2 might be related to Pi tolerance in maize genotypes.