The Mechanisms Of MicroRNA528 Regulates Lodging Resistance Of Maize Under Nitrogen-luxury Conditions

To obtain high yields,farmers often apply excessive quantities of chemical nitrogen(N)fertilizer.The excessive N fertilizer application resulted in decreasing N-use efficiency,environmental problems and also lodging under N-luxury conditions.However,the mechanisms remain to be explored.Our previous small RNA deep sequencing of maize seedlings under N-deficiency conditions showed that ZmmiR528 could response to N stress.miR528 is a monocot-specific miRNA.In rice,OsmiR528 contributes to the immune response by regulating an AO,however,the predicted potential targets of ZmmiR528 are different from those in rice,indicating that the function of ZmmiR528 may be different from rice.Thus,we use the molecular biology,the cell biology,and other techniques to further explore the function of miR528 in maize.The main results are as follows:1.We determined the lignin composition and total content by thioacidolysis and acetyl bromide(AcBr)method.Under N-luxury conditions,the generation of p-cumaryl alcohol H,coniferyl alcohol G and sinapyl alcohol S monomers was significantly suppressed,the S/G ratio was lower and total lignin content was reduced in shoots of maize seedlings.Under N-deficiency conditions,the generation of H,G and S monomers was induced,the S/G ratio was higher and total lignin content was enhanced.2.Stem-loop RT-qPCR results showed that the expression of ZmmiR528 in shoots was induced by N-luxury while reduced by N-deficiency,which was opposite to the responses of total lignin content to N supply.3.We conducted experiments to varify the predicted targets of ZmmiR528,and Zm LAC3 and ZmLAC5 showed opposite expression patterns to those of ZmmiR528 in response to N-luxury and N-deficient supply,indicating ZmLAC3 and ZmLAC5 are the authentic targets of ZmmiR528 in maize.Transient co-expression assays in Nicotiana benthamiana and 5?-RACE assay further verified this hypothesis.4.The RT-qPCR results showed that ZmmiR528 expressed highly in leaf,moderately in internodes,and at low levels in roots of adult maize,while ZmLAC3 transcripts showed an opposite expression pattern compared with ZmmiR528.In situ hybridization of ZmmiR528 and ZmLAC5 showed that ZmmiR528 was specifically detected in the phloem of maize leaves and stems,while ZmLAC5 accumulated in fibers of maize vascular,further indicating the negative regulation of ZmLACs by ZmmiR528.5.To further characterize the function of ZmmiR528,we generated ZmmiR528 overexpression and knock down transgenic maize.ZmmiR528 abundance was negatively correlated with the lignin content in hydroponically grown maize seedlings.Thus,we evaluated the lodging resistance of these transgenic maize plants in the mature period under N-luxury conditions.OE transgenic maize was more prone to lodging than the WT or TM transgenic maize under N-luxury conditions.In agreement with that phenotype,the rind penetrometer resistance and AcBr lignin content were much lower in OE transgenic maize than in the WT or TM transgenic maize.The cellulose and hemicellulose contents in TM or OE transgenic maize were similar to those in WT.These results indicate that ZmmiR528 affects lodging resistance in maize under N-luxury conditions by affecting lignin content.6.We also generated transgenic maize plants that overexpressed ZmLAC3.According to AcBr analysis,the lignin content and the rind penetrometer resistance in mature stems under N-luxury conditions was 11~20% higher in ZmLAC3 OE transgenic maize than in the WT,which was consistent with the observation in ZmmiR528 TM transgenic maize.7.We compared the whole-transcriptome profiles of WT and ZmmiR528 TM transgenic maize using RNA-seq,the results showed that ZmPAL7 and ZmPAL8 were differentially expressed in WT and ZmmiR528 knock-down transgenic maize.RT-qPCR results showed that ZmPAL7 and ZmPAL8 were down-regulated in ZmmiR528 OE transgenic maize and up-regulated in ZmmiR528 TM transgenic maize.The results were further verified in ZmLAC3 OE transgenic maize.ZmPAL7 and ZmPAL8 were also found to be regulated by nitrogen levels and were induced by N-deficiency.The transcripts of other ZmPALs were also determined in these transgenic maize lines by real-time RT-qPCR.Consistent with ZmPAL7 and Zm PAL8,the mRNA levels of ZmPAL1 and ZmPAL3 were regulated by N supply,ZmmiR528 and ZmLAC3 abundance.Thus,our results confirm that,the function of ZmmiR528 differs from rice.By regulating the expression of ZmLAC3 and ZmLAC5,ZmmiR528 affects the lignin content of transgenetic maize,and further affects maize lodging resistance under N-luxury conditions.