Molecular Mechanism Of ZmmiR169q/ZmNF-YA8 Regulating Primary Root Growth And Development In Maize

Maize is the largest crop in China,and it is also the world’s important food,feed and industrial raw material crop.The salinization and alkalization of soil significantly restrict the growth and yield of maize.mi RNAs are key regulatory factors in plant growth and stress response process.In our previous study,Zmmi R169q/ZmNF-YA8 regulatory module was identified to improve the resistance of maize to salt stress through ROS response.This study revealed that the growth and development of plant roots under salt stress would be significantly affected.In this study,we found that Zmmi R169q/ZmNF-YA8 module participated in the regulation of primary root growth of maize under salt stress by regulating auxin content in root tips,and found that ZmNF-YA8 overexpressed line could cope with the damage of salt stress by increasing proline content.The main results are as follows:1.By analyzing the members of NF-Y family,14 members of ZmNF-YA family,23 members of ZmNF-YB family and 15 members of ZmNF-YC family were identified.Analysis of gene structure,conserved motifs,evolutionary relationships and chromosome localization showed that ZmNF-YA,ZmNF-YB and ZmNF-YC have high structural conservation among each protein family members and functional diversity among different families.2.Protein structure and expression profile analysis of ZmNF-YA8 showed that ZmNF-YA8 consisted of two typical domains,which were mainly expressed in leaves,seeds and embryos.Salt stress could increase its expression level in maize.Gene expression analysis of overexpressed homozygous lines of Zmmi R169q and ZmNF-YA8 preserved in the laboratory,transient transformation of tobacco,and in-situ hybridization showed that Zmmi R169q targeted regulation of ZmNF-YA8 in the apical meristem of primary rooting.3.Through the observation of the root phenotypes of Zmmi R169q OE line,ZmNF-YA8 OE line and inbred line B104 under normal and salt stress conditions,as well as the analysis of the relative expression of ZmNF-YA8,it was found that the primary rooting growth of ZmNF-YA8 OE line was significantly inhibited.And similar to the root phenotype of inbred line B104 under salt stress,it was clear that Zmmi R169q/ZmNF-YA8 module was involved in the response of maize primary rooting to salt stress.Further cytological observation showed that the length of meristem in root tip of ZmNF-YA8 OE lines was significantly shorter than that in inbred line B104,and the number of meristem cells was decreased.The length of meristem and the number of cells in root tip of Zmmi R169q OE lines were significantly higher than that of inbred line B104 under salt stress,but there was no significant difference in normal growth conditions.The results demonstrated that Zmmi R169q/ZmNF-YA8 module regulates maize root growth mainly by influencing cell division in root regions.4.The determination of root auxin showed that more auxin was accumulated in the root tips of ZmNF-YA8 OE lines.The phenotype after Yucasin（the inhibitor of auxin）treatment further proved that Zmmi R169q/ZmNF-YA8 module regulated the growth of maize primary roots under salt stress by affecting the content of auxin in root.By transcriptomic analysis,it was determined that ZmERF1B was the target gene of ZmNF-YA8,and q-PCR shows that the expressions of ZmASA1 and ZmASA2,the downstream genes of ZmERF1B,were also up-regulated.Moreover,it was confirmed that ZmNF-YA8 could directly bind to P1 CCAAT-box in the ZmERF1B promoter and activate its expression by transient tobacco transformation assay,maize protoplast dual-luciferase quantitative assay,and EMSA validation in vitro.These results indicated that ZmNF-YA8 up-regulates the expression of auxin synthesis pathway genes ZmASA1 and ZmASA2 by up-regulating ZmERF1B,resulted in excessive accumulation of auxin in root tips,led to the inhibition of primary root growth and shortened length.5.Phenotype observation showed that although the primary root length of ZmNF-YA8 OE line was inhibited,the aerial parts showed salt-tolerant phenotype,while the aerial parts of Zmmi R169q OE line was sensitive to salt stress.The determination of proline content in root of OE lines showed that salt stress inhibited the expression of Zmmi R169q in the root tips,resulting in the increase of the expression of its target gene ZmNF-YA8,which accumulated more proline and developed a certain tolerance to salt stress.Enzyme activity assay showed that the increase of proline content in the root tips of ZmNF-YA8 OE lines was caused by the activity increase of pyrrolin-5-carboxylate synthetase（P5CS）,and the expression level of P5CS family gene Zm00001d010056 was increased in the root tips of ZmNF-YA8 OE lines.However,further experiments confirmed that ZmNF-YA8 could not directly target the promoter of Zm00001d010056.A variety of proteins interacting with ZmNF-YA8 were screened by yeast two-hybrid,and Zm00001d017563 expresses MYB transcription factor.It is speculated that the interaction between ZmNF-YA8 and MYB induces the accumulation of proline through ABA,but the specific mechanism remains to be studied,and the results indicated that ZmNF-YA8 gene was involved in various regulation of maize growth and development.6.Statistical analysis of yield traits such as plant height,ear height and hundred grain weight of transgenic strains showed that there was no significant difference in agronomic traits of Zmmi R169q and ZmNF-YA8 OE lines compared with the control under normal growth conditions.The ZmNF-YA8 OE lines was backcrossed with Z58 and F to obtain BC₆F₁,then self-pollination to obtain BC₆F₂,and then crossed with C72 and M respectively to obtain the hybrid Z^N9-8 and X^N9-8.The observation and analysis of salt stress treatment showed that ZmNF-YA8 gene overexpression was more tolerant to salt stress.Analysis of yield data of hybrid under normal growth in the field showed that ZmNF-YA8 inhibition of primary rooting did not affect the yield of hybrid.In summary,this study analyzed that Zmmi R169q/ZmNF-YA8 module participated in the regulation of primary root growth of maize under salt stress by regulating auxin content in root tips.ZmNF-YA8regulate ZmERF1B,resulting in the accumulation of root tip auxin.The growth of meristem and maize primary roots was inhibited.In addition,the contents of proline in ZmNF-YA8 OE were analyzed from the salt-tolerant mechanism of osmotic stress.It was revealed that ZmNF-YA8 OE could cope with the harm of salt stress by increasing the content of proline.Inlight of these findings,we speculate that Zmmi R169q/ZmNFYA8 maybe in a core regulatory position in plant responses to salt stress and play a vital role in homeostatic control of root growth and salt tolerance.