Function Mechanism Of ZmNF-YB16 In Maize Drought Stress Resistance

Maize(Zea may L.)is an important food and feeding crop,and plays important role in economic development.Drought is one of the major abiotic factors that affects the crop development and yield.It is important to explore the drought stress response network and key genes for drought-resistant maize breeding.NF-Ys are common transcription factors in eukaryotes,and compose of three subunits NF-YA,NF-YB and NF-YC.NF-Ys play important roles in plant growth and development,flowering time regulation,photosynthesis,ER stress response and abiotic stress response such as drought,high temperature,and salt stress.In the previous work of our laboratory,the expression level of ZmNF-YB16 in drought resistant line was higher than that in drought sensitive line after osmotic stress according to the comparative transcriptome.Transformed ZmNF-YB16 into maize and obtained the transgenic plants.Overexpression of ZmNF-YB16 improves drought stress resistance and crop yield.On this basis,we carried out a series work to explore the mechanism of ZmNF-YB16 in drought stress resistance.ZmNF-YB16,ZmNF-YC17 and ZmNF-YA1 heterotrimer regulates the expression of downstream genesA maize seedling cDNA yeast two-hybrid library with 9.5x 107 cfu/mL titer was constructed.By yeast two-hybrid screening,a NF-YC family gene ZmNF-YC17(GRMZM2G311316/Zm00001d024230)which could interact with ZmNF-YB16 was found.Direct interaction between ZmNF-YB16 and ZmNF-YC17 was verified by Bifc and pull-down assay.The interaction between them relied on the histone-like conserved domain.ZmNF-YA1-YB16-YC17 and ZmNF-YA7-YB16-YC7 trimer were found by Yeast three hybrid assay.The similarity between the longest transcript of ZmNF-YA1 and ZmNF-YA 7 was close.Subcellular localization found that vast majority of fluorescent signals were located in the cytoplasm,and few signals in the nucleus under normal conditions.After osmotic stress,some fluorescence signals were transferred to the nucleus,and ZmNF-YB16 and ZmNF-YC17 showed the co-location model.ZmNF-YA1 and ZmNF-YA7 were located in the nucleus no matter under normal or osmotic conditions,which implied that the heterotrimer formed and functioned in the nucleus.Gene expression levels of ZmNF-YA1 OE line and zmnf-ya1 mutant were analyzed by RNA sequencing.Under drought stress conditions,different express genes(DEGs)between ZmNF-YA1 OE lines from DH4866 and the DH4866(WT)and DEGs between zmnf-yal mutant from W22 and the W22(WT)were analyzed.Results showed the enrichements of two carbon metabolism GOs,five stress response GOs and twelve ribosome assembly GOs were signicifantly different between them.Combined the transcriptomic data,cis-acting elements assay,chip-qPCR and yeast one-hybrid assay,seven genes including GRMZM2G042895(ZmbHLH1116),AC205413.4_FG001(ZmPOD64),GRMZM2G058310(ZmAMY),GRMZM2G023081(Zm CNR9),GRMZM2G102760(ZmLOX5),GRMZM2G051135(ZmMBFl)and GRMZM2G089106(ZmLSD1)were directly regulated by ZmNF-YA1-YB16-YC17 heterotrimer.According to the EMSA assay,heterotrimer regulated the target genes by combining to the CCAAT cis-acting element in the promoter region of NF-YA subunit.Other proteins might exist in ZmNF-YB16 complexThree proteins(homeodomain transcription factor(GRMZM2G153263),serine/threonine phosphatase(GRMZM2G180691),60S ribosomal subunit(GRMZM5G868433))were identified by Co-IP in maize protoplast transformed with ZmNF-YB16-GFP.Yeast two-hybrid assay showed ZmNF-YB16 and its mutants with altered phosphorylation status could not interact with GRMZM2G153263 protein.However,the phosphorylation status changed the interaction between ZmNF-YB16 and ZmNF-YC17.When the 4th,8th,17th,18th phosphorylation sites changed,ZmNF-YB16 could not interact with ZmNF-YC17.GRMZM2G153263 protein could interact with ZmNF-YC17 and the heterodimer formed in nucleus.ZmNF-YC17 and GRMZM2G153263 had the similar organ expression patterns,with the highest expression levels in tassel,followed by root and leaf at V5 stage.And they were induced by osmotic stress.The similar expression pattern may have important roles in the heterodimer formation and function.Drought stress influence the transcriptome of different organs in maizeThe normal development of ears at V9 stage,leaves and kernels at 5DAP stage had great importance for yield formation of maize.Drought at V9 and 5DAP stage seriously reduced the maize yield.Drought stress at V9 stage affected the differentiation of the spikelets primordium and development of the spikelets,resulting in the decrease of the number of grains and the number of ear rows,and produced a significant decrease in yields.Drought stress at 5DAP stage affected the leaf photosynthesis,inhibited the normal growth and development of kernel,with abnormal development of transfer cells in the outer region of endosperm and decreased the accumulation of nutriment in kernels.292 common DEGs responded to drought stress were found according to the transcriptome analysis,but the changes of their expression levels showed no obvious consistency in the three organs.In the period suffered from drought stress,the organs endured different degree of stress at different development status showed the specificity of transcriptome in the organs.There were many genes related to stress response in these DEGs,including heat stress,oxidative stress.ethylene response and transcription factors of bZIP and WRKY family.Most of the 292 genes were heat,oxidative stress response genes,ethylene response genes and bZIP、WRKY transcript factor genes.Most of the genes related to heat stress response were heat-shock proteins,and four of the oxidative stress response genes were thioredoxin genes.And the stress response genes usually showed up-regulated expression after drought stress.Drought stress significantly influenced the gene expression involed in photosynthesis-antenna proteins,photosynthesis,protein processing in endoplasmic reticulum,plant hormone signal transduction pathways of ears at V9 stage.And genes in photosynthesis showed up-regulated after drought stress.DEGs in ER stress response were heat shock protein genes,such as HSP4,HSP20,HSP18,HSP70 etc.,with up-regulated expression after drought stress.DEGs in plant hormone signal transduction pathway were involved in ABA,auxin,cytokinin,ethylene,and gibberellin signaling pathway,which implied that drought stress had important influence on cell metabolism and fate.In leaf at 5DAP stage,drought stress influenced the expression levels of genes in photosynthesis,metabolic process,secondary metabolic process,photosynthesis-antenna protein and carbon metabolism pathways.DEGs in photosynthesis encoded the subunit proteins of photosystem I and photosystem II and showed down-regulated after drought stress,which indicated that drought stress seriously inhibited the photosynthesis and reduced the energy supply for growth and development.DEGs in carbon metabolism were concentrated in the TCA cycle,and the gene expression levels decreased after drought stress.These changes implied the energy and intermediates required in cell metabolism process would be reduced,and plant growth and development would be retarded.The development of kernels is sensitive to drought stress.Drought stress influenced the expression of some genes in mRNA surveillance pathway,photosynthesis-antenna proteins,oxidative phosphorylation,TCA cycle,and 2-Oxocaryboxylic acid metabolism of the kernels at 5DAP stage.Expression of many serine/threonine protein phosphatase PP1 and PP2A genes significantly changed,which might modify the protein phosphorylation status to affect cell metabolism and fate.The decreased expression of genes involved in energy metabolism would lead the poor energy supply for cells and poor kernel development.Overexpression of ZmNF-YB16 influence the gene expression in maize502 commom DEGs responded to drought stress were found in three organs.And these genes significantly enriched in stress response and metabolism process GO terms.DEGs related with stress response,such as EREBP112、EREBP126、DHN1,had the similar tendency in three organs.DEGs related with nucleosome assembly and carbon metabolism showed similar expression tendency in the ear and kernel,but not in the leaf.In the ears at V9 stage of ZmNF-YB16 OE lines,drought stress influenced gene expression in ribosome assembly,photosynthesis-antenna protein,TCA cycle,protein transport and fatty acid metabolism pathways.And the enrichment degree in ribosome assembly pathway was higher than that of others.The decreased expression of a large number in ribosomal assemble undoubtedly reduced the protein synthesis and influenced the development of ear.Genes responded to drought stress in leaf of ZmNF-YB16 OE lines were enriched in carbon metabolism,2-Oxocarboxylic acid metabolism,N-Glycan biosynthesis,glycosaminoglycan degradation and flavonoid biosynthesis.Genes responded to drought stress in ZmNF-YB16 OE lines were not enriched in photosynthesis,which indicated that overexpression of ZmNF-YB16 maintained the expression levels of genes in photosynthesis under drought conditions.Among the 122 DEGs in carbon metabolism,38 were in ear and 30 in kernel,which indicated that changes in carbon metabolism were obvious in leaf,but not in rapid development organs.Moreover,ZmNF-YB16 influenced the expression of genes in ER stress response and glutathione metabolism under drought stress conditions in leaf.ZmNF-YB16 improved drought stress resistance by maintaining photosynthesis,improving antioxidant capacity and stabling the protein processing of leaf at 5DAP stage.DEGs responded to drought stress in ZmNF-YB16 OE lines kernels at 5DAP stage enriched in metabolic pathways,biosynthesis of secondary metabolites,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,and phenylpropanoid biosynthesis pathways.Overexpression of ZmNF-YB16 influenced the sucrose and starch metabolism,flavonoids metabolism,glutathione metabolism pathways.Gene expression analysis in sucrose and starch metabolism showed the reduced sucrose degradation and enhanced starch synthesis played positive roles in maintaining high sucrose and low monosaccharide concentration.Effects of ZmNF-YB16 overexpression on newly synthesized transcripts in maizeAnalysis of newly synthesized transcripts was performed in wild type and ZmNF-YB16 OE lines under different conditions by 4-thiouridine(4 sU)labelling and separation.The proper parameters were determined for 4 sU labelling,which were 2 mM 4 sU for 2h under normal and heat treatment conditions,and 2 mM 4 sU for 6h under osmotic stress conditions.Compared to the wild type,the DSTs(differentially synthesized transcript)of the ZmNF-YB16 OE line under different conditions showed obvious differences in the newly synthesized transcripts.There were 3184,3196 and 2800 DSTs between WT and OE lines under normal,heat and osmotic conditions respectively.Under all three conditions,overexpression of ZmNF-YB16 influenced the new transcripts involved in redox,oxidative stress response and chemical stimulus stress response process,etc.It was interesting that DSTs between WT and ZmNF-YB16 OE lines were enriched in GO terms including stress response,programmed cell death,autophagy under normal and heat stress conditions.And under osmotic stress conditions,DSTs between WT and ZmNF-YB16 OE lines were enriched in DNA replication,nucleosome assembly,chromatin aggregation and depolymerization,chromosome assembly.Most of DSTs were histone coding genes including H1,H2A,H2B,H3and H4 and showed down-regulated tendency in ZmNF-YB16 OE lines.Expression changes of these genes resulted in reduced assembly of nucleosomes and increased chromosome activity.These results indicate that ZmNF-YB16 played complex and multi-target functions and may interact with different proteins under different environmental conditions.In summary,ZmNF-YB 16 formed heterotrimer with ZmNF-YC 17 and ZmNF-YA1.The heterotrimer regulated genes expression by binding to the CCAAT cis-acting element on the promoter region through ZmNF-YA1 subunit.Expression changes of genes related to stress resistance and plant growth improved drought stress resistance.Analysis of transcripts in different organs of wild-type and ZmNF-YB16 overexpression lines showed the function diversity of ZmNF-YB16 in drought stress response.Analysis of new transcripts in wild-type and ZmNF-YB16 overexpression lines under different stress conditions showed the function complicacies.