Functional Characterization Of ZmMYB95-and Its Implications In Regulation Of Maize Responses To Low Phosphorus

Plants have developed a wide array of adaptive responses to phosphorus(P)insuficiency at the morphological,physiological,and molecular levels over evolutionary history.Root system repatterning has profound impact on uptake and utilization of soil nutrients.The root/shoot ratio significantly increases in P-insufficient maize,and molecular mechanisms of maize responses to P insufficiency remain to be investigated.1.We analyzed root and shoot transcriptome in maize inbred B73 after 10-day P limitation and found distinct metabolic responses in the shoot and root.Under P limitation,the shoot underwent comprehensive remodeling of carbohydrate metabolism,glycolysis,TCA cycle to reduce energy consumption and provide carbon skeleton for plant growth and development.Adjustments of the ratio of phospholipids to glycolipids in the root also helped alleviate the low P stress.On the other hand,P uptake and reutilization shifted in the root and shoot.Hormone signaling and transcription factors play important roles in maize seedling responses to P limitation.2.Many MYB transcription factors had differential expression in low-P maize seedlings.To specifically analyze ZmMYM95's functions,phyologenetic analysis was performed with Amborella trichopoda as the outlier.ZmMYB95 is duplicated in the maize genome,and had an even earlier duplication event before grasseaceae emergence.ZmMYB95 is present in the basal monocot Spirodela polyrhiza,with three holomogues in model dicot Arabidopsis thaliana(AtMYB11,AtMYB12,AtMYB111).ZmMYB95 has two transcripts derived from alternative splicing.Two transcirpts(ZmMYB95-1 and ZmMYB95-2)had different abundance in different tissues and responded to low P quite differently,implying their different biological functions.GFP tagged ZmMYB95-1 and ZmMYB95-2 were localized to nucleus.ZmMYB95-1 had self-acitivation activities in the yeast system,while ZmMYB95-2 not likely due to truncation of the sonserved N terminus.Yeast one hybrid experiments showed that ZmMYB95-1 and ZmMYB95-2 recognizd and bound to the promoter region of CHS,CHI,F3H,and DFR,and ZmMYB95-2 had more binding sites than ZmMYB95-1.Expression levels of CHS,CHI,F3H,and DFR in the ZmMYB95-1 or ZmMYB95-2 overexpressed Arabidopsis plants were significantly higher than those in the WT,indicating that CHS,CHI,F3H,and DFR were probably target genes of ZmMYB 95 and that ZmMYB95 functioned in a different manner in terms of flavonoid metabolism compared to its homologous genes AtMYB12 and ZmMYB40.Yeast two hybrid was performed and indicated physical interaction of ZmMYB95-1 and ZmMYB95-2 with ZmTCP7.Heterologous expression of ZmMYB95-1 or ZmMYB95-2 in switchgrass significantly promote tillering.Together,ZmMYB95-1 and ZmMYB95-2 may regulate shoot branching via their interaction with ZmTCP7.Under low P,the shoot and root dry weights were significantly reduced in the ZmMYB95-1 expressed Arabidopsis lines compared to WT,and P concentration also significantly decreased.ZmMYB95-1 probably down-regulated AtPHR1 and AtPHT1;1 expression under low P,resulting in a significant decrease in P uptake and consequent sensitivity of transgenic plants to low P.Heterologous expression of ZmMYB95-2 in Arabidopsis had no significant impact on above physiological and molecular parameters.In summary,the shoot and root have complicated metabolic responses to P limitation in B73,and hormone signaling and transcription factors are crucial players in this process.ZmMYB95 is a novel gene generated by duplication in the maize genome.ZmMYB95-1 and ZmMYB95-2 were quite different in regard to expression and activation.ZmMYB95-1 and ZmMYB95-2 play a role in regulation of flavonoid metabolism,and may have novel functions compared to its homologues AtMYB12 and ZmMYB40.ZmMYB95-1 and ZmMYB95-2 regulate shoot branching possibly via their interaction with ZmTCP7,and ZmMYB95-1 is involved in regulation of maize responses to low P in PHR and PHT dependent manner.