ZmTCP7 Transcription Factor Is A Negative Regulator Of AGPase Encoding Gene Bt2 Involved In Maize Endosperm Starch Biosynthesis

Starch represents a vital source of calories in the human diet,livestock feed,and primary products for the food and biofuel industries.The process of endosperm starch biosynthesis is a major developmental event that determines the final grain yield and quality in maize,and transcriptional regulation plays a key role in modulating the expression of the main players of the pathway.Maize(Zea mays L.)brittle2(ZmBt2),which encodes the small subunits of AGPase,is a rate-controlling gene of the starch biosynthesis pathway;however,much remains unknown about its transcriptional regulation.Previous work in our lab identified a functional fragment of ZmBt2 promoter(approximately 394 bp),which has multiple cisacting regulatory elements,indicating that ZmBt2 expression is controlled by multiple transcription factors.Alteration of the level of expression of ZmBt2 leads to a significant variation in the amount of endosperm starch content in maize.To improve grain yield and quality of maize via planned modification of endosperm starch content,it is necessary to identify and characterize all transcription factors that affect ZmBt2 expression.In this study,the functional fragment of ZmBt2 promoter was used as a bait sequence to screen a library of maize endosperm c DNA in a yeast one-hybrid library screening assay and multiple independent c DNAs were isolated encoding a novel TCP transcription factor,ZmTCP7(GRMZM2G035944).DNA-protein interaction between the ZmBt2 promoter and ZmTCP7 transcription factor was further confirmed in an individual yeast one-hybrid assay.Based on ZmTCP7 expression data obtained from the Maize Genetics and Genome Database(Maize GBD),an expression heatmap was constructed to study the tissue expression profile of ZmTCP7.The results indicated that ZmTCP7 is primarily expressed in the endosperm.Furthermore,a tissue-specific expression pattern of ZmTCP7 was investigated by performing a q RT-PCR analysis on eight maize tissues,including anthers,silk,leaves,stems,roots,seeds,endosperms,and embryos,to validate the results of the ZmTCP7 expression heatmap.The q RT-PCR results confirmed that ZmTCP7 is predominantly expressed in maize endosperm,with relatively low transcript levels during early development: 3 to 8 days after pollination(DAP),but rapidly accumulates and maintains a relatively high level from 10 to 25 DAP during grain filling.Furthermore,the subcellular location of ZmTCP7 was investigated in maize endosperm protoplasts,and the result showed that ZmTCP7 is targeted and functions in the nucleus.However,a transactivation activity assay in the yeast system revealed that ZmTCP7 lacks potential for self-activation.Furthermore,ZmTCP7 was transiently overexpressed in maize endosperm protoplasts to examine its transcriptional regulatory influence on ZmBt2 expression in transfected protoplast cells by q RT-PCR.The expression of ZmBt2 expression was significantly suppressed in transfected protoplasts compared to non-transfected protoplasts(control).To study the mechanism by which ZmTCP7 regulates ZmBt2 transcription,ZmTCP7 and various deletion derivatives of the functional fragment of ZmBt2 promoter were transiently expressed in maize endosperm protoplasts and LUC and GUS activities were determined in an attempt to locate a region within the ZmBt2 promoter that contains ZmTCP7 binding sites.The results showed that the promoter region from-270 to-220(50-bp)is necessary for the transcriptional suppression of ZmBt2 by ZmTCP7.Subsequently,the entire promoter region from-270 to-220 was searched for previously reported TCP consensus motifs: GGNCCCAC(for class I TCPs)and GTGGNCCC(for class II TCPs).The ZmBt2 promoter region at position-238 contains a motif(GAACCCCAC)similar to the TCP class I consensus motif,a possible indication that ZmTCP7 binding is present at this position.Electrophoretic Mobility Shift Assay was performed and the results confirmed that ZmTCP7 specifically bind to the GAACCCCAC site in the ZmBt2 promoter to regulate its activity.Furthermore,ZmTCP7 was overexpressed in maize to study its role in starch biosynthesis.Developing endosperms from transgenic overexpression maize were subjected to an AGPase enzyme assay and q RT-PCR analysis to determine the AGPase activity and the abundance of starch biosynthesis genes and ZmBt2 transcripts,respectively.Additionally,the starch content of the mature kernels of transgenic maize was also determined.Overexpression of ZmTCP7 in transgenic maize induced a significant repression of the ZmBt2 gene approximately 77.58%,leading to a 14.2% decrease in AGPase activity and a 6.24% reduction in endosperm starch content.Moreover,the expressions of ZmBt1,Zm SSI,Zm SSIIa and Zm SSIIIa were significantly increased,while those of Zm Sh2 and Zm SSIV reduced significantly.The Zm GBSSI and Zm SBE2 b transcripts remain unchanged.Overall,this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation,providing new insights into the regulatory networks that govern the expression of ZmBt2 gene and starch biosynthesis pathway in maize endosperm.