Functional Analysis Of ZmADT2 Regulating Maize Kernel Development

Maize(Zea mays L.)is an important food crop and an important source of feed and industrial raw materials.Maize provides an estimated 15% of the world’s protein and 20% of the world’s calories.And the size and development of maize grain are the key to yield and quality,and the study of grain development mutants can provide theoretical reference for maize genetic improvement.In this study,we started with a maize mutant highly susceptible to U.maydis(mmsu)previously discovered in the laboratory.It was found that there was a phenotypic segregation of small grains in the heterozygous ear,and the segregation ratio was close to 3:1,indicating that the mutant phenotype was a recessive trait under the control of a single nuclear gene.This gene encodes arogenate dehydratase(ADT)and has a high homology with Arabidopsis At ADT2 and was named ZmADT2 according to its function.Using this mutant as the material,we used molecular biology,biochemistry and genetics methods to study the function of the gene ZmADT2 in regulating grain development.And the main results are as follows:The kernel structure of the mutant was significantly different from that of the wild type,with significantly smaller grain and lower 100-grain weight.Histological section experiments showed that the development of the mutant embryo was delayed,but the structure of the embryo was intact.The central starch endosperm cells were smaller,the content of starch grains and other nutrients was significantly decreased,and the cells in the aleurone layer were arranged irregularly.In addition,histological sections and transmission electron microscopy showed that basal endosperm transfer layer(BETL)of the mutant was retarded and wall-ingrowth was significantly reduced.We then analyzed the expression levels of wild type and mutant and found that the expression levels of BETL genes related to development and function were significantly decreased,which may affect the accumulation of storage materials in maize kernels.The further analysis of kernel traits showed that the content of starch and zein in mmsu mutant decreased.These results indicate that the mutant endosperm cells,especially BETL development defects,lead to impaired nutrient accumulation,resulting in a small-grain phenotype.In order to further study the biological function of ZmADT2 gene,we created ZmADT2 gene loss-of-function lines(ZmADT2-KO)and overexpression lines(ZmADT2-OE)and performed phenotypic analysis.The results showed that the small grain phenotype of the knocked out mutant was consistent with that of the mmsu mutant,and allelic cross analysis confirmed that the mutation of ZmADT2 gene was the cause of the small grain size of the mmsu mutant.However,there was no significant difference in the seeds of ZmADT2 overexpression strain compared with wild type.And ZmADT2 is a constitutive expression gene with relatively high expression in early grain development,suggesting that ZmADT2 may play a more important role in early grain development.Bioinformatics speculated that ZmADT2 protein had ADT and prephenate dehydratase(PDT)activities and was involved in the biosynthesis pathway of aromatic amino acids.According to laboratory ears metabolomics data,ZmADT2 mutation inhibited shikimic acid pathway and affected auxin synthesis.The transcriptomic analysis revealed that the expression levels of several auxin biosynthesis genes in the mutant were significantly downregulated.Transcriptomic analysis revealed that the expression levels of several auxin biosynthesis genes in the mutant were significantly down-regulated.Subsequently,through observing the signal of auxin response marker DR5 in the BETL site and quantitative detection of endosperm auxin,we found that the auxin content in the mutant’s endosperm,especially in the BETL site,was reduced.And In vitro grain culture experiments with auxin addition,the mutant grain development defect phenotype was recovered,indicating that the auxin synthesis defect does affect grain development.In addition,we also demonstrated that the lignin content in the BETL portion of the mutant kernel had no significant change compared with that of the wild type,which was not the cause of the mutant grain phenotype.And transcriptomic profiling revealed that most genes involved in carbon metabolism,glycolysis/gluconeogenesis,starch and sucrose metabolism,and amino acid biosynthesis pathways were significantly down-regulated in mmsu mutants.Taken together,the results suggest that the mutation of ZmADT2 gene affects the development of endosperm,especially BETL,by down-regulating the level of indole 3-acetic acid(IAA)in endosperm,further reducing the transport and accumulation of nutrients,and ultimately leading to small-seed phenotype.This gene plays a specific role in maize seed development and filling,which may provide a useful genetic resource for maize genetic improvement.