Cloning And Functional Analysis Of ZmNPF7.9 And ZmAPPI Regulating Maize Kernel Development

Maize(Zea mays L.)is one of the world's leading cereal crops,serving as a staple food,animal feed,and industrial raw materials.Kernel development is an important basis for yield and quality formation of maize.The study on maize kernel development can provide theoretical basis for maize genetic improvement.In this study,we obtained two maize kernel development mutants nrt1/ptr family 7.9(npf7.9)and a p-loop GTPase protein 1(app1)from a mutant library constructed by ethyl methane sulfonate(EMS)mutagenesis of maize inbred line B73pollen,and identified the genes that affect maize kernel development,namely Zm NPF7.9 and Zm APP1.Functions of these two genes was further analyzed by means of molecular biology,biochemistry and genetics.The main results are as follows:1.Cloning and functional analysis of Zm NPF7.9Compared with the wild type(WT),the mutant has smaller kernel with decreased hundred-grain weight and shows a slight decrease in plant height.Genetic analysis indicates that npf7.9is a recessive and nuclear monogenic mutation.The results of map-based cloning showed that one single nucleotide mutation(G to A)occurred in the open reading frame of the gene Zm00001d019294 in npf7.9-ref mutant,resulting in a TGC(Cys/C)to a TAC(Tyr/Y)codon change.We further examined the mutation of this gene in the allelic mutant npf7.9-1.Then we observed a G/A transition at the fourth exon that results in premature termination.The Zm00001d019294 gene is responsible for the phenotypes of npf7.9 and encodes a nitrate transporter that belong to the NRT1/PTR family(NPF).Therefore,the candidate gene was named Zm NPF7.9.Zm NPF7.9 is specifically expressed in the basal endosperm transfer layer(BETL)cells of maize endosperm.The BETL can be clearly identified in normal kernels as one or two layers of elongated transfer cells with extensive secondary cell wall ingrowth at 6 DAP(days after pollination).By contrast,the npf7.9-ref mutant had structurally irregular cells in the BETL,failed to develop elongated cells and delayed cell wall ingrowth,which may lead to less deposition of storage reserves in endosperm.Functional analysis of c RNA-injected Xenopus oocytes showed that Zm NPF7.9 is a low-affinity,p H-dependent bidirectional nitrate transporter.We further demonstrated that Zm NPF7.9 localizes in the plasma membrane by transient expression experiments in maize mesophyll cells using a fused construct p35S:Zm NPF7.9-GFP.Collectively,these suggest that Zm NPF7.9 may be targeted at the endosperm transfer cell layer is involved in delivering nitrate from maternal tissues to the developing endosperm.We determined the nitrate and starch content of mature kernels.Nitrate and starch accumulation in kernels of npf7.9-ref mutants was significantly lower than that in the WT,which might be responsible for the decrease of npf7.9 kernel weight.Moreover,differentially expressed genes and differential metabolites between WT and npf7.9 were analyzed by transcriptome and metabolomics methods.Most of the key genes associated with glycolysis/gluconeogenesis,carbon fixation,carbon metabolism and biosynthesis of amino acids pathways in the npf7.9 mutant were significantly down-regulated.In addition,most of the differential substances including lipids and amino acid that we found from the metabolomics level in npf7.9 mutant was also decreased.Thus,our results demonstrate that Zm NPF7.9 plays a specific role in kernel development and kernel weight by regulating nutrition transport and metabolism,which might provide useful information for maize genetic improvement.2.Cloning and functional analysis of Zm APP1Compared with the WT,the mutant kernels were smaller and shrunken with decreased hundred-grain weight.Histological sections of immature mutant and its WT siblings were analyzed found that app1 mutant endosperm transfer cells layer and aleurone layer structure differentiation abnormalities.BETLs showed structurally irregular cuboidal cell shape,the aleurone layer cells were disordered,the central starch endosperm cells were retarded,and the storage reserves accumulation was reduced.In addition,the embryo of app1 were significantly retarded.The embryo of app1 can establish the typical embryonic structure.The app1 mutant seedling obtained from embryo rescue in vitro were retarded in their growth and dead eventually.Genetic analysis indicated that app1 mutant was a recessive and nuclear monogenic mutant.Further analysis of kernel traits in WT and app1 mutant showed that the content of storage protein and starch of app1 endosperm were significant decreased compared with that of WT.In contrast,the content of soluble sugars and the ratio of carbon and nitrogen in app1 endosperm were increased.These results demonstrate that the defective mutant endosperm cell development lead to less nutrient accumulation,which are responsible for the decreased kernel size in app1.In the app1 mutant,we found one single nucleotide mutation(G to A)occurred in first exon of the gene Zm00001d047046 in the app1 mutant,resulting in a GAC(Asp/D)to an AAC(Asn/N)codon change.Meanwhile,we performed CRISPR/Cas9-based gene editing of Zm APP1 using Agrobacterium-mediated maize genetic transformation and allelism test confirmed that the app1 phenotypes are caused by mutation of Zm APP1 gene.Zm APP1 was constitutively expressed in all detected tissues and the highest transcripts was detected in early development kernels,which suggested that Zm APP1 plays a key role in the early development of kernels.Phylogenetic tree analysis and conserved protein sequence analysis revealed that Zm APP1 encodes an GTPase that belong to the superfamily of a P-loop GTPase with conserved GTP-binding domain.The enzyme activity experiment in vitro showed that Zm APP1 had GTP hydrolase activity.Meanwhile,transient expression experiments in maize mesophyll cells using a fused construct p35S:Zm APP1-GFP demonstrated that GFP signal was colocalized with the mitochondrial probe,Mito Tracker Red,indicating a main mitochondria localization of Zm APP1.Zm APP1 is homologous to ribosome assembly factor YIq F in Bacillus subtilis,and MTG1in humans,respectively.In addition,the transcripts of genes encoding mitochondrial ribosome proteins were accumulated in app1 mutants,which indicated that Zm APP1 may be involved in ribosome assembly.Meanwhile,we found that the expression of mitochondrial genes encoding components of electron transport chain complex were increased in the app1 mutant.Further detection of the expression levels of mitochondrial proteins revealed that the mitochondrial proteins were up-or down-regulated to varying degrees,indicating that the mutation of Zm APP1 protein affected the protein homeostasis in mitochondria.In addition,the abundance and activities of complex I+III₂ were declined in the app1 development endosperm.And the mitochondria of app1 had large internal spaces and lacked distinct cristae structures,which indicate that Zm APP1 is required for mitochondrial structure and function during kernel development.Due to mitochondrial dysfunction in the app1 mutant,excessive ROS were accumulated in the app1 endosperm,which promoted programmed cell death in the endosperm and resulted in defective kernel development phenotype.