Functional Validation And Genetic Analysis Of Phosphorus Deficiency Response Genes Of ZmARF Gene Family In Maize

Phosphorus(P)is one of the three essential nutrients easily fixed by cations to form insoluble complexes in soils.This phenomenon limits the availability of inorganic phosphate required by plants for growth and development.Maize(Zea mays L.)is one of the most important food crops in the word.Maize can also be utilized as feed and a bio-energy crop.Domestic demand for maize in China and other parts of the world continues to increase.Phosphorus deficiency is a major constraint to maximizing grain yield and quality of maize.This has triggered novel research involved in facilitating and enhancing the ability of Pi uptake in plants.Root morphology plays a vital role in water and nutrient absorption.Auxin response factors(ARFs)play an important status both in auxin response pathway and root development.The study on biological function of ZmARF and its regulatory mechanism therefore needs to be intensified,as it has proved helpful in improving maize root morphology,increase the absorption and utilization of nutrient elements,and ultimately serve for phosphorus efficient molecular breeding in maize.The present study was based on the members of ZmARF gene family identified within genome-wide in maize,using association analysis to mine phosphorus deficiency response genes,and combining genetic transformation experiments of Arabidopsis,rice and maize to explore the biological function of candidate genes and screening Y2 H library,split-LUC and Co-IP to analyze possible regulatory mechanisms of the target gene.The main findings are as follows:(1)Identification of ZmARF genes response to P deficiency stress in maize.A total of 38 ZmARF genes were identified in the genome-wide of maize.All members have a typical auxin response domain and were unevenly distributed in 10 chromosomes.Thirty-eight proteins were classified into four subgroups based on amino acid sequence analysis,of which subgroup IV is the simplest gene structure.An evolutionary tree analysis,constructed with rice and Arabidopsis ARF proteins revealed that ARF has a closer evolutionary relationship with monocots.Six of the 38 ZmARF genes failed to obtain the correct amplification product.Transcriptional activation assay revealed that only 7 of the 32 ZmARF transcription factors,ZmARF1,ZmARF3,ZmARF4,ZmARF20,ZmARF27,ZmARF30 and ZmARF34,had transcriptional activation activity.According to the physical location of 38 ZmARF genes in the chromosome,a total of 1166 SNPs had been extracted from previous RNA-seq data.Association analysis revealed that 573 loci were significantly associated with phosphorus deficiency related traits in the mixed linear model(MLM)in maize seedlings(P<0.01).Even though 6 genes did not detect significant association sites,the results indicated that most of the ZmARF genes responded to phosphorus starvation stress in maize seedlings.Twenty-six ZmARF members were significantly associated with root traits at seedling stage in three different statistical models.The result showed ZmARF23 was the most significant site,suggesting that ZmARF23 played an important role in the phosphorus stress response in maize.(2)Functional analysis of ZmARF23 with deficient phosphorus tolerance in maize.Association analysis found that ZmARF23 was significantly associated with RT and RSA under normal phosphorus supply and phosphorus deficiency stress conditions.Transient expression assays indicate that ZmARF23 is localized in the nucleus in tobacco leaves.q RT-PCR showed that ZmARF23 was strongly induced by phosphorus deficiency stress in roots of low-tolerant inbred 178,indicating that ZmARF23 responded to phosphorus starvation stress in maize.Three different g RNAs were designed for ZmARF23 sequence.DNA cleavage efficiency assays revealed significant differences in three g RNA-mediated DNA cleavage efficiencies in vitro.The genome editing ability of CRISPR/Cas9 vector was verified by transient expression of maize protoplast,which was constructed with the highest efficiency of g RNA.A number of different types of CRISPR/Cas9 with edited genome were detected in T1 generation transgenic maize.Overexpression analysis in rice showed that ZmARF23 should be a negative regulator of primary root growth and reduced rice yield.Under phosphorus deficiency stress,the tolerance of transgenic lines was significantly enhanced to phosphorus deficiency compared with wild-type Nipponbare.Screening Y2 H library experiment,combined with verification by split-LUC and Co-IP,revealed that the interaction between ZmARF23 and ZmLBD1 could be related to the promotion of adventitious root development.(3)Functional verification and favorable allelic variation of ZmLBD1,which is interacted with ZmARF23.Sequence polymorphism analysis of ZmLBD1 revealed that 32 SNPs and 40 In Dels were contained in the DNA sequence,which were amplified from 301 maize inbred lines.The nucleotide polymorphism(π)analysis revealed that exons were the lowest polymorphism(π = 2.16×10-3).Tajima’s D statistics test showed that ZmLBD1 significantly purified selection pressure during domestication.Eighty-seven,thirty-two,and twenty natural variations were identified in ZmLBD1 that were associated with P-deficiency-tolerance traits(P ≤ 0.01)by using the general linear model(GLM),GLM incorporated with population structure,and mixed linear model,respectively.In the MLM model,6 loci were found to be significantly associated with both root tip number(RT)and root-shoot ratio(RSR)under phosphorus starvation stress,which could explain up to 4.52% phenotypic variation.Transient expression in tobacco leaves and stable expression in transgenic plant of Arabidopsis indicates that ZmLBD1 is located in the nucleus.Expression analysis showed that ZmLBD1 was significantly induced by phosphorus deficiency stress in roots of low-tolerant inbred 178,nearly up-regulated to 10 times,indicating that ZmLBD1 responded to phosphorus deficiency stress in maize.Overexpression and mutant complementarity with lbd16 lbd18 analysis in Arabidopsis showed that ZmLBD1 could promote lateral root development and primary root growth,while resisting the stress of phosphorus deficiency.Screening Y2 H library experiment,combined with verification by split-LUC and Co-IP,revealed that the interaction between ZmLBD1 and ZmEXP7 could be related to the promotion of root growth and development.(4)Genetic variation of ZmARF31 and its association with phosphorus deficiency traits in maize.ZmARF31 is an important member of the gene family.Its homologous genes regulated by mi RNA participates in the morphogenesis of roots in Arabidopsis and soybean.However,the SNP of ZmARF31 could not be obtained in the RNA-seq data of related population.In the present study,a total of 331 diverse maize inbred lines were used to detect nucleotide diversity and favorable alleles of ZmARF31,which play a key role in low P responses and root architecture regulation.Sequence alignment analysis found that 30 single nucleotide polymorphisms(SNPs)and 14 insertion-deletions(In Dels)were detected in ZmARF31 among the 331 maize inbreds.The 5′-untranslated region(UTR)of ZmARF31 showed a small linkage disequilibrium(LD)block under significant purifying selection,whereas the 3′-UTR showed the most abundant diversity and a larger LD block.General linear model(GLM),GLM incorporated with population structure and mixed linear model were,respectively identified thirty,fourteen,and nine natural variations in ZmARF31 that were associated with P-deficiency-tolerance traits(P ≤ 0.01).Four SNPs were significantly associated with the total dry weight in the three models,of which SNPs S410 and S462 were located in a complete LD block.A further verification conducted in a recombinant inbred line population revealed that favorable allele G/G of nonsynonymous mutation S410 and favorable allele with a 38 bp insertion of In Del S1442 exhibited positive genetic effects on the total dry weight and total root tips,respectively.Expression analysis further confirmed that ZmARF31 was highly expressed in the roots of low P-tolerant inbred 178.The protein encoded by ZmARF31 was located both in the nucleus and cytoplasm.Haplotypes carrying more favorable alleles showed a greater effect on phenotypic variation than single loci.Such haplotypes should be helpful for developing valuable genetic markers and performing maize molecular breeding.