The Circadian Rhythm Gene ZmGI1 And ZmFKF1 Co-regulated Flowering Time In Maize

At various stages of maize growth and development,it is affected by a multitude of environmental factors such as light,temperature,moisture,nutrients,and salinity.There are complex and orderly cross interactions between these environmental factors in response signal pathways,which synergistically regulate plant growth and reproduction.Studying the genetic regulation mechanism of photoperiod in maize and exploring excellent stressresistant gene resources are the basis and core of introducing tropical and subtropical maize germplasm resources,broadening the genetic basis of existing germplasms and achieving diversified breeding goals.GIGANTEA is a nuclear protein unique to terrestrial plants,involved in the regulation of multiple genetic pathways and a variety of stresses.Further study of the biological functions and molecular regulatory mechanisms of Zm GI1 will help to improve photoperiodic sensitivity of maize,enrich genetic diversity of seed resources,fully coordinate and utilize the resistance advantages of Zm GI1 in multiple genetic resistant pathways for ultimate utilization in resistant molecular breeding in maize.In this study,photoperiodic sensitive material was used to analyze the expression pattern of Zm GI1,candidate gene association analysis was used to identify the candidate gene function and exploit its allelic variations.The biological functions of Zm GI1 and its interaction genes were studied using CRISPR/Cas9 and over-expressed genetic transformation experiments.These were combined with molecular biology techniques: Y2 H assay,split-LUC and Co-IP to screen and identify interactions among genome,to establish the molecular regulatory mechanisms.The main findings are as follows:1.Zm GI1 regulated flowering time and enhanced tolerance to salt stress in Arabidopsis.Analysis of the expression pattern of two GIGANTEA genes,Zm GI1 and Zm GI2,based on RNA-seq data of B73 maize revealed a typical rhythmic expression patterns.The combination of q RT-PCR and semi-quantitative RT-PCR revealed a relatively higher expression level of Zm GI1 in tropical photoperiod sensitive maize inbred lines,especially in long-daylight exposure(16 h-light /8 h dark).This suggested that Zm GI1 played an important regulatory role under long-day treatment conditions in the photoperiod sensitive maize materials.Combined with the re-sequencing data of 87 micro-core maize inbred lines and the relative position of Zm GI1 in the maize genome,a total of 143 SNPs loci were extracted,42 of which were located in the promoter region 3 kb before the transcription start site,the remaining 101 SNPs were located in the gene sequence.Candidate gene association analysis of Zm GI1 within GLM,GLM+Q and MLM models detected 29,125 and 40 natural variations significantly associated with flowering stage related traits(DMF/DFF/ASI)at P ≤0.01,respectively.There are 8 SNPs significantly associated with the same trait in three different models and up to explain 20.93% phenotypic variation.Overexpression of Zm GI1 gene showed no significant changes in flowering time under short-day conditions in Arabidopsis thaliana.While under long-day conditions the flowering time was significantly advanced,indicating that Zm GI1 is directly involved in the regulation of flowering pathway.Additionally,it was revealed that Zm GI1 overexpressing significantly enhanced the tolerance to salt stress,the germination rate of overexpressing lines was significantly increased,and mortality was significantly reduced in Arabidopsis.Combined with the Y2 H screening of maize genome and the experimental verification by split-LUC and Co-IP,it was found that Zm GI1 interacted with the blue receptor protein Zm FKF1 a,which may form a protein complex in Arabidopsis to regulate the expression of downstream flowering genes.At the same time,the interaction between Zm GI and Zm Thox may be related to its resistance to salt.2.Evolutionary Conservation and Functional Divergence of the LFK Gene Family.In this study,phylogenetic analysis revealed that the 171 LOV(Light,Oxygen,or Voltage)and Kelch repeat-containing F-box(LFK)gene family can be classified into two clades,ZTL/LKP2 and FKF1,with clear differentiation between monocots and dicots within each clade.The LFK family genes underwent strong purifying selection;however,signatures of positive selection to adapt to local conditions still existed in 18 specific codons.In 87 diverse maize inbred lines,significant differences were identified(P ≤ 0.01)for days to female flowering between the haplotypes consisting of eight positive selection sites at Zm FKF1 b corresponding to tropical and temperate maize groups of the phylogenetic tree,indicating a key role of Zm FKF1 b in maize adaptive evolution.In addition,positive coevolution was detected in the domains of the LFK family for long-term cooperation to targets.The Type-I and Type-II functional divergence analysis revealed subfunctionalization or neofunctionalization of the LFKs,and the ZTL subfamily is most likely to maintain the ancestral function of LFKs.Over 50% of critical amino acid sites involved in the functional divergence were identified in the Kelch repeat domain,resulting in the distinction of substrates for ubiquitination and degradation.3.The blue receptor protein Zm FKF1 a regulates Arabidopsis and maize flowering in the photoperiodic pathway.The tissue expression characteristics and subcellular localization analysis of four candidate genes of Zm LFKs gene family showed that Zm FKF1 a and Zm FKF1 b were located in the nucleus,whiles Zm ZTLa and Zm ZTLb were located in the nucleus and cytoplasm respectively.The four family members were not expressed in female flowers,but were expressed in roots,stems,leaves,male flowers and other tissues,though relative expression levels varied significantly.The rhythm expression pattern of the four genes was analyzed by using 6 photoperiod extreme maize inbred lines.It was found that Zm FKF1a/Zm FKF1 b had a typical rhythm expression pattern consistent with Zm GI1,while Zm ZTLa/Zm ZTLb had no obvious rhythm.Under long-day conditions,the expression of Zm FKF1 a in tropical photoperiod sensitive maize materials was significantly higher than that in temperate photoperiodic materials.Under short-day conditions,the expression level of Zm FKF1 a in photoperiodic sensitive/insensitive materials was not significant.The rhythm expression pattern of Zm FKF1 a and Zm GI1 was highly consistent,which further indicated that the interaction between Zm FKF1 a and Zm GI1 was involved in the same flowering regulatory pathway of maize photoperiodism.Overexpression of Zm FKF1 a in Arabidopsis had no significant effect on flowering time under long-day conditions,but significantly reduced the number of rosette leaves and significantly shortened the flowering time under short-day conditions.However,the homozygous mutant fkf1 of Arabidopsis thaliana significantly delayed flowering time under long-and short-day conditions.Zm FKF1 a was knocked out by CRISPR/Cas9,a variety of different gene edited types were detected in the T1 generation in maize.The homozygous line of T2 generation showed a delayed flowering in Sichuan in 2019.The interaction between Zm FKF1 a and candidate genes of multiple photoperiod regulatory pathways was verified by Y2 H assay,which can provide an insight for further analysis of the role of Zm FKF1 a in the regulation of photoperiod pathway in maize.