Identification Of Candidate Genes Response To Zinc Toxicity Tolerance In Roots Of Arabidopsis And Rice Using Genome-wide Association Study And Functional Analysis

Zinc（Zn）is an essential micronutrient for plants but is toxic in excess.Previous studies have improved the understanding of Zn homeostasis regulatory mechanisms,such as loading Zn into the xylem and translocating to the shoots,promoting sequestration into the root vacuolar,or cross-talk between Fe homeostasis and Zn tolerance.However,it is little known about other regulatory mechanisms in Zn homeostasis.And there are few reports on the fine mapping of major quantitative trait loci（QTL）and gene cloning in Zn toxicity tolerance in rice.Therefore,identifying the genes and revealing their regulatory mechanisms provide theoretical support for understanding why Zn toxicity inhibits plant growth,enrich the regulatory network of Zn toxicity,and provide valuable information for molecular marker-assisted selection to improve plant variety tolerance to high Zn.In this study,we explore the genetic mechanisms of root growth responses to Zn toxicity tolerance in Arabidopsis and rice.The main results are as follows:（1）There is a wide extensive natural variation among accessions in root growth responses to Zn toxicity.Using the relative root length from day1 to day 8,we detected 25 significant associated loci for Zn tolerance through a genome-wide association study（GWAS）,of which7 significant loci can be detected at different time points,indicating that there might be genetic factors regulating Zn toxicity in plants.（2）We then analyze the candidate genes in these significant loci,and found a known Zn tolerance gene Ferric Reductase Defective 3（FRD3）which was located at q RL3-3.FRD3showed significantly different expression in high Zn conditions between representative accessions containing either the G-variant or T-variant.Moreover,frd3 mutants showed a significant reduction of root growth and severe chlorosis compared to the wild type in response to high Zn.These results indicate that FRD3 is involved in regulating root growth response to Zn toxicity tolerance.（3）We then analyzed the candidate genes at the locus of q RL5-1 and found that the transcript level of Trichome Birefringence（TBR）was highly positively correlated with root growth response to high Zn when using the representative T-allelic accessions and A-allelic accessions.The root growth of both tbr mutants was more sensitive to high Zn compared to the wild type.These results demonstrated that TBR was expressed in the root and required for root growth tolerance to high Zn.We further measured the root length,analyzed the transcription level and protein level of different p TBR:TBR:HA-m CITRINE transgenic lines,and also measured the root length of p TBR:TBR_Col-0:HA-m CITRINE transgenic lines.The results indicated that the allelic variation in the TBR promoter,which causes TBR expression level variation,significantly contributes to the natural variation of Zn toxicity tolerance.Moreover,we tested the degree of pectin methylesterification and Zn content of the root cell wall in high Zn conditions.The results showed that tbr mutants exhibited a higher degree of pectin methylesterification and lower Zn content in the root cell wall compared to the wild type.Together,these results provide strong evidence that TBR,which regulates pectin de-methylesterification,is required to sequestrate excess Zn in the root cell wall and thereby protect the plant cells from their entry.Finally,this mechanism seems to be conserved across plant species such as Lotus japonicus and Oryza Sativa.（4）A total of 7 and 19 QTL were detected using root elongation and Zn tolerance index under high Zn conditions at the seedling stage of Ting’s core collection,respectively.Among them,q RE4 and q RRE9 were identical.q RRE15 was located in the same genomic region where 3QTL were reported previously.Furthermore,we found some candidate genes that are involved in Fe homeostasis,metal chelation,and phosphate transport in q RE2,q RRE1,q RRE8,and q RRE12 loci.Three accessions（S016,S024,and S095）in the core collection showed higher Zn toxicity tolerance and were associated with Zn tolerance in the analysis of allelic variation.The zinc tolerance alleles in these materials can be polymerized through molecular marker assisted selection,or edited by CRISPR-Cas9 technology and then cultivate excellent rice varieties tolerance to high Zn.