Natural Variation And Molecular Mechanism Of Cadmium Accumulation In Rice Grain

Cadmium?Cd?is a toxic heavy metal that not only affects plant growth and development,but also enters the human body through the food chain,posing a serious risk to human health.Large natural variations in grain cadmium concentration exist among rice germplasm,which is associated with the root-to-shoot translocation efficiency of cadmium.Os HMA3 encodes a cadmium transporter located in the vacuole membrane of rice roots and is responsible for transporting cadmium to the vacuole to restrict the transfer of cadmium to the shoot.Previous studies have identified two loss-of-function alleles of Os HMA3 harboring single amino acid mutations at positions 80^th?Arg to His?and 380^th?Ser to Arg?of amino acid sequence,both of which result in excessive accumulation of Cd in rice shoots and grains.In this work,we comprehensively analyzed the variation frequency and distribution characteristics of Os HMA3 in rice natural population,and identified a new allelic haplotype Os HMA3 resulting in high accumulation of cadmium in rice grains by means of positive genetics and molecular biology.The main research results are as follows:1. Through hydroponic test,the cadmium concentrations in roots and shoots of 529 rice varieties grown in a nutrient solution containing 0.5?mol/L Cd were measured,and their cadmium transfer rates from roots to shoot?cadmium concentrations in shoot/cadmium concentrations in root?were analyzed.It was found that despite the wide differences in cadmium transfer rates among different rice varieties,they were generally less than 0.16.Only 11 varieties showed extremely high Cd transfer rates?>0.2?,and 4other varieties had relatively high Cd transfer rates?0.16-0.18?;2. By analyzing the Os HMA3 of these 11 varieties with extremely high cadmium transfer rate,we found that 7 varieties are caused by two known Os HMA3 weak-function allelic haplotypes?natural variation of amino acids 80 or 380?,while the mechanism of the high cadmium transfer rate of the remaining 4 varieties is not yet known;3. C093?Xiannuo-2,code C093?,one of the remaining high cadmium transfer rate varieties,together with another low cadmium transfer rate variety C029?Huhui 628,code C029?,was used to construct a genetic population.The QTL mapping was carried out using the cadmium concentration in the grain as the trait,and it was found that there is a major QTL at the location of Os HMA3.We therefore speculated that Os HMA3 is possibly the effector gene underlying the QTL,that is,there may be a new Os HMA3 haplotype causing the high cadmium transfer rate in the parent C09;4. Through comparison of Os HMA3 expression levels between parents and co-segregation analysis between Os HMA3 expression level and cadmium transfer rate in BC₂F₃ population,we confirmed that Os HMA3 expression level in parent C093 with high cadmium transfer rate was significantly lower than that in parent C029 with low cadmium transfer rate;5. By comparing and analyzing the Os HMA3 coding region of the parents as well as Nipponbare,we found that the Os HMA3 from parent C093 had a non-synonymous natural variation at the 512^th position of amino acid sequence?glycine was replaced by arginine?compared to the parent C029 and Nipponbare.Moreover,a yeast heterologous expression experiment confirmed that this natural variation caused a significant reduction in the cadmium transport ability of Os HMA3 in C093;6. The analysis of Os HMA3 of the remaining 3 varieties with high cadmium transfer rate revealed that 2 of these 3 varieties possessed the same Os HMA3 haplotype as C093which showed significantly lower expression level than that of Nipponbare and other low cadmium varieties and harbored a non-synonymous variation at the 512^th position of amino acid sequence.In summary,we identified a new weak allele of Os HMA3 with natural variations containing both reduced expression levels and weakened protein function,which together resulted in a high root-to-shoot transfer rate of cadmium in a small amount of rice accessions.In addition,the Os HMA3 haplotype found in this study,together with two other Os HMA3 allelic haplotypes previously reported,explains the mechanism of high cadmium in most rice germplasm species?10/11?with extremely high cadmium transfer rate.