Functions And Mechanisms Of Cadmium Tolerance And Accumulation Of Rice(Ca²⁺/H⁺) Exchanger OsCAX2

Cadmium（Cd）in soil is highly mobile and easily absorbed by plants,resulting in toxic effects.Therefore,it can easily enter the food chain and endanger human health.Rice is the staple food for more than half of the world’s population,and its quality and safety are crucial to people’s health.Cd is absorbed from soil into roots and then transported to the aerial parts,seriously affects rice growth and development.Excess Cd accumulation in rice grains also influences metabolism,quality and safety.Therefore,reducing the accumulation of Cd in rice is of great significance to food safety and human health.To facilitate the development of Cd-safe rice varieties,extensive studies using forward and reverse genetics approaches have been conducted to identify genes controlling Cd tolerance and accumulation in rice and other crops.Although a few genes including OsNRAMP5 and OsHMA3 have been shown to be very promising,more genes with large effect on grain Cd content without or with little negative effect on other traits are urgently needed.In this study,（1）the functions of（Cation/H⁺）exchanger family（CAXs）in Cd tolerance and transport were explored through bioinformatics analysis,expression pattern analysis,heterologous yeast verification,histochemical localization and subcellular localization analysis.（2）The biological functions of OsCAX2 in rice Cd tolerance and accumulation were elucidated by analyzing the spatio-temporal expression pattern,tissue localization and subcellular localization,the transport activity in yeast of OsCAX2,and the Cd tolerance and transport at seedling stage,and the Cd distribution in aboveground tissues at maturity of OsCAX2 knockout mutants and overexpression lines.（3）The physiological mechanisms of OsCAX2-mediated Cd tolerance and accumulation through reactive oxygen species（ROS）and glutathione（GSH）-dependent phytochelatin（PC）pathway were investigated by transcriptome analysis（RNA-seq）,expression pattern analysis,fluorescence localization analysis of Cd,ROS and GSH,diaminobenzidine（DAB）and nitrogen blue tetrazolium（NBT）staining,and enzyme activity analysis of the OsCAX2 knockout and overexpression lines under different treatments.（4）The biochemical mechanism of OsCAX2 interaction with multiple important Cd transporters was demonstrated by expression level analysis,yeast two-hybrid assay（Y2H）,bimolecular fluorescence complementation（Bi FC）,glutathione sulfhydryl transferase（GST）pull-down technology and heterologous yeast verification analysis.The main results are as follows:1.The six rice CAX genes all have homologous genes in Arabidopsis thaliana.Phylogenetic analysis identified two subfamilies with three rice and three Arabidopsis thaliana genes each.OsCAX1a and OsCAX1c were localized in the vacuolar while OsCAX4 was localized in the plasma membrane in rice.qRT-PCR analysis showed that all the six genes strongly expressed in the leaves under the different Cd treatments.Their expression in roots increased in a Cd dose-dependent manner.GUS staining assay showed that all the six rice CAX genes strongly expressed in roots,whereas OsCAX1c and OsCAX4also strongly expressed in rice leaves.The yeast cells expressing OsCAX1a,OsCAX1c and OsCAX4 grew better than those expressing the vector control on SD-Ura medium containing Cd.OsCAX1a and OsCAX1c enhanced while OsCAX4 reduced Cd accumulation in yeast.No auto-inhibition was found for all the rice CAX genes.Therefore,OsCAX1a,OsCAX1c and OsCAX4 are likely to involve in Cd uptake and translocation in rice not through N-terminal regulation.2.OsCAX2 encodes a tonoplast localized protein,which is strongly up-regulated by Cd dose-dependently in roots and shoots.OsCAX2 was mainly expressed in roots,nodes and leaf sheaths.The fluorescence of p OsCAX2::OsCAX2-GFP was mainly expressed in root exodermis,parenchyma in cortex,endodermis and stele cells.Depletion of OsCAX2 resulted in enhanced Cd sensitivity and root-to-shoot translocation in rice,while overexpression of OsCAX2 significantly increased Cd tolerance and reduced Cd transport by promoting root Cd inflow,cytosol-to-vacuole transport and subcellular distribution in vacuole,which ultimately reduces Cd transport via xylem.Knockout and overexpression of OsCAX2significantly increased and decreased Cd accumulation in the above-ground tissues/organs including basal stems,nodes,internodes,leaf sheaths,leaves,and grains,respectively,but had no effects on grain yield and agronomic traits.The OsCAX2-overexpressing lines had more than 70%lower grain Cd accumulation,increased iron（Fe）,zinc（Zn）,and manganese（Mn）accumulation,and decreased copper（Cu）accumulation.Therefore,OsCAX2 is an important Cd transporter and ideal for developing Cd-safe rice varieties by transgenic approach.3.RNA-seq analysis showed that OsCAX2 co-regulated the expression of genes related to ROS synthesis pathway.qRT-PCR analysis showed that OsCAX2 was up-regulated by hydrogen peroxide（H₂O₂）treatment,and the GUS activity of OsCAX2 was also significantly enhanced under H₂O₂ treatment.On SD-Ura medium containing H₂O₂,the growth of yeast cells expressing OsCAX2 was better than that of the vector control,and OsCAX2 mutant and overexpressing plants were more sensitive and tolerant to H₂O₂in rice,respectively.Therefore,OsCAX2 enhances H₂O₂in rice.The addition of DMTU（a H₂O₂scavenger）complemented the Cd sensitivity and enhanced Cd tolerance of OsCAX2 mutant and overexpressing OE lines,respectively.Taken together these results suggested that OsCAX2mediates Cd tolerance and transport at least partially by regulating endogenous ROS homeostasis.Treatment of BSO（an inhibitor of GSH synthesis）treatment enhanced the Cd sensitivity and reduced Cd tolerance of OsCAX2 mutant and overexpressing lines,and decreased the content of thiols（Cys,GSH and PC）and the expression of PC synthesis-related genes in plants,indicating that OsCAX2 involves in Cd tolerance and transport in rice by coordinating GSH dependent PC synthesis pathway.4.qRT-PCR analysis showed that OsCAX2 affects the expression of multiple Cd uptake and transporter genes.Bi FC assay showed that OsCAX2 interacts with OsNRAMP1,OsNRAMP2 and OsNRAMP5.The interaction between OsCAX2 and OsHMA3 was verified by Y2H,Bi FC and GST pull-down assays.The interaction between these two transporters mediates Cd tolerance and transport in yeast.Thus,OsCAX2 interacts with other Cd transporters are important in to controlling Cd transport,tolerance and accumulation.In conclusion,this study identified OsCAX2 as a critical Cd transporter,which facilitates Cd sequestration.Physiologically,OsCAX2 mediates Cd tolerance and accumulation in rice through via the ROS homeostasis and PC chelation pathways.OsCAX2interacts with other Cd transporters to contribute to the regulation of Cd tolerance and accumulation,The research not only provided new insights into the genetic regulation of Cd uptake,transport and accumulation in rice,but also identified an ideal gene for low-Cd breeding in rice via transgenic approach.