A Preliminary Study Of OsFWL4 Cadmium Resistance And The Interaction With OsRbCS In Rice

Cadmium(Cd)is a widespread and toxic heavy metal that can severely inhibit the growth of plants and be harmful to human health.In recent years,with the use of Cd-containing substance in industry and agriculture,more and more cadmium was released to the environment,and the Cd-pollution has become an increasingly serious problem.Rice is a staple crops in the world,and has been seriously polluted by cadmium.So it has become one of the hottest research spots to study the mechanisms of cadmium absorption and transport in rice,to dig Cd-resistance genes,and then to breed low-cadmium rice.Previous work in our laboratory has identified a OsFWL gene family with eight numbers in rice by sequence alignment of FW2.2,and found that most members have the function of Cd-resistance.This study was aimed to explore the Cd-resistance mechanism of OsFWL4 and OsFWL2.OsFWL4 protein has two CCXXG domains and three putative phosphorylation sites(predicted by NetPhos 2.0 software).Through site-directed mutagenesis experiment,it was found that the capacity of Cd-resistance was significantly reduced when the two CCXXG domains were mutated to AAXXA,but mutation of the putative phosphorylation sites had no evident effects.Through yeast two hybrid library screening,OsFWL4 was found to interact with OsRbCS(Rubisco Small Subunit),and the interaction was further confirmed by yeast two hybrid point-to-point and BiFC experiments.In addition,the secondary structure of OsFWL2 protein was predicted to have two transmembrane domains.Then a series of truncations of OsFWL2 were made based on its secondary structure.We found that only the fragments containing two transmembrane domains had the capacity of Cd-resistance when expressed in yeast.The above results showed that OsFWL2 and OsFWL4 have important roles in cadmium resistance in rice.Their mechanisms may be complex.This study further unveiled the characteristics and functions of the OsFWL gene family,and provided clues for the future research on its mechanisms.