Molecular Mechanism Of ABC Transporter OsABCG36 In Rice Cadmium Tolerance

Cadmium(Cd)is a highly toxic heavy metal,which causes severe damage to plant growth and development.Cadmium is naturally present in the environment with low concentration,however,with the rapid development of industrial and agricultural production,the expansion of human activities such as mining and smelting of mineral products and the use of phosphorous fertilizers containing Cd lead to the increase of Cd pollution in soil.Rice is one of the most important food crops in our daily life,and its quality and security are vital to people’s health.In rice growing process,Cd was taken up into the roots and shoots from the Cd-polluted soil,which has a serious impact on its metabolism,quality and security.Therefore,it is an important subject for researchers to reduce the toxicity of excessive Cd in soil to the growth and development of rice and other crops.ATP-binding cassette(ABC)protein is a diverse family of transmembrane transporter,which is widely found in various organisms from bacteria to humans.ABC transporters mainly generate energy through hydrolysis of ATP to complete the transport of substrates,including hormones,lipids,inorganic acids,glutathione conjugates,polypeptides,heavy metals and so on.Many studies have reported that ABC transporters play a crucial role in Cd detoxification in plants.ABCG transporters belong to the G subfamily of ABC transporters.So far,few ABCG transporter are reported to be involved in Cd detoxification in rice.In this study,we used the CRISPR/Cas9 genome editing technology to construct the homozygous mutant of G-type ABC transporter gene OsABCG36 and analyzed its spatio-temporal expression pattern,subcellular localization,transport activity in yeast and physiological phenotypes of the knockout lines to clarify the biological function of OsABCG36 in rice.The main results of this study are as follows:(1)The expression levels of OsABCG36 were lower in roots and shoots,but its expression were significantly increased only in roots after Cd treatment.The expression level of OsABCG36 was similar in the root tips(0-1 cm)and the mature zones(1-2 cm)in both the presence and absence of Cd.Additionally,the expression of OsABCG36 was induced by Cd in a dose-dependent manner.A time-course analysis of Cd-treated roots showed that the expression of OsABCG36 was up-regulated rapidly by Cd and reached its highest level at 3 h.These results suggested that OsABCG36 is regulated by Cd in roots.(2)Immunostaining with a GFP antibody showed that the signal was weakly detected in the root cells under normal conditions.Furthermore,the signal in all root cells except the epidermal cells was significantly enhanced byexposure to Cd.(3)Transient expression of OsABCG36 in rice protoplast cells showed that the green fluorescence signal of GFP-OsABCG36 was merged with the red fluorescence signal of plasma membrane-localized marker.This result indicated that OsABCG36 was located at the plasma membrane.(4)When expressed OsABCG36 in yeast cells,the cells carrying pYES2-OsABCG36 exhibited better growth than those carrying the vector control pYES2 on SD-ura(Galactose)medium containing Cd.Cd efflux experiment showed that the OsABCG36-expressing yeast cells accumulate less Cd compared with the vector control during the Cd release period.These results indicated that OsABCG36 can export Cd from yeast cells,thus enhancing the tolerance to Cd in yeast.(5)Two mutants with different mutations,osabcg36-1 and osabcg36-2,were obtained by CRISPR/Cas9 technology.Under Cd treatment,the relative root elongation of the knockout lines was significantly shorter than that of the wild-type,showing a decreased tolerance to Cd,but did not alter tolerance to other metals,including Zn,Cu,Al,and Pb.In addition,mutation of OsABCG36 resulted in more Cd accumulation in roots and root cell sap.These results indicated that OsABCG36 is able to transport Cd out of the root cells for Cd detoxification,which is required for Cd tolerance in rice.(6)Wild-type and two knockout lines were grown in a nutrient solution with different Cd concentrations for 14 days.As a result,in the absence of Cd orat low Cd concentration(0.1 and 1 μM),the dry weight and Cd concentration of roots and shoots were similar between wild-type rice and two mutants.However,at a higher Cd concentration(5 μM),two knockout lines showed shorter root length,less dry weight of the roots,and higher Cd concentration in roots compared with the wild type.But these differences were not found in their aerial parts.Furthermore,the concentrations of Zn,Cu,Fe,and Mn in the roots and shoots were also similar in all lines.In addition,the Cd concentration in the xylem sap was also similar in all lines.These results indicated that the knockout lines contain more Cd in roots,which may be attributed to failure to transport Cd out of the root cells,resulting in more Cd accumulation in the cytosol.Besides,OsABCG36 is likely to be not involved in shoot growth,and Cd,Zn Cu,Fe,and Mn accumulation in the aerial parts in rice.In summary,this study indicated that OsABCG36 localized at the plasma membrane was involved in rice Cd tolerance through efflux of Cd or Cd conjugates out of the root cells.