The Role Of Cytokinin In Selenium Stress Response In Arabidopsis

Cytokinin?CK?regulate many developmental processes and environmental stress responses in plants.In this study,we used CK-deficient plant ipt1 3 5 7,CK-receptor-deficient mutants ahk2 and ahk3to study the role of cytokinin in selenite stress response in Arabidopsis.The specific results are listed as follows:1.We verified the cytokinin-related mutants and proved that the mutants used in this study were homozygous;Then we studied the role of cytokinin in selenium?Se?stress response in Arabidopsis.The ipt1 3 5 7 had longer roots compared with the WT under 30?M Na₂SeO₃ condition and showed more resistant in the presence of Se.however,under the Na₂SeO₃-containing media the root length of ahk2 and ahk3 were shorter than WT and the ahk2 and ahk3 were sensitive to Se.2.The CK content in ipt1 3 5 7,ahk2 and ahk3 treated with 30?M Na₂SeO₃ were measured.The CK contents of ipt1 3 5 7 were lower than that of WT in the presence of Se since loss-of function mutation in key enzyme of CK biosynthesis,which has severely decreased CK level.In contrast,the CK contents were higher in ahk2 and ahk3than in WT under control and treated condition.In addition,we tested the effect of exogenous CK?6-BA?application on Se stress.When 6-BA was added together with Se,root growth was inhibited severely in both WT and mutants and Se tolerance was abolished in ipt1 3 5 7.Further we studiedthe gene expression of IPT1,AHK2,AHK3,AHK4 and the type-A ARRs ARR3,ARR5,ARR7 under 30?M Na₂SeO₃ stress.The results suggest that Reduced CK level induced by Seapplication might be associated,at least in part,with negative feedbackof enhanced type-A ARR3,ARR5 and ARR7.3.We determined the Se content in WT,ipt1 3 5 7,ahk2 and ahk3 plants subjected to Se treatment and analyzed by ICP-MS.Se contents were lower in ipt1 3 5 7 mutant and higher in ahk2,ahk3 seedlings than in WT,respectively.The results suggested that enhanced Se resistant in ipt1 3 5 7 mutant might be relevant to decreased Se content,and Se sensitivity in ahk2 and ahk3 mutants may also be relevant to increased Se content.Selenite is absorbed by the phosphate?Pi?transporters in plant.Then we detected the transcription levels of PHT1;1,PHT1;8,PHT1;9 and PHT2;1 in the presence of Na₂SeO₃,PHT1;1 and PHT1;8 transcriptionswere lower,and PHT2;1transcription was higher in ipt1 3 5 7 than in WT.This reduced Se uptake and promoted Setranslocation form root,contributing to Se resistance in ipt1 3 5 7mutants.In contrast,the mutant ahk2 and ahk3 contained higher transcriptions of PHT1;1,PHT1;8 and PHT1;9 than the WT under 30?M Na₂SeO₃ stress,which increasing Se uptake and accumulation,and resulting in Se sensitivity.These results suggest that Seuptake and accumulation can be induced by endogenous CK.4.Selenocysteine methyltransferase?SMT?and Selenocysteinelyase?SL?play an important role in Se detoxification.We detected the transcription levels of SMT and SL by RT-PCR and qRT-PCR.SMT transcription level was higher in ipt1 3 5 7 than in WT,ahk2 and ahk3 mutants in the presence of Na₂SeO₃,while no obvious differences in SL transcriptions were observed among these accessions.suggesting Se detoxification is down-regulated by CK.5.We compared the growth of ipt1 3 5 7,ahk2 and ahk3 plants in media containing BSO.When BSO was added to the Na₂SeO₃ containing media,the resistance of ipt1 35 7 mutant was disappeared,while selenite-sensitive ahk2 and ahk3 were not affected by BSO treatment.In addition,the activities of antioxidant enzyme GPX,APX,CAT and POD in WT and mutants were analyzed.We found that enhanced CK levels could down-regulateantioxidant activityunder Se treatment condition.Moreover,the ipt1 3 57 showed higher increased GSH content and higher transcript level of GSH1relative to WT subjected to Se.These results indicate that GSH is required for Se tolerance of ipt13 5 7.In summary,the results presented here provide genetic evidencethat CK negatively regulates Arabidopsis Se stress response through controlling Se uptake and detoxification as well as antioxidant activity.