Transcription Factors Of OsHSF1,OsMYB45 And OsNAC300 Involved In Regulation Of Cd Tolerance In Rice

Transcriptional regulation plays an essential role in growth and resistance of plants.Using RNA-Seq,we selected some genes strongly induced by Cd from the rice genome.From these genes,we screened three transcription factors(OsHSF1,OsMYB45 and OsNAC300),which regulates rice tolerance to heavy metals.The main results of this paper are as follows:1.OsHSF1 was mainly expressed in the mature vascular bundles of root,lateral root,leaves and embryo of rice.The expression of OsHSF1 was strongly induced by Cd,JA or H2O2.OsHSF1 was a nuclear localization protein.Mutation of OsHSF1 resulted in an increase in Cd accumulation in roots and shoots of rice.At the seedling stage,the roots of oshsf1 mutants were more sensitive to Cd than the wild type'.Compared with the wild type,the contents of Cd,Cu and Zn in brown rice of oshsf1 mutants were significantly increased,but the content of Mn and Fe did not.The expression of OsNramp5 named the major transporter of Cd2+was tested,and was significantly up-regulated in two oshsf1 mutants.As yeast one hybridization shown,OsHSF1 could not bind directly to the promoter sequence of OsNramp5.These results indicated that OsHSF1 might reduce the accumulation of Cd by indirectly inhibiting the expression of OsNramp5.2.In the rice genome,OsHSFA4a was the highest homology with OsHSF1,The expression of OsHSFA4a was induced by Cd too.Yeast two hybrid,BiFC and Pulldown showed that there was an interaction between OsHSF1 and OsHSFA4a.The lower content of Cd are found in the aerial part of OsHSFA4a and OsHSF1 overexpression lines compared with the wild type.These results indicates that OsHSFA4a negatively regulates Cd accumulation in rice.However,yeast one hybrid experiment showed that OsHSFA4a could not bind directly to the promoter sequence of OsNramp5.Therefore,OsHSFA4a could not directly negatively regulate the expression of OsNramp5.3.Similar to OsHSF1,the expression of OsMYB45 was also induced by Cd and H2O2.GUS staining showed that OsMYB45 was mainly expressed in the embryo,lateral roots or leaves,and was also slightly expressed on the flower axis,palea,lemma,pistil arid stamens.OsMYB45 is a nuclear localization protein.The osmyb45 mutant was more sensitive than the wild type under Cd stress.The content of Cd in osmyb45 mutation line did not been changed in compare with the wild type'.The content of H2O2 and DAB staining showed that H2O2 accumulated in leaves of the mutant,especially under Cd stress.The activity of CAT in leaves of osmyb45 mutant line was significantly lower than that of the wild type under normal growth or Cd stress.However,activities of APX,SOD and POD did not show this difference.At the same time,we also found that activities of CAT and APX were inhibited by Cd stress.In the rice genome,CAT has three isomer genes(OsCATA,OsCATB and OsCATC).OsCATA and OsCATC had higher abundance in the leaves,and the expression level of OsCATA was five times higher than that of OsCATC and the expression of OsCATB was very low.In the osmyb45 mutant line,the expression levels of OsCATA and OsCATC were significantly lower than the wild type.The contents of H2O2,the activity of CAT and the expression level of OsCATA were complemented to these of wild-type in two independent complemental lines.These results indicated that osmyb45 mutation is responsible for the decrease in the expression of OsCATA and the activity of CAT,the increase in the content of HZO2,the Cd-sensitive phenotype.4.The expression of OsNAC300,the third transcription factor reported in this paper,was also strongly induced by Cd and H2O2.OsNAC300 was mianly expressed in roots of rice.OsNAC300 is a nuclear localization protein.Root elongation experiments showed that osnac300 knockout mutants were more sensitive to Cd.The accumulation of Cd,Mn and Zn increased in seeds of osnac300 mutants.Results of RNA-Seq and yeast one hybridization showed that osnac300 mutation affected the expression of OsCATA and OsHAK17,but OsNAC300 was not directly bound to promoter sequence of OsCA TA and OsHAK17.OsNAC300 might regulate the expression of some functional genes to influence rice tolerance to Cd stress,but its direct downstream gene remains unclear.In summary,transcriptional regulation plays an important role in the process of accumulation and tolerance of Cd to rice.