Study On Arsenate Tolerance Mechanisms In Rice And Arabidopsis

Arsenic(As)is a toxic metalloid element,which is widely present in the environment and is harmful to humans and plants.The main forms of arsenic in soil are arsenite[As(?)]and arsenate[As(?)].In aerobic conditions,As(?)is main chemical species present in the soil.Under flooded conditions,arsenic mainly exists in the form of As(?),although As(?)can still account for a certain proportion of the As in the soil solution due to the secretion of oxygen from the roots of rice and alternate wetting and drying irrigation regime.As(?)can be absorbed by rice through phosphate transporters.However,the contribution of different phosphate transporters(OsPT1-OsPT13)to As(?)uptake and tolerance is unclear.After As(?)is absorbed into the roots,it is readily reduced to As(?),which can be extruded from the roots,complexed with phytochelatins and sequestered into the vacuole for detoxification.However,some genes involved in the detoxification processes are still unknown.In addition,the regulation of As(?)uptake and detoxification genes in rice is still unclear.After translocation to the upper part,As can cause chlorosis,affect photosynthesis and inhibit shoot growth.However,the molecular mechanism of As affecting plant shoot growth is still unclear.To explore genes involved in As(?)uptake,detoxification and regulation processes,we performed the following experiments:(1)Through the forward genetic screening,several rice As(?)tolerant mutants were isolated,and the mutant genes were identified by sequencing and linkage analysis.(2)As(?)and As(?)tolerance of rice varieties Kasalath and Nipponbare was compared.Moreover,As(?)and phosphate uptake ability,as well as phosphate transporter gene expressions,were analyzed in the two varieties.Finally we compared As(?)tolerance and uptake in ospt8 mutants of these two varieties.(3)The expression pattern,subcellular localization and tissue localization of As(?)-induced transcription factor gene,OsWRKY28,were analyzed.The role of OsWRKY28 in As(?)uptake,growth and development was studied by using T-DNA insertion mutants.The transcriptomic analysis of mutant and wild type was carried out to explore the regulation mechanism of OsWRKY28.(4)Two Arabidopsis As(?)sensitive mutants were isolated The two mutants were identified as non-allelic mutations.Heavy metal tolerance analysis,As(?)uptake test,and chloroplast morphology and quantity observation were performed on the two mutants.The mutant genes were cloned by MutMap sequencing and confirmed by complementation test.Finally,via proteomic analysis,the functions of two genes and the mechanism of As(?)sensitivity were explored.(5)Double-gene mutant materials were obtained by hybridization of the two As(?)sensitive mutants of Arabidopsis.Chlorosis was observed in the double-gene mutant in the absence of As(?).The relationship between the two mutant genes and the underlying mechanisms were explored through subcellular localization analysis,protein interaction test and proteomic data comparison.The main results are summarized as follows:(1)Three As(?)tolerant rice mutants were isolated from an EMS-mutagenized library of Kasalath.The mutants showed no difference in the tolerance to As(?),indicating that the mutant genes may be involved in the absorption of As(?),instead of As(?)detoxification process.By sequencing the reported genes related to As(?)uptake,two mutants were found with mutations in OsPT8 and the other one had a mutation in OsPHF1.Since the mutation site in OsPHF1 gene of our mutant is identical to that reported in a previous study,we only performed genotypic and phenotypic linkage analysis on the backcross F2 population of the two ospt8 mutants.Finally,we confirmed that the mutant genes were OsPT8.This experiment suggested an important role of OsPT8 in As(?)tolerance.(2)Rice variety Kasalath was more tolerant to As(?)than Nipponbare,while there was no significant difference in As(?)tolerance between the two varieties,indicating that the difference in tolerance may be mainly due to the different As(?)uptake ability.The absorption experiments showed that Kasalath had lower uptake capacity for As(?)and phosphate,and lower expression levels of the phosphate transporter genes,OsPT2 and OsPT8,than Nipponbare.Mutation in OsPT8 abolished the varietal difference in As(?)uptake and tolerance between Kasalath and Nipponbare,suggesting that the difference in As(?)uptake and tolerance between the two varieties result from differences in OsPT8 gene expression.(3)OsWRKY28 expression was found to respond to As(?)and other oxidative stress.Subcellular localization and tissue localization analysis indicated that OsWRKY28 is localized in the nucleus and expressed in roots,leaves and reproductive organs.Physiological experiments showed that the concentrations of As(?)and Pi in the shoots of oswrky28 T-DNA mutants were significantly lower than those in the wild type,but there was no significant difference in the roots,suggesting that OsWRKY28 gene may be involved in the translocation of As(?)and Pi.In addition,OsWRKY28 mutation has a negative impact on root structure and grain yield.Transcriptomic analysis and comparison of wild type and mutants showed that the expression of jasmonic acid(JA)synthesis-related genes was generally elevated in oswrky28 mutants.In addition,exogenous JA treatment significantly inhibited root length and decreased As(?)uptake,similar to the phenotype of the mutants.These results suggest that OsWRKY28 may affect the uptake/translocation of As(?)and rice development though affecting JA synthesis.(4)An EMS mutagenesis library of Arabidopsis was established by using the phytochelatin synthase gene mutant(cad1-3)as the background.An As(?)sensitive mutant aicl(arsenate induced chlorosis 1)was isolated.There was no significant growth difference in aic1 cad1-3 and cad1-3 in the absence of As(?).In the presence of As(?),aicl cad1-3 showed leaf chlorosis and growth inhibition.The As concentrations in roots,shoots and chloroplasts of aic1 cad1-3 were not significantly different from those of cad1-3,but the morphology and quantity of chloroplasts were affected more severely than those of cad1-3 by As(?)treatment.Gene cloning and complementation experiments showed that the mutant gene was the AtTOC132 gene,encoding a chloroplast outer membrane translocon.The protein is responsible for transporting preproteins synthesized in the cytoplasm into the chloroplast.By proteomic analysis and comparison of aicl cad1-3 and cad1-3,we found that the As(?)sensitive phenotype of aicl cad1-3 resulted from the reduction of proteins associated-with chloroplast ribosomes,photosynthesis,compound synthesis,and thioredoxin systems in the mutant.(5)A second As(?)sensitive mutant,aic2 cad1-3 was isolated,which showed shoot growth inhibition leaf chlorosis phenotype in the presence of As(?).There were no significant differences between aic2 cad1-3 and cad1-3 in As concentrations in in roots,shoots and chloroplasts,but the morphology and quantity of chloroplasts were more sensitive to As(?)than cad1-3.Allelic analysis indicated that aic2 mutant gene was different from aicl mutant gene.By gene cloning and complementation verification,AtDJA4,which belongs to the J protein gene family,was confirmed as the mutant gene.The AtDJA4 protein was localized on the chloroplast,but showed no interaction with the chloroplast-localized heat shock protein Hsp70.(6)To further explore the function of AtDJA4,we obtained the dja4 toc132(aicl aic2)double gene mutant by hybridization.Under normal culture conditions,the dja4 toc132 double mutant showed chlorosis and growth inhibition.Under As(?)treatment,dja4 toc132 mutant was more sensitive than single gene mutant.These results suggest that AtDJA4 may also be involved in the transport of protein precursors into the chloroplast.However,there was no protein interaction between AtDJA4 and AtTOC132 or the major chloroplast inner membrane translocons.Finally,we compared the proteomic data of the two mutants,dja4 and toc132,to find proteins that were reduced in both mutants after As(?)treatment.These proteins are involved in chloroplast ribosome formation,photosynthesis,and thioredoxin systems.Like AtTOC132,AtDJA4 is also involved in the transport of these overlapping proteins.The loss of function of these proteins in the chloroplast affects the normal metabolism of the chloroplast and leads to the yellow leaf phenotype.Taken together,our study showed that OsPT8 plays an important role in As(?)uptake,which in turn affects As(?)tolerance.The difference in As(?)tolerance between Kasalath and Nipponbare mainly results from the difference in OsPT8 gene expression.OsWRKY28 can affect the uptake/translocation of As(?)and rice development by affecting the synthesis of JA.In Arabidopsis,AtTOC132 and AtDJA4 are involved in transporting preproteins into the chloroplasts and play an important role in protecting the chloroplasts against arsenic toxicity.