| Cadmium(Cd)is one of the most toxic and non-essential heavy metals and is widely distributed in the environment by natural processes and human activities.In many areas worldwide,soils are increasingly contaminated with Cd,raising concerns over food safety and environmental sustainability.Cadmium is relatively mobile in the soil and is readily taken up by plants.Cadmium not only has negative effects on plant growth and development,but also pose a risk to human health via the food chain.Cadmium can affect a wide range of physiological and biochemical processes in plants,including photosynthesis,transpiration,nutrient uptake,activities of many enzymes and generation of oxidative stress,resulting in growth inhibition.In humans,excessive exposure to Cd can cause renal dysfunction,osteoporosis and cancers.Recent studies have reported a number of genes involved in Cd accumulation or Cd detoxification in rice(Oryza sativa)and Arabidopsis(Arabidopsis thaliana).The most important mechanism for Cd detoxification in plants is that phytochelatins(PCs)are chelated with Cd2+ and the PC-Cd complexes are transferred to vacuoles for sequestration by some corresponding transporters.Therefore,defects in the synthesis of PCs can result in Cd sensitivity of plants.In order to further explore the mechanism about Cd accumulation or Cd detoxification in rice or Arabidopsis,an EMS(ethylmethylsulfone)-mutagenized population of rice or Arabidopsis was screened.We isolated a rice mutant with enhanced Cd tolerance,named cadt1(cadmium-tolerant 1),and an Arabidopsis Cd-hypersensitive mutant in the cad1-3(cadmium-sensitive 1-3)mutant background,which was defective in PCs synthesis,named cdsr1 cad1-3(Cd-induced short root 1 in cad1-3 background).Using whole genome resequencing and complementation tests,the causal gene was cloned,and detailed molecular genetics and physiological analyses were used to analyze the function of the gene.The main results are summarized as follows:1.A cadmium(Cd)-tolerant mutant of rice,named cadt1,was isolated from an EMSmutagenized population of an Indica rice cultivar(Zhongjiazao 17)widely grown in southern China.Without Cd treatment,no significant difference was detected in root growth of rice between wild type(WT)and cadt1 mutant.At 2 ?M Cd,the elongation of the primary roots was inhibited by 79%in the mutant and 96%in WT,and the mutant also accumulated less hydrogen peroxide in the roots.By element content analysis,the cadmium concentration in roots of cadt1 was higher than wild type,but Cd concentration in shoots or brown rice had no difference between WT and cadt1.Using genomic resequencing and complementation,OsSHM4 was identified as the causal gene for the mutant phenotype,which was annotated as encoding a putative serine hydroxymethyltransferase(SHM).By detecting the subcellular localization of 35S:OsSHM4:eYFP fusion protein,the OsSHM4 protein was localized to the nucleus.By detecting the GUS(glucuronidase)staining in OsSHM4pro-GUS transgenic plants,OsSHM4 gene was found to be expressed in roots and shoots.Although OsSHM4 mutation had no effects on Cd concentration in the brown rice,but it siganificantlly increased the sulphur(S)and selenium(Se)concentration in shoots and brown rice in hydroponic and paddy field experiments,and it also didn't affect the growth and grain biomass of rice.Under field conditions,the unpolished brown rice of cadtl contained 83-92%higher Se concentration than WT.By transcriptomic analyses of some genes involved in the uptake,assimilation and metabolism of S in rice,a significantly higher transcript level of the highaffinity sulphate transporter gene OsSULTR1;1 was found in roots of cadt1 mutant.The expression of sulphur-deficiency inducible gene OsSDI1 in the roots and shoots of cadt1 mutant were significantly increased.Under Cd stress,thiol compounds were significantly increased in both roots and shoots of cadt1 mutant,especially the phytochelatins,which play a critical role in Cd detoxcification in rice.In summary,these results indicate that the nuclear localised OsSHM4 is involved in the regulation of sulphur homeostasis.OsSHM4 mutation can significantly increased the uptake of sulphate and selenate,and promotes the synthesis of non-protein thiols,which enhanced the Cd tolerance of rice.More importantly,the mutant has important application values in increased Cd tolerance and elevated Se concentration in the unpolised brown rice.2.A Cd-hypersensitive mutant of Arabidopsis thaliana,named cdsr1 cad1-3,was isolated from an EMS-mutagenized population of the PC synthase mutant(cad1-3)background.Under the normal conditions without Cd,the double mutant cdsr1 cad 1-3 showed no significant difference from cad1-3 in the primary root length and plant biomass.In the presence of 10-20 ?M Cd,cdsr1 cad1-3 showed a significantly shorter primary root length(by 49-32%)and a smaller plant biomass(by 62-51%)than cad1-3.We backcrossed the double mutant cdsr1 cad1-3 to Col-0 to segregate the cdsr1 single mutant,and cdsr1 was also more sensitive to Cd than Col-0.Using whole genomic resequencing and complementation,we identified PP2A-4C as the causal gene for the mutant phenotype,which was annotated as encoding one of the isoforms of the catalytic subunit of protein phosphatase 2A(PP2A).A mutant of the PP2A scaffolding subunit 1A(PP2A-1A)was also more sensitive to Cd.PP2A-4C protein was localized in the cytoplasm and the nucleus through subcellular localization.Histochemical analysis of PP2A-4Cpro-GUS transgenic plants revealed that PP2A-4C was expressed in the main leaf veins,stamens,stigma and seeds.Because PP2A-4C is a catalytic subunit of PP2A,the PP2A-4C mutation resulted in the reduced PP2A enzyme activity and Cd stress also had a negative effect on PP2A enzyme activity.Ethylene production was significantly induced by Cd in Arabidopsis,and the expression of 1-aminocyclopropane-1-carboxylic acid(ACC)synthase genes ACS6 was also upregulated by Cd,which was a key gene in ethylene synthesis.Under Cd stress,ethylene production in cdsr1 and cdsr1 cad1-3 were much more than their respective wild type Col-0 and cad1-3,and the ACC content and the total activity of ACC synthase in cdsr1 cad1-3 were significantly increased.The results demonstrate that PP2A containing 1A and 4C subunits alleviates Cd-induced growth inhibition by modulating ethylene production in Arabidopsis.Taken together,we cloned the OsSHM4 gene in rice,and it can regulate S homeostasis and the synthesis of non-protein thiols,which affect Cd detoxcification in rice.In Arbidopsis,we found the PP2A-4C gene,and it can modulate ethylene production by affecting protein phosphatase PP2A to alleviate Cd-induced growth inhibition. |
PDF Full Text Request