The Mechanisms Of Nitric Oxide Scavenging Systems In Regulating Arabidopsis Response To Cadmium Stress

Cadmium contamination of agricultural soils is a serious problem of affecting food safety in China.A thorough understanding of how plants response to Cd stress,which include how plants accumulate Cd and the mechanism of Cd tolerance in plants,is a prerequisite for using of biotechnology or genetic breeding methods to generate new crops with low level of Cd accumulation.Previous studies have indicated that the signaling molecule nitric oxide(NO)plays important role in regulating Cd uptake by roots and tolerance of plant to Cd stress.However,the factor controlling NO level in plant cells under Cd stress remains unclear.Therefore,in this study,Arabidopsis plants were used as plant material to investigate the role and the mechanism of scavenging systems S-nitrosoglutathione reductase(GSNOR)and Arabidopsis thaliana hemoglobins(AHb1)in regulating NO level in plants response to Cd stress by using the physiological,genetic and molecular biological approaches.The main results are summarized as follows:Cadmium exposure stimulated the activity of GSNOR in roots of Arabidopsis plants by up regulating the expression of AtGSNOR gene and GSNOR protein.Analysis the effect of Cd exposure on NO level and GSNOR activity in roots of wild type(Col-0),loss of GSNOR function in gsnorl-3 mutants and the GSNOR over-expression transgenic plant GSNOROE3 and GSNOROE5 showed that GSNOR activity negatively regulated NO level in roots under Cd stress,suggesting that improvement of GSNOR activity induced by Cd exposure is beneficial to prevent NO over-accumulation in root cells.Meanwhile,by using Col-0,transgenic Arabidopsis of overexpressing AHb1(AHbl-H7)and transgenic Arabidopsis of expressing AHb1 in antisense orientation(AHb1-L3)as experimental plants,we found that NO level in roots under Cd exposure did not affected by AHbl expression in plants.It suggested that AHb1 is not involved in regulating NO accumulation in root cells under Cd stress.Based on the above findings,we investigated the role of GSNOR and AHb1 in plant Cd uptake.Here we found that Cd uptake is not affected by AHb1,but negatively regulated by GSNOR in plant.Further analysis shows that loss of GSNOR function in gsnorl-3 mutants lead to a further increase in Cd-induced NO burst in roots and resulted in improving expression of IRT1(IRON-REGULATED TRANSPORTER 1)and increased root uptake of Cd,while the opposite patterns were observed in the GSNOR over-expression transgenic plant GSNOROE5.These effects were reversed by NO scavenger cPTIO in gsnor1-3 mutants,and by NO donor S-nitrosoglutathione in GSNOROE5 plants,suggesting that a negative regulation of either IRT1 expression or Cd uptake by GSNOR depends on its negative regulation on NO accumulation.Furthermore,we also provide compelling evidence showing that the negative regulation of Cd uptake by GSNOR could be restrained by loss function of IRT1 in plants.Taken together,we therefore concluded that GSNOR negatively regulated Cd uptake by inhibiting IRT1 owing to its function of preventing NO accumulation,which thus decreased the level of Cd in plants.Interestingly,alt hough the concentration of Cd in shoots was significantly decreased by the elevated activity of GSNOR,the Cd toxicity positively correlated with GSNOR activity in shoots of Col-0,gsnorl-3 and GSNOROE5 plants.Moreover,it is found that Cd exposure stimulated the GSNOR activity in shoots at both transcriptional and translational levels.These results demonstrated that GSNOR activated by Cd stress does not favor the Cd tolerance in plants.Further analysis showed that enhancement of GSNOR activity inhibited the expression of CATs gene and CAT activity,and increased H2O2 concentration in plants.In addition,this phenomenon can be eliminated by treatment with CAT inhibitor 3-AT treatment.Taken together,we proposed that the Cd-induced increase of GSNOR activity in shoots inhibits CAT activity through repressing CATs gene expression,which results in more H2O2 accumulation in shoot cells and thus aggravates oxidative damage caused by Cd stress.