Functional Analysis Of MAN3 And ZAT6 In Regulation Of Cadmium Tolerance And Accumulation In Arabidopsis

Pollution of soil by the heavy metal cadmium (Cd) is a global environmental problem with high toxicity to animals and plants.In preliminary studies, we identified an Arabidopsis cadmium-tolerant dominant mutant xcd1-D (XVE system-induced cadmium-tolerance) and cloned MAN3 gene.MAN3 plays a positive role in the adaptation to Cd stress. The enhanced Cd-tolerance phenotype of xcdl-D was dependent on estradiol-induced overexpression of MAN3.Overexpression of MAN3 led to enhanced Cd tolerance, whereas loss-of-function of MAN3 resulted in decreased Cd tolerance, we further determine the role of MAN3 in Cd tolerance.MAN3 gene encodes an endo-β-mannanase, which is localized in the apoplastic space and possesses mannanase enzyme activity, leading a accumulation of mannose.Mannose play an important role in the regulation of MAN3-mediated Cd tolerance. Mannose can abolish the Cd-triggered growth inhibition in both WT and xcdl mutant.In order to understand the mechanism mannose-mediated Cd tolerance, We determined the effects of mannose on Cd accumulation, GSH and PC concentrations under Cd treatment, Also, The BSO, a GSH synthesis inhibitor, was used to examine the effect on mannose-triggered Cd tolerance. All the results suggest that mannose treatment increases the PC content by coordinated control of expression of Cd tolerance-related genes involved in GSH-dependent PC synthesis pathway.which results in enhanced Cd accumulation and tolerance.It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, thetranscription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown.Here, wereported a previously unknown function of ZAT6 in plant responses to Cd stress, which coordinately activates PC-synthesis related gene expression and directly targets GSH1 to regulate Cd tolerance in Arabidopsis.ZAT6, a C2H2-type zinc finger protein (ZFP) represent a large family of eukaryotic transcription factors, was cloned from a cadmium-toleranced mutant xcd2-D (XVE system-induced cadmium-tolerance) by Tail-PCR. We found that ZAT6 is involved in Cd tolerance inArabidopsis. The expression level of ZAT6 was strongly induced by Cd stress, Moreover, transgenic plants overexpressing ZAT6 showed enhanced Cd tolerance, while zat6 mutant plants displayed increased Cd sensitivity, suggesting that ZAT6 plays an important role in Cd tolerance.Enhanced expression of ZAT6 accelerates Cd accumulation in plants and the improved growth rate of the ZAT6-OE plants over WT plants to Cd stress disappeared in the BSO-containing media, these results suggest thatthe GSH-dependent PCs synthesis pathway is involved in regulation of ZAT6-mediated Cd tolerance. The data of GSH and PC content in ZAT6-OE plants, WT plants and zat6 mutant support the notion that ZAT6 positively regulates Cd tolerance through the GSH-dependent PC synthesis pathway.Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1 and PCS2. But ZAT6 is capable of specifically binding to GSH1 promoter in vivo by Chip-PCR assay. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6.Taken together, our data provide evidence that ZAT6 coordinately activates PC-synthesis related gene expression and directly targets GSH1 to positively regulate Cd accumulationand tolerance in Arabidopsis.