| With the increasing industrialization,heavy metal pollution has become a major threat to crop production and food safety.Heavy metal pollution contaminates soils and reduces the quality of water and atmospheric environment.Heavy metals in the soil are easily absorbed by plants,thus affecting crop growth and seriously threatening human health through the food chain.Remediation of heavy metal contaminated land and reduction of heavy metal accumulation in rice grains are two strategies that can help deal with heavy metal pollution.At the molecular level,understanding the absorption,transport and accumulation of heavy metals and the detoxification mechanism of plants requires digging out relevant genes in plants.Identification of genes potentialy involved in plant tolerance and detoxification of heavy metals has become a feasible approach to solve environmental pollution and food security.Heavy metal-associated isoprenylated plant proteins（HIPPs）are a subclass of heavy metal binding proteins namely metallochaperone family.They are characterized by one or two HMA domains and an isoprenoylation motif.The known heavy metal-related domain HMA is conserved in animal,plant and bacterial proteins,and its core sequence（M/L/IxCxxC）has two characteristic cysteines,which are involved in heavy metal binding.HIPPs play an important role in plant development and response to environmental stresses.In this paper,OsHIPP56,a member of the rice HIPP family with unknown functions,was studied.OsHIPP56 is a HIPP member selected by our laboratory from a transcriptome library.Bioinformatics analysis shows that it contains an HMA domain and an isoprenyl sequence,without transmembrane structure.Analysis of the expression pattern of OsHIPP56 in different tissues during the growth and development stages revealed that the gene was expressed in the roots,basal stem and leaf during the vegetative and reproductive stage;during the booting and flowering stage,it was highly expressed in immature panicle,followed by peduncle while at grain filling stage,the expression level in the leaf blade reached the highest,followed by husk and node I.OsHIPP56 expression was upregulated by excess heavy metals manganese（Mn）,cadmium（Cd）and zinc（Zn）.Tobacco subcellular localization test showed that OsHIPP56 was localized to the plasma membrane and nucleus.Expression of OsHIPP56 in yeast mutant pmr1 significantly enhanced the tolerance of yeast to Mn toxicity.Therefore,OsHIPP56 may be involved in detoxification of heavy metals in rice plants.OsHIPP56 Crispr/cas9 mutant and overexpressing lines were constructed and exposed to the heavy metals.The results showed that OsHIPP56-Crispr/cas9 mutants showed obvious symptoms of Mn poisoning characterized by yellow leaves,brown spots,and decreased chloropyll and biomass following Mn treatment.Compared with the wild type,the aboveground tissues of the overexpression lines had higher tolerance to Mn.Accumulation of Mn in mutant lines was higher than in that of wild type,wherase it was contrast with to the overexpression lines which were recorded with a lower accumulation of Mn relative to wild type.Under 250 μM Mn,the content of Mn in overground tissues of mutant lines was 1.1-1.3 times of WT,and the accumulation of Mn in roots was 1.2-1.3 times of WT,while the content of Mn in roots and overground tissues of OE lines was 61.4-72.4%and 65.7-71.4%of WT,respectively.After the treatment of rice seedlings with Cd,the growth of the Crispr/cas9 lines was inhibited and the plants were dwarfed with reduced shoot and root biomass and increased accumulation of Cd.However,the overexpression plants had improved tolerance to Cd toxicity to a certain extent,and plant height and biomass were significantly increased compared to wild type,wherase the accumulation of Cd content was lower than that of wild type.In summary,OsHIPP56 plays an important role in reducing accumulation of Mn and Cd in rice,thereby enhancing rice tolerance to Mn and Cd. |
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