OsNHAD is a chloroplast membrane-located transporter required for resistance to salt stress in rice (Oryza sativa).

Salt stress is one of the major environmental factors limiting crop productivity. Although physiological and molecular characterization of salt stress response in plants has been the focus for many years, research on transporters for sodium ion (Na+) uptake, translocation and accumulation in plants, particularly in food crops like rice is limited. In this study, we functionally identified an uncharacterized sodium ion transporter named OsNHAD which encodes a putative Na+ / H+ antiporter in rice. Homology search shows its close relation to the Arabidopsis Na+/H+ antiporter AtNHD1 with 72.74% identity of amino acids. OsNHAD transcripts mainly express in leaves and are induced by Na+ stress. Confocal laser scanning microscopy analysis of OsNHAD::GFP fusion in tobacco leaves shows that OsNHAD resides in the chloroplast envelop. Knock-down of OsNHAD by RNA interference led to increased rice sensitivity to Na+, manifested by stunted plant growth, enhanced cellular damage, reduced PSII activity and changed chloroplast morphology. Mutation of OsNHAD also resulted in accumulation of more Na+ in chloroplasts and in shoots as well, suggesting that OsNHAD is involved in mediating efflux and detoxification of Na+ but does not affect K+ accumulation in plant cells. Complementation test reveals that OsNHAD was able to functionally restore the Arabidopsis mutant atnhd1-1 growth phenotype. These results suggest that OsNHAD possibly mediates homeostasis of sodium ions in the subcellular compartments and tissues of the plants when challenged to salt stress.

Identification of epigenetic mechanisms in paddy crop associated with lowering environmentally related cadmium risks to food safety.

Cadmium (Cd) is a toxic metal that contributes to human diseases such as pediatric cancer and cardiovascular dysfunction. Epigenetic modification caused by Cd exposure is the major factor in etiology of environmentally-relevant diseases. However, the underlying epigenetic mechanism for Cd uptake and accumulation in food crops, particularly those growing in Cd-contaminated environments, is largely unknown. This study investigated uncharacterized regulatory mechanisms and biological functions of global DNA hypomethylation at CG sites that are associated with gene expression for Cd detoxification and accumulation in the food crop rice. Mutation of the CG maintenance enzyme OsMET1 confers rice tolerance to Cd exposure. Genome-wide analysis of OsMET1 loss of function mutant Osmet1 and its wild type shows numerous loci differentially methylated and upregulated genes for Cd detoxification, transport and accumulation. We functionally identified a new locus for a putative cadmium tolerance factor (here termed as OsCTF) and demonstrated that Cd-induced DNA demethylation is the drive of OsCTF expression. The 3'-UTR of OsCTF is the primary site of DNA and histone (H3K9me2) demethylation, which is associated with higher levels of OsCTF transcripts detected in the Osmet1 and Ossdg714 mutant lines. Mutation of OsCTF in rice led to hypersensitivity to Cd and the Osctf line accumulated more Cd, whereas transfer of OsCTF back to the Osctf mutant completely restored the normal phenotype. Our work unveiled an important epigenetic mechanism and will help develop breeding crops that contribute to food security and better human health.

OsZIP1 functions as a metal efflux transporter limiting excess zinc, copper and cadmium accumulation in rice.

BACKGROUND: Metal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown. RESULTS: In this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress. CONCLUSION: OsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.

Identification of novel rice (Oryza sativa) HPP and HIPP genes tolerant to heavy metal toxicity.

HPP (heavy metal associated plant protein) and HIPP (heavy metal associated isoprenylated plant protein) are a group of metal-binding metallochaperones playing crucial roles in metal homeostasis and detoxification. Up to now, only few of them have been functionally identified in plants. Here, we identified 54 HPP and HIPP genes in rice genome. Analysis of the transcriptome datasets of the rice genome exposed to cadmium (Cd) revealed 17 HPP/HIPP genes differentially expressed, with 11 being upregulated (>2 fold change, p < 0.05). Comprehensive analysis of transcripts by qRT-PCR showed that both types of genes displayed diverse expression pattern in rice under excess manganese (Mn), copper (Cu) and Cd stress. Multiple genomic analyses of HPPs/HIPPs including phylogenesis, conserved domains and motifs, genomic arrangement and genomic and tandem duplication were performed. To identify the role of the genes, OsHIPP16, OsHIPP34 and OsHIPP60 were randomly selected to express in yeast (Saccharomyces cerevisiae) mutants pmrl, cup2, ycf1 and zrc1, exhibiting sensitivity to Mn, Cu, Cd and Zn toxicity, respectively. Complementation test showed that the transformed cells accumulated more metals in the cells, but their growth status was improved. To confirm the functional role, two mutant oshipp42 lines defective in OsHIPP42 expression were identified under metal stress. Under normal condition, no difference of growth between the oshipp42 mutant and wild-type plants was observed. Upon excess Cu, Zn, Cd and Mn, the oshipp42 lines grew weaker than the wild-type. Our work provided a novel source of heavy metal-binding genes in rice that can be potentially used to develop engineered plants for phytoremediation in heavy metal-contaminated soils.