For the salt and hygromycin tolerance analysis in agar medium, the seeds were germinated and grown by following the method described by Bassil et al. . For the salt tolerance analysis in the pot filled with peat culture medium, the seedlings of uniform size after 2 weeks growth in a plate were selected and transplanted to the nutrient-enriched medium in a growth chamber at 23 °C with a 14 : 10 h L : D photoperiod. For salt treatment, NaCl was added to final concentrations as indicated in the figure legends. The rice hypotonic culture and soil pot culture were basically followed the protocols previously described by Li et al. and Yang et al. . Briefly, for hypotonic culture, 2-week-old seedlings of similar size were transferred to IRRI nutrient solution with specific treatment for additional 3 weeks. The nutrient concentrations of IRRI solution are as follows: N 2.5, P 0.3, K 1, Ca 1, Mg 1, Si 0.5, Fe 0.02, B 0.02, Mn 0.009, Zn 0.00077, Cu 0.00032, Mo 0.00039. For salt-stressed pot culture, 2-week-old seedlings were acclimated to the soil containing 0.1% NaCl for 1 week before being transplanted to the pot filled with 40 kg of soil which had been fertilized and mixture with indicated extra amount of salts for 2 weeks. For either K+ deficient or N + P deficient soil culture, 4-week-old seedlings were transplanted to the respective isolated paddy field and no extra fertilizer was added during the growth season. The soil properties used for the plant tolerance to salt stress, K+ deficiency or N+P deficiency were described in Table S2.Cadmium, arsenic, lead, and mercury are toxic metals that have no known role in animal or plant nutrition and are considered detrimental to human health and the environment. These substances are among the top 7 contaminants listed on the EPA Superfund’s list of priority hazardous substances, and can be found at high levels in soils and waters throughout the country.Metals are widely used in industrial processes and equipment,grow bench including battery production, electronics, paints, fertilizers and fuel production.
Industrialization has drastically increased emissions of toxic metals into the environment, which is a major concern for people living near industrial areas where toxic metal emissions are highest 3 . Metal contamination has become a serious worldwide environmental health issue due to two centuries of intense industrial activity combined with inappropriate waste disposal. Many diseases and disorders have been linked to high levels of toxic metal exposure, including high rates of lung cancer in workers from cadmium recycling and recovery facilities. The toxic effects of heavy metals have also been linked to hypertension, myocardial infarction, and diminished lung function. Plants and seeds are the main dietary source of many essential metals, including zinc, iron, manganese and copper, not only for humans but also for livestock. Plant based products are also the main entry point for toxic elements into the food chain, and many cases of heavy metal poisoning have been attributed to widespread consumption of contaminated products. Understanding the molecular mechanisms underlying plant uptake, transport and accumulation of both essential and non-essential metals will have two major impacts on human health. First, it will enhance the nutritional value and safety of plant products by enhancing the accumulation of essential metals while avoiding the retention of toxic metals. Second, it will allow us to use plants to restore and remediate heavy metal contaminated sites, which is a preferred alternative to physical removal of metals. The identification of genes and molecular mechanisms that allow plants to take up, tolerate and accumulate toxic metals will accelerate the engineering of plants for remediation purposes.The distribution of metals within a plant is a dynamic process that can be divided into the following processes: root uptake and intercellular mobilization, xylemloading/unloading and phloem-loading/unloading. In Arabidopsis, Fe, Cd, Mn and Zn are taken up from soil by IRT1, a member of the ZIP family of transporters, while arsenic has been shown to be taken up by inorganic phosphate transporters. Once inside the cell, metals can be sequestered into different cellular compartments or mobilized through the root for xylem loading and root to-shoot transport . Cadmium uptake by IRT1 and xylem loading mediated by HMA2 and HMA4 have been extensively studied. However, phloem transport and seed loading have been far less studied, perhaps due to the technical difficulties associated with phloem sampling and sap modification. Phloem plays a key role in delivering compounds to developing seeds where xylem-mediated transport is negligible due to the limited transpiration rate within reproductive tissues.
Phloem is a plant tissue composed of two highly specialized low-abundance cells called companion cells and sieve element. Companion cells transfer molecules into sieve elements for long-distance transport between mature leaves, younger leaves, roots and seeds. Thus, transporters expressed in companion cells are critical proteins regulating the long-distance movement of molecules, including toxic metals. Despite their importance, the identity and abundance of phloem-specific metal transporters as well as their regulation during plant development is largely unknown. The Arabidopsis genome is estimated to have approximately 25,500 open reading frames , of which 7% are predicted to encode transporters. Based on this large number of transporters and the potential for overlapping function between members of the same gene families, a forward genetic screen designed to identify genes mediating the mobilization of molecules into seeds would be time consuming and inefficient. Therefore, alternate approaches must be developed to identify and characterize the transporters involved in mobilizing metals throughout the plant. Phytochelatins are glutathione-derived peptides synthesized in the cytosol upon exposure to Cd, As, Zn, Hg or Cu. After being synthesized, they rapidly form PCmetal complexes that are transported into vacuoles, removing these toxic elements from the cytosol. More than 15 years ago, research suggested that vacuolar uptake of PCmetal complexes was mediated by ATP-binding cassette transporters. However, attempts to identify vacuolar PC transporters in plants were unsuccessful. By conducting a systematic analysis of the ABC transporter family in the fission yeast Schizosaccharomyces pombe, we were able to identify Abc2 as a novel vacuolar PC transporter. Our results indicated that S. pombe has two independent mechanisms for vacuolar sequestration of PC-Cd complexes, one mediated by Hmt1, a half-size ABC transporter and a different mechanism mediated by Abc2, a full-size ABC transporter. Notably, plants do not have Hmt1 homologues but they do have homologues of Abc2, which are the ABCC family of ABC transporters. Furthermore, Arabidopsis mutants carrying T-DNA insertions in genes displaying a high degree of similarity with S. pombe Abc2 were subsequently identified that led to the identification of ABCC1 and ABCC2 as the long-sought plant vacuolar PC transporters. Both, ABCC1 and ABCC2 are able to mediate the uptake of PCs in vacuolar preparations obtained from yeast expressing either ABCC1 or ABCC2 .
The single insertion mutants abcc1 and abcc2 are not sensitive to either Cd or As but the double mutant abcc1 abcc2 is both Cd, Hg and As hypersensitive. ABCC1 and ABCC2 are expressed approximately 3-fold higher in roots compared to shoots and in the Arabidopsis ecotype Col-0, roots are the main sink for Cd storage. Phytochelatins have long been considered part of an intracellular mechanism for Cd detoxification. However,plant nursery benches recent evidence suggests that PCs also play a key role in mobilizing cadmium from leaves to roots. Shoot-specific expression of PC-synthase in a PC-deficient mutant showed that despite being synthesized in leaves, PCs were preferentially accumulated in roots. Transport of molecules from leaves to roots occurs exclusively through the phloem and direct analysis of phloem and xylem sap by mass spectrometry demonstrated that PCs were more abundant in the phloem sap compared to the xylem sap and in sufficient quantities to chelate the Cd found in phloem sap. These results led to a model were Cd is removed from leaves to protect photosynthesis, which is extremely sensitive to Cd 23. Other metal ligand molecules found in phloem sap are nicotianamine and GSH.In fact, extended X-ray absorption fine structure analysis of seeds shows that 60% of Cd is coordinated with thiol-containing ligands . The mechanisms by which GSH, PCs and toxic metals are loaded into the phloem are not known. The identification of phloem transporters will allow us to address these questions and advance our understanding of how molecules are mobilized between leaves and roots and into seeds. Trace metals, such as iron, zinc, manganese and copper are essential micro-nutrients to all organisms and function as co-factors in a variety of enzymes and proteins. It has been estimated that one-third of all proteins require one of these metals for proper folding and activity. Trace metals are highly reactive and their intracellular concentration must be tightly regulated to prevent toxicity. Other metals, such as cadmium , lead, chromium, mercury and the metalloid arsenic are biologically non-essential but, because of their chemical similarity, can enter plants using the same transporters used for essential metals. Inside the cells, non-essential metals impair metabolism by displacing and interfering with the function of essential metals.Non-essential metals are toxic to plants at any level; however, essential metals can also be toxic if they accumulate to high levels. Traditional methods of remediating metal contaminated soil and water include excavation, transport, and reburial of contaminated soil and evaporation, filtration or electro-chemical removal from contaminated waters. These methods are both labor and energy intensive making them cost ineffective. Because plants are sessile and have little control over the soil or water they must survive in, they have developed unique strategies to cope with metal toxicity. This makes them well adapted for use in the bio-remediation of highly contaminated sites . Cadmium is an important pollutant due to its relatively high solubility and toxicity. Cadmium has no distinct function in human health and is extremely toxic at low concentrations.
The main oxidation state of cadmium is +2, which means that it can interfere with calcium, copper, iron, magnesium, and manganese containing enzymes by displacing these elements and competing with transport. While cadmium cannot undergo Fenton-type reactions, it is highly reactive with sulfhydryl groups and is thought to cause lipid oxidation. Arsenic is an extremely toxic metalloid, and arsenic pollution has been recognized as an environmental problem worldwide. Arsenate is a chemical analogue of phosphate and can disrupt phosphate metabolism in plants, while arsenite is highly reactive with the sulfhydryl groups of enzymes and proteins, causing oxidation of proteins, inhibition of cellular function and cell death. Because cadmium and arsenic are both highly reactive with sulfhydryl groups, they are detoxified by similar mechanisms in plants. Therefore, it is important to investigate the mechanisms by which plants take up and detoxify metals. Research over the past few decades indicates that uptake and subsequent accumulation of toxicants in the aerial portions of plant tissues could provide a cost effective approach for cadmium and arsenic removal and remediation.The mechanisms underlying cadmium and arsenic detoxification are biochemically well established. Exposure to cadmium and arsenic enhances the expression of sulfate assimilation genes for detoxification. Sulfur is a macro-nutrient in plants and is available primarily in the form of sulfate present in soil. Sulfate is actively transported into roots and then distributed throughout the plant. The high affinity sulfate transporter, SULTR1;2 is induced by both arsenic and cadmium. Once sulfate enters the root, it is reduced in a series of ATP-dependent reactions and incorporated into the amino acid cysteine . Cysteine can then be converted to methionine, glutathione , and other sulfur-containing metabolites. GSH, the most abundant thiol molecule in plant cells, is synthesized in two ATP-dependent steps catalyzed by γ-glutamylcysteine synthetase and glutathione synthetase. Upon toxic metal exposure, plants also produce thiol compounds called phytochelatins, which are polymers of GSH. Phytochelatins nGly. PCs have a very high affinity for cadmium and arsenic, which allow PCs to quickly chelate and sequester them in the vacuole.PC production can be induced by a wide range of ions, including Ag+ , As, Cd2+, Cu+ , Hg2+, Cu+ , Hg2+, and Pb2+ , with Cd2+ being the most potent. Unfortunately, our understanding of the cellular signaling underlying heavy metal uptake, transport, and accumulation in plants remains incomplete.