Physiological Mechanism Of Cadmium Hyperaccumulation In Amaranth (Amaranthus Mangostanus L.)

The potential harm of soil cadmium pollution to ecological environment and human health has been increasingly widely concerned. Phytoremediation, as a kind of new and effective technology, has become an important method for cleaning up cadmium in contaminated sites. Current documented cadmium hyperaccumulators were not widely used because of their low biomass, region-specific characters, or environmental dependence. Thus, screening cadmium hyperaccumulator that suits local ecological environment conditions has become the research focus. The amaranth (Amaranthus mangostanus L.) is widely distributed and has abundant varieties in China, its rapid growth and large biomass can be served as candidate for cadmium hyperaccumulators for phytoremediation. In this paper, based on screening of cadmium hyperaccumulator in amaranth from different ecological regions, the effect of root exudates (LMWOAs) and LMWOAs addition on Cd accumulation in amaranth, and characteristics of cadmium uptake, transport and their molecular mechanism in amaranth were investigated. The main findings obtained are as follows:1. To obtain cadmium hyper-accumulator, Cd uptake in 23 amaranth cultivars from different ecological region was investigated under hydroponic culture condition, and soil culture experiment was established to probe phytoremediation potentiality of Cd contaminated soil by amaranth. The results showed that under hydroponic culture with Cd 3 mg/L, the cadmium concentration in the shoots of the cultivar Tianxingmi reached 260mg/kg, and its total cadmium uptake was the highest among various cultivars. Pot experiment was carried out to investigate the effect of cultivar Tianxingmi phytoremediation on Cd contaminated soil. Three treatment (Cd5, 10 and 25 mg/kg) were imposed to red soil, yellow brown soil and vegetable soil. In the treatment Cd 25 mg/kg, the cadmium concentration in the shoots of the cultivar Tianxingmi reached 212 mg/kg, while bioconcentration factor and shoot purification rate reached 8.50 and 3.8 %, respectively. Further, the total biomass and shoot biomass were not decreased significantly under either Cd exposure. These results suggested that cultivar Tianxingmi is a typical Cd hyperaccumulator, and can be expected to be used in phytoremediation of Cd contaminated soil.2. To examine whether low- molecular weight organic acids (LMWOAs) in root exudates of amaranth play a role in Cd hyperaccumulation, we compared the amounts and composition of LMWOAs collected from two cultivars of amaranth(Amaranthus mangostanus L.), Zibeixian, a low Cd accumulator, and Tianxingmi, a high Cd accumulator. Plants were grown hydroponically and three treatment (Cd 0, 1 and 2.5 mg/L) were later imposed for 1 wk before collection of LMWOAs. With the increase of solution Cd concentration, the biomass of Zibeixian decreased dramatically, while those of Tianxingmi remained stable. In all treatment, total amount of LMWOAs in rhizosphere of Tianxingmi was significantly higher than that in Zibeixian, and the content of LMWOAs in two cultivars followed the same tendency, i.e. citric acid＞malic acid＞acetic acid＞propinonic acid＞butyric acid, and citric acid and malic acid predominated the composition of LMWOAs. The percent distribution of total LMWOAs of Zibeixian was very similar to Tianxingmi. With the increase of solution Cd supply, the content of LMWOAs in rhizosphere increased dramatically for both two cultivars, and the increment of LMWOAs of Tianxingmi was found to be greatly higher than that of Zibeixian. Meanwhile, Cd accumulation in roots, stems and leaves for both two cultivars increased obviously with the increase of LMWOAs, and increment of Cd in Tianxingmi was significantly higher than that of Zibeixian. Therefore, Cd accumulation in amaranth was highly related to the total amount of LMWOAs in rhizosphere, and the differential increment of LMWOAs in the rhizosphere induced by Cd exposure may played an important role in Cd bioaccumulation by the high and low Cd accumulating cultivars of amaranth.3. Pot experiment and laboratory culture were carried out to investigate the effect of malic acid and citric acid addition on transformation of Cd species in red soil and yellow brown soil, as well as Cd accumulation in amaranth (c.v. Tianxingmi, a high Cd accumulator). Compared to Cd 25mg/kg treatment, Cd 25mg/kg + malic acid, and Cd25mg/kg + citric acid treatment had no effect on biomass in amaranth, while significantly increased Cd accumulation in both root and shoot of amaranth. Further, malic acid and citric acid addition could lower concentration of specific adsorp.Cd, but increase exchangeable Cd, carbonate bound Cd and organic bound Cd significantly. Above results demonstrated that malic acid and citric acid addition could significantly modify Cd species to enhance Cd accumulation in amaranth.4. Radiotracer techniques were used to investigate kinetics of ¹⁰⁹Cd²⁺ influx and the influence of Fe deficiency on Cd²⁺ uptake and transport in roots of amaranth. It was found that concentration dependent ¹⁰⁹Cd²⁺-influx kinetics could be described by a curve, which could be dissected into a saturation section ([Cd²⁺]0-13μmol/L) and a linear section ([Cd²⁺]＞13μmol/L) The former represented carrier-mediated Cd influx across root-cell plasma membrane by Michaelis equation with 2.0 and 1.5μmol/L of constant (K_m) and 45 and 130 nmol/g (DW)/h of maximum initial velocity (Vmax) in both Fe-sufficient and Fe-deficient seedlings, respectively. Cd²⁺ uptake in Fe-deficient seedlings was nearly 3-fold higher than that of in Fe-sufficient, indicating that Cd²⁺ uptake can be induced by Fe-deficiency in roots of amaranth. Results also showed that only a linear kinetics phase of ¹⁰⁹Cd binding was observed with morphologically intact root cell wall preparations, La³⁺ partially inhibited both the saturated and the linear component of Cd²⁺ influx, and abolished the saturated component by the metabolic inhibitor CCCP. Therefore, the linear component reflected apoplastically bound Cd²⁺ in cell wall of roots.5. A cDNA encoding a putative iron-regulated transporter with the length of 662bp was isolated from Amaranthus mangostanus by using RACE method and designated AmIRT1. The sequence blast and phylogenetic tree analysis indicated that AmIRT1 protein shared a high sequence similarity with other IRT1 proteins (63.04%) and was clustered into Arabidopsis thaliana zinc transporter and iron transporter. The AmIRT1 protein contained a Pfam: Zip domain and three transmembrane motifs. Northern-blot analysis showed that the abundance of AmIRT1 mRNA increased greatly under Fe-deficient conditions. The present results suggest that the AmIRT1 protein may be a novel member of the ZIP family, AmIRT1 gene expression can be enhanced under Fe deficiency, and Cd uptake is regulated by this gene in amaranth.