Abstract
SaNramp6 in Sedum alfredii encodes a membrane-localized metal transporter. We isolated the SaNramp6h allele from the hyperaccumulating ecotype (HE) of S. alfredii. When this allele was expressed in transgenic yeast and Arabidopsis thaliana, it enhanced their cadmium (Cd) sensitivity by increased Cd transport and accumulation. We isolated another allele, SaNramp6n, from a nonhyperaccumulating ecotype (NHE) of S. alfredii. Amino acid sequence comparisons revealed three amino acid differences between SaNramp6h and SaNramp6n. We investigated the Cd transport activity of the Nramp6 allele, and determined which residues are essential for the transport activity. We conducted structure-function analyses of SaNramp6 based on site-directed mutagenesis and functional assays of the mutants in yeast and Arabidopsis. The three residues that differed between SaNramp6h and SaNramp6n were mutated. Only the L157P mutation of SaNramp6h impaired Cd transport. The other mutations, S218N and T504A, did not affect the transport activity of SaNramp6h, indicating that these residues are not essential for metal selectivity. Transgenic plants overexpressing SaNramp6hL157P showed altered metal accumulation in shoots and roots. Our results suggest that the conserved site L157 is essential for the high metal transport activity of SaNramp6h. This information may be useful for limiting or increasing Cd transport by other plant natural resistance associated macrophage protein (NRAMP) proteins.
Highlights
Cadmium (Cd) is a heavy metal with strong biological toxicity that is widely distributed in soil, water and the atmosphere
The coding regions of SaNramp6 from the two distinct ecotypes studied previously were sequenced and compared. This comparison showed that five nucleotides differ between SaNramp6h and SaNramp6n (Figure 1), corresponding to three amino acid differences between SaNramp6h and SaNramp6n at position 157 (Leu to Pro), 218 (Ser to Asn), and 504 (Thr to Ala)
There were no obvious differences in the transcript levels of SaNramp6 in response Itnot.CJ.dMtorl.eSacti.m20e2n0t, 2i1n, xeiFtOhRerPEeEcoRtRyEpVeI.ETWhe transcript levels were higher in shoots and lower in the 3rooof 1t2s Iinnt.HJ.EMpoll.aSncit.s20c2o0m, 2p1,a3r1e6d9 with the nonhyperaccumulating ecotype (NHE) plants
Summary
Cadmium (Cd) is a heavy metal with strong biological toxicity that is widely distributed in soil, water and the atmosphere. Such pathways include the synthesis of signaling molecules and stress-related proteins, such as antioxidant enzymes, metal chelators, osmotic regulators, and heavy metal transporters These proteins function in the absorption, transport, intracellular transport, and detoxification of heavy metals, which are necessary for plants to maintain intracellular metal homeostasis. It is of great theoretical significance and practical value to study the physiological response of plants to Cd stress and the molecular mechanism of related genes and stress signal transduction pathways. Each of the three amino acid residues that differ between SaNramp6h and SaNramp6n was altered by PCR site-directed mutation, and the effects of these mutations on Cd uptake and accumulation were test by expression in yeast and A. thaliana
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