Impairing both HMA4 homeologs is required for cadmium reduction in tobacco

In tobacco, the heavy metal P1B‐ATPases HMA4.1 and HMA4.2 function in root‐to‐shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root‐to‐shoot transfer by >90%. Analysis of plants with segregating null and wild‐type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild‐type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.     

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.     

Uptake of zinc by rye,bread wheat and durum wheat cultivars differing in zinc efficiency

Effect of zinc (Zn) nutritional status on uptake of inorganic ⁶⁵Zn was studied in rye (Secale cereale, cv. Aslim), three bread wheat (Triticum aestivum, cvs. Dagdas, Bezostaja, BDME-10) and durum wheat (Triticum durum, cv. Kunduru-1149) cultivars grown for 13 days in nutrient solution under controlled environmental conditions. The cultivars were selected based on their response to Zn deficiency and to Zn fertilization in calcareous soils under field conditions. When grown in Zn-deficient calcareous soil in the field, the rye cultivar had the highest, and the durum wheat the lowest Zn efficiency. Among the bread wheats, BDME-10 showed higher susceptibility to Zn deficiency and Bezostaja and Dagdas were less affected by Zn deficiency. Similarly to field conditions, in nutrient solution visual Zn deficiency symptoms (i.e. necrotic lesions on leaf blade) appeared to be more severe in Kunduru-1149 and BDME-10 and less severe in rye cultivar Aslim. Under Zn deficiency, shoot concentrations of Zn were similar between all cultivars. Cultivars with adequate Zn supply did not differ in uptake and root-to-shoot translocation rate of ⁶⁵Zn, but under Zn deficiency there were distinct differences; rye showed the highest rate of Zn uptake and the durum wheat the lowest. In the case of bread wheat cultivars, ⁶⁵Zn uptake rate was about the same and not related to their differential Zn efficiency. Under Zn deficiency, rye had the highest rate of root-to-shoot translocation of ⁶⁵Zn, while all bread and durum wheat cultivars were similar in their capacity to translocate ⁶⁵Zn from roots to shoots. When Zn²⁺ activity in uptake solution ranged between 117 p M and 34550 pM, Zn-efficient and Zn-inefficient bread wheat genotypes were again similar in uptake and root-to-shoot translocation rate of ⁶⁵Zn. The results indicate that high Zn efficiency of rye can be attributed to its greater Zn uptake capacity from soils. The inability of the durum wheat cultivar Kunduru-1149 to have a high Zn uptake capacity seems to be an important reason for its Zn inefficiency. Differential Zn efficiency between the bread wheat cultivars used in this study is not related to their capacity to take up inorganic Zn. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hair concentrations of calcium, iron, and zinc in pregnant women and effects of supplementation

In this investigation, the concentration levels of hair elements of calcium, iron, and zinc were measured in pregnant women from Tianjin metropolis, China. The subjects were 93 cases of pregnant women who had been suffering from calcium, iron, or zinc deficiency judged by blood tests at the mid-term of the second trimester or early in the third trimester. Of these 93 cases, 82 subjects had their hair element levels measured when the blood tests were conducted. Then, they were supplied with mineral element nutrients of gluconic acidic zinc (noted as Zn-nutrient), gluconic acidic calcium (Ca-nutrient), or/and ferrous sulfate (Fe-nutrient) which were correspondent to the deficient element(s) for more than 2 mo before 84 subjects returned to hospital for further diagnoses and had their hair element levels measured for the second time. Finally, in the third trimester or nearparturient phase, 13 subjects had their hair element levels measured again. Except for the deficiencies of calcium, iron, or/and zinc, these subjects were all healthy without symptoms of any diseases. The concentrations of hair Ca, Fe, and Zn were measured by X-ray fluorescence (XRF) spectrometry. These concentrations of the three hair elements measured at three different times were statistically analyzed. From the analyses, it was clear that hair concentrations of Ca, Fe, and Zn could reflect the effects of supplementation. Also, the mutual resistant effects among Ca-, Fe-, and Zn-nutrients were revealed. However, by appropriate combination, the mutual resistant effects could be depressed and mutual promotional effects might be enhanced. Finally, it could be concluded that mineral element deficiencies might be convalesced by adequate compensations of mineral element nutrients.     

Root Morphology and Zn2+ Uptake Kinetics of the Zn Hyperaccumulator of Sedum alfredii Hance

Root morphology and Zn2 uptake kinetics of the hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance were investigated using hydroponic methods and the radiotracer flux technique. The results indicate that root length, root surface area, and root volume of NHE decreased significantly with increasing Zn2 concentration in growth media, whereas the root growth of HE was not adversely affected, and was even promoted, by 500 μmol/L Zn2 . The concentrations of Zn2 in both ecotypes of S. alfredii were positively correlated with root length, root surface area and root volumes, but no such correlation was found for root diameter. The uptake kinetics for 65Zn2 in roots of both ecotypes of S. alfredii were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period of investigation. The concentration-dependent uptake kinetics of the two ecotypes of S. alfredii could be characterized by the Michaelis-Menten equation, with the Vmax for 65Zn2 influx being threefold greater in HE compared with NHE, indicating that enhanced absorption into the root was one of the mechanisms involved in Zn hyperaccumulation. A significantly larger Vmax value suggested that there was a higher density of Zn transporters per unit membrane area in HE roots.     

Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.     

Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys₂ His₂ zinc finger induces structural rearrangements of typical DNA base determinant positions

Among heavy metals, whose toxicity cause a steadily increasing of environmental pollution, cadmium is of special concern due to its relatively high mobility in soils and potential toxicity at low concentrations. Given their ubiquitous role, zinc fingers domains have been proposed as mediators for the toxic and carcinogenic effects exerted by xenobiotic metals. To verify the structural effects of zinc replacement by cadmium in zinc fingers, we have determined the high resolution structure of the single Cys₂His₂ zinc finger of the Arabidopsis thaliana SUPERMAN protein (SUP37) complexed to the cadmium ion by means of UV–vis and NMR techniques. SUP37 is able to bind Cd(II), though with a dissociation constant higher than that measured for Zn(II). Cd‐SUP37 retains the ββα fold but experiences a global structural rearrangement affecting both the relative orientation of the secondary structure elements and the position of side chains involved in DNA recognition: among them Ser17 side chain, which we show to be essential for DNA binding, experiences the largest displacement. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 801‐810, 2011.