Barley HvHMA1 Is a Heavy Metal Pump Involved in Mobilizing Organellar Zn and Cu and Plays a Role in Metal Loading into Grains

Heavy metal transporters belonging to the P_1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P_1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.     

Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.     

A genomics and multi-platform metabolomics approach to identify new traits of rice quality in traditional and improved varieties

Using a novel approach combining four complementary metabolomic and mineral platforms with genome-wide genotyping at 1536 single nucleotide polymorphism (SNP) loci, we have investigated the extent of biochemical and genetic diversity in three commercially-relevant waxy rice cultivars important to food production in the Lao People??s Democratic Republic (PDR). Following cultivation with different nitrogen fertiliser regimes, multiple metabolomic data sets, including minerals, were produced and analysed using multivariate statistical methods to reveal the degree of similarity between the genotypes and to identify discriminatory compounds supported by multiple technology platforms. Results revealed little effect of nitrogen supply on metabolites related to quality, despite known yield differences. All platforms revealed unique metabolic signatures for each variety and many discriminatory compounds could be identified as being relevant to consumers in terms of nutritional value and taste or flavour. For each platform, metabolomic diversity was highly associated with genetic distance between the varieties. This study demonstrates that multiple metabolomic platforms have potential as phenotyping tools to assist breeders in their quest to combine key yield and quality characteristics. This better enables rice improvement programs to meet different consumer and farmer needs, and to address food security in rice-consuming countries.     

Biosphere-atmosphere interactions of ammonia with grasslands: Experimental strategy and results from a new European initiative

A new study to address the biosphere-atmosphere exchange of ammonia (NH₃) with grasslands is applying a European transect to interpret NH₃ fluxes in relation to atmospheric conditions, grassland management and soil chemistry. Micrometeorological measurements using the aerodynamic gradient method (AGM) with continuous NH₃ detectors are supported by bioassays of the NH₃ `stomatal compensation point' (<sub>s</sub>). Relaxed eddy accumulation (REA) is also applied to enable flux measurements at one height; this is relevant to help address flux divergence due to gas-particle inter-conversion or the presence of local sources in a landscape.Continuous measurements that contrast intensively managed grasslands with semi-natural grasslands allow a scaling up from 15 min values to seasonal means. The measurements demonstrate the bi-directional nature of NH<sub>3</sub> fluxes, with typically daytime emission and small nocturnal deposition. They confirm the existence of enhanced NH<sub>3</sub> emissions (e.g. 30 g N ha<sup>–1</sup> d<sup>–1</sup>) following cutting of intensively managed swards. Further increased emissions follow fertilization with NH<sub>4</sub>NO<sub>3</sub> (typically 70 g N ha<sup>–1</sup> d<sup>–1</sup>). Measurements using REA support these patterns, but require a greater analytical precision than with the AGM.The results are being used to develop models of NH<sub>3</sub> exchange. `Canopy compensation point' resistance models reproduce bi-directional diurnal patterns, but currently lack a mechanistic basis to predict changes in relation to grassland phenology. An advance proposal here is the coupling of _s to dynamic models of grassland C–N cycling, and a relationship with modelled plant substrate-N is shown. Applications of the work include incorporation of the resistance models in NH₃ dispersion modelling and assessment of global change scenarios.     

Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status

Substantial concentrations of NH₄ ⁺ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with ¹⁵NH₄ ⁺ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH₄ ⁺ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH₄ ⁺ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH₄ ⁺ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH₄ ⁺. Withdrawal of NH₄ ⁺ supply for up to 96 h had little effect on mRNA expression or NH₄ ⁺ uptake; however, plants grown continuously at high NH₄ ⁺ levels exhibited decreased mRNA expression. BnAMT1;2mRNA expression was highest when NH₄ ⁺ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2may be regulated by similar mechanisms to GS2 in leaves.     

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.     

Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction

Regulation of the cytosolic isozyme of glutamine synthetase (GS(1); EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS(1) was the only active GS isozyme in senescing leaves. By use of [gamma-(32)P]ATP followed by immunodetection, it was shown that GS(1) is a phospho-protein. GS(1) is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS(1) is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS(1) changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS(1) interacts with 14-3-3 proteins as verified by two different methods: a His-tag 14-3-3 protein column affinity method combined with immunodetection, and a far-Western method with overlay of 14-3-3-GFP. The degree of interaction with 14-3-3-proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS(1). Thus, the results demonstrate that 14-3-3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post-translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14-3-3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.     

Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.