Targeted expression of HvHMA2 increases the mineral content of the inner endosperm in barley.

Cereals are a major source of dietary energy and protein but are nutritionally poor in micronutrients. Zinc (Zn) biofortification of staple crops has been proposed as a promising strategy to combat the global challenge of human Zn-deficiency. The aim of this study was to improve the Zn content in the edible part of the barley (Hordeum vulgare L.) grain by enhancing Zn translocation into the developing seeds. We demonstrate that the barley plasma membrane P-type ATPase Zn transporter, HvHMA2 is an efficient candidate for mineral biofortification of crops. Following a cisgenic approach to produce transgenic homozygous barley line over-expressing HvHMA2 in the transfer cells of the grain, resulted in a doubling of a wide range of nutrients including Zn, iron (Fe), and magnesium (Mg) in the inner endosperm. This article is protected by copyright. All rights reserved.

Approach to engineer tomato by expression of AtHMA4 to enhance Zn in the aerial parts.

The aim of this work was to assess the potential for using AtHMA4 to engineer enhanced efficiency of Zn translocation to shoots, and to increase the Zn concentration in aerial tissues of tomato. AtHMA4, a P1B-ATPase, encodes a Zn export protein known to be involved in the control of Zn root-to-shoot translocation. In this work, 35S::AtHMA4 was expressed in tomato (Lycopersicon esculentum var. Beta). Wild-type and transgenic plants were tested for Zn and Cd tolerance; Zn, Fe and Cd accumulation patterns, and for the expression of endogenous Zn/Fe-homeostasis genes. At 10µM Zn exposure, a higher Zn concentration was observed in leaves of AtHMA4-expressing lines compared to wild-type, which is promising in terms of Zn biofortification. AtHMA4 also transports Cd and at 0.25µM Cd the transgenic plants showed similar levels of this element in leaves to wild-type but lower levels in roots, therefore indicating a reduction of Cd uptake due to AtHMA4 expression. Expression of this transgene AtHMA4 also resulted in distinct changes in Fe accumulation in Zn-exposed plants, and Fe/Zn-accumulation in Cd-exposed plants, even though Fe is not a substrate for AtHMA4. Analysis of the transcript abundance of key Zn/Fe-homeostasis genes showed that the pattern was distinct for transgenic and wild-type plants. The reduction of Fe accumulation observed in AtHMA4-transformants was accompanied by up-regulation of Fe-deficiency marker genes (LeFER, LeFRO1, LeIRT1), whereas down-regulation was detected in plants with the status of Fe-sufficiency. Furthermore, results strongly suggest the importance of the up-regulation of LeCHLN in the roots of AtHMA4-expressing plants for efficient translocation of Zn to the shoots. Thus, the modifications of Zn/Fe/Cd translocation to aerial plant parts due to AtHMA4 expression are closely related to the alteration of the endogenous Zn-Fe-Cd cross-homeostasis network of tomato.

Expression of HvHMA2 in tobacco modifies Zn-Fe-Cd homeostasis.

HvHMA2 is a plasma membrane P1B-ATPase from barley that functions in Zn/Cd root-to-shoot transport. To assess the usefulness of HvHMA2 for modifying the metal content in aerial plant parts, it was expressed in tobacco under the CaMV35S promoter. Transformation with HvHMA2 did not produce one unique pattern of Zn and Cd accumulation; instead it depended on external metal supply. Thus Zn and Cd root-to-shoot translocation was facilitated, but not at all applied Zn/Cd concentrations. Metal uptake was restricted in HvHMA2-transformed plants and the level in the shoot was not enhanced. It was shown that HvHMA2 localizes to the plasma membrane of tobacco cells, and overloads the apoplast with Zn, which could explain the overall decrease in metal uptake observed. Despite the lower levels in the shoot, HvHMA2 transformants showed increased Zn sensitivity. Moreover, introduction of HvHMA2 into tobacco interfered with Fe metabolism and Fe accumulation was modified in HvHMA2-transformants in a Zn- and Cd-concentration dependent manner. The results indicate that ectopic expression of the export protein HvHMA2 in tobacco interferes with tobacco metal Zn-Cd-Fe cross-homeostasis, inducing internal mechanisms regulating metal uptake and tolerance.

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.

HvHMA2, a P(1B)-ATPase from barley, is highly conserved among cereals and functions in Zn and Cd transport.

Manipulation of crops to improve their nutritional value (biofortification) and optimisation of plants for removal of toxic metals from contaminated soils (phytoremediation) are major goals. Identification of membrane transporters with roles in zinc and cadmium transport would be useful for both aspects. The P(1B)-ATPases play important roles in heavy metal allocation and detoxification in Arabidopsis and it is now important to elucidate their roles in monocots. We identified nine P(1B)-ATPases in barley and this study focuses on the functional characterization of HvHMA2, providing evidence for its role in heavy metal transport. HvHMA2 was cloned using information from EST analysis and 5' RACE. It possesses the conserved aspartate that is phosphorylated during the reaction cycle of P-type pumps and has motifs and key residues characteristic of P(1B)-ATPases, falling into the P(1B-2) subclass. Homologous sequences occur in three major sub-families of the Poaceae (Gramineae). Heterologous expression in Saccharomyces cerevisiae demonstrates that HvHMA2 functions as a Zn and Cd pump. Mutagenesis studies show that proposed cation coordination sites of the P(1B-2) pumps are crucial for the metal responses conferred by HvHMA2 in yeast. HvHMA2 expression suppresses the Zn-deficient phenotype of the Arabidopsis hma2hma4 mutant indicating that HvHMA2 functions as a Zn pump in planta and could play a role in root to shoot Zn transport. When expressed in Arabidopsis, HvHMA2 localises predominantly to the plasma membrane.

Functional significance of AtHMA4 C-terminal domain in planta.

BACKGROUND: Enhancing the upward translocation of heavy metals such as Zn from root to shoot through genetic engineering has potential for biofortification and phytoremediation. This study examined the contribution of the heavy metal-transporting ATPase, AtHMA4, to the shoot ionomic profile of soil-grown plants, and investigated the importance of the C-terminal domain in the functioning of this transporter. PRINCIPAL FINDINGS: The Arabidopsis hma2 hma4 mutant has a stunted phenotype and a distinctive ionomic profile, with low shoot levels of Zn, Cd, Co, K and Rb, and high shoot Cu. Expression of AtHMA4 (AtHMA4-FL) under the CaMV-35S promoter partially rescued the stunted phenotype of hma2 hma4; rosette diameter returned to wild-type levels in the majority of lines and bolts were also produced, although the average bolt height was not restored completely. AtHMA4-FL expression rescued Co, K, Rb and Cu to wild-type levels, and partially returned Cd and Zn levels (83% and 28% of wild type respectively). In contrast, expression of AtHMA4-trunc (without the C-terminal region) in hma2 hma4 only partially restored the rosette diameter in two of five lines and bolt production was not rescued. There was no significant effect on the shoot ionomic profile, apart from Cd, which was increased to 41% of wild-type levels. When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect. When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL. CONCLUSION: The ionome of the hma2 hma4 mutant differs markedly from wt plants. The functional relevance of domains of AtHMA4 in planta can be explored by complementing this mutant. AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.

A combined zinc/cadmium sensor and zinc/cadmium export regulator in a heavy metal pump.

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

ECA3, a Golgi-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis.

Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the growth defect of K616 on low Ca. Promoter:beta-glucuronidase studies show that ECA3 is expressed in a range of tissues and cells, including primary root tips, root vascular tissue, hydathodes, and guard cells. When transiently expressed in Nicotiana tabacum, an ECA3-yellow fluorescent protein fusion protein showed overlapping expression with the Golgi protein GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly functioning in the transport of these ions into the Golgi. ECA3 is the first P-type ATPase to be identified in plants that is required under Mn-deficient conditions.

P(1B)-ATPases--an ancient family of transition metal pumps with diverse functions in plants.

P(1B)-ATPases form a distinct evolutionary sub-family of P-type ATPases, transporting transition metals such as Cu, Zn, Cd, Pb and Co across membranes in a wide range of organisms, including plants. Structurally they are distinct from other P-types, possessing eight transmembrane helices, a CPx/SPC motif in transmembrane domain six, and putative transition metal-binding domains at the N- and/or C-termini. Arabidopsis has eight P(1B)-ATPases (AtHMA1-AtHMA8), which differ in their structure, function and regulation. They perform a variety of important physiological tasks relating to transition metal transport and homeostasis. The crucial roles of plant P(1B)-ATPases in micronutrient nutrition, delivery of essential metals to target proteins, and toxic metal detoxification are discussed.

The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels.

The transition metal Zn is essential for many physiological processes in plants, yet at elevated concentrations this, and the related non-essential metal Cd, can be toxic. Arabidopsis thaliana HMA4, belonging to the Type P1B subfamily of P-type ATPases, has recently been implicated in Zn nutrition, having a role in root to shoot Zn translocation. Using Arabidopsis insertional mutants, it is shown here that disruption of AtHMA4 function also results in increased sensitivity to elevated levels of Cd and Zn, suggesting that AtHMA4 serves an important role in metal detoxification at higher metal concentrations. AtHMA4 and a truncated form lacking the last 457 amino acids both confer Cd and Zn resistance to yeast but a mutant version of the full-length AtHMA4 (AtHMA4-C357G) does not; this demonstrates that the C-terminal region is not essential for this function. Evidence is presented that AtHMA4 functions as an efflux pump.

Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass.

Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.