Recent developments in plant zinc homeostasis and the path toward improved biofortification and phytoremediation programs

Abstract

Zinc (Zn) is an essential micronutrient for all living organisms. Plants serve as a major entry point for this element into the food chain. Zn deficiency has become a widespread nutritional condition, which mirror the inadequate Zn reserves in significant proportion of the earth's arable land. A recent assessment by the World Health Organization revealed that one third of the world's population is at risk of Zn deficiency. To counter this alarming situation, substantial efforts have been made to increase Zn content and availability in staple crops and grains. Nevertheless, the absence of fundamental information has held back progress in this field. Developing a better understanding of how Zn homeostasis is regulated in plants, such as Zn transporters at loading bottlenecks, is of primary interest to biofortification and phytoremediation programs. Many reviews have been published on this subject, and here we briefly summarize the regulation of one limiting step in Zn distribution within plants — the loading of Zn into root xylem.

Keywords: :

• abiotic stress response
• heavy metals
• ion transport
• membrane transporters
• mineral nutrition
• stress
• xylem loading

In recent years, significant progress has been made toward our understanding of the regulation of Zinc (Zn) homeostasis in plants, and this phenomenon has been documented in many research publications and elegantly summarized in multiple reviews.^1-6 As well as being of great scientific interest, these advances have occurred because Zn is crucial for sustainable agriculture worldwide and phytoremediation programs. Zn is an essential micronutrient for all living organisms, and importantly, its involvement in many key biological processes in the cell can be adversely altered in situations where Zn is present in either too low (deficiency) or too high (toxicity) concentration. Zn serves as a highly effective cofactor for more than 300 enzymes, including the structural Zn-finger domains that mediate DNA-binding of transcription factors, as well as protein–protein interactions.⁴ Thus, it is of great interest for cells to tightly control Zn homeostasis, using complex regulatory pathways. Zn deficiency or toxicity manifests itself at physiological and molecular levels, and both conditions can alter plant growth capacity. Nevertheless, there are exceptions, such as the Zn hyperaccumulator plants. These plants can tolerate a spike in Zn content in excess of 10 mg Zn g⁻¹ dry weight when grown in their natural habitat, with an ability to detoxify excessive Zn²⁺ within the shoot.⁷ The transport of Zn within plants starts with its acquisition at the root periphery, followed by its loading into xylem. Interestingly, it has been reported that Zn hyperaccumulation presents unusually active mechanisms for Zn uptake and translocation to the shoot.^8-10 These are the first two limiting steps for the improvement of Zn content in many plant species. Once within the xylem sap, Zn can be complexed to organic acids^8,11 or histidine¹² and transferred to the above-ground parts of the plant. Therefore, enhancing the translocation of Zn from root to shoot has potential applications for biofortification and phytoremediation programs. It has been proposed that Zn may traverse the root to the xylem, either through symplastic or apoplastic pathways.^13,14 However, the relative contribution of the symplastic and apoplastic pathways to the loading of Zn into the xylem remains to be elucidated. The symplastic pathway requires membrane proteins that facilitate Zn transport. Numerous research groups worldwide have contributed to the identification and characterization of the various multigenic families that encode the transport proteins involved in the regulation of the homeostasis and transport of Zn and other essential heavy metal ions in plants; this includes the ABC, CDF, ZIP, ATPase and Nramp transporters.^2,15,16, Of particular importance here, the PIB-type ATPases transport Zn²⁺, Cd²⁺, etc across biological membranes.^17-20 Higher plants are the only eukaryotes where putative Zn²⁺-ATPases have been identified, and several members of the PIB-ATPase subfamily are present in plants. Both in vivo and in vitro metal transport studies have shown that PIB-ATPases drive the export of ions from the cell cytoplasm.^19,21-28 Here, we will focus on two out of the eight members of the P1B-ATPases (or HMAs) in Arabidopsis, HMA2 and its most related sequence in the HMA cluster, HMA4, which are involved in Zn root-to-shoot translocation.^29-34 The Zn transporters at loading bottlenecks, is of primary interest to biofortification and phytoremediation programs. The regulation of the expression Arabidopsis thaliana HMA2 and HMA4 and their structure-function relationships is briefly summarized.

In Arabidopsis thaliana, HMA2 and HMA4 are closely related in primary sequence and might have evolved as a result of gene duplication.³⁵ AtHMA2 and AtHMA4 gene expression profiling using the GUS reporter system revealed expression for both constructs, where GUS expression was predominantly observed in in roots in the pericycle cells surrounding the xylem, leaves and stems.²⁹ In a section of stem expressing HMA2p-GUS, GUS activity was observed in vascular bundles and appeared to be expressed in components of both the xylem and the phloem.²⁹ As for AtHMA4, it is predominantly expressed in the root stellar cells, the tissue that surrounds the vascular vessels. Such expression pattern is consistent with the involvement of AtHMA2 and AtHMA4 in the long distance transport of metal, and notably Zn, which is further reinforced by demonstrating their location in the plasma membrane.²⁹ The expression of the AtHMA2 and AtHMA4 promoters fused with GUS occurs also in inflorescences and changes during floral development.²⁹ Although the specific role of Zn in floral function remains unclear, the expression of these two Zn transporter proteins indicates the importance of Zn nutrition for normal flower development and reproduction. Our current knowledge on the transcriptional regulatory pathways that control the expression of AtHMA2 and AtHMA4 is rather limited. In this context and complementary to the published literature, multiple sets of Arabidopsis microarray data are accumulated in various available databases, providing a unique opportunity to further probe the regulation of HMA2 and HMA4 gene expression in response to various abiotic, chemical and biotic stresses. Ideally, this first-hand information could be utilized to discover the signals that are relayed to the transcription machinery and could preface further work that reconsiders the nature of signal(s) involved in regulating the expression of AtHMA2 and AtHMA4.

Both AtHMA2 and AtHMA4 are important for efficient translocation of Zn from roots to shoots in A. thaliana.^29-34It is worth noting that the roles of AtHMA2 and AtHMA4 are not exclusive to Zn transport, but also in cadmium translocation.^29-32,34 AtHMA2 was functionally characterized in yeast where it appeared as a Zn²⁺-dependent ATPase, and can also mediates Cd transport.³³ The Arabidopsis thaliana HMA4 (AtHMA4) has been demonstrated that it can also transport cadmium (Cd) in yeast, E. coli and in plants.^30-32It has been shown that QTL involved with Zn and Cd tolerance co-localize with HMA4 in Arabidopsis halleri, a plant that hyperaccumulates both Zn and Cd.³⁵ The HMA4 expression level seems to be one of the underlying genetic determinants for the hyperaccumulation phenotype in this plant species. By means of RNAi- mediated silencing in A. halleri, the Zn content was decreased in the shoots and increased in the roots.³⁶ It was also shown that HMA4 expression was higher in the hyperaccumulator A. halleri compared with its non- hyperaccumulator relative A. thaliana.³⁷ The higher expression in A. halleri compared with a non-tolerant relative (e.g., A. thaliana) is the tandem triplication and altered cis-regulation of the gene, and leads to in an increased capacity of this metallophyte to accumulate zinc in shoots.³⁶ Modifications of zinc accumulation in consumable part of the plants is noteworthy outcomes from the biofortification perspective and healthy food production. Recently, it has been shown that expression of Arabidopsis halleri HMA4 under its native promoter in tomato plants facilitates root-to-shoot Zn translocation and induces Zn uptake in a Zn supply-dependent manner.³⁸

In Arabidopsis, neither hma4 nor hma2 mutant plants show any observable distinctive morphological phenotypes.²⁹ The observation that none of the individual hma mutants exhibit any apparent phenotype in comparison with the wild type when grown in soil, along with the similarities in the expression patterns and their identical membrane localization,²⁹ raise questions about their equivalence, functional redundancy and/or interplay. Further research using innovative genetic studies is necessary to bring us closer to answering these questions in detail. Interestingly, the hma2 hma4 double mutant shows visible morphological alterations, a stunted phenotype and male sterility.²⁹ The hma2 hma4 double mutant accumulates zinc in root pericycle cells, which causes shoots to suffer from zinc deficiency. This observation largely in line with the suggested role for AtHMA2 and AtHMA4 in catalyzing zinc efflux from pericycle cells, thereby loading the xylem with zinc.^29,39 Increasing the levels of Zn²⁺ in the growth medium can compensate for these phenotypes. Expression of AtHMA4 under the CaMV-35S promoter partially rescued the stunted phenotype of hma2 hma4.⁴⁰ The ionome of the hma2 hma4 double mutant is markedly different from WT plants: the shoots exhibit low levels of Zn, Cd, Co, K and Rb, as well as a high level of Cu.⁴⁰ Decreased levels of Zn were detected in shoots of the hma2 hma4 double mutant and the hma4 single mutant, while increased levels of this metal were detected in roots of the hma2 hma4 double mutant. The hma2 hma4 double mutant forms sterile flowers without pollen resulting in a decrease in plant fertility.²⁹ This phenotype can be rescued by supplying the plants with high Zn. In this context, it will be interesting to pursue this further to get deeper into the biological relevance of the expression of the AtHMA2 and AtHMA4 in inflorescences and changes during floral development.²⁹

The hydropathy profile of the HMA2 and HMA4 proteins reveals the presence of three major domains: the hydrophilic N- and C-terminals, and a central membrane-spanning domain that is consistent with the location of the metal transporters in the plasma membrane.^41,42 It is predicted that the central domain have six to eight transmembrane segments responsible for metal ion coordination during transport. The C-terminal extensions AtHMA2 (244 amino acid residues) and AtHMA4 (~470 amino acid residues) are very different in length and show no sequence homology to each other.⁴² Identification of post-transcriptional mechanisms that regulate the activities of HMA2 and HMA4, especially that involves their N- and C-terminal domains, has become a major focal interest.^41-45 Functional analysis of HMA2 in Arabidopsis thaliana suggests that the N-terminal domain of HMA2 is essential for its function in planta.⁴³ The zinc-deficiency phenotype of the hma2 hma4 mutant can be restored with the expression of full-length AtHMA2, however mutated versions (either removal of the entire N-terminal domain or mutation of the Cys residues within the conserved sequence Cys¹⁷-Cys-X-X-Glu²¹) results in failure to complement.⁴² The role of the C-terminal domain of HMA2 protein, and particularly for its subcellular location, remains to be demonstrated. The ATP hydrolytic activity is affected by the deletion of the C terminus of AtHMA2, whereas deletion of the same region in AtHMA4 has no apparent effect on enzyme turnover but leads to more efficient zinc or cadmium pumping.^41,42 Similarly, the importance of N- and C-terminal domains of HMA4 for its function has been evaluated. The functional significance of the AtHMA4 C-terminal domain has been revealed in yeast and in planta.^40,42,44,45 Indeed, the functional complementation test in Arabidopsis hma2 hma4 double mutant with AtHMA4 protein lacking the C-terminal region can only restored partially the rosette diameter in two out of five lines while bolt production was not rescued.⁴⁰ Collectively, it is clear that HMA2 and HMA4 are post-transcriptionally regulated. Although there is evidence of a functional and/or regulatory roles for the N- and C-terminal domains in modulating the transport activity for both HMA2 and HMA4, the precise mechanisms of their regulation at the post-translational level is still unclear.

Zn loading into xylem involves two members of the P1B ATPases (or HMAs), namely HMA2 and HMA4. As we have shown here, much remains to be discovered regarding the regulation of HMA2 and HMA4 at the transcriptional level. Indeed, further work using innovative genetic, chemical and pharmacological studies is necessary to elucidate the nature of the signal(s) that control the regulation of these Zn transporters proteins. In addition, determining which transcription factors govern the expression of HMA2 and HMA4 will provide new insight into Zn-homeostasis signaling pathways. In this context, publicly available databases, data mining and analysis toolboxes such as TAIR, NASCArrays, Genevestigator, Stanford MicroArray Database, GEO, the Botany array resource and AthaMap could yield powerful clues for deciphering novel crosstalk networks.^46-52 Last but not least, additional levels of regulation that modulate HMA2 and HMA4 transporter activity must be determined, in order to fully appreciate the mechanisms that regulate Zn transport in plants. These efforts will all contribute to ameliorating the health risks in our society attributed to Zn.⁵³

Acknowledgments

The author would like to thank Professor Pierre Berthomieu and Dr. Françoise Gosti and Oriane Mith for their helpful discussion, and Brandon Loveall of Improvence for English proofreading of the manuscript. The author apologizes to any colleagues whose relevant work has not been mentioned.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.