Structure and evolution of the plant cation diffusion facilitator family of ion transporters

Background  

Members of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. The spectrum of cations known to be transported by proteins of the CDF family include Zn, Fe, Co, Cd, and Mn. Members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. CDF families range in size from nine members in Selaginella moellendorffii to 19 members in Populus trichocarpa. Phylogenetic analysis suggests that the CDF family has expanded within plants, but a definitive plant CDF family phylogeny has not been constructed.     

Sphingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana

Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.     

Multi-Element Bioimaging of Arabidopsis thaliana Roots

Better understanding of root function is central for the development of plants with more efficient nutrient uptake and translocation. We here present a method for multielement bioimaging at the cellular level in roots of the genetic model system Arabidopsis (Arabidopsis thaliana). Using conventional protocols for microscopy, we observed that diffusible ions such as potassium and sodium were lost during sample dehydration. Thus, we developed a protocol that preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, cryo-sectioned, and freeze dried. Samples are finally analyzed by laser ablation-inductively coupled plasma-mass spectrometry, utilizing a specially designed internal standard procedure. The method can be further developed to maintain the native composition of proteins, enzymes, RNA, and DNA, making it attractive in combination with other omics techniques. To demonstrate the potential of the method, we analyzed a mutant of Arabidopsis unable to synthesize the metal chelator nicotianamine. The mutant accumulated substantially more zinc and manganese than the wild type in the tissues surrounding the vascular cylinder. For iron, the images looked completely different, with iron bound mainly in the epidermis of the wild-type plants but confined to the cortical cell walls of the mutant. The method offers the power of inductively coupled plasma-mass spectrometry to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to Arabidopsis, the molecular and genetic approaches available in this system can now be fully exploited in order to gain a better mechanistic understanding of these processes.Investigations of the localization of inorganic elements in young plant roots may answer a range of important and unresolved questions with respect to root functionality and plant nutrient transport. To date, our understanding of how plants control the radial root transport of essential plant nutrients and toxic elements is mainly circumstantial, relying on changes in shoot or shoot-to-root concentration ratios or analyses of xylem sap composition. Roots of Arabidopsis (Arabidopsis thaliana) have a simple cellular organization and are unrivaled in their ability to be imaged by confocal microscopy, as they are very thin (diameter approximately 120 µm) and have a low background fluorescence. This has led to an amazingly detailed understanding of the growth and development of roots. Unfortunately, the fragile nature of these roots constitutes a major challenge when trying to understand the processes that drive nutrient uptake at the same level of detail. The method we present here for element bioimaging of Arabidopsis roots is a critical step in utilizing the potential of combining targeted genetic modifications and bioimaging at the cellular level in order to unravel the complexities of how roots selectively acquire and translocate mineral nutrients from the soil.The uptake and radial transport of inorganic ions is tightly controlled by transport proteins varying in selectivity, affinity, and capacity. However, it was demonstrated recently that physical barriers in the root systems also play a pivotal role in regulating ion uptake, for example by the epidermis at the root surface as well as by the lignin and suberin barriers adjacent to the endodermis (Hosmani et al., 2013; Pfister et al., 2014; Kamiya et al., 2015; Barberon et al., 2016). The cells of the endodermis are sealed by the Casparian strip, a lignin-based barrier that restricts the apoplastic movement of ions and water into the vascular bundles. Further control of ion movement into the stele (and ultimately to the leaves via the xylem) is achieved by suberin deposition along the cell walls of the endodermis (Baxter et al., 2009; Geldner, 2013). Radial transport of inorganic ions may also be impeded by the lack of proper ligands, for example by nicotianamine (NA) or other organic acids. The functional effects that root barriers and ligands have on nutrient acquisition are still elusive, and they probably constitute a combined response to various genetic determinants that are yet to be understood at the mechanistic level. A better understanding of the radial root transport of essential nutrients is a prerequisite to improve nutrient uptake efficiency in crops and optimize the use of natural resources in agriculture. Likewise, better knowledge about the transport of potentially toxic trace elements such as cadmium (Cd) and arsenic (As) will help improve food safety.Different techniques are available for elemental imaging, typically divided into mass spectrometry (MS)-based techniques (e.g. nano-secondary ion mass spectrometry [nano-SIMS] or laser ablation-inductively coupled plasma-mass spectrometry [LA-ICP-MS]) and synchrotron x-ray-based techniques (e.g. energy-dispersive x-ray microanalysis, proton-induced x-ray emission, x-ray fluorescence, or x-ray absorption spectrometry; Zhao et al., 2014). Relative to SIMS and the synchrotron x-ray-based techniques, LA-ICP-MS offers a range of advantages in terms of low detection limits and high sensitivity for many elements (Becker et al., 2010b; for details, see “Discussion”). LA-ICP-MS also is much more accessible and has substantially lower running costs than any of the competitive techniques.Because of the requirement for dry samples, the usefulness of LA-ICP-MS for root analysis has long been hampered. For this reason, LA-ICP-MS analysis of plant materials has to date mostly been applied to naturally dry samples, like seeds and cereal grains (Lombi et al., 2011b; Olsen et al., 2016). The analysis of hydrated samples, like roots, poses major sample preparation challenges, with respect to maintaining biological structure and the native composition of the elements therein, during drying. Uncontrolled drying of cross sections or longitudinal sections of young roots causes the disruption of most cells, mainly due to the sudden loss of turgor and the resulting ion leakage. In order to preserve sample integrity, specimens for microscopy are typically dehydrated with a slow, gradual exchange of water with ethanol, acetone, or tetrabutyl alcohol (Feder and Obrien, 1968; Beeckman and Viane, 2000). Following dehydration, it is a common practice to embed the specimens in a block, typically a resin or paraffin block, prior to sectioning. However, young plant roots, like the ones of the genetic model plant Arabidopsis, are extremely fragile, and they easily disintegrate during such sample preparation. In addition, for elemental bioimaging, these procedures are problematic, since some ions will leak out of the tissue during prolonged soaking in organic solvents (Fourie and Peisach, 1977; Davies et al., 1991). It is unclear, however, how dehydration affects the leakage and displacement of different elements and to what degree. In theory, ions with a low valence and little or no interaction with other compounds (e.g. potassium [K⁺] and sodium [Na⁺]) should be highly diffusible and easily lost from the tissue, whereas divalent and trivalent cations such as manganese Mn²⁺, zinc (Zn²⁺) and iron (Fe²⁺ and Fe³⁺) should be less prone to leakage due to covalent bonding or coordination with various ligands.In order to maintain not only the elements present in the root tissue but also the integrity of various chemical components (i.e. ligands, proteins, nucleic acids, and metabolites), rapid freezing of the root is an attractive alternative to dehydration. After freezing, samples can be sectioned on a cryotome and then freeze-dried prior to analysis; alternatively, they can be freeze-dried first and then sectioned (Bhatia et al., 2004). In order to be able to prepare very thin sections (less than 5 µm) while still maintaining cell structures and element composition, freeze-substitution also has been employed (Siegele et al., 2008; Smart et al., 2010). This technique is based on ultra-rapid freezing followed by slow substitution of the ice with acetone, then chemical fixatives like osmium tetroxide (Smart et al., 2010) or tetrahydrofuran (Pålsgård et al., 1994). It has been shown, however, that the localization of highly diffusible ions, like K⁺ and Na⁺, may be altered significantly during freeze substitution (Smart et al., 2010).For ordinary cryo-sectioning, the initial freezing and subsequent sectioning are typically done in OCT (Optimal Cutting Temperature) medium (Tissue-Tek; Sakura Finetek), which is a glycol-based freezing medium (containing polyvinyl alcohol and polyethylene glycol) that facilitates fast freezing and mechanical support of the specimen during the following sectioning. Upon transfer of the sections to glass slides, the section melts briefly, thereby adhering to the surface of the glass slide. In this critical process, the melted OCT medium may cover small specimens, partly or fully. OCT medium is highly water soluble and can be washed off easily, although with the risk of also washing off leachable ions. Also, the hygroscopic OCT medium has a high osmotic potential, which means that it may cause water and/or ion diffusion upon direct contact with the root during the time that passes from excision to freezing in liquid nitrogen, with the risk of ion displacement inside and/or outside of the specimen.In order to meet these challenges, we have developed a novel sample preparation method. We show that encapsulation of the fresh tissue with paraffin prior to freezing and cryo-sectioning is an essential step in order to avoid the displacement of elements. Furthermore, we also show that the method is applicable to the very small and fragile roots of Arabidopsis. The method was tested using the NA synthase quadruple mutant nas1nas2nas3nas4 (nas4x), which is unable to synthesize the metal chelator NA. Upon cultivation in hydroponics, the leaves of these mutants display symptoms typical for Fe deficiency (Schuler et al., 2012). Total element concentration and xylem sap analyses, in combination with elemental bioimaging of the roots, showed that NA deficiency has different effects on radial and long-distance transport of Fe compared to Zn and Mn, which in turn appears to be related to xylem-loading processes as well as the affinity of NA and other ligands to these different metal ions.By enabling the examination of Arabidopsis roots, the genetic model plant of choice in plant science, the methodological developments described here pave the way for a range of new possibilities for investigating ion uptake, transport, and compartmentation in root tissues. Moreover, the method works for any element present in the tissue, including any added isotope, be it an essential plant nutrient or any other element taken up by plant roots.     

The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.     

Robust cross-links in molluscan adhesive gels: testing for contributions from hydrophobic and electrostatic interactions

The cross-linking interactions that provide cohesive strength to molluscan adhesive gels were investigated. Metal-based interactions have been shown to play an important role in the glue of the slug Arion subfuscus (Draparnaud), but other types of interactions may also contribute to the glue's strength and their role has not been investigated. This study shows that treatments that normally disrupt hydrophobic or electrostatic interactions have little to no effect on the slug glue. High salt concentrations and non-ionic detergent do not affect the solubility of the proteins in the glue or the ability of the glue proteins to stiffen gels. In contrast, metal chelation markedly disrupts the gel. Experiments with gel filtration chromatography identify a 40 kDa protein that is a central component of the cross-links in the glue. This 40 kDa protein forms robust macromolecular aggregations that are stable even in the presence of high concentrations of salt, non-ionic detergent, urea or metal chelators. Metal chelation during glue secretion, however, may block some of these cross-links. Such robust, non-specific interactions in an aqueous environment are highly unusual for hydrogels and reflect an intriguing cross-linking mechanism.     

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 novel arsenate reductase from the arsenic hyperaccumulating fern Pteris vittata

Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.     

The Role of Metal Transport and Tolerance in Nickel Hyperaccumulation by Thlaspi goesingense Halacsy

Metal hyperaccumulators are plants that are capable of extracting metals from the soil and accumulating them to extraordinary concentrations in aboveground tissues (greater than 0.1% dry biomass Ni or Co or greater than 1% dry biomass Zn or Mn). Approximately 400 hyperaccumulator species have been identified, according to the analysis of field-collected specimens. Metal hyperaccumulators are interesting model organisms to study for the development of a phytoremediation technology, the use of plants to remove pollutant metals from soils. However, little is known about the molecular, biochemical, and physiological processes that result in the hyperaccumulator phenotype. We investigated the role of Ni tolerance and transport in Ni hyperaccumulation by Thlaspi goesingense, using plant biomass production, evapotranspiration, and protoplast viability assays, and by following short- and long-term uptake of Ni into roots and shoots. As long as both species (T. goesingense and Thlaspi arvense) were unaffected by Ni toxicity, the rates of Ni translocation from roots to shoots were the same in both the hyper- and nonaccumulator species. Our data suggest that Ni tolerance is sufficient to explain the Ni hyperaccumulator phenotype observed in hydroponically cultured T. goesingense when compared with the Ni-sensitive nonhyperaccumulator T. arvense.