The hyperaccumulator Alyssum murale uses complexation with nitrogen and oxygen donor ligands for Ni transport and storage
Graphical abstract
A detailed spectroscopic and microscopic investigation of the mechanisms involved in metal delivery and storage in the Ni hyperaccumulator Alyssum murale showed that Ni is transported in the stem and leaf xylem sap complexed with malate, tartrate, possibly histidine and low molecular weight organic acids (LMWOA) and/or counter-ions and stored in leaf epidermal vacuoles complexed primarily with malate.
Introduction
There is much to be learned about the biochemical or physiological mechanisms responsible for excess metal uptake and storage in hyperaccumulating plants. Understanding these mechanisms would be beneficial for increasing the efficiency of current hyperaccumulating cultivars or transferring this quality to higher biomass, faster growing and climactically adapted plant species for use in either phytoremediation or phytomining of metal-enriched soils across a variety of landscapes. Improving our understanding of metal physiology in hyperaccumulating plants will also assist in efforts to fortify staple agronomic food crops with essential metal micro-nutrients (e.g., Zn, Fe) (White and Broadley, 2009).
A component of deciphering the biochemical and/or physiological mechanisms responsible for metal hyperaccumulation is determining where and in what chemical form the metal is being stored and how it is translocated from the root zone to these locations. There have been a number of studies directed at elucidating these mechanisms which established that a plant’s ability to hyperaccumulate metals can depend on the plant species, the chemical properties of the element being detoxified (Assuncao et al., 2003) or, in some cases, tissue type and age (Kupper et al., 2004). For Ni (and other transition metals), most studies have implicated oxygen or nitrogen donor ligands such as citrate, malate, malonate or histidine as the compounds responsible for transport and storage of the metal. Histidine, in the Ni hyperaccumulator Alyssum lesbiacum, is the only amino acid shown to increase in xylem sap in response to increased metal concentrations in the growth media (Kramer et al., 1996). There is no evidence to support that endogenous overproduction of either organic or amino acids are a specific response resulting from elevated metal exposure. Instead, selected hyperaccumulators have been found to have a constant and unusually high organic and/or amino acid concentration which implies that excess metal accumulation in some species may be a constitutive property (Boyd and Martens, 1998, Ostergren et al., 1999). In this case, tolerance to internally high heavy metal concentrations, perhaps as an evolutionary adaptation to growing either on metal rich soils or in response to selective pressures from herbivores, would be a sufficient explanation for increased metal uptake.
In some plants, the cysteine-containing metal binding ligands metallothioneins (MT) and phytochelatins (PC) have been shown to play a role in metal tolerance when accumulated metals are at phytotoxic levels, but less so in metal tolerant plants (Schat et al., 2002). Phytochelatins are comprised of cysteine, glycine and glutamate and are used in algae and plants to detoxify excess cellular metal. PCs are readily produced in metal stressed, non-hypertolerant organisms (plants, insects), but their production in hypertolerant species is only inducible at extremely high exogenous metal concentration (Schat et al., 2002, Ebbs et al., 2002). Therefore, it is unlikely that they have a direct role in transition metal tolerance in hyperaccumulating plants. Phytochelatins have been found to have a role in the detoxification of arsenic in Pteris vittata (Chinese Brake fern), which several researchers have shown to have inducible PC production (Zhang and Cai, 2003).
Nickel hyperaccumulating plants have been the most studied of the hyperaccumulators, likely because over three quarters of the roughly 450 metal hyperaccumulators identified to date are hyperaccumulators of Ni. In the current paper, we investigated the biochemical mechanisms of Ni hyperaccumulation in Alyssum murale; one of the few hyperaccumulators to be developed as a commercial crop for the removal of metals from metal-enriched soils (Zhang et al., 2007). A. murale is native to Mediterranean serpentine soils and has the ability to hyperaccumulate Ni and Co (Broadhurst et al., 2004b), using different mechanisms to tolerate each.
Tappero et al. (2007), using a variety of techniques, examined the co-tolerance mechanisms in A. murale exposed to different combinations of Zn, Co and Ni. They showed that A. murale does not hyperaccumulate Zn and stores Co in the apoplasm of leaf mesophyll, in the leaf tips and possibly as extracellular precipitates on the leaf surface. They found that exposure of A. murale to either Zn or Co had no effect on the amount and localization of Ni within the plants, that Zn was not accumulated, nor did it interfere with Ni uptake, finally concluding that A. murale used different tolerance mechanisms for Co and Ni. Broadhurst et al., 2004b, Broadhurst et al., 2009, using scanning electron microscopy with energy dispersive X-ray detection (SEM/EDX), also reported that A. murale preferentially stores Mn in the basal compartments of the Ca rich leaf trichomes. From these studies, it is apparent that different biochemical mechanisms govern the way in which A. murale tolerates each of these metals.
More is known about the chemical mechanisms used for Ni tolerance in A. murale compared to either Mn or Co. Nickel accumulates primarily in the cell vacuoles of epidermal stem and leaf tissues and trichome basal compartments (Broadhurst et al., 2004a, McNear et al., 2005), with palisade mesophyll cells becoming important only at higher soil Ni concentrations probably resulting from overflow of the primary cellular storage compartments (Broadhurst et al., 2004a). The transport and storage of Ni in several Alyssum species has been attributed to organic acids and possibly histidine. Little information has been reported, however, on the mechanism used by A. murale to tolerate such high Ni concentrations, let alone the chemical mechanism used to tolerate Co, Mn and Ca. Montargès-Pelletier et al. (2008) using unfocused (bulk) synchrotron X-ray spectroscopy found that A. murale stored Ni in its leaves as Ni–malate and in the stems as Ni–citrate complexes. The bulk X-ray methods used in their studies relied on dried and homogenized tissues which provided a good measure of the average Ni speciation present in the plant tissues. One drawback, as pointed out by Sarret et al. (2002), when analyzing ground and homogenized plant samples, is the possibility that contents of different cellular compartments can come into contact and thus change the metal speciation. In addition, in heterogeneous materials like plant tissues, where the metal speciation can vary over microns depending on the tissue it is found in, bulk-EXAFS spectroscopy may not adequately ascertain the minor metal complexes which may have a major role in the plants mechanisms of detoxification, transport or storage. The authors themselves note this limitation and state that microbeam methods are necessary to “unravel the complete Ni pathway”.
The objectives of this research were to perform a detailed characterization of the chemical mechanisms responsible for the transport and storage of Ni in the ‘Kotodesh’ genotype of the Ni hyperaccumulator A. murale using a combination of synchrotron based microspectroscopic techniques, electron microscopy and Fourier transform infrared spectroscopy, respectively.
Section snippets
Element distribution and compartmentalization
Fig. 1a is a false colored confocal micrograph showing the primary fluorescence generated from the different tissue types within a leaf from A. murale and is provided here to highlight the location of the epidermal and vascular tissues (green) in relation to the mesophyll (red). Presented in Fig. 1b is the scanning electron micrograph (SEM) of an entire leaf cross-section with boxes highlighting examples of the regions analyzed in Fig. 1c and d. Fig. 1c and d presents the backscattered electron
Discussion
Determination of metal speciation in plant tissues is difficult due to the complex and dynamic matrix within which they reside. Identification of ligands responsible for metal detoxification in hyperaccumulating plants using solely chromatographic methods are limited by the typically low recovery of metals from the tissue extractions, the inability to specify which cellular compartments the complex comes from (e.g., vacuole vs vasculature), as well as potential mixing, degradation or
Conclusions
The findings contained herein contribute to the current understanding of metal tolerance and hyperaccumulation and provide an example of how coupling complementary techniques at multiple scales can provide a unique picture of metal sequestration and compartmentalization in biological tissues. While this study contributes to the growing evidence for the importance of organic/amino acids and counter-ions in the transport and storage of metals, there are still many questions to be answered
A. murale propagation
The ‘Kotodesh’ accession of the Ni hyperacumlator A. murale (Waldst. & Kit.) was grown in a glass house in nickel enriched soils collected from an area adjacent to an historic Ni refinery in Port Colborne, Ontario, Canada. The speciation of Ni in these soils and the soil physicochemical characteristics have been described elsewhere (Kukier and Chaney, 2001, McNear et al., 2005). Soils were sieved wet, mixed thoroughly and 1.5 kg (dry weight) of loam soil or 0.8 kg (dry weight) of muck soil were
Acknowledgements
The authors would like to thank Dr. Ken Livi of the Department of Earth and Planetary Sciences at Johns Hopkins University for help with EMPA data collection, analysis and interpretation. We thank Dr. Kirk Czymeck of the Delaware Biotechnology Institute for help collecting the confocal and SEM image of leaf cross-sections. We thank Dr. Ryan Tappero for assistance/instruction with plant propagation and Dr. Mike Borda for help with collecting ATR-FTIR spectra. We thank Dr. Timothy Strathman for
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2018, HydrometallurgyCitation Excerpt :A. murale accumulates Ni in its vacuoles while in R. bengalensis Ni is found in spongy mesophyll and in phloem at higher concentrations than in A. murale tissues (Van der Ent and Mulligan, 2015). In both cases, Ni is complexed by low molecular weight carboxylic acids (LMWCAs), which alleviates Ni toxicity (Callahan et al., 2006; McNear Jr et al., 2010; Montargès-Pelletier et al., 2008; Van der Ent et al., 2017a). The mechanisms of hyperaccumulation have been widely studied (Verbruggen et al., 2008) but still barely understood.