Microbeam Methodologies As Powerful Tools in Manganese Hyperaccumulation Research: Present Status and Future Directions

REVIEW ARTICLE published: 20 August 2013 doi: 10.3389/fpls.2013.00319 Microbeam methodologies as powerful tools in manganese hyperaccumulation research: present status and future directions

Denise R. Fernando 1*, Alan Marshall 2,AlanJ.M.Baker3 and Takafumi Mizuno 4

1 Department of Botany, La Trobe University, Bundoora, VIC, Australia 2 Analytical Electron Microscopy Facility, La Trobe University, Bundoora, VIC, Australia 3 School of Botany, The University of Melbourne, Parkville, VIC, Australia 4 Graduate School of Bioresources, Mie University, Mie, Japan

Edited by: Microbeam studies over the past decade have garnered unique insight into manganese Marc Hanikenne, University of (Mn) homeostasis in plant species that hyperaccumulate this essential mineral Liège, Belgium micronutrient. Electron- and/or proton-probe methodologies employed to examine tissue Reviewed by: elemental distributions have proven highly effective in illuminating excess foliar Mn Sebastien Thomine, Centre National de la Recherche Scientiﬁque, France disposal strategies, some apparently unique to Mn hyperaccumulating plants. When Tingsong Liu, The Samuel Roberts applied to samples prepared with minimal artefacts, these are powerful tools for extracting Noble Foundation, Inc., USA true ‘snapshot’ data of living systems. For a range of reasons, Mn hyperaccumulation *Correspondence: is particularly suited to in vivo interrogation by this approach. Whilst microbeam Denise R. Fernando, Department of investigation of metallophytes is well documented, certain methods originally intended for Botany, La Trobe University, Biological Sciences 1 Building, non-biological samples are now widely applied in biology. This review examines current Kingsbury Drive, Bundoora, knowledge about Mn hyperaccumulators with reference to microbeam methodologies, VIC 3086, Australia and discusses implications for future research into metal transporters. e-mail: [email protected] Keywords: Mn hyperaccumulator, microbeam analysis, Gossia, Maytenus cunninghamii, Alyxia rubricaulis

INTRODUCTION The possible benefits of seeking to understand lesser-known The intrinsic value of plant hyperaccumulation as a resource hyperaccumulators with restricted distributions and/or more for expanding fundamental knowledge is as equally well recog- unusual hyperaccumulated elements include: (a) further illu- nized as are its potential practical benefits (Brooks, 1987; Baker minating hyperaccumulation per se, (b) obtaining novel per- and Brooks, 1989; Baker et al., 1992; Brooks, 1998; Brooks and spective on the physiological roles of those hyperaccumulated Robinson, 2000; Chaney et al., 2000; Pollard, 2000; Reeves and elements essential to plant nutrition, (c) gaining new insights Baker, 2000; Lombi et al., 2001; Macnair, 2003; Whiting et al., into plant metal specificity and detoxification. Around 22 Mn 2004; Meharg, 2005; Reeves, 2005; Chaney et al., 2007). On a hyperaccumulators are known worldwide (Table 1), their highly global scale, it is clear that natural systems are increasingly under restricted collective distribution centered almost entirely over threat from human activities, with dire predictions for the future New Caledonia and Eastern Australia. Reports of two herbaceous survival of many biota. There is thus a pressing need to address Polygonum species described as Mn-hyperaccumulating on Mn habitat conservation of these extraordinary plants and to gain a mine sites in China (Wang et al., 2007; Deng et al., 2010)arediffi- better understanding of their ecology and evolutionary histories cult to access and may require further confirmation. Among well (Pollard, 2000; Reeves and Baker, 2000; Baker and Whiting, 2002; confirmed Mn hyperaccumulators are seven trees and a shrub Boyd, 2004; Whiting et al., 2004). Macnair (2003) points out that species from Australia (Bidwell et al., 2002; Fernando et al., 2009), notwithstanding the potential commercial benefit of hyperaccu- seven woody plants from New Caledonia (Brooks, 1998), a tree mulation yet to be realized in any real sense, “the phenomenon and a herb native to China (Xue et al., 2004; Yang et al., 2008), is of sufficient intrinsic interest that its investigation is justified aherbnativetotheUSA(Min et al., 2007; Pollard et al., 2009) without any immediate commercial applications.” Whilst eco- and a tree native to Japan (Mizuno et al., 2008). The single nomic and environmental factors are major drivers of scientific Malaysian tree species whilst documented has not been identi- research in this area, it is likely that accessibility and ease of fied below generic level (Proctor et al., 1989). The identities of propagation also influence species selection for study. Ubiquitous certain herbaceous Mn hyperaccumulators are yet to be veri- herbaceous Ni hyperaccumulators in the Brassicaceae remain the fied taxonomically at the species level, while several trees within most investigated subgroup to date (Brooks and Radford, 1978; the Australian group are listed as threatened due to habitat loss. Lee et al., 1978; Gambi et al., 1979; Baker, 1987; Baker and Brooks, Given that conservation and research are underpinned by cor- 1989; Baker et al., 1992, 2000; Krämer et al., 1997; Brooks, 1998; rect identification of hyperaccumulator species, plant taxonomy Nicks and Chambers, 2000; Mengoni et al., 2003; Asemaneh et al., is integral to the topic. Currently it is an uncommon practice 2006). to provide herbarium vouchers on hyperaccumulator plants in

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Table 1 | Current worldwide listing of Mn hyperaccumulators. weight (DW) (Baker et al., 2000). More recently, there has been argument supporting a downward revision (Baker et al., 2000; Plant (family) species (citation) Native country Fernando et al., 2009). It is widely accepted that Mn predominates + (Araliaceae) in its lowest ( 2) oxidation state in planta, and this has been con- Chengiopanax species formerly in Eleutherococcus firmed for several Mn hyperaccumulators, using X-ray absorption *Chengiopanax sciadophylloides (1) Japan near-edge spectroscopy (XANES) (Graham et al., 1988; Fernando et al., 2010). There are multiple physiological functions to which (Apocynaceae) Mn is essential, primarily photosynthesis and oxidative stress Alyxia rubricaulis (2) New Caledonia (NC) mitigation. Based on crop plants, “normal” nutritional requirements for Mn are met at ∼50–800 µgg−1 DW even though (Celastraceae) it is widely tolerated at concentrations above (Graham et al., Denhamia species formerly in Maytenus 1988; Marschner, 2002; Foulds, 2003). This is in marked con- *Denhamia fournieri (2) NC trast to other trace metal nutrients. Manganese crop toxicity is Denhamia cunninghamii (3) Australia (AU) a significant problem in certain regions of the world where soils (Clusiaceae) are Mn-enriched and acidic (Heenan and Carter, 1977; Temple- *Garcinia amplexicaulis (2) NC Smith and Koen, 1982; Hung et al., 1987; Davis, 1996; González et al., 1998; StClair and Lynch, 2005). The ability of tolerant crop (Myrtaceae) lines to sequester excess Mn, along with Mn sensitivity in other Eugenia sp.1 (2) NC varieties is not well understood, nor is the observed variation Eugenia sp. 2 (4) Malaysia in plant physiological responses. Therefore, Mn hyperaccumu- Gossia species formerly in Austromyrtus lators can be exploited to address these questions given they Gossia bamagensis (3) AU shine a novel light on Mn nutrition and could potentially yield *G. bidwillii (5) AU important new information regarding specific Mn chelators and G. gonocla (3) AU transporters. *G. fragrantissima (3) AU The first reports of Mn hyperaccumulation occurred over 35 G. lucida (3) AU years ago upon discovery of the New Caledonian group (Jaffré G. sankowsiorum (3) AU and Latham, 1974; Jaffré, 1977, 1979). At that time, the now little- G. shepherdii (3) AU used term “hypermangansphores” was introduced to describe Mn hyperaccumulators. In the early 1980s a group of Japanese (Phytolaccaceae) Mn-accumulating species was the subject of detailed physiolog- *Phytolacca acinosa (6) China (CH) ical investigation, including microprobe analysis (Memon and P. Americana (7) USA Yatazawa, 1980, 1981, 1982, 1984). Over the past decade it has (Polygonaceae) become apparent via in vivo microbeam studies at least that these Polygonum pubescens (8) Eurasia (E) are somewhat unusual among hyperaccumulators (Fernando P. hydropiper (9) E/USA et al., 2006a,b, 2008). Between 2002 and 2008, additional Mn hyperaccumulators were discovered in eastern Australia (Bidwell (Proteaceae) et al., 2002), China (Xue et al., 2004)andJapan(Mizuno et al., Beaupreopsis paniculata (10) NC 2008), that has now led to recognition of several new species and Virotia species formerly in Macadamia renewed interest in the phenomenon. To date, the woody species Virotia angustifolia (10) NC have only been investigated through a series of ecophysiological *V. neurophylla (10) NC studies, whereas the two herbaceous Phytolacca (Phytolaccaceae) species have mainly been examined in controlled experiments (Theaceae) (Xue et al., 2005; Fernando et al., 2006b; Mizuno et al., 2006; Schima superba (11) CH Xu et al., 2006a,b, 2009; Fernando et al., 2007a,b). Unlike *Species examined by microprobe analysis. their herbaceous metal-hyperaccumulating counterparts, Mn- References: 1, (Mizuno et al., 2008); 2, (Jaffré, 1977); 3, (Fernando et al., 2009); hyperaccumulating trees and shrubs are extremely slow growing 4, (Proctor et al., 1989); 5, (Bidwell et al., 2002); 6, (Xue et al., 2004); 7, (Min and often difficult to propagate. et al., 2007; Pollard et al., 2009); 8, (Deng et al., 2010); 9, (Wang et al., 2007); 10, Pioneering studies on plant hyperaccumulation using widely (Jaffré, 1979); 11, (Yang et al., 2008). accessible Ni-, Cd- and Zn-hyperaccumulating herbs provide an invaluable framework for current focus on Mn hyperaccumu- published research; however, it should be encouraged in order to lation. This has led to: (a) the discovery of several new Mn enable independent verification of species. The majority of Mn hyperaccumulators, which has augmented the overall biogeo- hyperaccumulators are woody plants with sclerophyllous leaves graphic knowledge base of hyperaccumulators by providing new and xerophytic anatomies, traits strongly favorable to retaining perspective on Mn-accumulating taxa analogous to those with leaf-tissue integrity for in vivo microprobe examination. already well-established links to other metals (Baker and Brooks, Plant Mn hyperaccumulation was originally defined by a 1989; Baker et al., 1992; Pollard et al., 2000, 2002; Fernando notional threshold foliar Mn concentration of 10,000 µgg−1 dry et al., 2009); (b) the discovery that foliar Mn detoxification varies

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specifically in a manner possibly unique to Mn hyperaccumula- reasoning that, (a) dermal tissues generally have a high apoplas- tors (Fernando et al., 2008); (c) indication that the physiological tic component, (b) the essentially non-photosynthetic status of mechanisms associated with excess Mn uptake and storage in dermal tissues renders them suited to storing potentially toxic herbaceous Mn hyperaccumulators are similar to those found concentrations of metals that might otherwise disrupt vital phys- in herbaceous hyperaccumulators of other metals (Küpper et al., iological processes in the mesophyll, and (c) excess metal storage 2000, 2001; Xu et al., 2006a,b); and (d) the pursuit to identify in the dermal layers may contribute to chemical defence against Mn-specific transporters, particularly in woody species where Mn animal and/or insect herbivory. In contrast to all other hyper- is compartmentalized in highly localized vacuolar concentrations accumulators examined to date, Mn hyperaccumulators exhibit (T. Mizuno unpublished data). a distinct variety of primary detoxification strategies, i.e., in photosynthetic tissues, in non-photosynthetic tissues, and simul- METAL LOCALIZATION METHODOLOGIES APPLIED TO taneously across all tissue types (Fernando et al., 2008). Currently HYPERACCUMULATORS AND OTHER PLANTS there is no clear explanation for this observation. It has been Early studies utilizing microbeam techniques to investigate excess argued that the highly vacuolated xerophytic leaf anatomies of Mn sequestration in certain tolerant crop lines established that woody Mn hyperaccumulators along with the essential role of dermal tissues were primary deposition sites (Blamey et al., Mn in photosynthesis might be associated with its concentra- 1986; Edwards and Van Steveninck, 1987, 1988). Scanning elec- tion in photosynthetic cells. Most recently however, PIXE/EDS tron microscopy energy dispersive spectroscopy (SEM/EDS), localization studies showed that primary foliar-metal sequestra- proton (or particle)-induced X-ray emission energy disper- tion in a Co-, Ni- and Zn-accumulating Mn hyperaccumulator sive spectroscopy (PIXE/EDS) and synchrotron methodologies tree Gossia fragrantissima (F. Muell. ex Benth.) N. Snow and are by far the most widely used techniques for localizing Guymer (Myrtaceae) occurred in photosynthetic cells for Mn, excess metal deposition within plant tissues. To obtain reli- Ni and Co; and in non-photosynthetic cells for Zn (Fernando able artifact-free data from in vivo methods such as these, et al., 2013). In further illuminating primary Mn-sequestration in it is imperative that sample preparations effectively immobi- the leaf mesophyll cells of a woody Mn hyperaccumulator, these lize cell metabolic and diffusional processes, thereby avoiding latest findings indicate involvement of tonoplastal metal trans- artifactual cell-content removal or relocation of diffusible ele- porters not exclusive to Mn since in this species at least, Ni and Co ments (Marshall, 1980c; Morgan, 1980; Echlin, 1992; Marshall were in addition to Mn, primarily located in photosynthetically and Xu, 1998). This subject will be addressed in greater detail important cells. below. Other techniques including 31P-nuclear magnetic reso- nance (NMR) (Roby et al., 1988; Quiquampoix et al., 1993a,b) LOCALIZATION STUDIES ON Mn HYPERACCUMULATORS radioactive tracer studies (Lasat et al., 1996), and cell frac- As discussed earlier, original in vivo examination of foliar Mn tionation (González and Lynch, 1999)havealsoprovideduse- microdistribution was performed on Mn accumulators from ful information regarding metal localization in plants. Mention Japan, including Chengiopanax sciadophylloides (Franch. and should also be made of histochemical techniques utilized by Sav.) C. B. Shang and J. Y. Huang (Araliaceae) now identified as Severne (1974) to locate foliar Ni in situ in a hyperaccu- a“true”Mnhyperaccumulator(Mizuno et al., 2008). However, mulator using dimethyl glyoxime stain; and by Horiguchi these samples were prepared by freezing leaf material directly (1987) to localize oxidized Mn in rice plants by staining with in liquid nitrogen, an approach now regarded as unreliable in benzidine. achieving adequate cryo fixation at the cellular level (see Echlin, In vivo microprobe investigation has been applied to hyper- 1992). About 25 years later, Gossia bidwillii (Benth.) N. Snow accumulating plants to reveal that with the exception of and Guymer (Myrtaceae), a Mn-hyperaccumulating Australian Mn hyperaccumulators, primary sequestration occurs in non- rainforest tree; and P. acinosa Roxb, a herbaceous Chinese Mn photosynthetic tissues (Vázquez et al., 1992; Krämer et al., 1997; hyperaccumulator were examined in separate Mn localization Küpper et al., 1999, 2000, 2001; Mesjasz-Przybylowicz et al., studies utilizing cryo-SEM/EDS and synchrotron radiation X-ray 2001; Lombi et al., 2002; Ager et al., 2003; Bhatia et al., 2003; fluorescence spectroscopy (SRXRF), respectively, (Fernando et al., Bidwell et al., 2004; Broadhurst et al., 2004; Fernando et al., 2006b; Xu et al., 2006a,b). Whilst the latter authors showed 2006a,b, 2010; Xu et al., 2006a,b; Smart et al., 2010). For exam- foliar Mn to be most strongly deposited in the upper epidermis ple, Küpper et al. (2000) described the accumulation of Zn and of P. acinosa, they also conceded that their sample preparation Cd in leaves of the hyperaccumulator Arabidopsis halleri (L.) might have been a confounding factor. Methodology lacking O’Kane and Al-Shehbaz Novon (Brassicaceae), and found the the essential initial step of immediate rapid-freezing of leaf tis- strongest deposition in trichomes, and to a lesser extent in the sue will likely yield unreliable data as a result of artifactual mesophyll. Bidwell et al. (2004) found Ni predominantly localized movement of cell solutes and water during subsequent sam- in the leaf epidermal vacuoles with some apoplastic localiza- ple processing and/or microbeam analysis. Studies on woody tion in the mesophyll tissues of Hybanthus floribundus (Lindley) Mn hyperaccumulators utilized appropriate sample fixation that F. Muell (Violaceae). These and numerous other studies demon- retained cellular content in close representation of the in vivo strated that leaf epidermal cell-layers and associated structures status. Leaf anatomical features of these species typically include such as hairs and trichomes are primary sequestration sites for substantial cuticles, particularly for the New Caledonian group; hyperaccumulated elements, i.e., where highest localized concen- multiple layers of large palisade mesophyll cells, and an over- trations were detected in vivo. Such findings are consistent with all distinct lack of intercellular spaces throughout (Fernando

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et al., 2006a,b, 2007b, 2008, 2012). These leaves are dermally Melting nitrogen, liquid propane and isopentane have previ- fortified around closely packed and highly vacuolated cells that ously been used to cryo-fix Mn hyperaccumulator samples for account for an overall high vacoular component of the total wet and dry sample preparations examined by qualitative and leaf volume. After field sampling, it is often necessary to main- quantitative electron and proton probe studies (Fernando et al., tain material in cool humid packaging over transit periods of 2006a,b, 2013). Whether applied to wet or dry samples, quan- least 24 h between remote field collection sites and laboratories titative approaches are more time consuming and expensive. where they are immediately cryo-fixed prior to storage or any Qualitative X-ray mapping used on freeze-dried bulk tissue cut subsequent processing. Their physical robustness combined with open to expose a clean cross-sectional surface has to date been highly localized cellular Mn concentrations that fall well within best exploited to locate primary Mn sinks in Mn hyperaccumu- the detection limits of widely used microprobe methodologies lator samples (Figure 1)(Fernando et al., 2006a, 2007b, 2008). render leaf samples of these woody Mn hyperaccumulators ideal Again, it is noteworthy that the sclerophyllous nature of these for in vivo microprobe localization studies on bulk specimens. leaves is a distinct advantage when hand-cutting freeze dried tis- Their characteristic leaf sclerophylly on the other hand poses sue. This is a relatively straightforward yet effective approach challenges to sample infiltration steps essential to ultrastruc- given that Mn hyperaccumulators harbour highly localized foliar tural examination by transmission electron microscopy (TEM), Mn. If necessary, these findings can then be followed up by quan- and/or analysis by scanning transmission electron microscopy titative techniques to characterize Mn sinks in greater detail, (STEM) EDS. using additional samples prepared appropriately. For example, SEM/EDS measurement of vacuolar elemental concentrations LEAF SAMPLE PREPARATION FOR In vivo MICROPROBE within individual cells can be carried out on frozen hydrated cryo- EXAMINATION OF HYPERACCUMULATORS planed samples treated to improve anatomical clarity by superfi- Appropriate sample preparation is mandatory to obtaining reli- cial surface-sublimation and metal coating (Figure 2)(Fernando able in vivo microprobe analytical data from biological material. et al., 2006b, 2012, 2013). A preparation such as this offers Reference to “sample” here should be taken to mean leaf tissues. minimal disruption to metabolic processes, and is the closest Sample preparation methodology can determine the degree of achievable representation of the true in vivo state for a fixed sam- anatomical resolution achievable by SEM, as can instrumenta- ple, provided it was correctly cryo-fixed at the outset. Among the tion. The latter also governs the limits of analytical resolution disadvantages of this approach however, is that the sample water and sensitivity, for example, factors such as operating condi- content has a “diluting” effect that renders cellular elements less tions, the choice of incident microbeam, detector and analyzer. detectable than in correspondingly dry tissue, although Mn is There is ample evidence that biological sample preparation for generally localized in sufficiently high concentrations to be eas- in vivo microprobe analysis requires initial rapid cryo-fixation ily detected even in fully hydrated tissue. Here, Figures 1, 2 are to immobilize metabolic processes and prevent delocalization presented side-by-side to reinforce the point that leaves of woody of diffusible elements (e.g., Echlin et al., 1980; Marshall, 1980a; Mn hyperaccumulators by virtue of their physical robustness, Echlin, 1992; Marshall and Xu, 1998). Plunging samples directly leaf anatomy combined with extraordinary foliar Mn concentra- into liquid nitrogen, while commonly used, does not achieve tions, are almost uniquely equally well suited to both wet and rapid cryo-fixation since the sample at room temperature upon dry sample preparation for in vivo analyses—be they qualita- contact with liquid nitrogen forms a self-enveloping “nitro- tive or quantitative. Hydrated samples are advantageous in that gen gas vapor cloud,” which has an insulating effect not con- the analytical resolution is higher than in freeze-dried samples ducive to rapid freezing. This is known as the “Leidenfrost since it is partially determined by sample depth. However, high effect”. Plunge- and metal-mirror contact-freezing methods can incident beam energies can lead to beam damage on the sample be employed to achieve rapid sample vitrification free of ice crystals, which can lead to cell content relocation and mem- brane rupture. It is desirable to maintain as small a sample size as possible to achieve artifact-free vitrification. Following appropriate cryo-fixation, samples can then be used for analysis after surface polishing or sectioning by cryo-ultramicrotomy. Bulk frozen tissue is cryo-planed to optimize X-ray collection by the detector since a rough sample surface causes irregu- lar absorption, which can then produce inconsistent analytical data. Planing is done cross-sectionally to enable examination of the in vivo spatial distribution patterns of elements including Mn through the leaf tissue. Samples can also be processed from a frozen cryo-fixed state to a dry state, depending on the constraints and requirements of an investigation. Whether FIGURE 1 | Dry specimen preparation. Figure taken from Fernando et al. wet or dry, samples are commonly surface-coated with a fine (2006a). Qualitative PIXE X-ray Mn maps of carbon-coated hand-cut leaf conductive layer of metal or carbon to avoid charge accumu- cross-sectional surfaces of cryo-fixed and freeze-dried Virotia neurophylla (LHS) and Gossia bidwillii (RHS) samples. High Mn deposition is lation from the incident beam provided it does not confound represented as orange to yellow (highest). analytical data.

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FIGURE 2 | Wet specimen preparation. Figure taken from Fernando et al. (2012). Cryo-ﬁxed frozen hydrated and planed leaf cross sectional surfaces of Virotia neurophylla (A) and Maytenus fournieri (B) lightly surface sublimed and Al-coated. LHS panels show cryo-SEM images (15 kV); and RHS panels show corresponding quantitative X-ray maps with localized vacuolar Mn highlighted in orange to yellow (highest).

(Marshall, 1980c). Küpper et al. (1999, 2000, 2001) and Robinson et al. (2003) have similarly employed cryo-SEM/EDS on cryo- fixed frozen samples to examine Ni, Cd, and Zn localization in herbaceous hyperaccumulators, and achieved subcellular spatial resolution of metal distribution in hydrated tissues. Freeze drying FIGURE 3 | Schematic summary of important steps in preparing (woody) Mn hyperaccumulator leaf-tissue for in vivo microprobe cryo-fixed material on the other hand, while elevating analytical analysis. detection limits, leads to further loss of analytical resolution when the water matrix is removed from cells. Analysis of sections using STEM enables examination of tissue ultrastructure with cell con- tents intact. This is achieved by anhydrous freeze-substitution as Mn TRANSPORTER STUDIES AND FUTURE DIRECTIONS used by Bidwell et al. (2004) to quantify foliar Ni sequestration The accumulation of shoot tissue Mn concentrations significantly in vivo in a herbaceous Ni hyperaccumulator. In an initial pro- above normal plant nutritional requirements is not an cess of cryo-substitution, tissue-water as ice is slowly replaced uncommon observation that raises questions about trans- with a non-polar solvent, subsequently followed by resin infiltra- porters associated with foliar Mn compartmentalization and tion (Pallaghy, 1973; Marshall, 1980b). Thick sections (1–2 µm) specificity at the root-soil interface. Plants that hyperaccumulate can then be used for energy dispersive analysis in STEM. It is Mn can be viewed as exceptional in this regard. There has been unlike TEM sample preparations routinely used for anatomi- little success in the search for Mn transporters responsible for cal/ultrastructural studies, in which cellular ions are washed out Mn acquisition by model plants. As reviewed by Pittman (2005), in polar solvents such as acetone and ethanol (Morgan, 1980). certain classes of divalent-metal transporters such as the ZIP The authors’ previous experience in attempting to infiltrate scle- (ZRT1/IRT1-related protein) and NRAMP (natural resistance- rophyllous leaf tissues of woody Mn hyperaccumulators for TEM associated macrophage protein) appear to be implicated in Mn studies were unsuccessful due to the apparent impermeabilty transport. Recently, a group of NRAMP transporters including of samples. Major considerations when preparing woody hyper- OsNramp5 in rice (Ishimaru et al., 2012; Sasaki et al., 2012), accumulator plant material for in vivo microprobe analysis is and AtNramp1 in Arabidopsis thaliana (Cailliatte et al., 2010) broadly summarized in Figure 3, however, caution needs to be were found to be involved in Mn uptake at the root surface. exercized when interpreting these steps for other material and The NRAMP group is recognized as being associated with heavy species. For example, “soft” herbaceous plant material while more metal transport and possibly contributing to plant adaptation to permeable to fixatives, requires far more rapid handling between metal homeostasis (Thomine et al., 2000). In certain instances collection sites and laboratory, and are considerably less suited to they are more highly expressed in metal-hyperaccumulating dry hand-sectioning. plants than in models such as A. thaliana (Weber et al., 2004;

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Oomen et al., 2009). This suggests that NRAMP transporters are Mn in hyperaccumulators by revealing remarkable tonoplas- important to metal hyperaccumulation either at the uptake level, tal specificity within certain cell types, particularly the palisade root to shoot translocation, or detoxification via vacuolar com- mesophyll (Fernando et al., 2006a,b, 2012). These findings pro- partmentation. Similarly, the ZIP transporters are known to be vide basis for future genetic studies to identify drivers of Mn highly expressed in metal hyperaccumulators. However, since nei- hyperaccumulation, which could ultimately contribute to dis- ther NRAMP nor ZIP transporters appear to be highly expressed cussiononplantMnaccumulationinthecontextofMncrop in Mn hyperaccumulators, there is little evidence of their involve- toxicity. Discovery that foliar Mn is variously co-localized with ment in Mn-hyperaccumulative uptake and/or accumulation (T. excess Zn, Co, and Ni in different palisade cell-layers of a Mn Mizuno, pers. comm.). Delhaize et al. (2003) identified ShMTP8 hyperaccumulator provides further insight into metal trans- as a vacuolar transporter responsible for Mn-accumulative tol- porters (Fernando et al., 2013). Currently there is no published erance in Stylosanthes hamata (Fabaceae), and also described research on the genetic basis of Mn hyperaccumulation. With AtMTP11 as a Mn transporter in prevacuolar compartments of a single exception, Mn transporter studies in yeast, plant mod- Arabidopsis (2007). Peiter et al. (2007) found that the Mn trans- els (Delhaize et al., 2003, 2007; Mizuno et al., 2005; Pittman, porter MTP11 was associated with Golgi bodies in Arabidopsis 2005), and the Mn hyperaccumulator C. sciadophylloides (T. mtp 11 mutants exhibiting an enhanced ability to accumulate Mn Mizuno, pers. comm.) have largely been unsuccessful in charac- in their shoots and roots. terizing the molecular basis of detoxification mechanisms asso- In vivo microprobe localization methodologies have expanded ciated with excess foliar Mn sequestration in Mn-accumulating knowledge about the compartmentalization of excess foliar plants.

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Environ. Pollut. 131, 393–399. doi: analysis. J. Environ. Sci. China 18, Conflict of Interest Statement: The This article was submitted to Plant 10.1016/j.envpol.2004.03.011 746–751. authors declare that the research Physiology, a section of the journal Xu,Shi,J.,Chen,Y.,Chen,X., Xu, X., Shi, J., Chen, X., Chen, Y., was conducted in the absence of any Frontiers in Plant Science. Wang, H., and Perera, A. (2006a). and Hu, T. (2009). Chemical commercial or financial relationships Copyright © 2013 Fernando, Marshall, Distribution and mobility of man- forms of manganese in the leaves that could be construed as a potential BakerandMizuno.Thisisanopen- ganese in the hyperaccumulator of manganese hyperaccumu- conflict of interest. access article distributed under the terms plant Phytolacca acinosa Roxb. lator Phytolacca acinosa Roxb. of the Creative Commons Attribution (Phytolaccaceae). Plant Soil 285, (Phytolaccaceae). Plant Soil 318, Received: 29 April 2013; accepted: 29 July License (CC BY). The use, distribution or 323–331. doi: 10.1007/s11104-006- 197–204. doi: 10.1007/s11104-008- 2013; published online: 20 August 2013. reproduction in other forums is permit- 9018-2 9829-4 Citation: Fernando DR, Marshall A, ted, provided the original author(s) or Xu, X. H., Shi, J. Y., Chen, Y. X., Xue, Yang, S. X., Deng, H., and Li, M. Baker AJM and Mizuno T (2013) licensor are credited and that the origi- S. G., Wu, W. B., and Huang, S. (2008). Manganese uptake Microbeam methodologies as power- nal publication in this journal is cited, in Y. (2006b). An investigation of and accumulation in a woody ful tools in manganese hyperaccumula- accordance with accepted academic prac- cellular manganese in hyper- hyperaccumulator, Schima tion research: present status and future tice. No use, distribution or reproduction accumulator plant Phytolacca superba. Plant Soil Environ. 54, directions. Front. Plant Sci. 4:319. doi: is permitted which does not comply with acinosa Roxb. using SRXRF 441–446. 10.3389/fpls.2013.00319 these terms.

www.frontiersin.org August 2013 | Volume 4 | Article 319 | 9

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Author/s: Fernando, DR; Marshall, A; Baker, AJM; Mizuno, T

Title: Microbeam methodologies as powerful tools in manganese hyperaccumulation research: present status and future directions

Date: 2013-08-20

Citation: Fernando, D. R., Marshall, A., Baker, A. J. M. & Mizuno, T. (2013). Microbeam methodologies as powerful tools in manganese hyperaccumulation research: present status and future directions. FRONTIERS IN PLANT SCIENCE, 4 (AUG), https://doi.org/10.3389/fpls.2013.00319.

Persistent Link: http://hdl.handle.net/11343/261628

File Description: Published version License: CC BY