Metal localization and elemental associations in plants were investigated with X-ray and electron microscopies and X-ray microtomography.Particular emphasis was placed on the phenomenon of ‘simultaneous hyper accumulation’and its relationship to metal co-tolerance.Metal localization and elemental associations in A.murale were investigated with SXRF, CMT, SEM-EDS and XAS.Two-dimensional SXRF images of A.murale leaves revealed a distinctive localization pattern for Co relative to Ni and Zn.The Ni distribution in leaves was essentially uniform, although the fluorescence intensity was slightly elevated in the midrib region.A uniform Ni distribution was anticipated for two-dimensional leaf images because Alyssum sequesters Ni within the epidermal layers.The Ni distributions were comparable for both young and old leaves, and Ni localization was not altered in A.murale plants exposed to mixed-metal systems.The Zn distribution in A.murale leaves appeared similar to that of Ni; however, elevated fluorescence intensity in the midrib region was more evident in SXRF images of Zn than Ni because the Zn fluorescence signal was not dominated by the epidermal cell layers.Zinc was not hyper accumulated by A.murale and would not be preferentially compartmentalized in epidermal This issue.The Zn distributions were comparable for both young and old leaves.In contrast to Ni and Zn distributions, Co was preferentially localized at the tips and margins of A.murale leaves.Similar Co localization patterns have been reported for various non accumulator plants investigated using autoradiography.Cobalt localization in A.murale was consistent for young and old leaves, but Co enrichment near the leaf tips was more common on older leaves than on younger leaves.
Spatial associations in leaves were visualized by combining fluorescence data from individual elements into a multi-color image and plotting a line profile.An SXRF image of Co, Ni and Ca localization in an A.murale leaf revealed a color gradient near the leaf tip as a result of the uniform Ni distribution and the irregular Co distribution in the leaf.A line profile generated for a segment from the leaf center towards the leaf tip showed that the substantial increase in the Co signal coincided with a subtle decrease in the Ni signal; Co and Ni were not preferentially co-localized in leaves.Spatial associations of Co with Mn and S coincided with statistically significant correlations in bulk shoot concentrations.Cobalt-treated plants had the highest shoot concentrations of Mn and Sand the highest S TFs.SXRF images of Co and Mn in A.murale leaves revealed co-localization of these elements at leaf trichomes.A Mn-rich zone surrounds the base of A.murale trichomes and could sequester Ni or Co.SXRF images of S and Co in A.murale leaves indicated co-localization of these elements near the leaf tips/margins.S and Co were spatially correlated,hydroponic bucket but preliminary XAS data did not indicate a direct chemical association.F-CMT images of A.murale fine-root segments revealed distinctive metal partitioning patterns as a function of distance from the root tip.Nickel, Co and Fe were localized in the root vasculature0.5 mm from the apex; the root tip appeared most active in the absorption of these metals.In the root segment 6 mm from the apex, these metals were predominantly confined to the epidermis, signifying sorption to surface functional groups, mucilage, bacterial bio-films, or metal oxide plaques on the root surface.Additionally, metal enrichment was discernible in the vasculature but was absent from the pith.Similar localization patterns were reported by Fellows et al., who investigated europiumup take in living roots of Avena sativa and observed maximal fluorescence intensities in the apical This issue at the root tip and within the zone of root maturation , whereas elevated fluorescence from highly differentiated root This issue was observed at the epidermis.Metal localization patterns in the A.murale root segment 3 mm from the apex were intermediate to those observed at 0.5 and 6 mm; elevated Ni and Co levels were observed in the vascular cylinder and at the root epidermis.
Zinc and Mn localization patterns in A.murale roots were distinct from Ni, Co and Fe and from one another.Zinc was predominantly localized in isolated domains within the root at 0.5, 3 and 6 mm from the apex, whereas Mn was sporadically localized at the epidermis.A separate experiment conducted with A.murale plants exposed to a Co-enriched nutrient solution verified that the Co localization phenomenon observed in the metal interaction study was not a result of simultaneous hyper accumulation; thus, Co localization had not been altered in plants exposed to elevated Ni and Zn concentrations.Cobalt accumulated by A.murale was ultimately deposited on leaf surfaces near the tips/margins.Identical Co localization patterns were observed for Co-treated A.corsicum and A.troodii , suggesting similar mechanisms exist in other Ni hyper accumulator species of Alyssum.Cobalt on the surface of leaves was visible by optical microscopy.BSE images showed a coating on the leaf surface near the tips/margins, and X-ray micro-analysis indicated that the coating was a Co-rich phase.The electron microprobe beam penetrated only a few microns into the sample and thus the recorded signals were emitted from the leaf surface or the cuticle layer; a comparison of EDS spectra from the Co-rich and bulk-leaf regions further supports the finding of a Co-rich phase deposited on the exterior of leaves.Leaf images from the optical microscope and SEM corroborate the Co distribution observed with SXRF.Micro-XAS spectra collected from hydrated A.murale leaves revealed that the oxidation state of Co in plants was Co; artificial Co oxidation was not observed in this study, but Co can result from sample alteration in the X-ray beam and by ligand stabilization with multi-dentate amine ligands.Spectra collected at the Co-rich region near the tip showed striking differences from spectra collected at the bulk-leaf region.The Co k3-weighted χ spectrum from the Co-rich region had a beat pattern near 5 Å–1 and a split oscillation between 7 and 8.5 Å–1, whereas the χ spectrum from the bulk-leaf region did not have these characteristic structural features ; spectra with several frequencies are indicative of a long-range ordered binding environment such as that in a mineral structure, whereas spectra dominated by a single frequency are indicative of a short-range ordered environment.
An evaluation of the Co-binding environment for Co-rich spot 1 provided distances and numbers of Co–Co pairs that are characteristic of an edge-sharing layered framework.A strong second-shell feature and a third metal shell at about twice this distance was present in the Fourier transform from the Co-rich region, but was absent in the FT from the bulk-leaf region.Cobalt accumulated by A.murale formed Co-rich mineral precipitate on the leaf surface.Detailed characterization of the Co phase formed on leaves and the ligands involved with Co transport and detoxification in Alyssum are beyond the scope of this study but will be reported in a future publication.DA-CMT images of a hydrated leaf from Co-treated A.murale revealed a lack of metal enrichment in epidermal This issue.Cobalt near leaf tips was localized predominantly on the leaf exterior.In addition to Co enrichment on the leaf surface, DA-CMT slices 20–30 µm below the leaf tip showed Co localized in isolated regions inside the leaf associated with the vascular system.Cobalt was consistently observed on the exterior of leaves, but deposition on leaf surfaces was less prevalent at greater distances from the leaf tip.Images from several hundreds to thousands of microns below the leaf tip showed a predominance of Co distributed around the leaf ground This issue ; the distribution between cells was interpreted as Co in the leaf apoplasm.Cobalt enrichment was more prevalent in the region composed of spongy mesophyll than palisade mesophyll; spongy mesophyll contains fewer chloroplasts and many intercellular spaces linked to the outside via stomata.Regions with elevated Co or Ni were frequently observed in proximity to leaf trichomes; however, Co enrichment was associated with the trichome structures on the leaf surface , whereas Ni was associated with the basal portion of trichomes.DA-CMT images of a hydrated leaf from Nitreated A.murale showed metal enrichment in the epidermis ; this result is consistent with other studies of Ni compartmentalization in Alyssum, which have shown Ni sequestration in epidermal cell vacuoles.In addition to epidermal localization, Ni was observed within leaf ground This issue.In contrast to Co, a fraction of Ni in ground This issue occupied the same spaces as mesophyll cells,stackable planters and this Ni distribution was interpreted as partial metal enrichment of mesophyll This issue.At a fundamental level, mechanisms of metal tolerance and hyper accumulation in Alyssum remain poorly understood.A.murale hyper accumulates Ni and Co, but Zn is not accumulated to abnormal levels.Elevated Co or Zn concentrations do not alter Ni accumulation or localization, and thus A.murale can be used to recover Ni from most metal-enriched soils containing these metal co-contaminants.A.murale is more tolerant to Ni than Co; nickel tolerance is attained via epidermal compartmentalization.A.murale does not sequester Co in epidermal cells; Co in the xylem or leaf apoplasm is excreted from leaves and subsequently sequestered on leaf surfaces as sparingly soluble precipitate.Therefore, the specialized biochemical processes linked to Ni tolerance in A.murale do not confer tolerance to Co.Cobalt is ultimately deposited at the leaf tips/margins, whereas Ni is sequestered in epidermal cells.Vacuolar sequestration is a key strategy for metal tolerance because leaf epidermal cells provide an effective sink for the accumulated metal.Metal concentrations exceeding 0.35 M were measured in epidermal cell vacuoles of a Zn hyperaccumulator.McNear et al.imaged Niin an A.murale leaf and reported, in addition to epidermal enrichment, elevated Ni accumulation on/in the leaf tip; furthermore, it was suggested that leaf tips function as an additional reservoir for Ni when concentrations exceed the finite capacity of cell vacuoles.However, McNear et al.did not consider that their leaf tip was curled such that the epidermal layers near the tip were oriented parallel to the incident X-ray beam regardless of the rotation angle; thus, the beam exclusively ‘sampled’ metal-enriched epidermal This issue in this region, leading to the erroneous impression of elevated Ni on/in the leaf tip.Upper slices from the DA-CMT movie revealed a lack of Ni enrichment near the point of the leaf ; nonetheless, these slices afford a rare glimpse of Ni localization across leaf epidermal This issue.
Clear differences between Ni and Co localization suggest that A.murale uses a different metal sequestration mechanism for Co than for Ni.Whereas Ni is redistributed to leaf epidermal cells and subsequently transported across the tonoplast for long-term sequestration in vacuoles , Co does not have an efficient route of entry into epidermal cells.Considering the serpentine origin of Alyssum, a cellular-level tolerance mechanism for Co may not have been naturally selected because the Ni : Co ratio in these soils is relatively large.Additionally, Co is typically retained more strongly by the soil components than Ni and thereby is less phytoavailable.Therefore, mechanisms other than vacuolar sequestration must be operating to cope with the elevated Co concentrations in plant This issue.A.murale leaves apparently lack the transport system needed to sequester Co in epidermal cells and thus accumulated Co resides in the xylem and the leaf apoplasm.Mass flow and diffusion gradients in the apoplasm will cause Co to redistribute in leaves.Water loss via transpiration will move Co towards the leaf surfaces and margins where transpiration is maximal.Cobalt principally follows the transpiration stream and results in Co enrichment at leaf tips/margins.When transpiration is low , root pressure will cause exudation of xylem sap from the ventilation poreslocated at the points of the leaf margin to where veins extend.Guttation fluids of plants from ultramafic soils have been reported to contain elevated metal concentrations.For example, Minuartia verna grown in metal-contaminated soil accumulated Cu and Zn in leaves and excreted these metals onto leaf surfaces via hydathodes.Cobalt accumulated by A.murale is ultimately deposited on leaf surfaces.Cobalt enrichment on leaf surfaces is evident from X-ray microtomography , SEM-EDS, optical microscopy and visual inspection of Co-treated A.murale leaves.Similar observations were made by Vergnano & Hunter , who noted red-colored leaf tips on plants exposed to Co-enriched nutrient solution.Cobalt deposition at A.murale leaf tips is most prevalent on older leaves.For many plant species, older leaves have the highest concentrations of elements such as Co because evapotranspiration continues as long as the leaf is attached to the plant.Deposition of sparingly soluble Co species near the tips/margins of A.murale leaves is corroborated by in situ microspectroscopic analyses , revealing that weight percent Co is sequestered on leaf surfaces and forms Co-rich mineral precipitate.Metal-tolerant Arabidopsis halleri , grown in a Zn- and Cu-contaminated soil, had mixed-metal precipitate on leaf surfaces.Elevated regions of Ni and Co occur consistently in proximity to the trichomes on A.murale leaves, and the Mn-rich zone surrounding the base of trichomes may be partially responsible for this metal enrichment.Simultaneous hyperaccumulation of Mn and Ni occurred in the basal compartment of the nonglandular trichomes on Alyssum leaves.