Denise R. Fernando

Zinc tolerance and the binding of zinc as zinc phytate in Lemna minor. X-ray microanalytical evidence

Summary Electron probe X-ray microanalysis of fracture faces of quench frozen hydrated bulk samples of Lemna minor fronds exposed to high levels of Zn (300 µM) has shown the presence of globular deposits up to 1 µm in diameter containing Zn, K and P in parenchyma cells of mature fronds, and Zn, K, Mg and P in immature parenchyma cells of daughter fronds (or turions). Although this method of direct analysis of bulk samples does not lend itself to a quantitative comparison of number and composition of globules in Zn-tolerant and Zn-sensitive clones of Lemna minor , it has the advantages of simplicity of specimen preparation, rapid detection of Zn-P-containing globules, and avoidance of diffusional artifacts. Comparisons with prepared K Mg phytate and Zn standards, and additional data obtained by X-ray microanalysis of freeze-substituted thin sections, established the elemental proportions of Zn, Mg, K and Ca relative to P, which indicate that generally up to 8 (molar ratio of Zn to phytic acid = 4) and sometimes up to 12 (molar ratio of Zn to phytic acid = 6) valencies can be occupied by Zn and up to 4 by K and/or Mg. Some of the Ca that appears to be associated with globules may result from elemental redistribution during the process of freeze-substitution especially when Ca oxalate crystals are present in neighbouring raphide cells. X-ray microanalysis provided evidence for the absence of Zn from these raphides.

Deposition of Zinc Phytate in Globular Bodies in Roots of Deschampsia caespitosa Ecotypes; a Detoxification Mechanism?

Summary Electron probe X-ray microanalysis of freeze-substituted thin sections (non-aqueous specimen preparation) has shown the presence of Zn, K, Mg and P in globular bodies up to 1 μm in diameter within small vacuoles of cortical cells in the elongation zone of root tips of Deschampsia caespitosa plants which had been exposed to toxic concentrations of Zn. Comparison of the spectra with those obtained from standards prepared from phytic acid and known quantities of various metal ions, strongly suggests that the globular bodies consist of phytate with Zn, Mg and K present in constant proportions relative to P. These proportions depend on Zn treatment and on whether the ecotype is sensitive or tolerant to Zn. In globules of the tolerant ecotype exposed to 1000μM Zn, up to six valencies are occupied by Zn, four by Mg and the remaining two by K, which indicates that the phytate consists of myo-inositol hexakisphosphate. The unusually high Zn content (20.1 % by weight) of the globules in the cortical cells of these roots suggests that they may act as a mechanism for detoxification of Zn, especially in the Zn tolerant ecotype of Deschampsia caespitosa .

The binding of zinc, but not cadmium, by phytic acid in roots of crop plants

Plant species adapted to soils enriched with heavy metals often accumulate these metals in their above or below ground organs. In this study, electron probe microanalysis of fractured, quench-frozen root specimens of common crop species shows that an appreciable quantity of Zn can be bound as Zn phytate (myo-inositolkis-hexaphosphate) within small vacuoles of cells in the root elongation zone of lucerne, soybean, lupins, tomato, rapeseed, cabbage, radish, maize and wheat exposed to high levels of Zn (80–300 μM). Globular deposits of Zn phytate are most frequently observed in the endodermis of dicotyledonous species and in the pericycle of monocotyledonous species, but may also occur in the stele and inner cortex after prolonged exposure to toxic levels of Zn. The deposits could not be found in Zn-treated sunflower, field peas and Italian ryegrass. In three crop species, lucerne, soybean and maize, Zn-induced phytate globules were frequent, but exposure of roots to 30 μM Cd did not induce the formation of Cd-containing globular deposits as observed inLemna minor (Van Steveninck et al., 1990a, 1992). Simultaneous Zn and Cd treatment induced the formation of Zn phytate globules as effectively as Zn alone, and Cd was not detected in the deposits.

Characterization of foliar manganese (Mn) in Mn (hyper)accumulators using X-ray absorption spectroscopy

Plant hyperaccumulation of the essential nutrient manganese (Mn) is a rare phenomenon most evident in the Western Pacific region, and differs from hyperaccumulation of other elements. Mn hyperaccumulators employ a variety of species-dependent spatial distribution patterns in sequestering excess foliar Mn, including primary sequestration in both nonphotosynthetic and photosynthetic tissues. This investigation employed synchrotron X-ray absorption spectroscopy (XAS) in a comparative study of Mn (hyper)accumulators, to elucidate in situ the chemical form(s) of foliar Mn in seven woody species from Australia, New Caledonia and Japan. Foliar Mn was found to predominate as Mn(II) in all samples, with strong evidence of the role of carboxylic acids, such as malate or citrate, as complexing ligands. Overall, the X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine-structure spectroscopy (EXAFS) data appeared weighted against previous observations that oxalate binds excess Mn in Mn-(hyper)accumulating species.

Heavy-metal (Zn, Cd) tolerance in selected clones of duck weed (Lemna minor)

Cryo-microprobe analysis of quench-frozen fronds of a Zn-tolerant clone of Lemna minor exposed to a high level of Zn (300 μM) showed the presence of cellular deposits consisting of Zn, Mg, K and P or Zn, K and P (Zn phytate). The same Zn-tolerant clone of Lemna minor, when exposed to a high level of Cd (30 μM), showed the presence of globular deposits consisting of Cd, K and P in mature fronds, but the immature cells of the enclosed daughter fronds contained relatively large deposits with Cd and S as the main components (Cd-phytochelatin?). Selection for Zn tolerance in a population of Lemna minor was easily achieved but selection for Cd tolerance has so far not been successful. The Zn-tolerant clone also tolerates high levels of phosphate.

Manganese phytotoxicity: new light on an old problem

BACKGROUND Manganese (Mn) is an essential micronutrient that is phytotoxic under certain edaphic and climatic conditions. Multiple edaphic factors regulate Mn redox status and therefore its phytoavailability, and multiple environmental factors including light intensity and temperature interact with Mn phytotoxicity. The complexity of these interactions coupled with substantial genetic variation in Mn tolerance have hampered the recognition of Mn toxicity as an important stress in many natural and agricultural systems. SCOPE Conflicting theories have been advanced regarding the mechanism of Mn phytotoxicity and tolerance. One line of evidence suggests that Mn toxicity ocurrs in the leaf apoplast, while another suggests that toxicity occurs by disruption of photosynthetic electron flow in chloroplasts. These conflicting results may at least in part be attributed to the light regimes employed, with studies conducted under light intensities approximating natural sunlight showing evidence of photo-oxidative stress as a mechanism of toxicity. Excessive Mn competes with the transport and metabolism of other cationic metals, causing a range of induced nutrient deficiencies. Compartmentation, exclusion and detoxification mechanisms may all be involved in tolerance to excess Mn. The strong effects of light, temperature, precipitation and other climate variables on Mn phytoavailability and phytotoxicity suggest that global climate change is likely to exacerbate Mn toxicity in the future, which has largely escaped scientific attention. CONCLUSIONS Given that Mn is terrestrially ubiquitous, it is imperative that the heightened risk of Mn toxicity to both managed and natural plant ecosystems be factored into evaluation of the potential impacts of global climate change on vegetation. Large inter- and intraspecific genetic variation in tolerance to Mn toxicity suggests that increased Mn toxicity in natural ecosystems may drive changes in community composition, but that in agroecosystems crops may be developed with greater Mn tolerance. These topics deserve greater research attention.

The binding of zinc in root cells of crop plants by phytic acid

Appreciable quantities of Zn are bound as Zn phytate (myo-inositol kis-hexaphosphate) within small vacuoles of cortical cells in the elongation zone of root tips of zinc tolerant Deschampsia caespitosa. These Zn/P-containing globular deposits have now been shown to occur in the roots of soybean, lucerne, lupins, tomato, rapeseed, cabbage, radish, wheat and maize. The globules are most frequent in the endodermis and pericycle but may also occur in the stele and inner cortex. The X-ray data again confirmed the presence of phytate with a relatively stable proportion of Zn and a species-dependent, variable, proportion of K, Mg and Ca to P.

Novel pattern of foliar metal distribution in a manganese hyperaccumulator

The primary sequestration of foliar manganese (Mn) in Mn-hyperaccumulating plants can occur in either their photosynthetic or non-photosynthetic tissues, depending on the species. To date, only non-photosynthetic tissues have been found to be the major sinks in other hyperaccumulators. Here, electron (SEM) and proton (PIXE) microprobes were used to generate qualitative energy dispersive (EDS) X-ray maps of leaf cross sections. Two Mn hyperaccumulators, Garcinia amplexicaulis Vieill. (Clusiaceae) and Maytenus fournieri (Panch. and Sebert) Loesn. (Celastraceae), and the Mn accumulator Grevillea exul Lindley (Proteaceae) were studied. PIXE/EDS data obtained here for M. fournieri were in agreement with existing SEM/EDS data showing that the highest localised foliar Mn concentrations were in the epidermal tissues. However, this is the first in situ microprobe investigation of G. amplexicaulis and G. exul. The Mn X-ray maps of G. amplexicaulis revealed a previously undescribed third spatial distribution pattern among Mn-hyperaccumulating species. Manganese was relatively evenly distributed throughout the leaf photosynthetic and non-photosynthetic tissues, while in G. exul it was most highly concentrated in the epidermal cells.

Multiple metal accumulation within a manganese-specific genus

PREMISE OF THE STUDY Plants that strongly accumulate metals may be practically beneficial, and also serve as novel resources for increasing fundamental understanding of plant biology. Australian Gossia (Myrtaceae) species are delineated by a conspicuous affinity for the heavy metal manganese (Mn), which is a micronutrient crucial to photosynthesis. This genus includes several Mn hyperaccumulators such as G. bidwillii. Unusually, in G. bidwillii foliar Mn is most highly concentrated in photosynthetic cells, an observation thus far restricted to foliar-Mn accumulation in Mn hyperaccumulators. Recent discovery that several of these Gossia species accumulate other metals in addition to Mn will enable investigation as to whether primary sequestration of metals in photosynthetic tissues is restricted to Mn. METHODS Gossia species known to accumulate nickel (Ni) or aluminum (Al) in addition to Mn were sampled in the field. Complementary proton- and electron-probe data were combined to evaluate in vivo microdistribution patterns of excessively accumulated foliar metals. KEY RESULTS It was discovered that in addition to Mn and Ni, Gossia fragrantissima accumulated foliar zinc (Zn) and cobalt (Co), with Mn, Ni, and Co most highly localized in mesophyll cells and Zn primarily located in the upper epidermis. In G. hillii, Mn and Al were highly concentrated in the palisade and epidermis, respectively. CONCLUSIONS This investigation provides evidence that the primary disposal of excess foliar metals in photosynthetic cells is not exclusive to Mn. It offers rare intrageneric perspective on metal compartmentation, pointing to significant variation among tonoplastal metal transporters associated with detoxification.

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

Microbeam studies over the past decade have garnered unique insight into manganese (Mn) homeostasis in plant species that hyperaccumulate this essential mineral micronutrient. Electron- and/or proton-probe methodologies employed to examine tissue elemental distributions have proven highly effective in illuminating excess foliar Mn disposal strategies, some apparently unique to Mn hyperaccumulating plants. When applied to samples prepared with minimal artefacts, these are powerful tools for extracting true ‘snapshot’ data of living systems. For a range of reasons, Mn hyperaccumulation is particularly suited to in vivo interrogation by this approach. Whilst microbeam investigation of metallophytes is well documented, certain methods originally intended for non-biological samples are now widely applied in biology. This review examines current knowledge about Mn hyperaccumulators with reference to microbeam methodologies, and discusses implications for future research into metal transporters.