Low-Temperature X-ray Microanalysis Sheds New Light on Mineral Nutrition Aspects of Insect Leaf Galling.

Manipulation of host plant physiology by leaf-galling insects is a multifaceted process. Among fundamental knowledge gaps surrounding this scientifically intriguing phenomenon is the appropriation of plant mineral nutrients and moisture for galling advantage. Small, soluble mineral ions and watery cell contents in dense gall tissues risk disruption during routine sample preparations. In this study, an X-ray microanalysis was applied to investigate gall mineral nutrition. Morphologically diverse leaf galls were sampled from three Australian rainforest tree species. Using cryo-analytical scanning electron microscopy, real-time X-ray analytical maps of cellular mineral nutrients and water were integrated with anatomical images of gall and leaf cross-sectional surfaces. A comparison of host-leaf and gall anatomies bore direct evidence of drastic changes to leaf cells through the galling process. Distinct "wet" and "dry" regions within galls were anatomically and/or chemically differentiated, suggesting specific functionality. "Wet" regions comprising hydrated cells including soft gall-cavity linings where larvae are known to feed contained soluble plant mineral nutrients, while C-rich "dry" tissues largely devoid of mineral nutrients likely contribute structural support. Mapping immobile nutrients such as Mn may provide a means of "matching" specific gall cell types to those in ungalled host-leaf tissues. The findings here provided otherwise inaccessible insights into leaf-gall mineral nutrition.

Cellular-level distribution of manganese in Macadamia integrifolia, M. ternifolia, and M. tetraphylla from Australia.

Macadamia integrifolia and M. tetraphylla, unlike M. ternifolia, are known for their edible nuts. All three species over-accumulate the trace metal nutrient manganese (Mn) in their shoots. This study seeks to examine tissue- and cellular-level distribution of Mn and other plant nutrients in the three Macadamia species. The distribution of Mn, calcium, iron, and potassium were investigated in whole leaves and cross-sections of roots, petioles, and leaves using synchrotron-based X-ray fluorescence microscopy (XFM) in M. integrifolia, M. tetraphylla, and M. ternifolia. The results show Mn sequestration primarily in the leaf and midrib palisade mesophyll cells of all three species. Leaf interveinal regions, root cortical cells, and phloem cells were also found to be Mn loaded. The current study confirms earlier findings but further reveals that Mn is concentrated in the vacuoles of mesophyll cells owing to the exceptional resolution of the synchrotron XFM data, and the fact that fresh hydrated samples were used. New insights gained here into Mn compartmentalization in these highly Mn-tolerant Macadamias expand knowledge about potentially toxic over-accumulation of an essential micronutrient, which ultimately stands to inform strategies around farming edible species in particular.

Incidence of hyperaccumulation and tissue-level distribution of manganese, cobalt, and zinc in the genus Gossia (Myrtaceae).

The rare phenomenon of plant manganese (Mn) hyperaccumulation within the Australian flora has previously been detected in the field, which suggested that the tree genus Gossia (Myrtaceae) might contain new Mn hyperaccumulators. We conducted the first growth experiment on Gossia using a multi-factorial dosing trial to assess Mn, cobalt (Co), and zinc (Zn) (hyper)accumulation patterns in selected Gossia species (G. fragrantissima and G. punctata) after a systematic assessment of elemental profiles on all holdings of the genus Gossia at the Queensland Herbarium using handheld X-ray fluorescence spectroscopy. We then conducted detailed in situ analyses of the elemental distribution of Mn, Co, Zn and other elements at the macro (organ) and micro (cellular) levels with laboratory- and synchrotron-based X-ray fluorescence microscopy (XFM). Gossia pubiflora and Gossia hillii were newly discovered to be Mn hyperaccumulator plants. In the dosing trial, G. fragrantissima accumulated 17 400 µg g-1 Mn, 545 µg g-1 Co, and 13 000 µg g-1 Zn, without signs of toxicity. The laboratory-based XFM revealed distinct patterns of accumulation of Co, Mn, and Zn in G. fragrantissima, while the synchrotron XFM showed their localization in foliar epidermal cells, and in the cortex and phloem cells of roots. This study combined novel analytical approaches with controlled experimentation to examine metal hyperaccumulation in slow-growing tropical woody species, thereby enabling insight into the phenomenon not possible through field studies.

Does foliage metal accumulation influence plant-insect interactions? A field study of two sympatric tree metallophytes.

Gossia (Myrtaceae) is a highly restricted tree genus most speciose in New Caledonia and eastern Australia. The latter group accumulates above-normal foliar manganese (Mn) concentrations, with some individuals exhibiting the rare Mn-hyperaccumulative trait. Whether foliar metals contribute to chemical defence has been addressed via numerous feeding experiments and very few field studies. This investigation exploited specifically different insect activities on the foliage of sympatric Gossia grayi (N.Snow & Guymer) and Gossia shepherdii (F.Muell.) N.Snow & Guymer, endemic to north-eastern Australia, to test for direct and indirect effects of foliar Mn enrichment on plant-insect interactions. Leaf organic and inorganic chemistries, specific weight, surface damage, gall infestation and occupancy were quantified. Discovery that both species are Mn hyperaccumulators augments the world listing by 5-7%. Highly elevated gall-Mn concentrations coupled with negligible gall parasitisation suggested chemical fortification and adaptation by the host insect - a Cecidomyiidae fly. Linear mixed modelling (LMM) showed differences in leaf Mn, phenolics, toughness and surface damage across tree species and leaf age. There was no direct relationship between leaf Mn and insect impact. However, LMM did resolve indirect effects, i.e. between insect impact and certain foliar elements, consistent with nutritional dynamics in a physiologically novel plant system where Mn is vastly overaccumulated.

Cellular ion interactions in two endemic tropical rainforest species of a novel metallophytic tree genus.

Gossia, a small genus of physiologically novel rainforest trees restricted to the Western Pacific and belonging to a key neotropical Southern Hemisphere family, the Myrtaceae, is characterized by high foliar manganese (Mn) concentrations. This field study provides a quantitative in planta snapshot detailing cellular localization of foliar Mn and other mineral nutrients in sympatric Gossia grayi N. Snow & Guymer and Gossia shepherdii N. Snow & Guymer endemic to far northeastern Australia, and previously not examined. Elements localized in the cells of fresh hydrated leaf tissues were quantified via in vivo cryo-scanning electron microscopy energy dispersive spectroscopy, a non-invasive method that effectively immobilizes cell contents. Leaf anatomical differences were found between species, along with foliar Mn spatial distribution patterns. Localized cellular Mn concentrations exceeding 600 mmol kg-1 were detected in G. shepherdii, whose Mn accumulation across different mesophyll cell types was heterogeneous compared with G. grayi. In both species there was little evidence to support previous findings on other Gossia species of carboxylate association with excess Mn. The analytical X-ray data strongly implicated chloride as a counter-ion to Mn in the two species examined here. The key findings align with the hypothesis that Mn disposal in the mesophyll is a generic trait in Gossia. This research has forged an emerging view of Gossia as being characterized by unusual cellular metal and mineral accumulation patterns that vary at the species level. It contributes to current limited knowledge about generic plant metallophyty, highlighting that assimilating a broader perspective of the phenomenon demands evaluation of individual taxa through field studies.

Foliar Nutrient Distribution Patterns in Sympatric Maple Species Reflect Contrasting Sensitivity to Excess Manganese.

Sugar maple and red maple are closely-related co-occurring tree species significant to the North American forest biome. Plant abiotic stress effects including nutritional imbalance and manganese (Mn) toxicity are well documented within this system, and are implicated in enhanced susceptibility to biotic stresses such as insect attack. Both tree species are known to overaccumulate foliar manganese (Mn) when growing on unbuffered acidified soils, however, sugar maple is Mn-sensitive, while red maple is not. Currently there is no knowledge about the cellular sequestration of Mn and other nutrients in these two species. Here, electron-probe x-ray microanalysis was employed to examine cellular and sub-cellular deposition of excessively accumulated foliar Mn and other mineral nutrients in vivo. For both species, excess foliar Mn was deposited in symplastic cellular compartments. There were striking between-species differences in Mn, magnesium (Mg), sulphur (S) and calcium (Ca) distribution patterns. Unusually, Mn was highly co-localised with Mg in mesophyll cells of red maple only. The known sensitivity of sugar maple to excess Mn is likely linked to Mg deficiency in the leaf mesophyll. There was strong evidence that Mn toxicity in sugar maple is primarily a symplastic process. For each species, leaf-surface damage due to biotic stress including insect herbivory was compared between sites with acidified and non-acidified soils. Although it was greatest overall in red maple, there was no difference in biotic stress damage to red maple leaves between acidified and non-acidified soils. Sugar maple trees on buffered non-acidified soil were less damaged by biotic stress compared to those on unbuffered acidified soil, where they are also affected by Mn toxicity abiotic stress. This study concluded that foliar nutrient distribution in symplastic compartments is a determinant of Mn sensitivity, and that Mn stress hinders plant resistance to biotic stress.

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.

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.

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.

Plant homeostasis of foliar manganese sinks: specific variation in hyperaccumulators.

Plant manganese (Mn) hyperaccumulation provides unusual insight into homeostasis of this essential micronutrient, in particular its excessive storage in shoot tissues. The compartmentation of hyperaccumulated foliar Mn appears exceptional among metal hyperaccumulators, since it occurs via specific microdistribution patterns. Here, three associated Mn hyperaccumulators, Virotia neurophylla, Maytenus fournieri, and Garcinia amplexicaulis exhibiting distinctly different Mn detoxification strategies were examined. Non-invasive sample preparation in conjunction with cryo scanning electron microscopy (SEM) was used to obtain in vivo quantitative microprobe X-ray and anatomical data from fully hydrated cells. Highly vacuolated large palisade mesophyll cells in V. neurophylla leaves were found to contain around 650 mM Mn. The large non-photosynthetic hypodermal cells of M. fournieri leaves, also with high vacuolar content, and the main site for Mn disposal, had an estimated mean vacuolar Mn concentration of around 600 mM. Previous qualitative X-ray mapping had shown Mn to be almost evenly sequestered across the entire leaf cross section of G. amplexicaulis. However, quantitative data obtained here showed a marked variation in localised concentrations that ranged between ~15 and >800 mM. Notable among these were mean values of >600 mM in spongy mesophyll cells, and ~800 mM within cells of a narrow sub epidermal layer preceding the palisade mesophyll. This study demonstrated the extraordinary Mn carrying capacities of different types of leaf cell vacuoles.

Foliar Mn accumulation in eastern Australian herbarium specimens: prospecting for 'new' Mn hyperaccumulators and potential applications in taxonomy.

BACKGROUND AND AIMS: The analysis of herbarium specimens has previously been used to prospect for 'new' hyperaccumulators, while the use of foliar manganese (Mn) concentrations as a taxonomic tool has been suggested. On the basis of their geographic and taxonomic affiliations to known Mn hyperaccumulators, six eastern Australian genera from the Queensland Herbarium collection were sampled for leaf tissue analyses. METHODS: ICP-OES was used to measure Mn and other elemental concentrations in 47 species within the genera Austromyrtus, Lenwebbia, Gossia (Myrtaceae), Macadamia (Proteaceae), Maytenus and Denhamia (Celastraceae). KEY RESULTS: The resulting data demonstrated (a) up to seven 'new' Mn hyperaccumulators, mostly tropical rainforest species; (b) that one of these 'new' Mn hyperaccumulators also had notably elevated foliar Ni concentrations; (c) evidence of an interrelationship between foliar Mn and Al uptake among the Macadamias; (d) considerable variability of Mn hyperaccumulation within Gossia; and (e) the possibility that Maytenus cunninghamii may include subspecies. CONCLUSIONS: Gossia bamagensis, G. fragrantissima, G. sankowsiorum, G. gonoclada and Maytenus cunninghamii were identified as 'new' Mn hyperaccumulators, while Gossia lucida and G. shepherdii are possible 'new' Mn hyperaccumulators. Of the three Myrtaceae genera examined, Mn hyperaccumulation appears restricted to Gossia, supporting its recent taxonomic revision. In the context of this present investigation and existing information, a reassesment of the general definition of Mn hyperaccumulation may be warranted. Morphological variation of Maytenus cunninghamii at two extremities was consistent with variation in Mn accumulation, indicating two possible 'new' subspecies. Although caution should be exercised in interpreting the data, surveying herbarium specimens by chemical analysis has provided an effective means of assessing foliar Mn accumulation. These findings should be followed up by field studies.

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.

In vivo localization of manganese in the hyperaccumulator Gossia bidwillii (Benth.) N. Snow & Guymer (Myrtaceae) by cryo-SEM/EDAX.

Gossia bidwillii (Myrtaceae) is a manganese (Mn)-hyperaccumulating tree native to subtropical eastern Australia. It typically contains foliar Mn levels in excess of 1% dry weight. However, in G. bidwillii and other Mn-hyperaccumulating species, the cellular and subcellular localization of Mn has not been measured. Quantitative in vivo cryo-scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDAX) was used to localize Mn and other elements in tissue collected from mature trees growing in a natural population. Cryo-SEM showed that the leaf mesophyll is differentiated as a double-layer palisade mesophyll above spongy mesophyll. Transmission electron microscopy (TEM) revealed that the palisade and epidermal cells are highly vacuolated. EDAX data were used to estimate in situ vacuolar Mn concentrations of all cell types in fresh cryo-fixed leaf tissues. The highest average vacuolar Mn concentration of over 500 mM was found in the upper-layer palisade mesophyll, while the lowest concentration of around 100 mM was found in the spongy mesophyll. Qualitative in vivo cryo-SEM/EDAX was employed to further investigate the spatial distribution of Mn in fresh leaf tissues and young bark tissue, which was also found to have a high Mn concentration. It is concluded that Mn distribution in G. bidwillii is quantitatively different to metal distribution in other hyperaccumulating species where the highest localized concentrations of these elements occur in non-photosynthmetic tissues such as epidermal cells and associated dermal structures including trichomes and leaf hairs.

Manganese uptake and accumulation by the hyperaccumulator plant Phytolacca acinosa Roxb. (Phytolaccaceae).

The perennial herb Phytolacca acinosa Roxb. (Phytolaccaceae), which occurs in Southern China, has been found to be a new manganese hyperaccumulator by means of field surveys on Mn-rich soils and by glasshouse experiments. This species not only has remarkable tolerance to Mn but also has extraordinary uptake and accumulation capacity for this element. The maximum Mn concentration in the leaf dry matter was 19,300 microg/g on Xiangtan Mn tailings wastelands, with a mean of 14,480 microg/g. Under nutrient solution culture conditions, P. acinosa could grow normally with Mn supplied at a concentration of 8000 micromol/l, although with less biomass than in control samples supplied with Mn at 5 micromol/l. Manganese concentration in the shoots increased with increasing external Mn levels, but the total mass of Mn accumulated in the shoots first increased and then decreased. At an Mn concentration of 5000 micromol/l in the culture solution, the Mn accumulation in the shoot dry matter was highest (258 mg/plant). However, the Mn concentration in the leaves reached its highest value (36,380 microg/g) at an Mn supply level of 12,000 micromol/l. These results confirm that P. acinosa is an Mn hyperaccumulator which grows rapidly, has substantial biomass, wide distribution and a broad ecological amplitude. This species provides a new plant resource for exploring the mechanism of Mn hyperaccumulation, and has potential for use in the phytoremediation of Mn-contaminated soils.