Damien L.
Callahan
*a,
Dominic J.
Hare
b,
David P.
Bishop
b,
Philip A.
Doble
b and
Ute
Roessner
c
aDeakin University, School of Life and Environmental Sciences, Centre for Chemistry and Biotechnology, Burwood, Geelong, Victoria 3125, Australia. E-mail: Damien.Callahan@deakin.edu.au
bElemental Bio-imaging Facility, University of Technology Sydney, Australia
cMetabolomics Australia, School of BioSciences, The University of Melbourne, Parkville, Victoria 3010, Australia
First published on 22nd December 2015
Elemental imaging using laser ablation inductively coupled plasma mass spectrometry was performed on whole leaves of the hyperaccumulating plant Noccaea caerulescens after treatments with either Ni, Zn or Cd. These detailed elemental images reveal differences in the spatial distribution of these three elements across the leaf and provide new insights in the metal ion homeostasis within hyperaccumulating plants. In the Zn treated plants, Zn accumulated in the leaf tip while Mn was co-localised with Zn suggesting similar storage mechanisms for these two metals. These data show a Zn concentration difference of up to 13-fold higher in the distal part of the leaf. Also, there was no correlation between the S and Zn concentrations providing further evidence against S-binding ligands. In contrast, Ni was more evenly distributed while a more heterogeneous distribution of Cd was present with some high levels on leaf edges, suggesting that different storage and transport mechanisms are used for the hyperaccumulation of these two metals. These results show the importance of correct sampling when carrying out subcellular localisation studies as the hyperaccumulated elements are not necessarily homogenously distributed over the entire leaf area. The results also have great implications for biotechnological applications of N. caerulescens showing that it may be possible to use the mechanisms employed by N. caerulescens to increase the Zn concentration in nutrient poor crops without increasing the risk of accumulating other toxic elements such as Ni and Cd.
Most hyperaccumulators have developed a tolerance for a particular metal ion and this tolerance is typically related to the soil substrate. The mechanisms behind this specificity and the detoxification mechanisms are not completely understood. Part of the challenge in studying hyperaccumulators is the great diversity in the types of plants which have these traits since they range from large rainforest trees such as Pycnandra acuminata (previously Sebertia acuminata) endemic to New Caledonia to small species belonging to the Brassicaceae family found throughout Europe.1 For hyperaccumulation to occur the metal ions in above ground tissue must be sequestered and stored in subcellular structures in order to inhibit disruption of normal metabolic processes within the leaf.
Noccaea caerulescens (J & C Presl.) F. K. Mey. Brassicaceae (formerly Thlaspi caerulescens) has been identified as a model species for studying hyperaccumulation.8,9 This species has the ability to hyperaccumulate Ni, Zn and Cd. Of note is that ecotypes from different metalliferous soils throughout Europe exhibit large variations in their accumulation abilities and tolerances for Ni, Zn or Cd.10–13 An example of this is the Prayon ecotype which can accumulate more than 30000 mg kg−1 Zn without showing any typical phenotypical responses of heavy metal toxicity such as chlorosis or reduced growth, however, it does not accumulate Cd to the same amounts as other ecotypes.12,14
Hyperaccumulating plants have considerable potential for remediation of contaminated soils through metal decontamination and re-establishment of vegetation cover. The potential applications of these plants have been recognised for some time and is one of the key drivers for research on these plants, therefore numerous reviews and books have been written for example the book by Chaney et al. nearly 20 years ago.15 They also have great potential for biotechnological applications through the transfer of metal transport mechanisms into nutrient poor crops, known as biofortification. For review on the challenges of biofortification see Antosiewicz et al.16
In this study, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to investigate the spatial distribution of the hyperaccumulated metal ions Ni2+, Zn2+ and Cd2+ as well as other key micronutrients in whole N. caerulescens leaves from plants that had been treated with these metal ions individually. This imaging technique rasters a laser beam across samples and the ablated material is transported to the ICP-MS using an Ar carrier gas. The ICP-MS then detects ionised elements based on the mass-to-charge ratio. A particular strength of this technique is that multiple micronutrients may be measured in a single acquisition allowing the study of metal ion homeostasis and direct comparisons of micronutrient distribution in the same leaf. This technique has been used to study a broad range of matrices from geological samples to an array of different biological samples.17,18 Although it does not have the fine spatial resolution of scanning electron microscopy or X-ray microanalysis, LA-ICP-MS is highly sensitive, has a large linear dynamic range as well as the ability for isotopic analysis. For a review of in situ elemental analysis in plants see Lombi et al.19 There have been a number of studies using LA-ICP-MS on metal accumulation in plants, for example, it was used to coarsely map (750 μm2) Ni in the roots from the Ni-hyperaccumulator Berkheya coddii Roessler, in leaves of Helianthus annuus (sunflower) with a spatial resolution of 200 μm, leaves from Cu treated Elsholtzia splendens leaves, Zn and Cu in roots of cucumber plants, boron in Poplar leaves and biofortification and localisation studies in wheat grains.20–25 However, this is the first study that has spatially mapped metal ion distributions across whole leaves of a metal hyperaccumulating plant. This work reveals new information on the spatial distribution of hyperaccumulated metal ions and also illustrates the strength of this technique for studying micronutrients and metal ion homeostasis in plants.
Fig. 1 (Left) Map showing the origin of seeds for each ecotype; (right) different ecotypes after three months growing in Jiffy Netted Pots (control group) showing clear morphological differences. |
Seeds were germinated and grown in Jiffy pots (sphagnum peat moss and wood pulp) in a glass house maintained at a maximum temperature of 22 °C with 14 hour day (supplemented with artificial grow lights) and 10 hour night. Plants were then watered with a 1/5 Hoagland's solution containing either NiSO4 (250 μM), ZnSO4 (500 μM) or CdSO4 (10 μM) as well as a non-supplemented control group. These treatment concentrations allow normal growth of plants in relation to control and were selected based on a previous laboratory screening trial. Plants were treated one week after germination and were harvested after 3 months of growth. At least five individual replicate plants were grown from each ecotype in each of the four treatment regimes (control, Ni, Zn, Cd). The amounts which plants accumulate also depend on the growth conditions and bioavailability of metals supplied. In order to confirm the accumulation capabilities of the four selected ecotypes quantitative elemental analysis using an ICP-optical emission spectrometer (OES) was carried out on dried leaf tissues from these plants. The oldest leaf was selected for elemental imaging, while remaining leaves were immediately frozen in liquid nitrogen and stored at −80 °C. The leaves for imaging were washed with deionised water (18.2 MΩ) and methanol, placed between Whatman filter paper and stored under ambient conditions prior to imaging analysis. Minimal sample handling was undertaken in order to try and maintain metal ion distribution.
Leaf sections were also made to quantitatively confirm the spatial distributions found by elemental imaging. The leaves that were snap frozen and stored were rinsed with de-ionised water then immediately sectioned into thirds (tip, middle, base). Each section was then placed into a separate pre-weighed 2 mL Eppendorf tube. This approach removes the chance for re-distribution of metal ions within the leaf tissues. Fresh weights and dry weights were recorded. The samples were then acid digested as above and elements quantified by ICP-MS (PerkinElmer NexION 350-X).
Each scan line produces a (.csv) file, which was collated using a Visual Basic macro (Microsoft) and imported into ENVI (Exelis, Boulder, Co., USA) for image analysis.27
Images based on the elemental intensity were produced for each measured element and are represented here either normalised to the 13C isotope or plotted based on the raw signal intensity. The fine resolution of these images show obvious features in the leaves, in particular leaf venation (Fig. 3–5). A striking accumulation pattern was observed from the Zn-treated plants (Fig. 3). Significantly higher concentrations of Zn were clearly identified in the leaf tip. A Zn concentration gradient is present from the leaf tip to approximately half way down the lamina. The relative differences in elemental concentrations were determined using the mean signal intensity from key spatial regions of the leaf. For the Zn image, this represents a 13-fold higher concentration of Zn at the leaf tip compared to the base of the leaf (Fig. 3). The bottom half of the lamina (closest to the petiole) contained a relatively homogenous Zn concentration. The Zn-concentration supplied to the plants was constant throughout the 3 month growth period and therefore the plant is actively concentrating Zn at the leaf tip. It is also clear from these images that Zn was lower in concentration in the vascular tissues, where the leaf veins are darker and therefore lower in Zn-concentration. The Zn treated Prayon ecotype was also imaged had the same Zn-accumulation pattern as the Puente Basadre ecotype suggesting similar storage mechanisms for Zn (ESI Fig. S2†). Examination of other images showed a very clear co-localisation of Mn and Zn (Fig. 3). From previous work in our laboratory, a clear reduction in Mn was observed when the plant Zn concentration exceeded 10000 mg kg−1 (ESI Fig. S3†).28 This suggests that Zn and Mn have similar uptake mechanisms and also explains why the Mn concentration decreases when N. caerulescens accumulates extremely high concentrations of Zn. No other elements showed the same striking co-localisation patterns in the Zn-treated plants. Sulphur showed some increase in intensity (2-fold difference) at the leaf tip but not to the same extent of Zn providing further evidence against S-binding ligands as part of storage in N. caerulescens.29 The Zn image of the non-treated control leaf on the same scale of the treated plants is hardly discernible (Fig. 3). However, in the leaf from a control plant, which has a 3.5-fold lower total leaf Zn concentration, a similar accumulation pattern was observed with higher concentrations in the leaf tip (2-fold). No other accumulation patterns were observed for the other elements in the control plants (ESI Fig. S4†). From the Fe and Cu images it appears that some leaf contamination occurred which was not removed by washing. This most likely occurred during the supply of nutrient solutions to the plants causing the precipitation of insoluble Fe and Cu salts on the leaf surface (Fig. 5, ESI Fig S4†). However it can still be observed in the Fe and Cu images that concentrations of these elements are not effected by high localised concentrations of Zn. The same homogenous distribution is also observed in leaves from plants treated with Ni and Cd.
Fig. 4 Ni (top) and Cd (bottom) images from the different treatments from the Puente Basadre population. The Ni elemental image shows a relatively homogenous distribution, while Cd shows a more heterogeneous distribution with some increased Cd concentration on the leaf edges. Note in the signal intensity from the plants not treated with the corresponding metal are outlined due to the low relative intensity. Non-normalised images are in ESI.† |
Fig. 5 Elemental images based on the raw signal intensity of potassium, phosphorous, magnesium, sulfur, carbon, and copper from Zn treated plant showing a homogenous distribution of these macro elements. The intensity values of these images illustrated on the bar to the right of each image. Not some contamination of leaf surface can be seen in the copper image. All images available in ESI 4.† |
As expected the elemental images from the Ni and Cd treated plants show a huge contrast in signal intensities relative to the non-treated plants (Fig. 4). The elemental images in the Ni and Cd treated plants did not show any distinct accumulation patterns. Ni was relatively evenly distributed across the whole leaf with lower concentrations in leaf vascular tissue (Fig. 4). No clear pattern could be observed for Cd distribution apart from having higher concentrations in leaf vascular tissue and some of the leaf edges (Fig. 5 and 6). The more heterogeneous Cd distribution is in contrast to Zn and Ni. These images reveal that even though multiple elements are hyperaccumulated by N. caerulescens, different storage strategies are used for Ni, Zn and Cd. It is evident that in N. caerulescens Zn accumulation begins at the distal part of the lamina. In contrast, there is no regional concentration for Ni. A closer examination of the Cd and Cu images show higher concentrations in the vascular tissues of the lamina in comparison to surrounding tissue which also contrasts to Zn and Ni. This is most apparent in the zoomed in elemental images of Zn versus Cd (Fig. 6). As discussed above the elemental imaging technique used here cannot produce images which would enable conclusions to be made at the subcellular level. Molecular studies have shown that Zn is transported symplastically by various highly expressed membrane transporters such as the HMA4 (Heavy Metal ATPase 4) and ZIP (Zn regulated transporter, iron-regulated transporter-related protein) gene families.32,33 With regards to Cd there is no solid evidence for apoplastic localisation of Cd in leaves. A very early study which claimed apoplastic storage was most likely due to sample preparation artefact due to the chemical fixation technique used.30 It was subsequently shown that Cd is actively taken up into the epidermal storage cells.31,34
Fig. 6 Zoomed in image of Zn (top) and Cd (bottom). Higher Zn concentrations are located outside the vascular tissue in contrast to Cd. Images are produced on the raw signal intensity. |
The distribution of macro elements such as Na, K, Mg, S and Fe show a homogenous distribution throughout the leaves in all treatments. This shows that the concentrations of these key elements are not affected by the presence of high concentrations of the hyperaccumulated metal ion (Fig. 5).
As the imaging data showed differences elemental spatial distributions, quantitative measurements were made to confirm that the patterns observed were not an artefact of sample preparation. These quantitative measurements were also carried out in triplicate to provide replication of these observations. The leaves were sectioned into thirds: tip, middle and base, oven dried, acid digested then total elemental concentrations determined by solution ICP-MS. These measurements confirm the elemental imaging data (Table 1) with 4–10 fold higher concentrations of Zn in the leaf tip and more homogenous distributions of Ni and Cd.
Base | Mid | Tip | |
---|---|---|---|
Zn (mg kg −1 ) dw | |||
Zn-1 | 420 | 1400 | 1700 |
Zn-2 | 239 | 600 | 1200 |
Zn-3 | 150 | 640 | 1600 |
Zn-4 | 1500 | 3700 | 6500 |
Ni (mg kg −1 ) dw | |||
Ni-1 | 370 | 550 | 630 |
Ni-2 | 590 | 700 | 960 |
Ni-3 | 410 | 1900 | 1300 |
Cd (mg kg −1 ) dw | |||
Cd-1 | 210 | 370 | 340 |
Cd-2 | 150 | 300 | 380 |
Cd-3 | 190 | 220 | 230 |
Only a few published studies to date have examined the macro leaf spatial distribution of elements in hyperaccumulators. One study analysed leaf cross sections using micro-PIXE and found a homogenous distribution of Ni in different cell types in the Australian Ni hyperaccumulator Stackhousia tryonii, suggesting that a similar accumulation pattern was observed to this study.35 The radioactive 109Cd isotope was used previously to image Cd compartmentation at the leaf scale in the hyperaccumulators N. caerulescens, Arabidopsis halleri and willow leaves.36–38 These studies agree with the results described here. In the three species referenced the authors showed a heterogeneous Cd distribution in the older leaves, in particular, high concentrations in vascular tissues and leaf edges. The paper by Cosio et al. (2005) is of particular relevance to this work. The Cd autoradiography was carried out on two contrasting ecotypes of N. caerulescens. This work clearly showed Cd localised at leaf edges as well as points of higher concentration spread out over the whole leaf surface. X-ray microanalysis of the Cd cellular distribution showed that Cd was stored in less metabolically active areas of leaf cells. Unfortunately this study did not carry out a similar analysis for Zn. An early study comparing Cd and Zn uptake also showed different distribution patterns for these metals based on leaf age.39 The study by Perronnet et al. (2003) showed that Cd concentrations remained relatively constant with increasing biomass while Zn concentrations had a small decrease with time. It has also been shown that Cd is not re-distributed from old to young organs during leaf senescence suggesting that Cd ions are fixed in place once accumulated.40 This is in contrast to Zn which is actively take up and concentrated in leaf tissues by N. caerulescens.41 Other studies have shown that the Cd co-ordination environment changes with tissue age. For example, tissue age differences in the concentration and the co-ordination environment of Cd were observed N. caerulescens leaf tissue using X-ray absorption spectroscopy.42
The determination of the spatial distribution of elements is important for understanding how hyperaccumulators transport and store the large amounts of metals in a way that does not interfere with essential metabolic processes and maintains elemental homeostasis. The findings here also have implications for tissue sampling. For example, a recent study focussed on trace elements in hyperaccumulators from New Caledonia, it was shown concentrations of Mn increase in the older leaves of the hyperaccumulator Grevillea meisneri.43 These findings illustrate the importance of correct sampling and the consideration of leaf age when determining the ability and the mechanisms of plants to accumulate trace elements.
Most research on metal ion distribution in hyperaccumulators has focused on cellular and sub-cellular distributions as opposed to whole tissues as was carried out in this research. For a review on elemental distribution in plants see Zhao et al. 2014.44 There have been some conflicting reports of the cellular metal ion distribution in N. caerulescens and other closely related species using X-ray microanalysis techniques. For example, organelle isolation found that 65–70% of the total leaf Cd and Zn was located in vacuoles of mesophyll tissues in N. caerulescens.45 However, other studies have shown Zn storage in the epidermis cells instead of the metabolically active mesophyll layer.46–48 The conflict here may be due to different sample preparation techniques. The paper by Ma et al. (2005) removed the epidermal layer.45 This may have resulted in the rupture of epidermal cells with the cell contents transferring to the mesophyll layer.
Here we have shown the importance of selecting the correct areas within the leaf to study subcellular localisation and to first confirm if there is a difference on the spatial distribution of elements. A single leaf cross section may not represent the same distribution across the whole leaf. The spatial resolution of the LA-ICP-MS does not allow imaging of subcellular structures, however it is highly sensitive and whole leaf elemental images still provide useful information on the metal-ion homeostasis in plants.
Finally, as Zn is stored differently to Ni, and Cd it may be possible to utilise the selectivity of the Zn transporters used by N. caerulescens in future biofortification studies without the risk of increasing Ni and Cd concentrations.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra23953b |
This journal is © The Royal Society of Chemistry 2016 |