Effects of Dielectric Barrier Ambient Air Plasma on Two Brassicaceae Seeds: Arabidopsis thaliana and Camelina sativa.

We investigated low-temperature plasma effects on two Brassicaceae seeds (A. thaliana and C. sativa) using dielectric barrier discharge in air. Comparisons of plasma treatments on seeds showed distinct responses on germination rate and speed. Optimal treatment time giving optimal germination is 15 min for A. thaliana with 85% increase compared to control after 48 h of germination and 1 min for C. sativa with 75% increase compared to control after 32 h of germination. Such germination increases are associated with morphological changes shown by SEM of seed surface. For better understanding at the biochemical level, seed surfaces were analyzed using gas chromatography-mass spectrometry which underlined changes of lipidic composition. For both treated seeds, there is a decrease of saturated (palmitic and stearic) fatty acids while treated C. sativa showed a decrease of unsaturated (oleic and linoleic) acids and treated A. thaliana an increase of unsaturated ones. Such lipid changes, specifically a decrease of hydrophobic saturated fatty acids, are coherent with the other analyses (SEM, water uptake and contact angle). Moreover, an increase in A. thaliana of unsaturated acids (very reactive) probably neutralizes plasma RONS effects thus needing longer plasma exposure time (15 min) to reach optimal germination. For C. sativa, 1 min is enough because unsaturated linoleic acid becomes lower in treated C. sativa (1.2 × 107) compared to treated A. thaliana (3.7 × 107).

Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling.

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

The relationship between cuticular lipids and associated gene expression in above ground organs of Thellungiella salsugineum (Pall.) Al-Shehbaz & Warwick.

The cuticle plays a critical role as barrier between plant and environment. Here, cuticular wax morphology, cuticular wax and cutin monomer composition, and expression of associated genes in five above ground organs were examined in model extremophyte Thellungiella salsugineum. Alkanes, ketones, and 2-alcohols were the predominant wax constitutes in rosette leaves, inflorescence stem leaves, stems, and siliques, whereas alkanes and acids were the predominant cuticular lipids in whole flowers. Unsubstituted acids were the most abundant cutin monomers in vegetative organs, especially C18:2 dioic acids, which reached the highest levels in stems. Hydroxy fatty acids were the predominant cutin monomers in flowers, especially 16-OH C16:0 and diOH C16:0. High-throughput RNA-Seq analysis using the Hiseq4000 platform was performed on these five above organs of T. salsugineum, and the differentially expressed lipid-associated genes and their associated metabolic pathways were identified. Expression of genes associated in previous reports to cuticle production, including those having roles in cuticle lipid biosynthesis, transport, and regulation were examined. The association of cuticle lipid composition and gene expression within different organs of T. salsugineum, and potential relationships between T. salsugineum's extreme cuticle and its adaptation to extreme environments is discussed.

Plant evolution in alkaline magnesium-rich soils: A phylogenetic study of the Mediterranean genus Hormathophylla (Cruciferae: Alysseae) based on nuclear and plastid sequences.

Habitats with alkaline edaphic substrates are often associated with plant speciation and diversification. The tribe Alysseae, in the family Brassicaceae, epitomizes this evolutionary trend. In this lineage, some genera, like Hormathophylla, can serve as a good case for testing the evolutionary framework. This genus is centered in the western Mediterranean. It grows on different substrates, but mostly on alkaline soils. It has been suggested that diversification in many lineages of the tribe Alysseae and in the genus Hormathophylla is linked to a tolerance for high levels of Mg+2 in xeric environments. In this study, we investigated the controversial phylogenetic placement of Hormathophylla in the tribe, the generic limits and the evolutionary relationships between the species using ribosomal and plastid DNA sequences. We also examined the putative association between the evolution of different ploidy levels, trichome morphology and the type of substrates. Our analyses demonstrated the monophyly of the genus Hormathophylla including all previously described species. Nuclear sequences revealed two lineages that differ in basic chromosome numbers (x = 7 and x = 8 or derived 11, 15) and in their trichome morphology. Contrasting results with plastid genes indicates more complex relationships between these two lineages involving recent hybridization processes. We also found an association between chloroplast haplotypes and substrate, especially in populations growing on dolomites. Finally, our dated phylogeny demonstrates that the origin of the genus took place in the mid-Miocene, during the establishment of temporal land bridges between the Tethys and Paratethys seas, with a later diversification during the upper Pliocene.

Silique valves as sails in anemochory of Lunaria (Brassicaceae).

The generally held opinion that seeds of Lunaria remain at the replum after detachment of the two valves and then wind causes a shaking or rattling of the replum with its diaphragm, thus launching the seeds, is challenged. In a sparse forest in the Swabian Alb, the first author noticed flying valves of Lunaria rediviva to which the narrow-winged flat seeds are attached. Investigations with SEM and histology have shown that the valves secrete a glue only at those sites where the seeds rest on the valves before valve tissues die. Further analysis has shown (using the periodic acid-Schiff reaction) that the glue consists of polysaccharides. After detachment and dispersal of the valves, the adhesive strength continuously decreases. This is the first report for a sticky valve exudate in the Brassicaceae. Because of the adhesion of Lunaria seeds to their valves for some time, the 1st order diaspore is a mericarp, in a broad sense, and can be interpreted as an adaptation to long-distance dispersal by stronger winds. In this context, the 'flying carpets' of Lunaria are more effective and transport more than one seed. Molecular studies assigned Lunaria to the tribe Biscutelleae, which now contains the angustiseptate genera Biscutella and Megadenia as well as the latiseptate genera Lunaria and Ricotia. The valves in Ricotia can easily be detached (studied in herbarium material and a living plant), but, in contrast to Lunaria, the ripe seeds remain at the replum and its diaphragm, respectively.

Seasonal Regulation of Petal Number.

Four petals characterize the flowers of most species in the Brassicaceae family, and this phenotype is generally robust to genetic and environmental variation. A variable petal number distinguishes the flowers of Cardamine hirsuta from those of its close relative Arabidopsis (Arabidopsis thaliana), and allelic variation at many loci contribute to this trait. However, it is less clear whether C. hirsuta petal number varies in response to seasonal changes in environment. To address this question, we assessed whether petal number responds to a suite of environmental and endogenous cues that regulate flowering time in C. hirsuta We found that petal number showed seasonal variation in C. hirsuta, such that spring flowering plants developed more petals than those flowering in summer. Conditions associated with spring flowering, including cool ambient temperature, short photoperiod, and vernalization, all increased petal number in C. hirsuta Cool temperature caused the strongest increase in petal number and lengthened the time interval over which floral meristems matured. We performed live imaging of early flower development and showed that floral buds developed more slowly at 15°C versus 20°C. This extended phase of floral meristem formation, coupled with slower growth of sepals at 15°C, produced larger intersepal regions with more space available for petal initiation. In summary, the growth and maturation of floral buds is associated with variable petal number in C. hirsuta and responds to seasonal changes in ambient temperature.

A comparative study of the seed structure between resynthesized allotetraploid and their diploid parents.

Brassicaceae is at the forefront of evolution because of its frequent hybridization. Hybridization is responsible for the induction of widespread genetic and phenotype changes, making it important in agricultural production. In this study, we obtained resynthesized allotetraploid Brassica napus by performing interspecific crossing of B. rapa × B. oleracea combined with embryo rescue. We applied light microscopy and electronic microscopy to analyze the microstructure and ultrastructure of seeds of diploid parents and their allotetraploid progeny. Results showed that pigments in the seed coat were mainly distributed in the palisade layer. B. rapa presented the highest amount of pigment followed by B. napus and B. oleracea. B. napus had the thickest palisade layer followed by B. rapa and B. oleracea. The seed coat microsculpturing in B. rapa and B. napus was characterized as reticulate or reticulate-foveate, whereas that in B. oleracea was observed to be rugose and sulcate. The area index of the protein body was higher in central meristematic cells than in parenchyma cells. By contrast, the area index of the oil body was the lowest in central meristematic cells. Protein bodies were found to be heterogeneous with crystal globoids in two diploid parents and resynthesized allotetraploid progenies. Oil bodies consisted of large and small oil bodies, the sizes of which differed between two parents and allotetraploid progenies. Small oil bodies were spheroid, whereas large oil bodies were ovoid in shape. The quantity of oil bodies indicated that oil bodies were spheroid in two parents, ranging in size from 0.12 to 1.18 µm. In comparison, the size of large oil bodies in allotetraploid progenies exceeds 2.0 µm. These findings suggest that the anatomy of resynthesized allotetraploid seeds remarkably differs from that of two diploid parents, and these differences definitely affect the nutritional components of rapeseeds.

Developmental Control and Plasticity of Fruit and Seed Dimorphism in Aethionema arabicum.

Understanding how plants cope with changing habitats is a timely and important topic in plant research. Phenotypic plasticity describes the capability of a genotype to produce different phenotypes when exposed to different environmental conditions. In contrast, the constant production of a set of distinct phenotypes by one genotype mediates bet hedging, a strategy that reduces the temporal variance in fitness at the expense of a lowered arithmetic mean fitness. Both phenomena are thought to represent important adaptation strategies to unstable environments. However, little is known about the underlying mechanisms of these phenomena, partly due to the lack of suitable model systems. We used phylogenetic and comparative analyses of fruit and seed anatomy, biomechanics, physiology, and environmental responses to study fruit and seed heteromorphism, a typical morphological basis of a bet-hedging strategy of plants, in the annual Brassicaceae species Aethionema arabicum Our results indicate that heteromorphism evolved twice within the Aethionemeae, including once for the monophyletic annual Aethionema clade. The dimorphism of Ae. arabicum is associated with several anatomic, biomechanical, gene expression, and physiological differences between the fruit and seed morphs. However, fruit ratios and numbers change in response to different environmental conditions. Therefore, the life-history strategy of Ae. arabicum appears to be a blend of bet hedging and plasticity. Together with the available genomic resources, our results pave the way to use this species in future studies intended to unravel the molecular control of heteromorphism and plasticity.

Following the Time-Course of Post-pollination Events by Transmission Electron Microscopy (TEM): Buildup of Exosome-Like Structures with Compatible Pollinations.

In the Brassicaceae, the dry stigma is an initial barrier to pollen acceptance as the stigmatic papillae lack surface secretions, and consequently rapid cellular responses are required to accept compatible pollen. Regulated secretion with secretory vesicles or multivesicular bodies is initiated in the stigmatic papillae towards the compatible pollen grain. In self-incompatible species, this basal compatible pollen response is superseded by the self-incompatibility signaling pathway where the secretory organelles are found in autophagosomes and vacuole for destruction. In this chapter, we describe a detailed protocol using the Transmission Electron Microscope to document the rapid cellular changes that occur in the stigmatic papillae in response to compatible versus self-incompatible pollen, at the pollen-stigma interface.

Clade-specific positive selection on a developmental gene: BRANCHLESS TRICHOME and the evolution of stellate trichomes in Physaria (Brassicaceae).

Positive selection is known to drive the evolution of genes involved in evolutionary arms races, but what role does it play in the evolution of genes involved in developmental processes? We used the single-celled epidermal trichomes of Brassicaceae as a model to uncover the molecular evolutionary processes that contributed to the transition from dendritic trichomes, as seen in most species of Brassicaceae, to the distinctive stellate trichomes of the genus Physaria. We explored the role of positive selection on the evolution of BRANCHLESS TRICHOME (BLT), a candidate gene for changes in trichome branching pattern. Maximum likelihood models of codon evolution point to a shift in selective pressure affecting the evolution of BLT across the entire Physaria clade, and we found strong evidence that positive selection has acted on a subset of Physaria BLT codons. Almost all of the 10 codon sites with the highest probability of having evolved under positive selection are clustered in a predicted coiled-coil domain, pointing to changes in protein-protein interactions. Thus, our findings suggest that selection acted on BLT to modify its interactions with other proteins. The fact that positive selection occurred throughout the radiation of Physaria could reflect selection to stabilize development in response to an abrupt switch from the dendritic form to the stellate form, divergent selection for diversification of the stellate form, or both. These results point to the need for evolutionary developmental studies of BLT and its interacting proteins in Physaria.

Floral nectar production and carbohydrate composition and the structure of receptacular nectaries in the invasive plant Bunias orientalis L. (Brassicaceae).

The data relating to the nectaries and nectar secretion in invasive Brassicacean taxa are scarce. In the present paper, the nectar production and nectar carbohydrate composition as well as the morphology, anatomy and ultrastructure of the floral nectaries in Bunias orientalis were investigated. Nectary glands were examined using light, fluorescence, scanning electron and transmission electron microscopy. The quantities of nectar produced by flowers and total sugar mass in nectar were relatively low. Total nectar carbohydrate production per 10 flowers averaged 0.3 mg. Nectar contained exclusively glucose (G) and fructose (F) with overall G/F ratio greater than 1. The flowers of B. orientalis have four nectaries placed at the base of the ovary. The nectarium is intermediate between two nectary types: the lateral and median nectary type (lateral and median glands stay separated) and the annular nectary type (both nectaries are united into one). Both pairs of glands represent photosynthetic type and consist of epidermis and glandular tissue. However, they differ in their shape, size, secretory activity, dimensions of epidermal and parenchyma cells, thickness of secretory parenchyma, phloem supply, presence of modified stomata and cuticle ornamentation. The cells of nectaries contain dense cytoplasm, plastids with starch grains and numerous mitochondria. Companion cells of phloem lack cell wall ingrowths. The ultrastructure of secretory cells indicates an eccrine mechanism of secretion. Nectar is exuded throughout modified stomata.

ESEM-EDS: In vivo characterization of the Ni hyperaccumulator Noccaea caerulescens.

Environmental scanning electron microscopy (ESEM) permits to analyze samples in their native-hydrated state, allowing a broad spectrum of biological applications. In this study, ESEM equipped with energy dispersive X-ray spectrometer (EDS) was used as a fast method to analyze tissue morphology and to investigate metal distribution in the Ni hyperaccumulator Noccaea caerulescens, an established model to study the adaptation of plants to metalliferous soils. The low vacuum and wet mode operative conditions required the proper choice of experimental parameters both for morphological and compositional characterization of plant tissues. The calibration strategy for semi-quantitative analysis involved the use of Ni fortified agar as standard and signal normalization respect to endogenous carbon, chosen as internal standard. The obtained results are in accordance with present literature, showing a preferential Ni distribution in the epidermal cells respect to near the stomata for leaves and in the cotyledon epidermidis respect to cotyledon parenchyma area for seeds. Thanks to the absence of any time consuming sample treatment steps, ESEM-EDS technique can be proposed as valid strategy for in vivo high-throughput analysis of plant tissues and for a rapid screening and identification of other hyperaccumulator plants in a selected contaminated area.

Cytological studies of Brassicaceae burn. (Cruciferae juss.) from Western Himalayas.

Cytological studies have been carried out on 12 species of Brassicaceae Burn. on population basis from different geographical areas of Kashmir and Himachal Pradesh in the Western Himalayas. Variable chromosome reports for Barbaraea intermedia (n = 16), Cardamine loxostemonoides (n = 8), Nasturtium officinale (n = 8), Sisymbrium orientale (n = 14) on world-wide basis have been added to the previous reports of these species. The chromosome numbers in seven species as Barbaraea intermedia (n = 8), B. vulgaris (n = 8), Capsella bursa-pastoris (n = 8), Descuriania sophia (n = 10), Rorippa islandica (n = 8), Sisymbrium strictum (n = 7) and Thlaspi alpestre (n = 7) have been worked out for the first time from India. The meiotic course in the populations of seven species such as Barbaraea intermedia, Capsella bursa-pastoris, Coronopus didymus, Descuriania sophia, Nasturtium officinale, Sisymbrium orientale and S. strictum varies from normal to abnormal while all the populations of two species Barbaraea vulgaris and Sisymbrium irio show abnormal meiotic course. Meiotic abnormalities are in the form of cytomixis, chromosomal stickiness, unoriented bivalents, inter-bivalent connections, formation of laggards and bridges, all resulting into abnormal microsporogenesis. Heterogenous sized fertile pollen grains and reduced reproductive potentialities have invariably been observed in all the meiotically abnormal populations. However, the meiotic course in all the populations of Cardamine loxostemonoides, Rorippa islandica and Thalspi alpestre is found to be normal with high pollen fertility.

Imaging translocation and transformation of bioavailable selenium by Stanleya pinnata with X-ray microscopy.

Selenium hyperaccumulator Stanleya pinnata, Colorado ecotype, was supplied with water-soluble and biologically available selenate or selenite. Selenium distribution and tissue speciation were established using X-ray microscopy (micro-X-ray fluorescence and transmission X-ray microscopy) in two dimensions and three dimensions. The results indicate that S. pinnata tolerates, accumulates, and volatilizes significant concentrations of selenium when the inorganic form supplied is selenite and may possess novel metabolic capacity to differentiate, metabolize, and detoxify selenite concentrations surpassing field concentrations. The results also indicate that S. pinnata is a feasible candidate to detoxify selenium-polluted soil sites, especially locations with topsoil polluted with soluble and biologically available selenite.

Possible contributions of TERMINAL FLOWER 1 to the evolution of rosette flowering in Leavenworthia (Brassicaceae).

Leavenworthia crassa is a rosette flowering species that differs from inflorescence flowering species, such as Arabidopsis thaliana, in having elongated pedicels and shortened interfloral internodes on the main axis. Based on previous experiments, we hypothesized that changes to the L. crassa TFL1 ortholog, LcrTFL1, were important in the evolution of rosette flowering. We isolated LcrTFL1 and introduced a genomic construct into tfl1 mutant A. thaliana plants. We also generated and analyzed EGFP-LcrTFL1 reporter-fusion lines, and LcrTFL1/LcrLFY doubly transgenic lines. The transgene rescued the mutant defects, but manifested gain-of-function phenotypes. However, LcrTFL1 lines differed from 35S:TFL1 lines in several regards. Defects in floral meristem identity establishment were observed, as was the production of flowers with extra petals. We also noted features that resemble rosette flowering: LcrTFL1 lines produced significantly shorter interfloral internodes and significantly longer pedicels than either wild-type or 35S:TFL1 plants. Our data show that there are substantive differences in the regulation and/or function of TFL1 orthologs between A. thaliana and L. crassa. These may reflect changes that occurred during the evolution of rosette flowering in Leavenworthia, but, if so, our results show that additional, as-yet-unidentified genes were involved in this instance of architectural evolution.

[Geometry and mechanics of the morphogenesis of active membranes on the example of plant trichome cells of the genus Draba L].

The stages of the early morphogenesis of simple (unbranched) and complex (branched) unicellular trichomes are studied in two species of the genus Draba--D. sibirica (Pall.) Thell. and D. daurica DC. The geometry of morphogenesis is estimated by analyzing intraindividual variation of quantitative morphological characteristics of the developing leaf blade and peduncle trichomes. The surface of all types oftrichome cells first acquires a spherical shape, followed by a U-shaped configuration with cylindrical proximal and spherical distal regions. In the development of complex trichomes, the area of the distal zone grows at a higher rate, which leads to separation of its volume into individual spherical regions, the morphogenesis of which repeats the early morphogenetic stages of the overall trichome cell, forming simple (unbranched) or complex (branched) trichome rays. As a rule, the lateral polarity of a trichome cell coincides with the proximodistal polarity of the leaf. Quantitative morphological data make it possible to infer an algorithm of the changes in shape common for all trichome cells, namely, the growth cycle comprising alternation of the phases of increase and decrease in the curvature of the outer cell surface. This surface is an active membrane expanded by the internal pressure and concurrently capable of actively increasing its area by incorporation of new structural elements. A distinctive feature of the proposed model is the geometrical inhomogeneity of the surface movement, changing the radius of curvature and creating internal (active) mechanical stresses in this membrane. A decrease in the ratio of the membrane surface area to the volume deprives the spatially homogeneous shape of its stability; correspondingly, the transition from elastic resistance to internal pressure to active resistance with the help of curvature differentiation becomes more energetically favorable. The source for growth and morphogenesis of the active membrane is alternation of the phases of local curvature leveling, which "charges" the membrane with active mechanical stresses and "discharge" of these stresses, leading to differentiation of the membrane's local curvatures.

High-resolution elemental localization in vacuolate plant cells by nanoscale secondary ion mass spectrometry.

By combining the capabilities of advanced sample preparation methodologies with the latest generation of secondary ion mass spectrometry instrumentation, we show that chemical information on the distribution of even dilute species in biological samples can be obtained with spatial resolutions of better than 100 nm. Here, we show the distribution of nickel and other elements in leaf tissue of the nickel hyperaccumulator plant Alyssum lesbiacum prepared by high-pressure freezing and freeze substitution.

Salt stress induces programmed cell death in Thellungiella halophila suspension-cultured cells.

Thellungiella halophila (T. halophila) suspension-cultured cells were used to gain knowledge of the pathway of programmed cell death (PCD) in halophytes under salt stress. Several apoptotic-like features occurred in T. halophila cells after exposure to 300 mM NaCl, including the retraction of the plasma membrane from the cell wall, nuclear condensation, DNA laddering and the release of cytochrome c accompanying the increase of caspase 3-like protease activity. This process resulted in ultrastructural changes of mitochondria and Golgi bodies, and autophagy was also induced by high salinity stress. DNA laddering and caspase 3-like activity were inhibited prior to the inhibition of cell death by a specific caspase 3 inhibitor, Ac-DEVD-CHO. The results indicate that 300 mM NaCl stress-induced PCD in T. halophila is similar to animal apoptosis, and this process occurs partly through a caspase 3-like dependent pathway.

The role of two LEAFY paralogs from Idahoa scapigera (Brassicaceae) in the evolution of a derived plant architecture.

Idahoa scapigera produces solitary flowers in the axils of rosette leaves without elongation of the shoot axis, a rosette-flowering architecture. Previous work with one of the two I. scapigera LFY paralogs, IscLFY1, showed that this gene caused aerial flowering rosettes in Arabidopsis thaliana. In this paper, we report that after three generations IscLFY1 transgenic lines are phenotypically indistinguishable from wild-type Arabidopsis, indicating that IscLFY1 protein is able to replace normal LFY function. Additionally, we found that ectopic LFY expression late in development can phenocopy aspects of the aerial rosette phenotype, suggesting that shoot compression caused by IscLFY1 could be caused by localized overexpression of a functional IscLFY protein. We also characterized the expression and function of the second I. scapigera LFY paralog, IscLFY2, in A. thaliana. In contrast to IscLFY1, this paralog was expressed in floral meristems and the shoot apical meristem (SAM). In I. scapigera, LFY-specific antibodies detected high protein levels in developing flowers but not in the apex, suggesting trans-regulatory differences between I. scapigera and A. thaliana. Most IscLFY2 transgenic A. thaliana plants were indistinguishable from wild type, but in a minority of lines the SAM was converted to a terminal flower as would be expected from the reporter-expression pattern. Taken together these results show that both I. scapigera paralogs have conserved LFY function, both proteins can rescue lfy and both can modify inflorescence architecture in an A. thaliana background: either by affecting internode elongation (IscLFY1) or by causing homeotic conversion of shoots into flowers (IscLFY2).

Cellular and subcellular compartmentation of Ni in the Eurasian serpentine plants Alyssum bracteatum, Alyssum murale (Brassicaceae) and Cleome heratensis (Capparaceae).

This study investigated the cellular and subcellular compartmentation of Ni in the Eurasian serpentine species Alyssum murale, Alyssum bracteatum and Cleome heratensis and a non-serpentine population of A. murale (as a control) grown in hydroponic culture. Plant growth responses and Ni uptake clearly revealed the higher Ni tolerance of serpentine plants than the non-serpentine plants. Serpentine A. murale and A. bracteatum grew better at elevated (0.01 mM) Ni in the nutrient solution, supporting the view that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Low shoot Ni content of C. heratensis in response to the high Ni treatments indicated that this species employs an avoidance strategy for Ni tolerance. Energy-dispersive X-ray microanalysis showed that Ni was highly concentrated in the cell walls and cell lumen, most likely the vacuoles, of leaf epidermis of A. murale and A. bracteatum rather than in the mesophyll cells. EDX spectra from leaves of the non-serpentine A. murale suggested that Ni accumulated in both epidermal and mesophyll cells but not in the epidermal cell walls. Growth reduction and Ni toxicity in plants of the non-serpentine A. murale could be due to accumulation of Ni in the lumen of leaf mesophyll cells. Our data suggest that cellular and subcellular compartmentation are both possible mechanisms for Ni tolerance employed by the serpentine A. murale and A. bracteatum.