Small Epidermal Cells Research Articles

Age-dependent changes in leaf size in apple are governed by a cytokinin-integrated module.

Plants undergo various age-dependent changes in leaf morphology during the transition from the juvenile to the adult stage. However, the precise molecular mechanisms governing these changes in apple (Malus domestica) remain unknown. Here, we showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5), an age-dependent gene, encodes a functional CKX enzyme and serves as the common downstream target of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MdSPL14 and WRKY transcription factor MdWRKY24 to control the degradation of cytokinin (CK). As the target of mdm-microRNA156a, MdSPL14 interacts with MdWRKY24 to coordinately repress the transcription of MdCKX5 by forming the age-mediated mdm-miR156a-MdSPL14-MdWRKY24 module, which regulates age-dependent changes in CK during the juvenile-to-adult phase transition. We further demonstrated that MdARR6, a type-A ARABIDOPSIS RESPONSE REGULATOR (ARR), is a negative feedback regulator in the CK signaling pathway. Silencing of MdARR6 in apple resulted in large leaves with smaller epidermal cells and a greater number of epidermal cells. Biochemical analysis showed that the mdm-miR156a-MdSPL14-MdWRKY24 module acts as a transcriptional repressor to directly regulate MdARR6 expression, thus controlling the age-dependent changes in leaf size by reducing CK responses. These findings established a link between the age pathway and CK signaling and revealed the molecular mechanism underlying age-dependent changes during the juvenile-to-adult phase transition; our results also provide targets for the genetic improvement of the vegetative phase transition in apple.

Cell size differences affect photosynthetic capacity in a Mesoamerican and an Andean genotype of Phaseolus vulgaris L.

The efficiency of CO2 flux in the leaf is hindered by a several structural and biochemical barriers which affect the overall net photosynthesis. However, the dearth of information about the genetic control of these features is limiting our ability for genetic manipulation. We performed a comparative analysis between three-week-old plants of a Mesoamerican and an Andean cultivar of Phaseolus vulgaris at variable light and CO2 levels. The Mesoamerican bean had higher photosynthetic rate, maximum rate of rubisco carboxylase activity and maximum rate of photosynthetic electron transport at light saturation conditions than its Andean counterpart. Leaf anatomy comparison between genotypes showed that the Mesoamerican bean had smaller cell sizes than the Andean bean. Smaller epidermal cells in the Mesoamerican bean resulted in higher stomata density and consequently higher stomatal conductance for water vapor and CO2 than in the Andean bean. Likewise, smaller palisade and spongy mesophyll cells in the Mesoamerican than in the Andean bean increased the cell surface area per unit of volume and consequently increased mesophyll conductance. Finally, smaller cells in the Mesoamerican also increased chlorophyll and protein content per unit of leaf area. In summary, we show that different cell sizes controls the overall net photosynthesis and could be used as a target for genetic manipulation to improve photosynthesis.

Stomatal patchiness and cellular computing

Control of carbon dioxide and water vapor exchange between a leaf's interior and the surrounding air is accomplished by variations in the turgor pressures in the small epidermal and guard cells that cover the leaf's surface. These pressures respond to changes in light intensity and wavelength, temperature, CO2 concentration, and air humidity. The dynamical equations that describe such processes are formally identical to those that define computation in a two-layer, adaptive, cellular nonlinear network. This exact identification suggests that leaf gas-exchange processes can be understood as analog computation and that exploiting the output of two-layer, adaptive, cellular nonlinear networks might provide new tools in applied plant research.

Hormone released by the microalgae Neochlorisaquatica and alkalinization influence growth of terrestrial and floating aquatic plants

The microalgae Neochloris aquatica were previously evaluated as a potential biological control agent and source of bioactive compounds against immature stages of Culex quinquefasciatus. Larvae reared on microalgae suspension showed mortality or drastic effects with morphological alterations and damage in the midgut. N. aquatica have nutritional and toxic effects, resulting in delayed life cycle and incomplete adult development. Given the possibility of its use as a biological control agent, in this work we evaluate the effect of microalgae on other organisms of the environment, such as plants. Arabidopsis thaliana, a terrestrial plant, and Lemna sp., a floating aquatic plant, were selected as examples. Interaction assays and compound evaluations showed that the microalgae release auxins causing root inhibition, smaller epidermal cells and hairy root development. In Lemna sp., a slight decrease in growth rate was observed, with no deleterious effects on the fronds. On the other hand, we detected a detrimental effect on plants when interactions were performed in a closed environment, in a medium containing soluble carbonate, in which microalgae culture rapidly modifies the pH. The experiments showed that alkalinization of the medium inhibits plant growth, causing bleaching of leaves or fronds. This negative effect in plants was not observed when plants and microalgae were cultured in carbonate-free media. In conclusion, the results showed that N. aquatica can modify plant growth without being harmful, but the rapid alkalinization produced by carbon metabolism of microalgae under CO2-limiting conditions, could regulate the number of plants.

Anatomical studies of Brazilian Amazonian Isoëtes species: inferences on habitat adaptation

Abstract Isoëtes is a cosmopolitan genus of aquatic lycophytes, containing more than 200 species. In Brazil, the genus comprises 29 species, with three occurring in Pará state, Amazon. Isoëtes cangae and I. serracarajensis are endemic to the ferruginous outcrops of Serra dos Carajás, and I. amazonica occurs on the inundated shores of the Tapajós River. Despite the great diversity of quillworts in South America, their anatomy remains unknown. This study discusses Brazilian Amazon species’ leaf and root anatomical traits in relation to habitat and genetic diversity. The amphibious I. amazonica and I. serracarajensis were observed to have similar stomata and cuticular ornamentations. Isoëtes cangae, a fully aquatic species, had smaller epidermal cells and a smooth cuticle and showed slight differences regarding the lacuna diaphragm. The genetically closer species from Carajás both lacked peripheral fiber strands on the leaves. Our study complements current knowledge regarding the morphoanatomy of Amazonian species and provides a better understanding of their biology, contributing to the development of conservation strategies for these species.

Diverse mangroves deviate from other angiosperms in their genome size, leaf cell size and cell packing density relationships.

While genome size limits the minimum sizes and maximum numbers of cells that can be packed into a given leaf volume, mature cell sizes can be substantially larger than their meristematic precursors and vary in response to abiotic conditions. Mangroves are iconic examples of how abiotic conditions can influence the evolution of plant phenotypes. Here, we examined the coordination between genome size, leaf cell sizes, cell packing densities and leaf size in 13 mangrove species across four sites in China. Four of these species occurred at more than one site, allowing us to test the effect of climate on leaf anatomy. We found that genome sizes of mangroves were very small compared to other angiosperms, but, like other angiosperms, mangrove cells were always larger than the minimum size defined by genome size. Increasing mean annual temperature of a growth site led to higher packing densities of veins (Dv) and stomata (Ds) and smaller epidermal cells but had no effect on stomatal size. In contrast to other angiosperms, mangroves exhibited (1) a negative relationship between guard cell size and genome size; (2) epidermal cells that were smaller than stomata; and (3) coordination between Dv and Ds that was not mediated by epidermal cell size. Furthermore, mangrove epidermal cell sizes and packing densities covaried with leaf size. While mangroves exhibited coordination between veins and stomata and attained a maximum theoretical stomatal conductance similar to that of other angiosperms, the tissue-level tradeoffs underlying these similar relationships across species and environments were markedly different, perhaps indicative of the unique structural and physiological adaptations of mangroves to their stressful environments.

Morphological and Biochemical Factors Associated with Constitutive Defense to Thrips in Alfalfa

Plants have evolved a series of inducible or constitutive defense mechanisms in response to herbivore attack. Constitutive plant defenses are morphological and biochemical traits of the plants themselves, regardless of the presence of herbivores. We bred an alfalfa variety (Caoyuan No.4) with high thrips resistance, but the mechanisms underlying Caoyuan No.4 resistance to thrips are not well understood. To explore the constitutive defense of Caoyuan No.4, the morphological and biochemical traits associated with constitutive defense to thrips in alfalfa were analyzed using a thrips-susceptible alfalfa accession (Caoyuan No.2) as a control. The results showed that Caoyuan No.4 had thicker palisade tissue and parenchyma tissue, wider collenchyma, phloem, cambium and lignin layer, and smaller epidermal cells and stomatal aperture compared to Caoyuan No.2. Moreover, Caoyuan No.4 showed more non-glandular trichomes in both leaves and stems, but less glandular trichomes and more wax in stems. In addition, the results of the widely targeted metabolomics analysis showed that metabolites related to flavonoid, isoflavonoid, flavone and flavonol biosynthesis, as well as cysteine and methionine metabolism, differed between CaoyuanNo.2 and Caoyuan No.4. These findings shed new light on the constitutive insect defense of plants associated with physical or biochemical traits and may provide convenient markers for breeding thrips-resistant alfalfa cultivars.

Ethylene-independent functions of the ethylene precursor ACC in Marchantia polymorpha.

The plant hormone ethylene has many roles in growth and development1. In seed plants, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is converted into ethylene by ACC oxidase (ACO), and treatment with ACC induces ethylene responses2. However, non-seed plants lack ACO homologues3-8, which led us to examine the relationship between ACC and ethylene in the liverwort Marchantia polymorpha. Here, we demonstrate that ACC and ethylene can induce divergent growth responses in Marchantia. Ethylene increases plant and gemma size, induces more gemma cups and promotes gemmae dormancy. As predicted, Mpctr1-knockout mutants display constitutive ethylene responses, whereas Mpein3-knockout mutants exhibit ethylene insensitivity. Compared with the wild type, Mpctr1 gemmae have more and larger epidermal cells, whereas Mpein3 gemmae have fewer and smaller epidermal cells, suggesting that ethylene promotes cell division and growth in developing gemmae. By contrast, ACC treatment inhibits gemma growth and development by suppressing cell division, even in the Mpein3-knockout alleles. Knockout mutants of one or both ACC SYNTHASE (ACS) gene homologues produce negligible levels of ACC, have more and larger gemma cups, and have more-expanded thallus branches. Mpacs2 and Mpacs1 Mpacs2 gemmae also display a high frequency of abnormal apical notches (meristems) that are not observed in ethylene mutants. These findings reveal that ethylene and ACC have distinct functions, and suggest that ACC is a signalling molecule in Marchantia. ACC may be an evolutionarily conserved signal that predates its efficient conversion to ethylene in higher plants.

Xeromorphic Features of the Leaves of Liriodendron Tulipifera L. (Magnoliaceae) in the Arid Climate of Central Asia

High irradiance of a habitat in arid areas is usually associated with high temperature and dry air, which can cause water deficiency in plants. The Tulip tree (Liriodendron tulipifera) is a light-loving mesophytic species. This study showed that L. tulipifera forms shade or sun leaves depending on the light conditions. Some xeromorphic features were found in leaves of the trees in illuminated habitats. These sun leaves are smaller, denser, and thicker, with smaller epidermal cells and a thick cuticle; the spongy parenchyma consists of a larger number of rows of small cells; the cells of the columnar parenchyma are larger than the shade leaves. The sun leaf contains more total chlorophyll (by 20–30%), and the guard cells of the stomata of the sun leaf contain chloroplasts. The increased concentration of chlorophyll probably maintains the energy balance in the conditions of inhibited photosynthesis under high illumination and temperature. Decreased sizes of the sun leaf and epidermis cells, thickening of the cuticle and cell wall of epidermal cells, sunken stomata, compact mesophyll with small intercellular spaces, and multirow spongy parenchyma protect from overheating and passive loss of water and can be considered as xeromorphic features. Large numerous stomata and vascular bundles contribute to effective cooling of the surface by transpiration at high temperatures under sufficient water supply. Thus, the leaves of L. tulipifera have an ecological plasticity in relation to illumination and temperature, which contributed to the acclimation of the species to the habitat conditions in arid climates.

Asclepiadospermum gen. nov., the earliest fossil record of Asclepiadoideae (Apocynaceae) from the early Eocene of central Qinghai-Tibetan Plateau, and its biogeographic implications.

Apocynaceae is common in the fossil record, especially as seed remains from the Neogene of Europe and North America, but rare in Asia. Intrafamilial assignment is difficult due to the lack of diagnostic characters, and new fossil and modern data are needed to understand the paleobiogeography of this group. We studied three Apocynaceae seed impressions from the Lower Eocene Niubao Formation, Jianglang village, Bangor County, central Qinghai-Tibetan Plateau. Morphological data from living and fossil species were phylogenetically mapped to enable systematic assignment. We describe a new genus, Asclepiadospermum gen. nov., and two new species, A. marginatum sp. nov. and A. ellipticum sp. nov. These species are characterized by an elliptical seed, a margin surrounding the central part of the seed, and polygonal, irregular, and small epidermal cells, and differ mainly in terms of the size of the margin and the shape of the apex. All these characters indicate that this new genus belongs to the subfamily Asclepiadoideae (Apocynaceae). These fossils represent the earliest fossil seed records of Asclepiadoideae. Asclepiadospermum indicates a humid tropical to subtropical flora during the early Eocene in central Tibet. Moreover, our discoveries indicate a close floristic connection between Eurasia and Africa during the early Eocene, which expands our knowledge of the floristic linkage between Tibet and other regions at that time.

Natural Root Cellular Variation in Responses to Osmotic Stress in Arabidopsis thaliana Accessions.

Arabidopsis naturally occurring populations have allowed for the identification of considerable genetic variation remodeled by adaptation to different environments and stress conditions. Water is a key resource that limits plant growth, and its availability is initially sensed by root tissues. The root’s ability to adjust its physiology and morphology under water deficit makes this organ a useful model to understand how plants respond to water stress. Here, we used hyperosmotic shock stress treatments in different Arabidopsis accessions to analyze the root cell morphological responses. We found that osmotic stress conditions reduced root growth and root apical meristem (RAM) size, promoting premature cell differentiation without affecting the stem cell niche morphology. This phenotype was accompanied by a cluster of small epidermal and cortex cells with radial expansion and root hairs at the transition to the elongation zone. We also found this radial expansion with root hairs when plants are grown under hypoosmotic conditions. Finally, root growth was less affected by osmotic stress in the Sg-2 accession followed by Ws, Cvi-0, and Col-0; however, after a strong osmotic stress, Sg-2 and Cvi-0 were the most resilience accessions. The sensitivity differences among these accessions were not explained by stress-related gene expression. This work provides new cellular insights on the Arabidopsis root phenotypic variability and plasticity to osmotic stress.

Morphophysiological and Biochemical Responses of Lippia grata Schauer (Verbenaceae) to Water Deficit

In the present study, we investigated the morphophysiological and biochemical responses of the species Lippia grata Schauer to water deficit. Plants from cuttings were submitted to two water regimes, irrigation (control) and suspended irrigation (stress) for 15 days and were then rehydrated for 10 days. During water regimes, we performed physiological (gas exchange, growth parameters, and biochemical) and anatomical analyses. The water deficit negatively affected growth of the aerial part of the plant with reduction of approximately 72%. On the contrary, root system growth was stimulated by water deficit (increase of 205%). The drought caused a reduction in the physiological parameters (gs, E, and A), while leaf temperature and internal and external CO2 concentration ratio (Ci/Ca) increased. Plants subjected to drought had higher intrinsic efficiency of water use on the 10th day of deficit (A/gs). The levels of total chlorophyll, carotenoids, and soluble carbohydrates increased on the 10th and 15th days of water suspension. The levels of H2O2, proteins, and MDA were high in plants on the 15th day of water deficit. The levels of H2O2 in the control plants increased on the 20th and 25th day of rehydration, along with the activation of some enzymes of the antioxidative system SOD, CAT and APX. Plants under water deficit increased SOD activity on the 10th day and CAT on the 10th and 15th day under stress. Plants submitted to water deficit showed reduction of mesophilic thickness, smaller epidermal cells, reduction of intercellular spaces, more vascular bundles, greater lignification of xylem cells, morphological modification of cortical cells, and have more glandular trichomes in the abaxial face compared to plants always irrigated. During rehydration, the plants submitted to the water deficit recovered in all variables analyzed and have a higher density of glandular and the tector trichomes in both epidermises, adaxial and abaxial. We conclude that the rapid recovery of the parameters evaluated for Lippia grata are related to the absence of irreversible damage in the cellular structures, due to the efficiency of the antioxidative system, and metabolic processes involved with the dissipation of the excess energy because of the imposed water stress. In addition, other important morphophysiological strategies such as increased root growth, increased vascular bundles and density of trichomes improved soil water absorption and maintained the cellular water content in Lippia grata plants submitted to drought.

FossilNewhousia imbricata(Dictyotales, Phaeophyceae) from Postglacial Coral Reef Deposits in Tahiti

Abstract. The calcareous brown alga Newhousia imbricata (Dictyotales, Phaeophyceae) was found from postglacial reef deposits collected during Integrated Ocean Drilling Program Expedition 310 from Tahiti. The alga is composed of successive tiers of bilayered laminae, each consisting of a basal cuboid hypodermal cell bearing smaller epidermal cells (one or two in cross-sectional profiles and three or four in long-sectional profiles). The laminae are separated by extracellular cement. Modern N. imbricata is known only from the Hawaiian Islands; this is the first reported occurrence of fossil N. imbricata.

Leaf anatomy and its contribution to the systematics of Aechmea subgenus Macrochordion (de Vriese) Baker (Bromeliaceae)

The leaf anatomy of the species Aechmea subgenus Macrochordion was analyzed to obtain valuable data on their taxonomic delimitation and to identify anatomical adaptations to their respective habitats and habits. All leaves of these species are hypostomatic, and present: peltate trichomes on both surfaces; stomata sunk in epidermal depressions; small epidermal cells with thick walls and inclusions of silica bodies; a mechanical hypodermis; an aquiferous parenchyma; chlorenchyma with fibrous clusters and air channels; and vascular bundles surrounded by a parenchymatic sheath and a cap of fibers. The results are evaluated within an adaptive and taxonomic context. Variations in hypodermic thickening, amount of water parenchyma, position of the air channels and shape of the cells filling the air channels are useful for delimiting groups of species, strengthening the relationships suggested by their external morphology.

Micromorphology of the floral elements, the structure of the nectary, and the apicultural value of Elaeagnus commutata Bernh. ex Rydb.

The investigations were carried out using light and scanning electron microscopy. The flowers of <i>Elaeagnus commutata</i> grow in clusters of 1-4 in the leaf axils. They are actinomorphic, four-lobed, with a single perianth that is yellow from the adaxial side, while the abaxial side is silvery-white. Peltate hairs of different structure are found on both surfaces of the sepals. The conical epidermal cells of the lobes are covered with a thick striated cuticle. Cylindrical hairs were observed on the edges of the lobes. Peltate hairs also grew on the style. The dish-shaped nectary gland is located at the base of the style. Nectar is secreted through numerous, evenly distributed stomata located above or at the level of other epidermal cells. Different stages of stomatal development are evidence of the asynchronous functioning of the stomata. The nectary consists of small epidermal cells and 5-6 layers of secretory parenchyma. The deeper layers of the gland are composed of larger cells of subglandular parenchyma in which vascular bundles supplying the nectary run. Honey bees were the main pollinators of silverberry. Ten silverberry flowers produced an average of 12 g of nectar with a sugar concentration in the 29.5-34.5% range. The weight of pollen produced by 10 flowers was 3.33 mg.

Effect of sowing dates and vernalization on Betavulgaris L. cv. Univers C-leaf structure

This research was conducted to study the effect of three different sowing dates (15th October, 15th November and 15th December) and two vernalization treatments (5 °C and −20 °C) on leaf structure of Beta vulgaris L. cv. Univers. The obtained data are summarized as follows: The maximum values of the most studied parameters; lower epidermis + spongy tissue thickness, midrib, mesophyll tissue, vascular bundle, collenchymatous tissue and number of xylem vessels per arm were found as a result of 15th October sowing date treatment compared with the two other sowing dates. Furthermore, effect of the cooling treatments varied according to the recorded character, sowing date and cooling degree. Most of the vernalization treatments at early sowing dates increased the mesophyll tissue, midrib, number of vascular bundles per transverse section, vascular bundle thickness and number of xylem arms per transverse section. The two studied cooling treatments at 15th October sowing date increased both stomatal index and average number of stomata: average number of epidermis cells compared with the control. Furthermore, 15th October under −20 °C treatment led to small epidermal cells and stomata formation, straight epidermal cell walls and closed stomata in comparison to the control.

Alcantarea (Bromeliaceae) leaf anatomical characterization and its systematic implications

Alcantarea (Bromeliaceae) has 26 species that are endemic to eastern Brazil, occurring mainly on gneiss–granitic rock outcrops (‘inselbergs’). Alcantarea has great ornamental potential and several species are cultivated in gardens. Limited data is available in the literature regarding the leaf anatomical features of the genus, though it has been shown that it may provide valuable information for characterizing of Bromeliaceae taxa. In the present work, we employed leaf anatomy to better characterize the genus and understand its radiation into harsh environments, such as inselbergs. We also searched for characteristics potentially useful in phylogenetic analyses and in delimiting Alcantarea and Vriesea. The anatomical features of the leaves, observed for various Alcantarea species, are in accordance with the general pattern shown by other Bromeliaceae members. However, some features are notable for their importance for sustaining life on rock outcrops, such as: small epidermal thick-walled cells, uneven sinuous epidermal walls, hypodermis often differentiated into lignified layers with thick-walled cells, aquiferous hypodermis bearing collapsible cells, and the presence of well developed epicuticular stratum. Alcantarea leaves tend to show different shapes in the spongy parenchyma, and have chlorenchymatous palisade parenchyma arranged in more well-defined arches, when compared to Vriesea species from the same habitat.

The Signaling Peptide EPF2 Controls Asymmetric Cell Divisions during Stomatal Development

Stomata are pores in the plant epidermis that control carbon dioxide uptake and water loss. They are major regulators of global carbon and water cycles [1]. Several signaling components that regulate stomatal development have been characterized. These include a putative secretory peptide EPF1, LRR receptor components TMM and ER, and a peptidase SDD1 [2-4]. We have identified EPF2, a peptide related to EPF1 that is expressed in proliferating cells of the stomatal lineage, known as meristemoids, and in guard mother cells, the progenitors of stomata. EPF2 expression during leaf development affects stomatal density on the mature leaf. In the absence of EPF2, excessive numbers of cells enter the stomatal lineage and produce numerous small epidermal cells that express stomatal lineage reporter genes, whereas plants overexpressing EPF2 produce virtually no stomata. Results from genetic experiments indicate that EPF2 regulates a different aspect of stomatal development to EPF1 and are consistent with EPF2 acting in a pathway to regulate stomatal density that involves ER and TMM, but not SDD1. We propose that EPF2 is expressed earlier in leaf development than EPF1 and is involved in determining the number of cells that enter, and remain in, the stomatal lineage.

Features related to anther opening inSolanumspecies (Solanaceae)

The mode of anther opening and the morphological and histological variability of the stomium are described in 30 Solanum species. Poricidal, poricidal-longitudinally dehiscing and longitudinally dehiscing anthers are observed. In the three types, the stomium may be diverse with regard to shape and histological characteristics before opening, but is always composed of small epidermal cells as the sole anther wall layer; the stomial cells may be differentiated only in part of the anther length. Particular crescent-shaped structures in the epidermis, called ‘ridges’, are observed to line the stomium in most species. These ridges may be related to the stomium opening, working together with the cells with thickened walls of the anther. Cells with thickened walls are developed in the endothecium, middle layers and/or connective tissue at the apical end of the anther, surrounding the pore; only in the longitudinally dehiscing anthers of S. nitidum does an endothecium with thickened cell walls develop along its entire length. At least two histological features (the differentiation of small stomial epidermal cells as a unique layer, and the distribution of cells with thickened walls) seem to constrain the form of the open stomium. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 344–354.

Distribution of cadmium in leaves of Thlaspi caerulescens

Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 microM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cells.