Abstract: Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers (>10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid–fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing ∼8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type–specific biological processes.

Abstract: The C4 pathway is a complex combination of both biochemical and morphological specialisation, which provides an elevation of the CO2 concentration at the site of Rubisco. We review the key parameters necessary to make the C4 pathway function efficiently, focussing on the diffusion of CO2 out of the bundle sheath compartment. Measurements of cell wall thickness show that the thickness of bundle sheath cell walls in C4 species is similar to cell wall thickness of C3 mesophyll cells. Furthermore, NAD-ME type C4 species, which do not have suberin in their bundle sheath cell walls, do not appear to compensate for this with thicker bundle sheath cell walls. Uncertainties in the CO2 diffusion properties of membranes, such as the plasmalemma, choroplast and mitochondrial membranes make it difficult to estimate bundle sheath diffusion resistance from anatomical measurements, but the cytosol itself may account for more than half of the final calculated resistance value for CO2 leakage. We conclude that the location of the site of decarboxylation, its distance from the mesophyll interface and the physical arrangement of chloroplasts and mitochondria in the bundle sheath cell are as important to the efficiency of the process as the properties of the bundle sheath cell wall. Using a mathemathical model of C4 photosynthesis, we also examine the relationship between bundle sheath resistance to CO2 diffusion and the biochemical capacity of the C4 photosynthetic pathway and conclude that bundle sheath resistance to CO2 diffusion must vary with biochemical capacity if the efficiency of the C4 pump is to be maintained. Finally, we construct a mathematical model of single cell C4 photosynthesis in a C3 mesophyll cell and examine the theoretical efficiency of such a C4 photosynthetic CO2 pump.

Abstract: Polysaccharides containing β-1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.

Abstract: The molecular mechanisms that control the ordered patterning of vascular tissue development in plants are not well understood. Several models propose a two-component system for vascular differentiation. These components include an inducer of vascular tissue development and an inhibitor that prevents the formation of vascular bundles near pre-existing bundles. We have identified two recessive allelic mutants in Arabidopsis , designated continuous vascular ring ( cov1 ), that display a dramatic increase in vascular tissue development in the stem in place of the interfascicular region that normally separates the vascular bundles. The mutant plants exhibited relatively normal vascular patterning in leaves and cotyledons. Analysis of the interaction of cov1 with a known auxin signalling mutant and direct analysis of auxin concentrations suggests that cov1 affects vascular pattering by some mechanism that is independent of auxin. The COV1 protein is predicted to be an integral membrane protein of unknown function, highly conserved between plants and bacteria. In plants, COV1 is likely to be involved in a mechanism that negatively regulates the differentiation of vascular tissue in the stem.

Abstract: Ions can enhance water flow through the xylem via changes in the hydraulic resistance at border pit membranes. Because flow between adjacent xylem vessels occurs primarily via bordered pit fields, it is hypothesized that xylem sap ion concentrations would affect lateral movement of water more than longitudinal flow. Using tomato as a model system, evidence is presented for ion-mediated changes in xylem hydraulic resistance and the lateral transport of water. Water flow between adjacent xylem bundles increased by approximately 50% in the presence of ions while longitudinal flow only increased by approximately 20%. However, the enhancement of lateral exchange due to ions was magnified by the presence of a pressure difference between vascular bundles. These results indicate that the degree of nutrient-sharing among sectors of a plant may depend on both nutrient concentration and the availability of water in the root zone.

Abstract: Carbon assimilation in maize follows the C4 mechanism. This requires the tissue-specific and light-induced expression of a set of different genes involved in CO2 fixation as well as adaptations in the leaf anatomy including a reduced distance between vascular bundles compared to C3 plants. However, several maize tissues exist with larger bundle distances and there is significant evidence that CO2 fixation follows the C3 mechanism in these tissues. We isolated maize C3 and C4 tissues and quantified the accumulation of mRNAs encoding PEPC, ME, the small subunit of Rubisco, and PPDK. For this, primer systems for the specific and sensitive detection by real-time PCR were established. The observed patterns show the expected distribution for foliar leaf tissues. Also in total husk leaves, all transcripts under investigation were detected, albeit at a lower level. When mesophyll cells which are located distant from bundles were isolated from husk leaves, only accumulation of RbcS was observed. Comparing the expression of two genes encoding for isoenzymes of the small subunit of RbcS in the different tissues differential patterns of relative transcript abundance were observed. Transcripts for the DOF1 transcription factor involved in the activation of photosynthetic genes in maize were found in leaf tissues performing both C4 and C3 photosynthesis with highest accumulation levels in C4 mesophyll cells, whereas the homologous DOF2 gene was not expressed in any of the investigated samples. The results provide novel insights into the regulation of C3 and C4 carbon fixation pathways in maize.

Abstract: The leaf of the NADP-malic enzyme type C 4 grass, Arundinella hirta, has not only mesophyll cells (MCs) and bundle sheath cells (BSCs, usual Kranz cells) but also another type of Kranz cells (distinctive cells; DCs) that are not associated with vascular bundles. We investigated photosynthetic enzyme accumulation along the base-to-tip maturation gradient of developing leaves by immunogold electron microscopy. In mature leaves, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were detected in the MC cytosol and in the BSC and DC chloroplasts, respectively. Pyruvate, P i dikinase (PPDK) was present in the chloroplasts of all photosynthetic cells but with higher levels in the MCs. Rubisco was first detected in the basal region of emerging leaf blades where the BSCs and DCs became discernable. Subsequently, the accumulation of PEPC and PPDK was initiated in the region where the granal proliferation in the chloroplasts was conspicuous; and, suberized lamellae were formed in the cell walls of the Kranz cells. There was no difference in the patterns of cellular development and enzyme accumulation between the BSCs and DCs or between the MCs adjacent to each type of Kranz cells. These results demonstrate that, although the DCs are not associated with veins, they behaved like BSCs with respect to enzyme induction and cellular differentiation.

Abstract: Asparagus spears are rapidly growing structures supplied with abundant vascular bundles. After extended periods of water loss, the stem surface becomes flaccid over a core of turgid tissue. We have used magnetic resonance imaging (MRI) coupled with a paramagnetic contrast reagent (Mn2+) to visualise water and ion movement within spear tissues. Using this technique we estimated the rate of water flow in the xylem and lateral movement out of the xylem into the surrounding tissues. Longitudinal flow rates of at least 3.7 mm min–1 and lateral diffusion rates of at least 17 μm min–1 were recorded. The outer parenchyma was difficult to label with vascular-supplied Mn2+, suggesting the presence of an apoplastic barrier. Stem shrinkage was greater in the outer parenchyma than in the inner cortex and pith parenchyma. Feeding the cut spear with 70 mM sucrose reduced the rate of lateral movement of Mn2+. The MRI technique highlighted differences in transport rates between adjacent vascular bundles and identified structural features with a resolution of 78 μm. This non-invasive technique is useful both for observing details of the transport paths in living tissue and for setting minimal estimates of transport rates.

Abstract: Development of characteristics of the C4 syndrome was studied in cotyledons and leaves of NADP‐ME type C4 Salsola richteri, a desert shrub. This species has seeds in which the cotyledons contain chloroplasts, storage proteins, and lipid bodies but no starch. Following imbibition (day 0), tissue types are already apparent in the cotyledons, and the chloroplasts have extensive grana stacking, but Kranz type anatomy and C4 photosynthesis have not developed. At day 0, there is high Rubisco and low phosphoenolpyruvate carboxylase (PEPC) content, distributed throughout all tissue. After 15 d of development in the dark, the cotyledons were in a C3‐like default condition with three distinct layers of chlorenchyma (hypodermis, mesophyll, and bundle sheath [BS]); all contained Rubisco in their chloroplasts. Light was required for development of the C4 syndrome, including differentiation of chloroplasts in mesophyll and BS cells, development of thick BS cell walls, selective compartmentation of Rubisco in BS cells a...

Abstract: The extended bundle sheath (EBS) is a specialized layer of cells that enhances the lateral transport of photoassimilates within the leaf. This little-known tissue is often considered to be legume-specific. We identified an EBS in cotyledons and leaves of the non-legume Ricinus communis L. By means of cytological and immunological studies and using the localization of the iron-chelator nicotianamine as an established indicator for mass transport, we confirmed its role as a transport tissue and a temporal sink. Observations on cotyledons of Ricinus seedlings further proved that the EBS carries out these tasks from a very early stage of development onwards. This is the first time that information has been obtained on the physiological role of an EBS in a non-legume. Our results support the idea of its widespread occurrence among higher plants.

Abstract: C4 plants have many attractive traits for crops, but their structural and functional relationships are complicated. C4 plants are different in bundle sheath cell (BSC) chloroplast location (centrifugal or centripetal) among species. The effects of light intensity on the centrifugal location of BSC chloroplasts were investigated in four grass species of NADP malic enzyme (NADP-ME) type (Zea mays, Echinochloa utilis, Sorghum bicolorand Eriachne aristidea)by light and electron microscopy. Furthermore, the degree of granal development was examined to investigate the relation between BSC chloroplast location and dependence of BSC chloroplasts on the reducing power of mesophyll cells. We investigated BSC chloroplast location grown under high intensity light (HL) (600 μ mol m-2s-1), low intensity light (LL) (2.5 μ mol m-2s-1) and dark conditions and counted the number of granal thylakoids per granum. Although BSC chloroplasts of maize maintained the centrifugal position under all light conditions, the ce...

Abstract: Cyperus giganteus shows Kranz anatomy of the clorocyperoid type or with two sheaths, one internal, adjacent to the vascular system and known as Kranz sheath, with thin-walled cells and a large number of organelles, mainly chloroplasts; and an external sheath, the mestome sheath or endodermis, the cells of which present thickened walls, are without chloroplasts and possess a suberin lamella, together with the casparian strip which are detected in early stages of differentiation. The development of the vascular bundles shows the Kranz sheath originating from the procambial as well as the mestome sheath. The chloroplasts of the Kranz cells are relatively larger, with convoluted thylakoids and a prominent peripheral reticulum, while the chloroplasts of the mesophyll cells are relatively smaller, with thylakoids forming grana and a sparse peripheral reticulum. These ultrastructural characteristics show similarities to those of other species of the genus Cyperus.

Abstract: Leaves from three_year_old solar greenhouse nectarine trees ( Prunus persica L. var. nectarina Ait. “Zao Hong Yan”) were used as materials in this study. It was the first time that the ultrastructural characteristics of phloem tissues of source leaves were observed and compared in normal and weak light intensities using the transmission electron microscopy. Results showed that the average diameters of companion cells (CC) and sieve elements (SE) of all kinds of veins were bigger in normal than that in weak light intensity, indicating that light could influence the cell development and growth. Dense cytoplasm with abundant mitochondria, endoplasmic reticulums, multivesicular bodies, vesicles and plastids were observed in normal light intensity. On the contrary, CC with small vacuolar structures and few mitochondrias, endoplasmic reticulums were shown in weak light. Misalignment of grana thylakoid margins of nectarine leaves also was seen in weak light. The sieve pores of SEs were obstructed in weak light. Chloroplasts with numerous starch grains and few mitochondrias were noticed in the mesophyll cell (MES) surrounding the bundle sheath in weak light. The storage of starch grains appeared to result from an unbalance between photosynthate production and export of photosynthates. This observation provided a strong support to the point that most leaves export the most of assimilates in the light time. Plasmodesmal densities between SE/CC, CC/PP (phloem parenchyma cell), PP/PP and PP/BSC (bundle_sheath cell) decreased in weak light. Plasmodesmata were observed between CC/SE (NS) (nacreous_walled sieve element), PP/BSC in branch veins in normal light intensity, but not in weak light. Thus apoplasmic pathway may be the main mode of transport of assimilates in weak light, however symplasmic pathway may be the main mode of transport of assimilates in normal light intensity. These results demonstrated that the solar greenhouse nectarine trees could be adapted to the weak light via the ultrastructure variation of phloem tissues of the source leaves.

Abstract: The effect of a direct electric current on electrolyte transport through plant tissues was studied by applying it to 10-mm fragments of the mesocotyls of etiolated maize seedlings, similar fragments of one-year linden shoots with the normal conducting system and without vascular bundles, and isolated elements of the xylem and cell wall segments. At the current density and voltage of 9-38 microA/mm2 and 10-20 V, electrolyte solutions in plant tissues always moved toward the cathode. The results suggest that electroosmosis is one of the factors responsible for changes in solution transport through the conducting plant tissues that occur under the effect of electric current.

Abstract: Possible presence of vascular bundle sheath cells and its relation to photosynthetic pathway, leaf anatomy and carbon isotope composition (d13C) were examined in six species of genus Coffea: C. arabica (cvs. Catuai Vermelho, Mundo Novo, Bourbon Vermelho and Icatu Amarelo), C. canephora (cvs. Apoata and Guarini), C. liberica, C. dewevrei, C. salvatrix and C. stenophylla. In all genotypes, the vascular bundle was surrounded by a layer of cells with numerous chloroplasts in a centrifugal position. Visually no differences could be seen between the spongy parenchyma cells and the bundle sheath cells, neither in size nor in chloroplast number. The leaf d13C values ranged between a maximum of - 26.2 ‰ in C. salvatrix and a minimum of -29.7 ‰ in C. liberica. A strong correlation (r = 0.972, p = 0.001) between d13C and anatomical characteristics was observed in coffee species. C. salvatrix exhibited the highest d13C values and the most compact mesophyll, with more palisade and spongy parenchyma cells in contact with the vascular bundle sheath.

Abstract: Leaf anatomy and eco-physiology of Elymus repens, a temperate loess grassland species, were determined after seven years of exposure to 700 μmol (CO2) mol−1 (EC). EC treatment resulted in significant reduction of stomatal density on both surfaces of couch-grass leaves. Thickness of leaves and that of the sclerenchyma tissues between the vessels and the adaxial surfaces, the area of vascular bundle, and the volumes of phloem and tracheary increased at EC while abaxial epidermis and the sclerenchyma layer between the vessel and the abaxial surface were thicker at ambient CO2 concentration (AC). Stomatal conductance and transpiration rates were lower in EC, while net CO2 assimilation rate considerably increased at EC exposure. Contents of soluble sugars and starch were higher in EC-treated couch-grass leaves than in plants grown at AC.

Abstract: The leaf structure, content and the storage location of aloin in the leaves of six species of Aloe L. were studied by means of semi_thin section, high performance liquid chromatography (HPLC) and fluorescent microscope. Results showed that all leaves consisted of epidermis, chlorenchyma, aquiferous tissue and vascular bundles. The leaves had the xeromorphic characteristics, including thickened epidermal cell wall, thickened cuticle, sunken stomata and well_developed aquiferous tissue. With the exception of this, there were remarkable differences in leaf structure among the six species. The chlorenchyma cells were similar to palisade tissues in Aloe arborescens Mill. and A. mutabilis Pillans, but isodiametric in A. vera L., A. vera L. var. chinensis Berg., A. saponaria Hawer and A. greenii Bak. A. arborescens , A. mutabilis , A. vera and A. vera var. chinensis included large parenchymatous cells at the vascular bundles, whereas no such cells were observed at the vascular bundles of A. saponaria and A. greenii . In A. arborescens , A. mutabilis and A. vera , the aquiferous tissue sheaths were present and composed of a layer of small parenchymatous cells without chloroplasts around the aquiferous tissue. While there were no aquiferous tissue sheaths in A. vera var. chinensis , A. saponaria and A. greenii . The HPLC revealed that the content of aloin was high in A. arborescens , low in A. vera , and very low in A. saponaria among the six species. The fluorescent microscopy showed that the yellow_green globule only appeared in the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath, but not in the chlorenchyma and aquiferous tissue. Consequently, the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath were the storage location of aloin. They were positively correlated with the content of aloin.

Abstract: Pre-treatment with DL-3-aminobutyric acid (BABA) in the cucumber plants caused the decrease of disease severity after inoculation with anthracnose pathogen Colletotrichum orbiculare. In this study, ultrastructures of the vascular bundle and the infection structures in the leaves of BABA-treated as well as untreated cucumber plants were observed after inoculation with the anthracnose pathogen by electron microscopy. The ultrastructures of vascular bundle in the leaves of BABA-treated plants were similar to those of the untreated plants except plasmodesmata. In the BABA-treated plants, the plasmodesmata were more numerous than in the untreated plants, suggesting that the BABA treatment may cause the active transfer of metabolites through the vascular bundle. In the leaves of untreated plants, the fungal hyphae were spread widely in the plant tissues at 5 days after pathogen inoculation. Most cellular organelles in the hyphae were intact, indicating a compatible interaction between the plant and the parasite. In contrast, in the leaves of BABA pre-treated plants the growth of most hyphae was restricted to the epidermal cell layer at 5 days after inoculation. Most hyphae cytoplasm and nucleoplasm was electron dense or the intracellular organelles were degenerated. The cell walls of some plant cells became thick at the site adjacent to the intercellular hyphae, indicating a mechanical defense reaction of the plant cells against the fungal attack. Furthermore, hypersensitive reaction (HR) of the epidermal cells was often observed, in which the intracellular hyphae were degenerated. Based on these results it is suggested that BABA causes the enhancement of defense mechanisms in the cucumber plants such as cell wall apposition or HR against the invasion of C. orbiculare.

Abstract: Studies on stem (and leaf) structure and histology of a semi-natural grassland community, permanently growing in mini-FACE rings under elevated concentrations of atmospheric CO2 (560 μmol mol−1) are presented. Histochemical analysis of stem sections from legume plants grown under high CO2 concentration revealed both a reduction of lignin deposition in spring vascular bundles of Trifolium repens L., and a decrease in size of the xylem vessels in Vicia hybrida L. and Vicia sativa L. Thus, the effects of elevated CO2 on the stem histology of the species investigated are rather species-specific and/or organ-specific, and of major account especially in the early phases of vegetative growth, in particular as regards lignin deposition mechanisms. In leaves, neither differences as to lignification nor any other anatomical structure modification were found under CO2 enrichment.

Abstract: The present invention relates to one kind of newly found DNA sequence, capable of being used as promoter to regulate destination gene specifically expressed in plant vascular bundle tissue. Angrpl gene and its upstream sequence are cloned from Aijiaonante rice genome, the promoter activity expressed by 5' sequence deletion segment driven GUS gene in Angrpl gene upstream sequence is analyzed via the instantaneous tobacco expression system on the basis of determining transcription start site, and one basic promoter region of 1021 bp segment is determined. Subsequently, it is proved via chemicalstaining of GUS tissue in transgenic tobacco and transgenic rice that the gene promoter can direct the specific expression of reporter gene in vascular bundle tissue.

Abstract: The tip of rhizome of Dioscorea zingiberensis C.H.Wright is surrounded by lepises.The cells derived from primordial meristem forms protoderm,ground meristem and dispersed procambium strands.The primary structure of the rhizome is includes epidermis,ground tissue and dispersed vascular bundles.There is primary thickening meristem at the lower part of rhizome tip under protoderm.The increase of cell number derived from primary thickening meristem and the enlargement of cells volume make the rhizome thicken rapidly.The mature rhizome is composed of periderm,ground tissue and vascular bundles.The periderm consists of phellem,phellogen and phelloderm;the ground tissue is of parenchyma;vascular bundle belongs to closed vascular bundle.Dioscin exists mainly in parenchyma cells of ground tissue.The primordial meristem and the procambium strand do not contain dioscin and the xylem and the phloem fiber of the vascular bundle do not contain dioscin either,but the life cells of phloem and the cells of vascular bundle sheath accumulate dioscin.Dioscin does not exist in the form of granule in ground meristem near the tip of rhizome,but granulous dioscin is formed in the cells of mature ground tissue.Dioscin that accumulated and distributed in ground tissue with small vascular bundle is the most abundant in the rhizome.The amount of dioscin of biennial is higher than that of annual rhizome.

Abstract: Radopholus similis, the burrowing nematode colonizes and reproduces in roots and rhizomes of Anthurium andraeanum Linden ex Andre in Madeira, Portugal. Histological examination of nematodeinfected rhizomes shows necrosis and cavities extending from the cortical parenchyma into the vascular bundles. Nematode feeding and migration caused disruption of the vascular bundles. All nematode life stages, coiled juveniles, adults, including gravid females, and eggs, occurred in the phloem. Nematode specimens were also observed in metaxylem elements.