Solute sorting in grass leaves: the transpiration stream

Solutes distribute differentially between leaf tissues and cells. The present study tested the hypothesis that certain solutes are supplied preferentially to the epidermis in the transpiration stream, by-passing mesophyll cells along bundle sheath extensions. Using energy dispersive X-ray analysis of extracted cell sap, the distribution of solutes was studied in the emerged zone (transpiring) and the elongation zone (non-transpiring) of the developing leaf three of barley (Hordeum vulgare L.). The basic distribution of Cl, K, P and Ca between epidermis and bulk tissue, and between cells within the epidermis, was similar in the two leaf regions. However, in the emerged zone differences in solute concentrations between tissues and cells were greater. A local reduction in transpiration rate along the emerged portion of the blade specifically prevented Ca from accumulating to high levels in epidermal cells close to stomata. It is concluded that differences in solute concentrations between epidermal cells and other leaf tissues can be established in the absence of transpiration, but that they require transpiration for their full expression. Peristomatal transpiration appears to be responsible for high Ca in interstomatal cells.Abbreviations EDX-analysis Energy-dispersive X-ray analysis - IS-cell Interstomatal cell - R-cell Ridge cell - TR-cell Trough cell     

A study of the transpiration surfaces of Avena sterilis L. var. Algerian leaves using monosilicic acid as a tracer for water movement

Summary The sites and pathways of transpiration from leaves of Avena sterilis L. var. Algerian were studied using the accumulation of monosilicic acid as a tracer for water movement. Seedlings of Algerian oats were grown under silicon free conditions and fed monosilicic acid, in a normal nutrient solution, via the roots. The silicon component of monosilicic acid was located in freeze substituted tissue by means of x-ray microprobe analysis. Methods of tissue fixation preventing post treatment movement of tracer were developed and it was determined that monosilicic acid is a suitable tracer for water.Sites of water loss were marked by accumulation of silicon. Internal evaporating surfaces having a high intensity of water loss were demonstrated. Evaporation from epidermal surfaces was most intense over the guard and subsidiary cells with very little evaporation from the cuticular surfaces of normal epidermal cells. Moderately high evaporation occurred from epidermal fibre cells located above the veins. Evaporation from all exposed walls of guard cells including the wall adjacent to the pore was intense. Smaller amounts of tracer were located in the unexposed anticlinal walls of epidermal cells as well as within the unexposed walls of mesophyll cells. The results are interpreted as demonstrating the extent of internal transpiration surfaces and that cuticular epidermal transpiration is low. Strong support is given to the existence of peristomatal transpiration. Internal pathways of water movement are defined and the occurrence of these is discussed in relation to cuticular transpiration and lateral water movement in the epidermis.     

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype.     

ELECTRON PROBE ANALYSIS OF SILICON AND OTHER ELEMENTS IN LEAF EPIDERMAL CELLS OF THE RICE PLANT (ORYZA SATIVA L.)

By means of electron probe analysis, the effects of significant amounts of accumulation of silicon on the accumulation of calcium, potassium, magnesium, manganese, phosphorous, iron, and sodium in the silica cells of rice leaves are described. The silica cells of both the surfaces of the leaf blade and leaf sheath were studied. Silicon accumulation in the silica cells appears to decrease the amount of accumulation of potassium on both the surfaces of the leaf blade and sheath. The effect of significant amounts of silicon accumulation on the accumulation of other elements in a particular cell varies in different organs or on different surfaces of the organ of the same plant. Magnesium, manganese, iron, and phosphorus could not be detected in the adaxial epidermis of the leaf sheath and magnesium and iron in the adaxial epidermis of the leaf blade. Manganese, magnesium, and phosphorus were not detected in the abaxial epidermis of the leaf blade nor iron in the abaxial epidermis of the leaf sheath. Sodium was not revealed in either surface of the leaf blade and leaf sheath. Possible mechanisms for the effects of silicon accumulation on the accumulation of these elements in rice leaf epidermal cells are discussed.     

Electron Microprobe Analysis of Silicon and Other Elements in Developing Silica Cells of the Leaf and Internode of Cyperus alternifolius

This study is an analysis of changes in accumulation of Ca,K, Mg, Mn, Fe, P, and Na involved during the development ofsilica cells in the leaf and internode of Cyperus alternifolius.Electron microprobe analysis shows that with the accumulationof silicon in the silica cells, there appears to be a decreasein the accumulation of potassium and phosphorus; in contrast,the accumulation of calcium and iron is increased. Manganeseand magnesium were found in non-detectable quantities in thesedeveloping epidermal and silica cells. The effect of silicondeposition on the accumulation of these elements is discussed.     

THE DEVELOPMENT OF APOSPOROUS GAMETOPHYTES AND REGENERATED SPOROPHYTES FROM EPIDERMAL CELLS OF EXCISED FERN LEAVES: AN ANATOMICAL STUDY

Three different types of outgrowths develop from epidermal cells of excised juvenile leaves of Microgramma vacciniifolia: aposporous gametophytes, intermediates, and regenerated sporophytic plantlets. The gametophytes and intermediates arise from derivatives of epidermal cell divisions which are developed to the exterior of the leaf surface, whereas the sporophytic regenerants originate from derivatives produced by cell divisions to the interior of the leaf. Anatomical observations of excised leaves grown in vitro demonstrate that only the epidermal cells are stimulated to divide and give rise to the various types of outgrowths. Incorporation of tritiated thymidine by the nuclei of leaf epidermal cells gives further evidence for the metabolic activity of these cells.     

ELECTRON MICROPROBE ANALYSIS OF SILICA IN EPIDERMAL CELLS OF EQUISETUM

Patterns of silica deposition on the outer epidermal cell walls of Equisclum arvense and E. hycmalc var. affine were examined by means of electron microprobe analysis. Silica is deposited primarily in discrete knobs and rosettes on the epidermal surface in E. arvense and essentially in a uniform pattern on and in the entire outer epidermal cell walls of E. hyemale var. affine. This markedly contrasts with patterns of silica deposition in internodal epidermal cells of Avena saliva (Gramineae) where silica is deposited primarily in cell walls and cell lumina, and to a much lesser extent, on the outer epidermal surface. Semi-quantitative analysis with the electron microprobe shows that in intercalary meristematic cells of E. aruense, silicon is not present in any cells, but that in mature epidermal cells above the intercalary meristem it is present in significant quantities. The study thus suggests that silica deposition must be a very rapid process in Equisclum and Avena.     

SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY ANALYSIS OF CHALK SECRETING LEAF GLANDS OF OF PLUMBAGO CAPENSIS

Chalk secreting leaf glands of Plumbago capensis Thunb. were examined by polarized reflected light microscopy, scanning electron microscopy, x-ray diffraction, and energy dispersive x-ray analysis. There are about 23 glands per mm² on the lower leaf surface and none on the upper surface. Each gland measures 20–30 μm in diam and consists of four small secretory cells surrounded by four subsidiary cells. Secretion of chalk is apparently through a pore in the surface of each secretory cell. The secreted material was crystalline and x-ray diffraction confirmed the presence of two minerals: calcite (CaCO₃) and nesquehonite (MgCO₃ · 3H₂O). Energy dispersive x-ray analysis revealed the presence of magnesium, silicon, phosphorus, sulfur, chlorine, potassium, and calcium in the epidermal cells. However, only calcium and magnesium and traces of silicon were detected in the secreted material. A distribution analysis showed calcium and magnesium to be uniformly distributed through the secreted material.     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

Relationships between xylem anatomy,root hydraulic conductivity,leaf/root ratio and transpiration in citrus trees on different rootstocks

The aim of the study was to determine the extent in which leaf and whole plant transpiration (Tp) were influenced by root hydraulic conductance (K_r), leaf to root ratio and leaf mass. Also, the relationships between the anatomic characteristics of roots and K_r were investigated. To this end, 9‐month‐old seedlings of the citrus rootstocks Cleopatra mandarin (CM), Poncirus trifoliata (PT), and their hybrids Forner‐Alcaide no 5 (FA‐5) and Forner‐Alcaide no 13 (FA‐13) and 15‐month‐old trees of Valencia orange budded on these four rootstocks were tested. The hybrid FA‐13 and PT had higher values of K_r and leaf transpiration rates (E) than FA‐5 and CM. There was a positive curvilinear correlation between E and K_r. Furthermore, E levels in the different types of plants decreased with increased leaf/root (L/R) ratios. Pruning of the roots and defoliation confirmed that transpiration rates were strongly influenced by the L/R ratio. However, variations in E because of differences in L/R ratios were less pronounced in trees budded on FA‐13 and PT than on the other two rootstocks. In addition, there was a positive correlation between Tp and leaf biomass, although differences between rootstocks may be attributed to differences in K_r. The average lumen diameter of xylem vessels was greater in rootstocks with high K_r. Size of epidermal and hypodermal cells of fibrous roots may also restrict K_r.     

Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice

We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants.     

Cellular basis for dimethipin-induced loss of leaf turgor and desiccation

Dimethipin-induced increase in transpiration from kidney bean leaves (Phaseolus vulgaris L. cv. Black Valentine) was not correlated with stomatal aperture. From analysis of the kinetics of water loss from excised kidney bean leaves, it was concluded that the increase in transpiration was due almost entirely to an increase in the cuticular component. Both light and scanning electron microscopic analysis of dimethipin-treated leaves indicated that the first cells to be affected by dimethipin were the epidermal cells. These results suggest that dimethipin causes a loss of leaf turgor and desiccation by disrupting epidermal cells, thereby removing a major barrier for water loss from the leaf.Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L

A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed.     

THE EFFECT OF MODERATE SALT STRESS ON LEAF ANATOMY IN HIBISCUS CANNABINUS (KENAF) AND ITS RELATION TO LEAF AREA

Kenaf responded to salt stress in a manner that was typical of moderately salt tolerant non-halophytes. Increase in leaf area was more sensitive to salinity than either leaf emergence rate or dry matter accumulation. Dry weight was reduced only above a threshold of approximately 37 mM NaCl while leaf area was already significantly reduced at this salt concentration. Measurement of epidermal cell cross sectional area and epidermal cell numbers showed that the salt induced reduction in leaf area was due primarily to smaller epidermal cell size. Epidermal cell numbers were also significantly reduced by salinity. Stomatal density increased with increasing salt stress and there was no effect on leaf thickness.     

The role of epidermal turgor in stomatal interactions following a local perturbation in humidity

Humidity in a small area of a Vicia faba L. leaf was perturbed with a flow of dry air from an 80 µm (inside diameter) needle, while the remainder of the leaf was maintained at high and constant humidity. The influence of the needle flow on the humidity at the leaf surface was quantified by using a spatially explicit dewpoint hygrometer to observe condensation patterns. When the dry air from a needle was applied to the leaf, stomata within the influence of the needle opened within the first few minutes of the perturbation, and local epidermal turgor pressure declined within the same time frame. When the needle flow was removed from the leaf, these responses were reversed, but with more variable kinetics. Stomata and epidermal cells outside the influence of the needle flow, which were exposed to a constant and high humidity, showed similar, but smaller, responses when the needle flow was applied to the leaf. Since the opening of these stomata should have had only a small effect on transpiration (because of the high humidity), it is likely that the reduction in epidermal turgor was the cause (rather than the result) of the stomatal opening. The magnitude of the turgor response was only loosely related to the distance from the needle flow up to distances of almost 400 µm. The data support the idea that neighbouring stomata can interact through the influence of transpiration on epidermal turgor.     

Electron-probe microanalysis of silicon in the epidermis of rice (Oryza sativa L.) internodes

Summary Electron-probe X-ray microanalysis showed that significant amounts of silicon are accumulated in the entire epidermal system of the rice internode except in the stomatal apparatuses. Thus, there is a lack of specific sites for Si deposition from levels just above the base to the tip of the rice internode. In the intercalary meristem region, 1 cm above the base of the internode, point-count data indicate more Si accumulation in the dumb-bell shaped silica cells than in the long epidermal cells. Above this region, Si is accumulated essentially in a uniform pattern in all epidermal cells. Such a pattern for Si accumulation in rice internodes markedly contrasts with that for Avena internodes and may explain, in part, why rice plants have a higher percentage Si (dry weight basis) in their shoots. The adaptive significance of this silicification pattern in rice is discussed.     

Silicification of the adaxial epidermis of leaves of a panicoid grass in relation to leaf position and section and environmental conditions

Many studies relate silica content in plants with internal or external factors; however, few works analyse the effect of these factors on the silicification of different cell types. In this study, we examined the effect of leaf section and leaf position, and environmental conditions on the percentages of silicified epidermal cells of a native Pampean panicoid grass, Bothriochloa laguroides D. C. Pilger. Two different environmental situations were selected for the collection of plants: a natural wetland and a quartzite quarry, located in the southeast Buenos Aires province, Argentina. Clarification and staining methodologies were applied so as to study the distribution of silicified cells in different sections of leaves of the plants collected. Two and three-factor anovas were applied to the data. Between 13% and 19% of total cells of the adaxial epidermis of leaf blades were silicified. Typical silica short cells were the largest contributor to total silicified cells (53-98%), while the second largest contributor was bulliform cells (0-30%). Percentages of total silicified cells were higher in superior than in inferior leaves, while values from leaf sections varied. When collection sites were compared, plants growing in Los Padres pond, where the silica content in soils is higher, had the higher percentage of silicified cells. Among all types of cell, bulliform cells showed differences in the proportion of silicified cells between leaf position and section and collection site. These results show that silica availability in soils is an important factor that conditions silica accumulation and overlaps with the transpiration effect.     

Heterogeneity of epidermal cells in relation to nickel accumulation in hyperaccumulator plants belonging to the genus <Emphasis Type="Italic">Alyssum</Emphasis> L.

Epidermal cells of some plants are able to accumulate high levels of heavy metals (Zn, Ni, Cd). We studied this ability in plants in the genus Alyssum L. distinguished by tolerance to nickel (Ni). It was established that the predominant Ni accumulation occurred in epidermis, whereas in other tissues lower concentrations of the metal were revealed. It was also found that epidermal cells were characterized by heterogeneity in relation to Ni accumulation. The highest metal amount was accumulated in ordinary epidermal cells and in trichomes. Species-specific features of Ni distribution in leaf tissues in Alyssum spp. were shown. The reasons for the heterogeneity of epidermal cells in relation to Ni accumulation were discussed. We have attempted to resolve the contradictions encountered in the literature concerning the distribution and accumulation of Ni in the leaf tissues of plants belonging to the genus Alyssum L.     

Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants

For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and F_ab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and F_abfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeF_ab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or F_ab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream. ⁴Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be     

枫香变红过程中叶片组织结构、光合特性及色素含量变化研究

枫香(Liquidambar formosana)因其叶片入秋后逐渐变红而极具观赏价值,是优良的景观生态树种。为了解枫香叶片结构变化与叶色的关系,该文通过连续监测枫香叶片变红过程中组织结构、光合特性及色素含量的变化,分析叶片结构与其光合特性和色素的关系。结果表明：(1)叶片变色过程中,表皮细胞均为椭圆形,紧密排列,未观察到明显的细胞变异,表面未附着绒毛和蜡质,且上表皮细胞与栅栏组织细胞间排列紧密,未出现较大的气室。(2)随着叶片逐渐变红,叶片结构变化显著,其中叶片、上表皮、栅栏组织和海绵组织厚度及气孔开度均逐渐减小,而气孔器长和宽、单个气孔器面积则逐渐增大。(3)随着叶片结构的变化,其叶绿素含量逐渐减少,致使净光合速率逐渐减小,在出现光破坏时,叶片通过在栅栏组织细胞液泡内合成花色苷来自我保护,而大量的花色苷致使叶片表面呈现红色。综上认为,叶绿素含量降低,花色素苷大量积累是导致枫香叶片变红的直接原因,而枫香叶色变红则是其一系列生理结构特征综合作用的结果。