Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.     

Structure of the Photosynthetic Apparatus in Leaves of Freshwater Hydrophytes: 2. Quantitative Characterization of Leaf Mesophyll and the Functional Activity of Leaves with Different Degrees of Submersion

The structure of leaf photosynthetic elements was investigated on 42 boreal plant species characterized by different degrees of submergence (helophytes, neustophytes, and hydatophytes). Six main types of mesophyll structures were identified. Quantitative characteristics for the mesostructure of the photosynthetic apparatus in these groups were determined, such as the size and abundance of cells and chloroplasts in the mesophyll and epidermis, the number of plastids per cell in each tissue, the total surface area of the mesophyll cells, epidermal cells, and chloroplasts per unit leaf area. Analysis showed that quantitative characteristics of the photosynthetic apparatus in hydrophytes are determined by two factors: (a) the degree of leaf submergence and (b) the type of mesophyll structure. With an increasing degree of immersion in water, the mesophyll types change in a sequence isopalisade dorsoventral homogeneous. The leaves become thinner, their weight per unit area diminishes, cells and chloroplasts become less numerous (on a per unit leaf area basis), but their dimensions become larger. Adaptation to aquatic medium is also manifested in the increasing contribution of the epidermal tissue to the overall photosynthesis: in submerged leaves, the epidermis accounts for more than 50% of the photosynthetic activity. The occurrence of six structural types of leaves contrasting in their characteristics was confirmed by discriminatory analysis according to the qualitative parameters of mesophyll.     

Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development

Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10-20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000-4000 anticlinal divisions for the adaxial epidermis, was 2-3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.     

Genome Expression during Normal Leaf Development : I. CELLULAR AND CHLOROPLAST NUMBERS AND DNA, RNA, AND PROTEIN LEVELS IN TISSUES OF DIFFERENT AGES WITHIN A SEVEN-DAY-OLD WHEAT LEAF

Changes in genome expression during normal cellular and plastid development in the first leaf of young (7-day-old) wheat (Triticum aestivum var. Maris Dove) were investigated by examining homogeneous populations of leaf cells and plastids of several developmental ages present in the same leaf. The cells were characterized over a period immediately following the last cell division. All of the leaf cells had cytoplasmic contents and nuclei, and between 44% (young tissue) and 54% (older tissue) of the leaf cells were mesophyll cells. Chloroplast development was complete 36 hours after the chloroplasts had ceased dividing. Extremely large changes occurred in cellular constituents over a very short period of leaf development. Maximum rates of accumulation of ribulose bisphosphate carboxylase per mesophyll cell (80 picograms/hour), chlorophyll per mesophyll cell (9 picograms/hour), and 70S ribosomes per mesophyll cell (19 × 10⁵/hour) were recorded.     

Structure of the Photosynthetic Apparatus in Leaves of Freshwater Hydrophytes: 1. General Characteristics of the Leaf Mesophyll and a Comparison with Terrestrial Plants

The structure of photosynthetic elements was investigated in leaves of 42 boreal plant species featuring different degrees of submergence (helophytes, neustophytes, and hydatophytes). The mesophyll structure types were identified for all these species. Chlorenchyma tissues and phototrophic cells were quantitatively described by such characteristics as the sizes of cells and chloroplasts in the mesophyll and epidermis, the abundance of cells and chloroplasts in these tissues, the total surface area of cells and chloroplasts per unit leaf area, the number of plastids per cell, etc. The hydrophytes typically had thick leaves (200–350 m) with a well-developed aerenchyma; their specific density per unit area (100–200 mg/dm²) was lower than in terrestrial plants. Mesophyll cells in aquatic plants occupied a larger volume (5–20 × 10³m³) than epidermal cells (1–15 × 10³m³). The number of mesophyll cells per unit leaf area was nearly 1.5 times higher than that of epidermal cells. Chloroplasts were present in the epidermis of almost all species, including emergent leaves, but the ratio of the chloroplast total number to the number of all plastids varied depending on the degree of leaf submergence. The total number of plastids per unit leaf area (2–6 × 10⁶/cm²) and the surface of chloroplasts per unit leaf area (2–6 cm²/cm²) were lower in hydrophytes than in terrestrial plants from climatically similar habitats. The functional relations between mesophyll parameters were similar for hydrophytes and terrestrial plants (a positive correlation between the leaf weight per unit area, leaf thickness, and the number of mesophyll cells per unit leaf area), although no correlation was found in hydrophytes between the volume of mesophyll cells and the leaf thickness. Phototrophic tissues in aquatic plants contributed a larger fraction to the leaf weight than in terrestrial plants, because the mechanical tissues were less developed in hydrophytes. The CO₂assimilation rates by leaves were lower in hydrophytes than in terrestrial plants, because the total surface area of chloroplasts per unit leaf area is comparatively small in hydrophytes, which reduces the conductivity for carbon dioxide diffusion towards the carboxylation sites.     

Structural Adaptation of the Leaf Mesophyll to Shading

Structural characteristics of the mesophyll were studied in five boreal grass species experiencing a wide range of light and water supply conditions. Quantitative indices of the palisade and spongy mesophyll tissues (cell and chloroplast sizes, the number of chloroplasts per cell, the total cell and chloroplast surface area per unit leaf surface area) were determined in leaves of each of the species. The cell surface area and the cell volume in spongy mesophyll were determined with a novel method based on stereological analysis of cell projections. An important role of spongy parenchyma in the photosynthetic apparatus was demonstrated. In leaves of the species studied, the spongy parenchyma constituted about 50% of the total volume and 40% of the total surface area of mesophyll cells. The proportion of the palisade to spongy mesophyll tissues varied with plant species and growth conditions. In a xerophyte Genista tinctoria, the total cell volume, cell abundance, and the total surface area of cells and chloroplasts were 30–40% larger in the palisade than in the spongy mesophyll. In contrast, in a shade-loving species Veronica chamaedris, the spongy mesophyll was 1.5–2 times more developed than the palisade mesophyll. In mesophyte species grown under high light conditions, the cell abundance and the total cell surface area were 10–20% greater in the palisade mesophyll than in the spongy parenchyma. In shaded habitats, these indices were similar in the palisade and spongy mesophyll or were 10–20% lower in the palisade mesophyll. In mesophytes, CO₂ conductance of the spongy mesophyll accounted for about 50% of the total mesophyll conductance, as calculated from the structural characteristics, with the mesophyll CO₂ conductance increasing with leaf shading.     

Visualisation of plastid degradation in sperm cells of wheat pollen

Like most angiosperms, wheat (Triticum aestivum) shows maternal inheritance of plastids. It is thought that this takes place by cytoplasmic stripping at fertilisation rather than the absence of plastids in sperm cells. To determine the fate of plastids during sperm cell development, plastid-targeted green fluorescent protein was used to visualise these organelles in nuclear transgenic wheat lines. Fewer than thirty small 1–2-μm plastids were visible in early uninucleate pollen cells. These dramatically increased to several hundred larger (4 μm) plastids during pollen maturation and went through distinct morphological changes. Only small plastids were visible in generative cells (n?=?25) and young sperm cells (n?=?9). In mature sperm cells, these green fluorescent protein (GFP)-tagged plastids were absent. This is consistent with maternal inheritance of plastids resulting from their degradation in mature sperm cells in wheat.     

Changes in Ultrastructure of Phaseolus Vulgaris L. Cotyledons Associated with their Modulated Life Span

French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged.     

Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia

Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C₄ dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4×, 6×, and 8×, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area.     

Plant response to overcrowding – Lemna minor example

Plants adopt various strategies in response to increasing density. We tested that response in two populations of Lemna minor L. – a free floating aquatic plant that frequently experiences intraspecific competition for space. Surface area of fronds and colonies, colony size (the number of fronds per colony), the rate of reproduction (based on the number of produced fronds) and growth rate (enlargement of surface area of all colonies) were the analysed factors presumably affected by density. The study was performed in natural stands and in experimental conditions with the use of two contrasting plant densities. Plants growing in natural conditions produced fronds of smaller and less variable surface area as a response to overcrowding but the number of fronds per colony was unrelated to plant density. Stable experimental conditions facilitated formation of fronds and colonies larger than in the field but frond detachment decreasing colony size was more intensive at high than at low density. This strategy allowed plants to more efficiently occupy limited available space. No self-thinning was observed during experimental cultures. Due to increasing frond area in cultures, growth rate was always higher than the rate of plant reproduction. Both were strongly negatively affected by high density. Performed calculations indicate that density-dependent growth inhibition starts when L. minor colonies cover the available water surface with a mono-layer mat. Some types of responses were found to significantly differ between analysed populations, which was also shown by genetic differences tested with he ISSR-PCR technique. Possible causal relationship between plant strategies and their genomic structure needs, however, further studies.     

Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat

The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2×), Triticum dicoccum (4×), and Triticum aestivum (6×). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2×, 4×, and 6× ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content.     

Shading-induced changes in the leaf mesophyll of plants of different functional types

Changes in the structural characteristics of mesophyll induced by shading were investigated in ten species of wild plants of diverse functional types. In all plant types, shading reduced leaf thickness and density by 30–50% and total surface of mesophyll, by 30–70%. The extent and mechanisms of mesophyll structural rearrangement depended on the plant functional type. In the ruderal plants, integral parameters of mesophyll, such as the surface of cells and chloroplasts and mesophyll resistance, changed threefold predominantly because of changes in the dimensions of the cells and chloroplasts. In these plants, shading reduced the volume of chloroplasts by 30%, and the chloroplast numbers per cell declined. The competitor plants showed a twofold increase in mesophyll resistance due to a decrease in the number of photosynthesizing cells per leaf area unit. Moreover, these plants maintained constant dimensions of mesophyll cells, ratios mesophyll surface/mesophyll volume and chloroplast surface/cell surface. In stress-tolerant plants, diffusion resistance of mesophyll remained the same irrespective of the growing conditions, and mesophyll rearrangement was associated with inversely proportional changes in the dimensions of the cells and cell volume per chloroplast. Noteworthy of these plants were relatively constant chloroplasts number per cell, per leaf area unit and total surface area of chloroplasts. The nature of relationship between the mesophyll diffusion resistance and structural parameters of leaf mesophyll differed in plants of diverse functional types.     

The plant growth regulator activity of the fungicide,epoxiconazole, on Galium aparine L. (cleavers)

The plant growth regulator activity of epoxiconazole, a new triazole fungicide, was investigated by time-course, dose-response and histology experiments with Galium aparine L. (cleavers). Seven days after treatment with 125g ai ha^–1 epoxiconazole (field rate), plant height was reduced by 43%. After seventeen days, leaflet area was reduced by 27% but leaflet fresh weight was not significantly influenced. This was partly because leaflet thickness had increased by 20% following epoxiconazole application. Chlorophyll concentrations were also increased on a unit area basis. Examination of leaflet anatomy showed that epoxiconazole elongated palisade, spongy mesophyll and upper epidermal cells. For example, 125g ai ha^–1 caused a 35% increase in the length of spongy mesophyll cells. Epoxiconazole also prevented cell separation as there were significantly more palisade and spongy mesophyll cells per unit area than in leaflets sprayed with water. Stem development was reduced and 125g ai ha^–1 inhibited the elongation of pith cells in stem tissue by 53%. However, the simultaneous application of gibberellin A₃ (GA₃) with epoxiconazole resulted in stem elongation similar to that of control plants. These observations are consistent with the expected effects following the inhibition of cytochrome P-450 dependent enzyme activity.Abbreviations GA₃ gibberellin A₃ - g ai ha^–1 grams of active ingredient per hectare - L ha^–1 litres per hectare - PPFD photosynthetic photon flux density - RH relative humidity - SE standard error     

Light-Induced Adaptive Responses under Greenhouse and Controlled Conditions in the Fern Pteris cretica var. ouvrardii

The influence of different growth irradiance conditions on plant development and foliar features were assessed in a shade-adapted fern variety, Pteris cretica var. ouvrardii. A comparison of frond morphology, anatomy, chloroplast infrastructure and chlorophyll content of plants cultivated in both greenhouse and in controlled growth chambers under moderate light, low light (control) and extreme shade revealed pronounced phenotypic modifications. Moderate light induced decreased frond surfaces, thicker leaves, lower chlorophyll content per surface unit, and a markedly reduced density of intraplastidial membranes. In contrast, morphological responses to extreme shade included the formation of larger, thinner fronds, increased chlorophyll content; and a higher membrane density in chrloplasts. A dorsi-ventral distribution of starch-gorged chlroplasts (lower mesophyll cell layers) and essentially starch-free chloroplasts (upper cell layer) characterizes low-light and moderate light fronds, while homogenous starch-free chloroplasts are present in all cell layers of extreme shade fronds. The light-induced modifications are discussed as adaptive responses.     

Increases in cell elongation,plastid compartment size and phytoene synthase activity underlie the phenotype of the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant of tomato

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.     

Subcellular Localization of a UDP-Glucose:Aldehyde Cyanohydrin beta-Glucosyl Transferase in Epidermal Plastids of Sorghum Leaf Blades

Epidermal and mesophyll protoplasts, prepared from leaf blades of 6-day-old light-grown Sorghum bicolor seedlings were separated by differential sedimentation and assayed for a number of enzymes. The epidermal protoplasts contained higher levels of NADPH-cytochrome c reductase (EC 1.6.2.4), triose phosphate isomerase (EC 5.3.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and a UDP-glucose:cyanohydrin β-glucosyl transferase (EC 2.4.1.85), but lower levels of NADP⁺ triosephosphate dehydrogenase (EC 1.2.1.13) than did mesophyll protoplasts. When protoplast preparations were lysed and applied to linear sucrose density gradients, triosephosphate isomerase was found to be present in epidermal plastids. A significant fraction (41%) of the glucosyl transferase activity was also associated with the epidermal plastids.     

Light fluence rate and chloroplasts are sources of signals controlling mesophyll cell morphogenesis and division

As part of the acclimation of the photosynthetic apparatus to high fluence rates of light, mesophyll (photosynthetic) leaf cells change in morphology (they elongate anticlinally or perpendicular to the leaf surface) and undergo extra cell divisions. This results in increased leaf thickness and internal, protective shading among chloroplasts. Here we have examined whether the chloroplasts themselves are sources of intracellular signals that trigger these changes, by monitoring the Arabidopsis thaliana chm1 variegated mutant, in which albino (chloroplast-defective) and green (with functional chloroplasts) sectors coexist in one leaf. Our results have uncovered two separable responses. The increase in mesophyll cell elongation was substantially reduced but still observable in albino sectors, indicating that chloroplasts contribute to the cell morphogenesis response, but a chloroplast-independent light sensory mechanism must exist. In contrast the change in number of mesophyll cell layers was completely abolished when plastids were dysfunctional, indicating that plastids are sole sources of signals for the cell division response. These data highlight the importance of plastid-derived signals in the cellular responses associated with photosynthetic acclimation.     

Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration

Mesophyll protoplasts were isolated from Nicotiana tabacum L. cv. Xanthi, and cell-colony formation induced in liquid culture. The plastid changes associated with the morphogenetic sequence from mesophyll protoplast to whole plant were examined. Minor ultrastructural changes in the plastids were evident after 1 d of culture, but by 8 d (four-to-eight-cell stage) the plastids were small, there was much less thylakoid membrane appression, and many prominent plastoglobuli were also present. Plastid-division figures were evident at this point of time and it was common to find plastids clustered around the nucleus. A typical proplastid was the dominant plastid type in the cultured cells from about 11 d until about five weeks when large amyloplasts and pregranal plastids were observed. Normally structured chloroplasts were present in the regenerated plant. There was no plastid division until the four-cell stage, with plastid numbers per cell approximately halving at each cell division, then stabilising around 12 per cell during cell-colony development, a number typical of meristematic cells. Though nucleoids were always present, their numbers in the plastids were reduced by the eight-cell stage.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): ULTRASTRUCTURE,PLASMODESMATAL FREQUENCY,AND SOLUTE CONCENTRATIONS

The minor veins and contiguous tissues of mature leaves of Populus deltoides Bartr. ex Marsh. were examined with the electron microscope to determine the ultrastructural characteristics of the component cells and to determine the structure, distribution, and frequency of plasmodesmata between the various cell types. In addition, plasmolytic studies were carried out to determine the solute concentrations of the various cell types of the minor veins and contiguous tissues. The cells comprising the mesophyll and bundle sheath contain all the components typical of photosynthetic cells. Paraveinal mesophyll cells and bundle-sheath cells have fewer microbodies and smaller chloroplasts than do palisade parenchyma cells. Vascular parenchyma and companion cells tend to intergrade with one another structurally but can be distinguished from one another by their characteristic plastids. The mature, enucleate sieve-tube member is lined by a parietal layer of cytoplasm consisting of plasmalemma, endoplasmic reticulum, mitochondria, plastids, and P-protein. Plasmodesmata occur along all possible routes from the palisade parenchyma cells to the sieve tubes of the minor veins, and their frequency increases with increasing proximity to the sieve-tube members. Plasmolytic studies revealed that the paraveinal mesophyll cells had a higher C₅₀ (estimated mannitol concentration plasmolyzing, on the average, 50% of a given cell type) than any other cell type of the leaf. Concentration gradients existed along the palisade cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route as well as along the paraveinal mesophyll cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route. Considering the frequency of plasmodesmata along these routes, it is conceivable that photosynthate diffuses from palisade cells to the companion cells along concentration gradients. Within the minor veins, the C₅₀ was higher for sieve-tube members than for either companion cells or vascular parenchyma cells, indicating that loading of the sieve tubes is an active, energy-dependent process.