Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass)

Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C₄ grass Digitaria sanguinalis (L.) Scop. Two-dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.

Protein patterns were also compared with C₄ plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C₄ species were found to be more like bundle sheath proteins of another C₄ species than like mesophyll proteins of the same species.

     

EXPRESSION OF THE C4 PATTERN OF PHOTOSYNTHETIC ENZYME ACCUMULATION DURING LEAF DEVELOPMENT IN ATRIPLEX ROSEA (CHENOPODIACEAE)

Immunolocalization of the bundle sheath-specific enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase), and of the mesophyll-specific enzyme, phosphoenolpyruvate carboxylase (PEPCase), was used to follow development of the C₄ pattern of photosynthetic enzyme expression during leaf growth in Atriplex rosea. The leaf tissue used for this characterization was also used in a parallel ultrastructural study, so that the temporal coordination of developmental changes in enzyme expression and cell structure could be monitored. Bundle sheath-specific accumulation of RuBPCase occurs early, at the time that bundle sheath tissue is delimited from the ground meristem, and follows the order of vein initiation. PEPCase proteins were detected 2–4 days after the first appearance of RuBPCase. PEPCase accumulation is restricted to ground meristem cells that are in direct contact with bundle sheath tissue and that will become C₄ mesophyll; PEPCase was never found in more distant ground tissue. This pattern suggests that, while bundle sheath-specific accumulation of RuBPCase coincides with formation of the appropriate precursor cells, PEPCase expression is delayed until mesophyll tissue reaches a critical developmental stage. Cell-specific expression of both photosynthetic enzymes occurs well before the striking anatomical divergence of bundle sheath and mesophyll tissues, suggesting that biochemical compartmentation might serve as a developmental signal for subsequent structural differentiation.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis

The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C₄ plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C₄ dicarboxylic acid pathway—phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase—were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase—enzymes of the reductive pentose phosphate pathway—were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.     

Distribution of carboxylation and decarboxylation enzymes in isolated mesophyll cells and bundle sheath strands of C 4 plants

Mature leaves of Cyperus rotundus, Cyperus polystachyos, Digitaria decumbens, and Digitaria sanguinalis were separated, using pectinase and cellulase, into pure preparations of mesophyll cells and bundle sheath strands. Assays on these distinct leaf cell types show a clear compartmentation of phosphoenolpyruvate carboxylase, >98%, into mesophyll cells and of ribulose-1, 5-diphosphate carboxylase and malic enzyme, >98%, into the bundle sheath strands. The results clearly establish that the major CO₂ uptake in mesophyll cells is via a β-carboxylation and that both a decarboxylation and a carboxylation reaction occurs in the bundle sheath strands of plants using C₄-dicarboxylic acid photosynthesis.     

Low bundle sheath carbonic anhydrase is apparently essential for effective c(4) pathway operation

Bundle sheath cells from leaves of a variety of C₄ species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C₄ pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO₂] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C₄ function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C₄ pathway.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2)

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO₂ compensation points at different O₂ levels, which is typical of C₄ plants, yet it does show about 4% inhibition of net photosynthesis by 21% O₂ at 30°C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C₄ pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C₃ cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial ¹⁴C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO₂ is fixed into C₄ acids (malate and aspartate), whereas about 20% of the CO₂ directly enters the C₃ cycle. This is consistent with the high activity of enzymes for CO₂ fixation by the C₄ pathway and the substantial activity of enzymes of the C₃ cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C₃ photosynthesis in the mesophyll cells and should be considered a C₄-like species.     

Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C₄ groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.     

CO2 donation by malate and aspartate reduces photorespiration in Panicum milioides,A C3C4 intermediate species

Oxygen inhibition of leaf slice photosynthesis in Panicum milioides increased from 20% to 30% at 21% O₂ in the presence of maleate, a phosphoenolpyruvate carboxylase inhibitor. The increased O₂ sensitivity was completely reversed by the addition of malate and aspartate, the stable products of the phosphoenolpyruvate carboxylase reaction. The C₄ acids, malate and aspartate, also reduced O₂ inhibition of photosynthesis by isolated bundle sheath strands, but not mesophyll protoplasts. Similarly, only bundle sheath strands exhibited an active C₄ acid-dependent O₂ evolution. Compartmentation of C₄ cycle enzymes, with pyruvate, Pi dikinase in the mesophyll and NAD-malic enzyme in the bundle sheath, was demonstrated. It is concluded that reduced photorespiration in P. milioides is due to a limited potential for C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath ribulosebisphosphate carboxylase.     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.     

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.     

Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation

Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C₃ and C₄ cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C₄ plants, the activities of C₄ pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C₃ plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C₃ or C₄ plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C₄ acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species.     

Isolation of Mitochondria from Leaf Tissue of Panicum miliaceum, a NAD-Malic Enzyme Type C(4) Plant

A mechanical isolation procedure was developed to study the respiratory properties of mitochondria from the mesophyll and bundle sheath tissue of Panicum miliaceum, a NAD-malic enzyme C₄ plant. A mesophyll fraction and a bundle sheath fraction were obtained from young leaves by differential mechanical treatment. The purity of both fractions was about 80%, based on analysis of the cross-contamination of ribulose bisphosphate carboxylase activity and phosphoenolpyruvate carboxylase activity.

Mitochondria were isolated from the two fractions by differential centrifugation and Percoll density gradient centrifugation. The enrichment of mitochondria relative to chloroplast material was about 75-fold in both preparations.

Both types of mitochondria oxidized NADH and succinate with respiratory control. Malate oxidation in mesophyll mitochondria was sensitive to KCN and showed good respiratory control. In bundle sheath mitochondria, malate oxidation was largely insensitive to KCN and showed no respiratory control. The oxidation was strongly inhibited by salicylhydroxamic acid, showing that the alternative oxidase was involved. The bundle sheath mitochondria of this type of C₄ species contribute to C₄ photosynthesis through decarboxylation of malate. Malate oxidation linked to an uncoupled, alternative pathway may allow decarboxylation to proceed without the restraints which might occur via coupled electron flow through the cytochrome chain.

     

Correlations between photosynthetic enzymes,CO2-fixation and plastid structure in an albino mutant of Zea mays L.

Summary An albino seedling of Zea mays L. was investigated for its potential for CO₂-assimilation. In the mesophyll the number, dimensions and fine structure of chloroplasts are drastically reduced but to a lesser extent in the bundle sheath. Chlorophyll concentration is zero and carotenoid concentration almost zero. Albinism also exerts a strong influence on the stroma of bundle sheath chloroplasts; ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) activity and glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) activity is not detectable. The C₄-enzymes phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (decarboxylating) (EC 1.1.1.40) and the non-photosynthetic linked enzymes malate dehydrogenase (NAD) (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1.) and glyceraldehyde-3-phosphate dehydrogenase (NAD) (EC 1.2.1.1.) are present in the albino seedling with activities comparable to those in etiolated maize seedlings. The potential for CO₂ fixation of the albino seedlings exceeds that of comparable dark seedlings considerably. The results are discussed with regard to enzyme localization of the C₄ pathway of photosynthesis.Abbreviations Aspartate aminotransferase L-aspartate-2-oxoglutarate aminotransferase-EC 2.6.1.1. - GAPDH (NAD) glyceraldehyde-3-phosphate dehydrogenase (NAD dep.)-EC 1.2.1.12 - GAPDH (NADP) glyceraldehyde-3-phosphate dehydrogenase (NADP dep.)-EC 1.2.1.13 - malic enzyme malate dehydrogenase (NADP dep., decarboxylating)-EC 1.1.1.40 - MDH malate dehydrogenase (NAD dep.)-1.1.1.37 - PEP carboxylase phosphoenolpyruvate carboxylase-EC 4.1.1.31 - RuDP carboxylase ribulose-1.5-biphosphate carboxylase-EC 4.1.1.39     

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C₄ plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.