Bundle Sheath Strands Research Articles

Isolation of Envelope Membranes from Bundle Sheath Chloroplasts of Maize

Bundle sheath strands were isolated from maize (Zea mays L.) leaves treated with preparations of cellulase, hemicellulase, and pectinase. A three-phase discontinuous gradient yielded two fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm(-3). The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of electron microscopy. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic, and electron microscopic examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes, and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56-g leaf sections. The Mg(2+)-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 mumoles Pi released hr(-1) mg protein(-1), a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants.

C 4 acid decarboxylation and CO 2 donation to photosynthesis in bundle sheath strands and chloroplasts from species representing three groups of C 4 plants

The mechanism of decarboxylation of C 4 acids and carboxyl donation to the Calvin pathway was studied in bundle sheath strands and chloroplasts isolated from leaves of species representing three groups of C 4 plants: Digitaria sanguinalis (NADP-malic enzyme type), Panicum miliaceum (NAD-malic enzyme type), and Eriochloa borumensis (phosphoenolpyruvate-carboxykinase type). High rates of 14CO 2 fixation and HCO 3− dependent O 2 evolution were exhibited by bundle sheath strands and chloroplasts of P. miliaceum and E. borumensis, while those of D. sanguinalis (the NADP-malic enzyme type) required the addition of ribose 5-phosphate and malate for maximum activity. In all three species, 3-(3-4-dichlorophenyl)-1,1-dimethylurea inhibition of 14CO 2 fixation was partially overcome by the addition of malate, suggesting that malate can serve as a source of reductive power for carbon assimilation. Decarboxylation of 4- 14C-labeled C 4 acids was followed in the presence of glyceraldehyde, an inhibitor of the Calvin pathway. A large light-dependent stimulation of malate decarboxylation occurred by the addition of 3-phosphoglycerate (6 m m) with bundle sheath cells of all three groups and with bundle sheath chloroplasts of D. sanguinalis and E. borumensis. Although malate is suggested to be decarboxylated through the respective primary decarboxylase of each species, the 3-phosphoglycerate enhancement suggests that pyridine nucleotide reduction from malate oxidation is used during the reductive phase of the Calvin pathway. Bundle sheath strands from all three species decarboxylated aspartate with severalfold stimulation by the addition of either α-ketoglutarate (10 m m) or pyruvate (1 m m), with the lowest rate in D. sanguinalis. Bundle sheath chloroplasts from either of the three species could not decarboxylate aspartate. A light-independent decarboxylation of aspartate is suggested through NAD-malic enzyme in bundle sheath mitochondria in all three types of C 4 species, the rates being highest in P. miliaceum. A light-dependent portion of aspartate decarboxylation in E. borumensis is suggested to be through phosphoenolpyruvate carboxykinase. C 4. acid decarboxylation in all three species was inhibited by HCO 3− (CO 2 may be the active species). Consistent with the C 4 acid decarboxylation studies, the bundle sheath strands of the three species gave light-dependent O 2 evolution with aspartate or malate, the bundle sheath chloroplasts of D. sanguinalis and E. borumensis exhibited light-dependent O 2 evolution only with malate, and the bundle sheath chloroplasts of P. miliaceum showed no C 4 acid-dependent O 2 evolution. In all three C 4 types, malate decarboxylation, though by different decarboxylases, interacted with the Calvin pathway by providing both CO 2 and reducing power, while aspartate decarboxylation provided only CO 2.

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C4-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH4Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C4-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C4-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C4-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C4-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed (14)CO2 at high rates and exhibited C4-acid-dependent O2 evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.

Inhibition of oxalacetate decarboxylation during C4 photosynthesis by 3-mercaptopicolin acid.

3-Mercaptopicolinic acid specifically inhibits phosphoenolpyruvate carboxykinase in leaves of the C4 plant Panicum maximum. Both the ATP- and ADP-dependent decarboxylation of oxalacetate and the carboxylation activity of phosphoenolpyruvate carboxykinase are inhibited by 3-mercaptopicolinic acid while phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase are not inhibited. 3-Mercaptopicolinic acid inhibits the fixation of 14CO2 by illuminated P. maximum bundle sheath strands which is dependent upon oxalacetate and ATP but does not affect C3 photosynthesis in bundle sheath strands nor C4 photosynthesis in mesophyll cells. 3-Mercaptopicolinic acid treatment reduced P. maximum leaf photosynthesis 25% while raising the photosynthetic CO2 compensation point from near zero to 18 to 45 mul of CO2/liter of air.

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

AbstractVariable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C3 gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C4 grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl2, 1 mM KH2PO4, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO2 fixation by the C4 mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C3 mesophyll protoplasts supported CO2 fixation without added substrates. Rates of CO2 fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C4 mesophyll protoplasts were stable when stored at 25°C.

Carbon dioxide assimilation and photosystem II deficiency in bundle sheath strands isolated from C<sub>4</sub> plants

The activities of Hill reaction and photosynthetic 14CO2 fixation in bundle sheath strands enzymatically isolated from millet (Panicum miliaceum) were 3–15 times as high as those observed in corn (Zea mays). In both preparations, 3-phosphoglycerate was the initial 14CO2 fixation product and the radioactivity was incorporated into sucrose and insoluble compounds (glucose-polymers) during the later period. After 20 sec of photosynthetic 14CO2 fixation, the percent of 14C incorporated into sugar phosphates in millet was about 3 times as high as that in corn, while in corn, the percent of 14C in 3-phosphoglycerate was higher than that observed in millet throughout the experimental period. When 14C-phosphoglycerate was added to the isolated bundle sheath strands, the rates of transfer of the radioactivity to dihydroxyacetone phosphate and sugar diphosphates in millet were significantly higher than those in corn. These results indicate that in the bundle sheath strands isolated from corn in which photosystem II activity is deficient, the reductive pentose cycle is impaired at the reduction step of 3-phosphoglycerate to glyceraldehyde phosphate due to the limited supply of NADPH through the photoelectron transport system. In contrast, the bundle sheath strands isolated from millet which have adequate photosystem II activity can carry out normal photosynthetic CO2 fixation.

14CO 2 fixation and glycolate metabolism in the dark in isolated maize ( Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO 2 per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO 2-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg 2+ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO 2-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO 2 uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO 2. Glycolate is synthesized in this dark system in the presence of [U- 14C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO 2. These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.

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 C 4-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. 14CO 2 Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C 4 photosynthesis is compartmentalized into mesophyll CO 2 fixation via PEP carboxylase and bundle sheath CO 2 fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C 4-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.

Assimilation of carbon dioxide in mesophyll chloroplasts and bundle sheath strands mechanically isolated from corn leaves

Assimilation of carbon dioxide in mesophyll chloroplasts and bundle sheath strands mechanically isolated from corn leaves

Conversion of carbon 4 of malate into products of the pentose cycle by isolated bundle sheath strands of Digitaria sanguinalis (L.) Scop. leaves

Summary Isolated bundle sheath strands of crabgrass, incubated with malate 4- 14 C, were shown to take up malate and to incorporate 14 C into products of the pentose cycle. The conversion of 14 C into pentose cycle products was stimulated by light and by the addition of ribose-5-phosphate, ADP, and NADP + . Malate was taken up near 0.25 μmoles/mg chlorophyll and this appeared to be the limiting factor for the conversion processes in isolated bundle sheath cells.

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.

Photosynthetic Carbon Metabolism in Isolated Maize Bundle Sheath Strands

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO(2) per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic (14)CO(2) fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath (14)CO(2) fixation. After 5 minutes of photosynthesis in (14)C-bicarbonate, 95% of the incorporated (14)C was found in compounds other than C(4)-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of (14)C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of "malic" enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP(+)-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize.

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 CO 2 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 C 4-dicarboxylic acid photosynthesis.

The Warburg effect in maize bundle sheath photosynthesis

Bundle sheath strands isolated from maize are capable of photosynthesizing glycolate in the presence of an inhibitor of glycolate oxidase. Glycolate synthesis is stimulated by high oxygen concentrations and suppressed by high levels of CO 2. This data, together with the reversible oxygen inhibition of bundle sheath photosynthesis reported earlier [Ref. 6], demonstrates the occurrence of the Warburg effect in maize bundle sheath photosynthesis. These results also suggest a mechanism to account for the inhibition of photo-respiration in intact maize leaves.

Oxygen inhibits maize bundle sheath photosynthesis

Oxygen inhibits photosynthetic CO 2 fixation in isolated maize bundle sheath strands. This inhibition is rapidly and completely reversible and is relieved by increased levels of CO 2. These observations provide evidence that the relative oxygen-insensitivity of photosynthesis in intact maize leaves results from a CO 2 concentrating mechanism.

Isolation of Mesophyll Cells and Bundle Sheath Cells from Digitaria sanguinalis (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology

A technique is described for the separation of mesophyll and bundle sheath cells from Digitaria sanguinalis leaves and evidence for separation is given with light and scanning electron micrographs. Gentle grinding of fully differentiated leaves in a mortar releases mesophyll cells which are isolated on nylon nets by filtration. More extensive grinding of the remaining tissue yields bundle sheath strands which are isolated by filtration with stainless steel sieves and nylon nets. Further grinding of bundle sheath strands in a tissue homogenizer releases bundle sheath cells which are collected on nylon nets. Percentage of purity derived from cell counts and yield data on a chlorophyll basis are given.The internal leaf cell morphology is presented in scanning electron micrographs and compared with light micrographs of fully-differentiated D. sanguinalis leaves. In leaves of plants which possess the C(4)-dicarboxylic acid cycle of photosynthesis, the relationship of leaf morphology to photosynthesis in mesophyll and bundle sheath cells is considered, and the hypothesis is presented that as atmospheric CO(2) enters a leaf about 85% is fixed by the C(4)-dicarboxylic acid cycle in the mesophyll cells and 10 to 15% is fixed by the reductive pentose phosphate cycle in the bundle sheath cells.A technique also is given for the isolation of mesophyll cells from spinach leaves.