Estimation of Bundle Sheath Cell Conductance in C4 Species and O2 Insensitivity of Photosynthesis

Low conductance to CO2 of bundle sheath cells is required in C4 photosynthesis to maintain high [CO2] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Elevated [CO2] allows high CO2 assimilation rates by this enzyme and prevents Rubisco oxygenase activity and O2 inhibition of carboxylation. Bundle sheath conductance to CO2 was estimated by chemically inhibiting phosphoenolpyruvate carboxylase and calculating the slope of the linear response of leaf CO2 uptake to [CO2]. The inhibitor 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate was supplied to detached leaves of Panicum maximum, Panicum miliaceum, and Sorghum bicolor at 4 mM. Uptake of CO2 was measured at 210 mL L-1 O2 over the CO2 concentration range of 0.34 to 28 mL L-1. Without the inhibitor, CO2 uptake increased steeply at low [CO2] and saturated at about 1 mL L-1. After inhibition, CO2 uptake was a linear function of [CO2] over much of the range tested. The slope of this CO2 response, taken as bundle sheath conductance, was 2.35, 1.96, and 1.13 mmol m-2 s-1 for P. maximum, P. miliaceum, and S. bicolor, respectively, on a leaf area basis. Conductance based on bundle sheath area was 0.76, 0.93, and 0.54 mmol m-2 s-1, respectively. Uptake of CO2 by leaves of P. maximum supplied with the inhibitor was not affected by reduction of [O2] from 210 to 20 mL L-1 over the range of [CO2] used. Because [CO2] in bundle sheath cells of inhibited leaves is likely to be much lower than ambient, the lack of O2 sensitivity of CO2 uptake cannot be ascribed to lack of O2 reaction with ribulose bisphosphate and is probably due to the low conductance of bundle sheath cells, especially at low ambient [CO2]. The likely result of reducing [O2] from 210 to 20 mL L-1 is to stimulate carboxylation of ribulose bisphosphate, thus further reducing [CO2] in bundle sheath cells and increasing CO2 diffusion to these cells from the mesophyll. However, the increase in diffusion is greatly limited by low conductance of the bundle sheath cell walls. Calculations based on estimated bundle sheath conductance show that changes in bundle sheath [CO2] of 0.085 to 0.5 mL L-1, which might be associated with reduced [O2], would have a negligible effect on CO2 uptake.     

Photosynthetic carbon metabolism in Panicum milioides, a C3-C4 intermediate species: evidence for a limited C4 dicarboxylic acid pathway of photosynthesis

Panicum milioides, a naturally occurring species with C4-like Kranz leaf anatomy, is intermediate between C3 and C4 plants with respect to photo-respiration and the associated oxygen inhibition of photosynthesis. This paper presents direct evidence for a limited degree of C4 photosynthesis in this C3-C4 intermediate species based on: (a) the appearance of 24% of the total 14C fixed following 4 s photosynthesis in 14CO2-air by excised leaves in malate and aspartate and the complete transfer of label from the C4 acids to Calvin cycle intermediates within a 15 s chase in 12CO2-air; (b) pyruvate- or alanine-enhanced light-dependent CO2 fixation and pyruvate stimulation ote- or alanine-enhanced light-dependent CO2 fixation and pyruvate stimulation of oxaloacetate- or 3-phosphoglycerate-dependent O2 evolution by illuminated mesophyll protoplasts, but not bundle sheath strands; and (c) NAD-malic enzyme-dependent decarboxylation of C4 acids at the C-4 carboxyl position, C4 acid-dependent O2 evolution, and 14CO2 donation from (4-14C)C4 acids to Calvin cycle intermediates during photosynthesis by bundle sheath strands, but not mesophyll protoplasts. However, P. milloides differs from C4 plants in that the activity of the C4 cycle enzymes is only 15 to 30% of a C4 Panicum species and the Calvin cycle and phosphoenolpyruvate carboxylase are present in both cell types. From these and related studies (Rathnam, C.K.M. and Chollet, R. (1979) Arch. Biochem. Biophys. 193, 346-354; (1978) Biochem. Biophys. Res. Commun. 85, 801-808) we conclude that reduced photorespiration in P. milioides is due to a limited degree of NAD-malic enzyme-type C4 photosynthesis permitting an increase in pCO2 at the site of bundle sheath, but not mesophyll, ribulose-bisphosphate carboxylase-oxygenase.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

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.     

C4 Photosynthesis (The CO2-Concentrating Mechanism and Photorespiration)

Despite previous reports of no apparent photorespiration in C4 plants based on measurements of gas exchange under 2 versus 21% O2 at varying [CO2], photosynthesis in maize (Zea mays) shows a dual response to varying [O2]. The maximum rate of photosynthesis in maize is dependent on O2 (approximately 10%). This O2 dependence is not related to stomatal conductance, because measurements were made at constant intercellular CO2 concentration (Ci); it may be linked to respiration or pseudocyclic electron flow. At a given Ci, increasing [O2] above 10% inhibits both the rate of photosynthesis, measured under high light, and the maximum quantum yield, measured under limiting light ([phi]CO2). The dual effect of O2 is masked if measurements are made under only 2 versus 21% O2. The inhibition of both photosynthesis and [phi]CO2 by O2 (measured above 10% O2) with decreasing Ci increases in a very similar manner, characteristically of O2 inhibition due to photorespiration. There is a sharp increase in O2 inhibition when the Ci decreases below 50 [mu]bar of CO2. Also, increasing temperature, which favors photorespiration, causes a decrease in [phi]CO2 under limiting CO2 and 40% O2. By comparing the degree of inhibition of photosynthesis in maize with that in the C3 species wheat (Triticum aestivum) at varying Ci, the effectiveness of C4 photosynthesis in concentrating CO2 in the leaf was evaluated. Under high light, 30[deg]C, and atmospheric levels of CO2 (340 [mu]bar), where there is little inhibition of photosynthesis in maize by O2, the estimated level of CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the bundle sheath compartment was 900 [mu]bar, which is about 3 times higher than the value around Rubisco in mesophyll cells of wheat. A high [CO2] is maintained in the bundle sheath compartment in maize until Ci decreases below approximately 100 [mu]bar. The results from these gas exchange measurements indicate that photorespiration occurs in maize but that the rate is low unless the intercellular [CO2] is severely limited by stress.     

Comparative analysis of biochemical properties of mesophyll and bundle sheath chloroplasts from various subtypes of C4 plants grown at moderate irradiance

The photochemical characteristics of mesophyll and bundle sheath chloroplasts isolated from the leaves of C4 species were investigated in Zea mays (NADP-ME type), Panicum miliaceum (NAD-ME type) and Panicum maximum (PEP-CK type) plants. The aim of this work was to gain information about selected photochemical properties of mesophyll and bundle sheath chloroplasts isolated from C4 plants grown in the same moderate light conditions. Enzymatic as well as mechanical methods were applied for the isolation of bundle sheath chloroplasts. In the case of Z. mays and P. maximum the enzymatic isolation resulted in the loss of some thylakoid polypeptides. It was found that the PSI and PSII activities of mesophyll and bundle sheath chloroplasts of all species studied differed significantly and the differences correlated with the composition of pigment-protein complexes, photophosphorylation efficiency and fluorescence emission characteristic of these chloroplasts. This is the first report showing differences in the photochemical activities between mesophyll chloroplasts of C4 subtypes. Our results also demonstrate that mesophyll and bundle sheath chloroplasts of C4 plants grown in identical light conditions differ significantly with respect to the activity of main thylakoid complexes, suggesting a role of factor(s) other than light in the development of photochemical activity in C4 subtypes.     

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

A comparative anatomical and biochemical analysis in salsola (Chenopodiaceae) species with and without a Kranz type leaf anatomy: a possible reversion of C4 to C3 photosynthesis

Leaf anatomy was studied by light and electron microscopy and the leaf activities of RUBP carboxylase, PEP carboxylase, and malic enzyme were assayed in: Salsola australis and S. oreophila grown on the West Pamirs at 1800 m altitude; in S. australis grown on the East Pamirs at 3860 m; and in S. arbusculiformis grown in the Kisil-Kum desert in Middle Asia near 500 m. Carbon isotope fractionation ratio values also were measured on whole leaf tissue for 18 Salsola species field collected in these and other regions of the former USSR. S. australis leaves are cylindrical and in cross section exhibit a peripheral ring of mesophyll and then an inner ring of bundle sheath type cells; and its biochemical characteristics and deltaC values are typical of a C4 species of the NADP-malic enzyme malate-forming group. These traits were expressed independent of the plant growth altitude up to 4000 m. C4 type deltaC values were obtained in 14 of the Salsola species. Anatomical, structural, and biochemical features typical of the C4 syndrome were absent in S. oreophila and S. arbusculiformis. Four Salsola species, including these two, had C3-type deltaC values. Their cylindrical leaves in cross section exhibited two to three peripheral rings as layers of palisade parenchyma. Although their vascular bundles were surrounded by green bundle sheath cells, their organelle numbers were comparable to those in mesophyll cells. Neither bundle sheath cell wall thickenings nor dimorphic chloroplasts in two leaf cell types were observed. In S. oreophila, there was a high activity of RuBP carboxylase, but a low activity of C4 cycle enzymes. Interpretation of these data lends evidence to the hypothesis that a small group of C3 Salsola species, including S. oreophila, S. arbusculiformis, S. montana, and S. pachyphylla, arose as the result of a reversion of a C4 to a C3 type of photosynthetic CO2 fixation in the cooler climates of Middle Asia.     

The impact of elevated carbon dioxide on the growth and gas exchange of three C4 species differing in CO2 leak rates

Recent work has suggested that the photosynthetic rate of certain C₄ species can be stimulated by increasing CO₂ concentration, [CO₂], even under optimal water and nutrients. To determine the basis for the observed photosynthetic stimulation, we tested the hypothesis that the CO₂ leak rate from the bundle sheath would be directly related to any observed stimulation in single leaf photosynthesis at double the current [CO₂]. Three C₄ species that differed in the reported degree of bundle sheath leakiness to CO₂, Flaveria trinervia, Panicum miliaceum, and Panicum maximum, were grown for 31–48 days after sowing at a [CO₂] of 350 μl l^?1 (ambient) or 700 μl l^?1 (elevated). Assimilation as a function of increasing [CO₂] at high photosynthetic photon flux density (PPFD, 1 600 μmol m^?2 s^?1) indicated that leaf photosynthesis was not saturated under current ambient [CO₂] for any of the three C₄ species. Assimilation as a function of increasing PPFD also indicated that the response of leaf photosynthesis to elevated [CO₂] was light dependent for all three C₄ species. The stimulation of leaf photosynthesis at elevated [CO₂] was not associated with previously published values of CO₂ leak rates from the bundle sheath, changes in the ratio of activities of PEP-carboxylase to RuBP carboxylase/oxgenase, or any improvement in daytime leaf water potential for the species tested in this experiment. In spite of the simulation of leaf photosynthesis, a significant increase in growth at elevated [CO₂] was only observed for one species, F. trinervia. Results from this study indicate that leaf photosynthetic rates of certain C₄ species can respond directly to increased [CO₂] under optimal growth conditions, but that the stimulation of whole plant growth at elevated carbon dioxide cannot be predicted solely on the response of individual leaves.     

In situ immunofluorescent localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in mesophyll of C4 dicotyledonous plants

The location of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in the leaf mesophyll of some dicotyledonous C4 plants was confirmed by immunofluorescent labelling. The anti-RuBPCO immune serum was obtained by inoculating a rabbit with commercially obtained RuBPCO. Specificity of these antibodies was tested by immunodiffusion, immunoelectrophoresis, and Western blotting. Fresh hand-cuts of leaves from dicotyledonous C4 plants, Amaranthus caudatus, A. dubius, Gomphrena globosa, and Portulaca oleracea, were incubated with the conjugated anti-RuBPCO immune serum and then with a commercial FITC-anti-rabbit IgG conjugate. Nerium oleander was used a control C3 plant pattern and Zea mays as a C4 plant pattern. The immunofluorescent label was distributed in both mesophyll and bundle sheath in all the C4 plants tested. It is an unequivocal proof that in the C4 dicotyledonous plants the RuBPCO is not only located in the chloroplasts of the bundle sheath cells but also in the chloroplasts of the mesophyll cells. In these plants therefore, the C4 pathway cannot exclusively be viewed as an intercellular level concentration mechanism. In the mesophyll cytoplasm, phosphoenolpyruvate carboxylase traps CO2, while in the mesophyll chloroplasts, RuBPCO operates with atmospheric CO2 and CO2 from the C4 decarboxylation step at an intracellular level, which could mean a significant energetic economy. The CO2 from photorespiration could be saved and reincorporated. Location of RuBPCO in the mesophyll and/or bundle sheath chloroplasts is a matter of inter- and intracellular compartmentation which makes another variation of C4 photosynthetic pathway possible. This revised version was published online in September 2006 with corrections to the Cover Date.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

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.     

Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae)

Certain members of the family Chenopodiaceae are the dominant species of the deserts of Central Asia; many of them are succulent halophytes which exhibit C4-type CO2 fixation of the NAD- or NADP-ME (malic enzyme) subgroup. In four C4 species of the tribe Salsoleae, the Salsoloid-type Kranz anatomy in leaves or stems was studied in relation to the diversity in anatomy which was found in cotyledons. Halocharis gossypina, has C4 NAD-ME Salsoloid-type photosynthesis in leaves and C3 photosynthesis in dorsoventral non-Kranz cotyledons; Salsola laricina has C4 NAD-ME Salsoloid-type leaves and C4 NAD-ME Atriplicoid-type cotyledons; Haloxylon persicum, has C4 NADP-ME Salsoloid-type green stems and C3 isopalisade non-Kranz cotyledons; and S. richteri has C4 NADP-ME Salsoloid-type leaves and cotyledons. Immunolocalization studies on Rubisco showed strong labelling in bundle sheath cells of leaves and cotyledons of organs having Kranz anatomy. The C4 pathway enzyme phosphoenolpyruvate carboxylase was localized in mesophyll cells, while the malic enzymes were localized in bundle sheath cells of Kranz-type tissue. Immunolocalization by electron microscopy showed NAD-ME is in mitochondria while NADP-ME is in chloroplasts of bundle sheath cells in the respective C4 types. In some C4 organs, it was apparent that subepidermal cells and water storage cells also contain some chloroplasts which have Rubisco, store starch, and thus perform C3 photosynthesis. In non-Kranz cotyledons of Halocharis gossypina and Haloxylon persicum, Rubisco was found in chloroplasts of both palisade and spongy mesophyll cells with the heaviest labelling in the layers of palisade cells, whereas C4 pathway proteins were low or undetectable. The pattern of starch accumulation correlated with the localization of Rubisco, being highest in the bundle sheath cells and lowest in the mesophyll cells of organs having Kranz anatomy. In NAD-ME-type Kranz organs (leaves and cotyledons of S. laricina and leaves of H. gossypina the granal index (length of appressed membranes as a percentage of total length of all membranes) of bundle sheath chloroplasts is 1.5 to 2.5 times higher than that of mesophyll chloroplasts. In contrast, in the NADP-ME-type Kranz organs (S. richteri leaves and cotyledons and H. persicum stems) the granal index of mesophyll chloroplasts is 1.5 to 2.2 times that of the bundle sheath chloroplasts. The mechanism of photosynthesis in these species is discussed in relation to structural differences.     

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.     

Roles of the bundle sheath cells in leaves of C3 plants

This review considers aspects of the structure and functions of the parenchymatous bundle sheath that surrounds the veins in the leaves of many C(3) plants. It includes a discussion of bundle sheath structure and its related structures (bundle sheath extensions and the paraveinal mesophyll), its relationship to the mestome sheath in some grasses, and its chloroplast content. Its metabolic roles in photosynthesis, carbohydrate synthesis and storage, the import and export of nitrogen and sulphur, and the metabolism of reactive oxygen species are discussed and are compared with the role of the bundle sheath in leaves of C(4) plants. Its role as an interface between the vasculature and the mesophyll is considered in relation to the movement of water and assimilates during leaf development, export of photosynthates, and senescence.     

Photosynthetic Metabolism of Aspartate in Mesophyll and Bundle Sheath Cells Isolated from Digitaria sanguinalis (L.) Scop., a NADP-Malic Enzyme C(4) Plant

Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4)-dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate.