NAD-malic Enzyme Research Articles

Climate and the U.S. distribution of C4 grass subfamilies and decarboxylation variants of C4 photosynthesis

I compared the C(4) grass flora and climatic records for 32 sites in the United States. Consistent with previous studies, I found that the proportion of the grass flora that uses the NADP malic enzyme (NADP-ME) variant of C(4) photosynthesis greatly increases with increasing annual precipitation, while the proportion using the NAD malic enzyme (NAD-ME) variant (and also the less common phosphoenolpyruvate carboxykinase [PCK] variant) decreases. However the association of grass subfamilies with annual precipitation was even stronger than for the C(4) decarboxylation variants. Analysis of the patterns of distribution by partial correlation analysis showed that the correlations between the frequency of various C(4) types and rainfall were solely due to the association of the C(4) types with particular grass subfamilies. In contrast, there was a strong correlation of the frequency of the different subfamilies with annual precipitation that was independent of the influence of the different C(4) variants. It therefore appears that other, as yet unidentified, characteristics that differ among grass subfamilies may be responsible for their differences in distribution across natural precipitation gradients.

Occurrence of C(3) and C(4) photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae).

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C(4) photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C(3) and C(4) pathways, (14)C labeling of primary photosynthesis products and (13)C/(12)C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C(4) type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C(3) and C(4) types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C(4) type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C(4) type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C(3) type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C(4) photosynthesis in the family Chenopodiaceae.

Assessing photosystem I and II distribution in leaves from C4 plants using confocal laser scanning microscopy

ABSTRACTImages of chlorophyll fluorescence emitted at wavelengths above and below 700 nm were recorded from leaf sections of C4 species using confocal laser scanning microscopy (LSM). We investigated species exhibiting both NAD‐malic enzyme (NAD‐ME) C4 photosynthesis and NADP‐malic enzyme (NADP‐ME) C4 photosynthesis. Comparing LSM fluorescence of leaf sections with flow‐cytometrically determined fluorescence from individual chloroplasts revealed that LSM fluorescence was distorted by the optical properties of leaf sections. Leaf section fluorescence, when corrected by transmission data derived from light transmission images, agreed with flow cytometry data. The corrected LSM fluorescence yielded information on the distribution of the individual photosystems in the C4 leaf sections: PSII concentrations in bundle sheath cells were elevated in NAD‐ME species but diminished in most of the NADP‐ME species investigated. The NADP‐ME species, Arundinella hirta, however, showed normal PSII and increased PSI concentration in bundle sheath chloroplasts. Finally, a gradient of PSI was observed within the bundle sheath cells from Euphorbia maculata.

Study of the non-photochemical dark rise in chlorophyll fluorescence in pre-illuminated leaves of various C3 and C4 plants submitted to partial anaerobiosis

Changes in chlorophyll fluorescence yield were studied during a dark period in pre-illuminated leaves of various C3 and C4 plants. The oxygen content in the gaseous atmosphere was either normal (21 kPa) or low (1.5 or 0.36 kPa). C3 and C4 plants of the NAD malic enzyme subgroup showed an initial rise in fluorescence at the onset of the dark period with an amplitude depending on the O 2 level in the gas. In C4 plants belonging to the other two subgroups, the slow rise was absent or of very low size. At high [O 2], the fluorescence level decreased in some minutes to the initial F 0 level (determined in dark-adapted leaves). Conversely at low [O 2], the fluorescence yield remained higher than F 0 in all the C4 plants studied, whereas it decreased slowly to the F 0 level in the different C3 plants. At low [O 2], the fluorescence level decreased rapidly to F 0 when introducing for 30 s, a high O 2 level or when giving a 15-s far-red illumination. At the end of these treatments, the fluorescence level re-increased. These results demonstrate the presence at low [O 2] of highly fluorescent `closed' photosystem II centres containing Q - A in equilibrium with reduced plastoquinone molecules of the chloroplastic pool. Reoxidation of the plastoquinone pool would be dependent on the functioning of an oxidase probably dependent on a chlororespiration process fully active at O 2 levels higher than 2 kPa. The source of reducing equivalents for the plastoquinone pool is discussed.

Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.

The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme.

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].

The isolation and characterisation of a mutant of the C 4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C4 leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F2 plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO2 assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C4 enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C4 photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME.

Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017.

An NAD-malic enzyme was purified to homogeneity from Bradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids' carbon metabolism is discussed.

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C 4 grasses (three NADP-malic enzyme (NADP-ME) subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C 4 sedges (NADP-ME subtype species) and two C 4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1 ,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C 4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.

Induction of kranz anatomy and C4-like biochemical characteristics in a submerged amphibious plant by abscisic acid

The amphibious leafless sedge Eleocharis vivipara develops C4-like traits as well as Kranz anatomy under terrestrial conditions, but it develops C3-like traits without Kranz anatomy under submerged conditions. When submerged plants are exposed to aerial conditions, they rapidly produce new photosynthetic tissues with C4-like traits. In this study, experiments were performed to determine whether abscisic acid (ABA), a plant stress hormone, could induce the formation of photosynthetic tissues with Kranz anatomy and C4-like biochemical traits under water in the submerged form. When the submerged plants were grown in water containing 5 &mgr;M ABA, they developed new photosynthetic tissues with Kranz anatomy, forming well-developed Kranz (bundle sheath) cells that contained many organelles. The ABA-induced tissues accumulated large amounts of phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, and NAD-malic enzyme at the appropriate cellular sites. The tissues had 3.4 to 3.8 times more C4 enzyme activity than did tissues of the untreated submerged plants. Carbon-14 pulse and carbon-12 chase experiments revealed that the ABA-induced tissues fixed higher amounts of carbon-14 into C4 compounds and lower amounts of carbon-14 into C3 compounds as initial products than did the submerged plants and that they exhibited a C4-like pattern of carbon fixation under aqueous conditions of low carbon, indicating enhanced C4 capacity in the tissues. This report provides an example of the hormonal control of the differentiation of the structural and functional traits required for the C4 pathway.

Photosynthetic Characteristics of an Amphibious C4 Plant, Eleocharis retroflexa ssp. chaetaria

Eleocharis retroflexa (Poir.) Urban ssp. chaetaria (Roem. & Schult.) T. Koyama, an amphibious leafless sedge, grows not only under terrestrial conditions but also under completely submerged aquatic conditions. We investigated the photosynthetic traits and structural features of the culms, which are the photosynthetic organs, in the terrestrial and submerged forms of this species and compared them with those of other amphibious species of Eleocharis which are known to change the photosynthetic modes. The culms of the terrestrial form had Kranz anatomy with well-developed Kranz (bundle sheath) cells and high levels of C4 enzyme activity typical of the NAD-malic enzyme (NAD-ME) subtype of C4 metabolism. They also had a δ 13C value typical of C4 plants, indicating that the terrestrial form fixes carbon through the C4 pathway. The culms of the submerged form had not only a Kranz-like anatomy but also revealed anatomical traits typical of leaves of submerged aquatic plants. The activities of the C4 enzymes in the submerged form were lower than those in the terrestrial form, but were still in the range typical of G4 plants, 14C pulse-12C chase experiments with the submerged form indicated that almost all of the fixed 14C was incorporated into G4 compounds, and subsequently the raioactivity was transferred into C3 compounds and sucrose. The submerged form showed no diurnal fluctuation in malate level. These data demonstrate that a C4 metabolism is operative even in the submerged form. This unique amphibious C4 plant provides an intriguing example of the physiological and ecological adaptability of C4plants.

NaCl-induced changes of NAD(P) malic enzyme activities in Eucalyptus citriodora leaves

Three-month-old plants of Eucalyptus citriodora were grown in a greenhouse in a culture medium adjusted either to 50 or 100 mM NaCl. Sodium was rapidly absorbed by the plants and as early as 3 weeks after treatment, an increased Na+ level was observed in shoots of 50 mM NaCl plants. At this stage of treatment, the growth of plants was not reduced but the malate metabolism was modified. The malate content decreased in leaves while the specific activities of NAD and NADP-malic enzymes increased. The stimulation in enzyme activity was more pronounced for NADP-malic enzyme but for both enzymes, enzyme activity diminished as early as 5 weeks after treatment. The immunological study showed that the higher activity of NAD-malic enzyme was linked to an increase in the protein amount. By contrast, a more active form of the NADP-malic enzyme mainly accounted for the higher activity of this enzyme without involving modification in the electrophoretic mobility of the protein. The 100 mM NaCl treatment also induced changes in the malic enzyme behaviour, the magnitude of the response however being less than for the 50 mM treatment. These results are discussed, mainly considering the role of these enzymes in the reducing power of the leaf cell.

Expression, Purification, and Characterization of the Recombinant NAD-Malic Enzyme fromAscaris suum

The cDNA encoding the 65-kDa subunit of malic enzyme fromAscaris suumwas cloned into the bacterial expression vector pKK223-3 and overproduced inEscherichia coli.A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS–PAGE. The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an overall yield of 51%. The bacterially expressed enzyme exhibits kinetic constants identical to those measured for nativeA. suumNAD-malic enzyme.

Leaf Anatomy and Carbon Discrimination in NAD-malic EnzymePanicumSpecies and their Hybrids Differing in Bundle Sheath Cell Ultrastructure

The relationship between leaf anatomy, ultrastructure and carbon discrimination was investigated in leaves of two F1hybrids (F1−1 and F1−2) between two different types of the grassPanicum[an NAD-malic enzyme (ME) C4species], which differ in bundle sheath ultrastructure. The female parent was Kabulabula grass, which has centrifugal chloroplasts in bundle sheath cells and is designated an NAD-ME(F) species, while the male parent was Makarikari grass, which has centripetal chloroplasts in the bundle sheath cells and is designated an NAD-ME(P) species. Suberin lamellae are present in Kabulabula grass but are lacking in Makarikari grass. Both F1hybrids had the same chromosome number (2n=36) as the parents but exhibited both univalent (about 45%) and bivalent (about 55%) chromosome pairing which was the major basis for the identification of F1hybrids. In F1−1, elongated bundle sheath cell chloroplasts are arranged mainly in a centripetal position, similar to those in the male parent, Makarikari grass. In contrast, most of the bundle sheath cells in F1−2 are packed with starch-containing chloroplasts, although in some cells chloroplasts tended to be centripetally arranged. In both F1hybrids, suberin lamellae were found in the bundle sheath cell walls, similar to the female parent, Kabulabula grass. The δ13C values of both F1hybrids were −11.4 to −11.7‰, almost the same as those of Kabulabula grass (−11.4‰), but significantly higher than those of Makarikari grass (−12.7‰). These results indicate that the chloroplast orientation in the bundle sheath cells and the presence of suberin lamellae are not obligatorily linked in their expression and suggest that suberin lamellae may play an important role in discrimination against13C.

Post-illumination CO2 Exchange and Light-induced CO2 Bursts during C4 Photosynthesis

Detailed kinetics of the post-illumination CO2 exchange, and darklight transients following post-illumination exchange, were measured in leaves of Sorghum bicolor, a NADP-malic enzyme (NADP-ME), and Amaranthus cruentus, a NAD-malic enzyme (NAD-ME) type C4 plant using a gas system that has a full-response time of 3.5 s. The amount of CO2 fixed in the dark (assimilatory charge, AC) was up to 200 µmol m-2 for A. cruentus and 350-450 µmol m-2 for S. bicolor. AC was at its maximum value at CO2 concentrations close to the inflection of the CO2 response curve, and decreased when photosynthesis was limited by low light intensity. The kinetics of post-illumination CO2 fixation indicate that the rate of carboxylation in the C4 cycle is limited by the supply of phosphoenolpyruvate. In A. cruentus, under saturating CO2 the post-illumination CO2 uptake was replaced by a burst (68 µmol m-2). In S. bicolor, the dark-light induction commenced with a rapid CO2 burst (less than 5 s) of 46 µmol m-2, followed by a gulp. The observed CO2 transients show imbalances in the C4 and C3 cycles. In S. bicolor the lack of a post-illumination burst, and the presence of the light- induced CO2 burst is taken as evidence for strict coupling of malate decarboxylation to PGA reduction in NADP-ME species; the opposite response in A. cruentus indicates the lack of strict coupling between the C4 and C3 cycle in NAD-ME species.

NaCl-induced changes of NAD(P) malic enzyme activities in

NaCl-induced changes of NAD(P) malic enzyme activities in

Parallelism and Diversity in Multiple Origins of C 4 Photosynthesis in the Grass Family

C4 photosynthesis is thought to be an adaptation to warm environments, involving complex changes in the expression of genes governing photosynthesis, intermediary metabolism, and leaf anatomy and histology. Such complexity should be difficult to evolve, yet the pathway has arisen multiple times in the history of the flowering plants and at least four times in the grass family alone. We have used immunolocalization techniques to compare photosynthetic gene expression across all four origins, to determine which genetic changes occur in parallel and which are unique to a particular lineage. The only gene expression patterns common to all origins of the pathway are up-regulation of PEP carboxylase and down-regulation of RuBisCO in mesophyll cells. Both NAD-malic enzyme and NADP-malic enzyme are expressed in bundle sheaths. Expression patterns of light-harvesting chlorophyll a/b binding proteins and pyruvate orthophosphate dikinase appear to be lineage specific, and may be localized to bundle sheaths or to mesophyll or expressed throughout the photosynthetic tissue of the leaf. We suggest that future studies of parallel origin of the C4 pathway concentrate on regulation of the two carboxylases, as well as the increased density of vascular tissue, which is the only histological characteristic common to all origins of the pathway.

Patterns of Carbon Partitioning in Leaves of Crassulacean Acid Metabolism Species during Deacidification.

Carbohydrates stored during deacidification in the light were examined in 11 Crassulacean acid metabolism (CAM) species from widely separated taxa grown under uniform conditions. The hypothesis that NAD(P) malic enzyme CAM species store chloroplastic starch and glucans, and phosphoenolpyruvate carboxykinase species store extrachloroplastic sugars or polymers was disproved. Of the six malic enzyme species examined, Kalanchoe tubiflora, Kalanchoe pinnata, Kalanchoe daigremontiana, and Vanilla planifolia stored mainly starch. Sansevieria hahnii stored sucrose and Agave guadalajarana did not store starch, glucose, fructose, or sucrose. Of the five phosphoenolpyruvate carboxykinase species investigated, Ananus comosus stored extrachloroplastic carbohydrate, but Stapelia gigantea, Hoya carnosa, and Portea petropolitana stored starch, whereas Aloe vera stored both starch and glucose. Within families, the major decarboxylase was common for all species examined, whereas storage carbohydrate could differ both between and within genera. In the Bromeliaceae, A. comosus stored mainly fructose, but P. petropolitana stored starch. In the genus Aloe, A. vera stored starch and glucose, but A. arborescens is known to store a galactomannan polymer. We postulate that the observed variation in carbohydrate partitioning between CAM species is the result of two principal components: (a) constraints imposed by the CAM syndrome itself, and (b) diversity in biochemistry resulting from different evolutionary histories.

Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C3 and C4 metabolism

Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C4 plants while the submerged form develops structural and biochemical traits similar to those of C3 plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.

Mitochondrial Respiration in Relation to Photosynthetic C4 Acid Decarboxylation in C4 Species

Certain respiratory features of bundle sheath cells isolated from the C4 species Urochloa panicoides (phosphoenolpyruvate carboxykinase (PCK)-type)), Panicum miliaceum (NAD malic enzyme (NAD-ME)-type) and Zea mays (NADP malic enzyme (NADP-ME)-type) were examined in relation to the requirements of the C4 acid decarboxylation step of C4 photosynthesis. Cells from both PCK-type and NAD-ME-type species showed high rates of malate-dependent respiration; with ADP or uncoupler the rates were in the range 2-3 μatom O min-1 mg-1 chlorophyll, about 5-10-times the rates with other respiratory substrates. Studies with inhibitors of cytochrome oxidase and the alternative oxidase indicated negligible alternative oxidase-mediated malate respiration in cells from Z. mays, a minor contribution in U. panicoides cells, but possibly a major role for this oxidase in the respiration of P. miliaceum cells. These differences were related to the different roles of respiration in photosynthetic C4 acid decarboxylation. Oxaloacetate strongly suppressed malate-dependent respiration in P. miliaceum bundle sheath cells but not in U. panicoides cells. This difference in the response to oxaloacetate was not due to different kinetic features of the mitochondrial malate dehydrogenase but was apparently largely due to the much lower activity of the enzyme in U. panicoides bundle sheath mitochondria. We propose that insensitivity of respiration to oxaloacetate in bundle sheath cells of PCK-type species may be essential for maintaining the C4 acid decarboxylation process. The reverse may be true for NAD-ME- type species.