Mitochondria In Bundle Sheath Cells Research Articles

A scheme for C4 evolution derived from a comparative analysis of the closely related C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

A comparative analysis of the genus Flaveria showed a C4 evolutionary process in which the anatomical and metabolic features of C4 photosynthesis were gradually acquired through C3-C4 intermediate stages. C4 photosynthesis has been acquired in multiple lineages of angiosperms during evolution to suppress photorespiration. Crops that perform C4 photosynthesis exhibit high rates of CO2 assimilation and high grain production even under high-temperature in semiarid environments; therefore, engineering C4 photosynthesis in C3 plants is of great importance in the application field. The genus Flaveria contains a large number of C3, C3-C4 intermediate, C4-like, and C4 species, making it a good model genus to study the evolution of C4 photosynthesis, and these studies indicate the direction for C4 engineering. C4 photosynthesis was acquired gradually through the C3-C4 intermediate stage. First, a two-celled C2 cycle called C2 photosynthesis was acquired by localizing glycine decarboxylase activity in the mitochondria of bundle sheath cells. With the development of two-cell metabolism, anatomical features also changed. Next, the replacement of the two-celled C2 cycle by the two-celled C4 cycle was induced by the acquisition of cell-selective expression in addition to the upregulation of enzymes in the C4 cycle during the C3-C4 intermediate stage. This was supported by an increase in cyclic electron transport activity in response to an increase in the ATP/NADPH demand for metabolism. Suppression of the C3 cycle in mesophyll cells was induced after the functional establishment of the C4 cycle, and optimization of electron transport by suppressing the activity of photosystem II also occurred during the final phase of C4 evolution.

Revealing diversity in structural and biochemical forms of C4 photosynthesis and a C3-C4 intermediate in genus Portulaca L. (Portulacaceae).

Portulacaceae is one of 19 families of terrestrial plants in which species having C4 photosynthesis have been found. Representative species from major clades of the genus Portulaca were studied to characterize the forms of photosynthesis structurally and biochemically. The species P. amilis, P. grandiflora, P. molokiniensis, P. oleracea, P. pilosa, and P. umbraticola belong to the subgenus Portulaca and are C4 plants based on leaf carbon isotope values, Kranz anatomy, and expression of key C4 enzymes. Portulaca umbraticola, clade Umbraticola, is NADP-malic enzyme (NADP-ME)-type C4 species, while P. oleracea and P. molokiniensis in clade Oleracea are NAD-ME-type C4 species, all having different forms of Atriplicoid-type leaf anatomy. In clade Pilosa, P. amilis, P. grandiflora, and P. pilosa are NADP-ME-type C4 species. They have Pilosoid-type anatomy in which Kranz tissues enclose peripheral vascular bundles with water storage in the centre of the leaf. Portulaca cf. bicolor, which belongs to subgenus Portulacella, is an NADP-ME C4 species with Portulacelloid-type anatomy; it has well-developed Kranz chlorenchyma surrounding lateral veins distributed in one plane under the adaxial epidermis with water storage cells underneath. Portulaca cryptopetala (clade Oleracea), an endemic species from central South America, was identified as a C3–C4 based on its intermediate CO2 compensation point and selective localization of glycine decarboxylase of the photorespiratory pathway in mitochondria of bundle sheath cells. The C4 Portulaca species which were examined also have cotyledons with Kranz-type anatomy, while the stems of all species have C3-type photosynthetic cells. The results indicate that multiple structural and biochemical forms of C4 photosynthesis evolved in genus Portulaca.

Physiological, anatomical and biochemical characterisation of photosynthetic types in genus Cleome (Cleomaceae)

C4 photosynthesis has evolved many times in 18 different families of land plants with great variation in leaf anatomy, ranging from various forms of Kranz anatomy to C4 photosynthesis occurring within a single type of photosynthetic cell. There has been little research on photosynthetic typing in the family Cleomaceae, in which only one C4 species has been identified, Cleome gynandra L. There is recent interest in selecting and developing a C4 species from the family Cleomaceae as a model C4 system, since it is the most closely related to Arabidopsis, a C3 model system (Brown et al. 2005). From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ13C) in leaves, we have identified two additional C4 species, C. angustifolia Forssk. (Africa) and C. oxalidea F.Muell. (Australia). Several other species have δ13C values around -17‰ to -19‰, suggesting they are C4-like or intermediate species. Eight species of Cleome were selected for physiological, anatomical and biochemical analyses. These included C. gynandra, a NAD-malic enzyme (NAD-ME) type C4 species, C. paradoxa R.Br., a C3-C4 intermediate species, and 6 others which were characterised as C3 species. Cleome gynandra has C4 features based on low CO2 compensation point (Γ), C4 type δ13C values, Kranz-type leaf anatomy and bundle sheath (BS) ultrastructure, presence of C4 pathway enzymes, and selective immunolocalisation of Rubisco and phosphoenolpyruvate carboxylase. Cleome paradoxa was identified as a C3-C4 intermediate based on its intermediate Γ (27.5 μmol mol-1), ultrastructural features and selective localisation of glycine decarboxylase of the photorespiratory pathway in mitochondria of BS cells. The other six species are C3 plants based on Γ, δ13C values, non-Kranz leaf anatomy, and levels of C4 pathway enzymes (very low or absent) typical of C3 plants. The results indicate that this is an interesting family for studying the genetic basis for C4 photosynthesis and its evolution from C3 species.

Photosynthesis in phospho enolpyruvate carboxykinase-type C 4 plants: Activity and role of mitochondria in bundle sheath cells

Mitochondria from bundle sheath cells of the phospho enolpyruvate carboxykinasetype C 4 species Urochloa panicoides were shown to have metabolic properties consistent with a role in C 4 photosynthesis predicted from earlier studies. The rate of O 2 uptake in response to added malate plus ADP was at least five times the activity observed with NADH, glycine, or succinate. With malate plus ADP the O 2 uptake rate averaged about 150 nmol O 2 min −1 mg −1 protein, equivalent to about 0.6 μmol min −1 mg −1 of extracted chlorophyll. About half of this activity was apparently phosphorylation-linked with ADP O 2 ratios of about 4. Studies with electron transport inhibitors suggested that about 65% of this malate oxidation is cytochrome oxidase-terminated with a minor component mediated via the alternative oxidase. These mitochondria supported rapid rates of pyruvate production from malate and this activity was also stimulated by ADP but blocked by inhibitors of electron transport. Adding oxaloacetate increased pyruvate production but inhibited O 2 uptake. The results were consistent with the notion that in this subgroup of C 4 species mitochondrial-located NAD malic enzyme contributes substantially to total C 4 acid decarboxylation. This enzyme is apparently also the primary source of NADH necessary to generate the ATP required for phosph oenolpyruvate carboxykinase-mediated oxaloacetate decarboxylation.

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C3-C4 intermediate species Moricandia arvensis (L.) DC has been compared with that of the C3 species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO2 and hence a lower rate of photorespiration.

Isolation of leaf bundle sheath protoplasts from C 4 dicot species and intracellular localization of selected enzymes

A new source of cell wall digestive enzymes, Onozuka Cellulase RS (from Trichoderma viride), was evaluated for the effectiveness of leaf digestion and the release of bundle sheath protoplasts (BSP) in a variety of C 4 monocots and dicots. Several dicot species were identified from which BSP, as well as mesophyll protoplasts (MP), can readily be isolated by using the RS cellulase. Assays of marker enzymes, plus chlorophyll measurements, permitted analysis of protoplast yield and purity. BSP and MP have been purified (90–100%) from Atriplex spongiosa (NAD-malic enzyme (ME) subgroup) and from Falveria trinervia (NADP-ME subgroup), with final yields ranging from 6–24%. Enzyme localization studies indicate that a substantial amount of NADP-malate dehydrogenase (DH) in F. trinervia occurs not only in the mesophyll chloroplasts, but also in the bundle sheath chloroplasts. Similar activities of an apparent NADP-utilizing malate dehydrogenase (but not activated by dithioerythritol, DTE) occurs in the mitochondria of bundle sheath cells of A. spongiosa. Aspects of the potential role of this enzyme in photosynthetic metabolism are further discussed.

Intraspecifíc variability in <i>Alloteropsis semialata</i> (R. Br.) Hitchc.

Intraspecific variability in Alloteropsis semialata (R. Br.) Hitchc. is shown ultrastructurally with particular regard to the structure of the bundle sheath and the development of the dimorphic chloroplasts in both the C3 and C4 forms of this species. A. semialata is known to possess two distinct anatomical forms — Kranz and non-Kranz — within one species and occurring within a single ecological niche. Kranz and non-Kranz anatomy is known to be correlated with C3 and C4 physiology respectively. Transverse sections of leaf portions taken at midsheath, at the ligule and at midlamina show plastids with different morphologies at different ontogenetic stages. Plastid form is related to the stage of development, the influence of light on the emerging leaf and the C3 or C4form of A. semialata. Stages from amyloplast to chloroplast are investigated with regard to fine structure. Leaf transverse sections are examined microscopically and formation of new bundles, chiefly in the lamina is traced. Differences in anatomy and distribution of vascular bundles are more evident in the lamina than in the colourless leaf sheath. Both C3 and C4 forms of A. semialata are found to show chloroplast dimorphism in vascular bundle sheath and mesophyll cells. This is shown to differ in the two forms. The specialized chloroplasts of the Kranz sheath are shown to develop in the inner or mestome sheath, and not in the parenchyma sheath as in some other members of the Kranz Panicoideae. Features of A. semialata, such as the double bundle sheath, granal chloroplasts and large numbers of mitochondria in bundle sheath cells, in the C4 form, question the current classification of this grass as a malate former.

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C(3), C(4) or C(3)-C(4) intermediate were undertaken to examine features associated with C(3) and C(4) photosynthesis. The C(3) 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 C(3)-C(4) species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C(3) 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 C(3)-C(4) species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C(4) species Panicum prionitis Griseb. and the C(3)-C(4) species, but in C(3) species the organelles were similar in size or were smaller in the bundle sheath cells. The C(3)-C(4) 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 C(3)-C(4) species.

Influence of Nitrate and Ammonia on Photosynthetic Characteristics and Leaf Anatomy of Moricandia arvensis

The leaf anatomy and certain photosynthetic properties of nitrate- and ammonia-grown plants of Moricandia arvensis (L.) DC., a species previously reported to be a C(3)-C(4) intermediate, were investigated. Nitrate-grown plants had a high level of malate in the leaves while ammonia-grown plants had low levels of malate. In young leaves of nitrate-grown plants, there was a diurnal fluctuation of malate content, increasing during the day and decreasing during the night. Titratable acidity remained low in leaves of both nitrate- and ammonia-grown plants.In nitrate-grown plants, the activity of phosphoenolpyruvate (PEP) carboxylase was about 2-fold higher than in ammonia-grown plants, the latter having activity typical of C(3) species. Also, in nitrate-grown plants, the ratio of activities of ribulose 1,5-bisphosphate (RuBP) carboxylase/PEP carboxylase was lower than in ammonia-grown plants. Nitrate reductase activities were higher in nitrate- than in ammonia-grown plants and the greatest activity was found in younger leaves.With nitrate-grown plants, during a pulse-chase experiment the label in malate, as a percentage of the total labeled products, increased from about 7% after a 10-second pulse with (14)CO(2) up to 17% during a 5-minute chase with (12)CO(2). The pattern of (14)C labeling in various metabolites suggests the primary carboxylation is through RuBP carboxylase with a secondary carboxylation through PEP carboxylase. In similar experiments, with ammonia-grown plants, the percentage label in malate was only 0% to 4% with no increase in malate labeling during the chase period. The CO(2) compensation point was lower in nitrate-grown than ammonia-grown plants.There was no evidence of Kranz-like anatomy in either the nitrate or ammonia-grown plants. Mitochondria of bundle-sheath cells were strikingly positioned along the inner tangential wall. This might allow the chloroplasts of these cells to fix the mitochondrial photorespired CO(2) more effectively and contribute to the low CO(2) compensation point in the species. Chloroplasts of bundle-sheath cells and contiguous mesophyll cells were similar in size and structure in plants grown on different media, although chloroplast thylakoids and stromata of the ammonia-grown plants stained more intensely than those of nitrate-grown plants. In addition, irregular clusters of phytoferritin particles occurred in the chloroplasts of the ammonia-grown plants.The results indicate that the substantial activity of PEP carboxylase, incorporation of CO(2) into malate, the high malate content, and in part the relatively low CO(2) compensation point in Moricandia arvensis may be accounted for by metabolism of nitrate rather than by a state of C(3)-C(4) intermediacy.

Subdivision of C4-Pathway Species Based on Differing C4 Acid Decarboxylating Systems and Ultrastructural Features

A selection of C4 species was surveyed to determine the relationship between their content of C4 acid decarboxylating enzymes, the activities of several other enzymes implicated in the C4 pathway, and their anatomical and ultrastructural features. The species examined clearly fell into three groups according to whether they contained high levels of either NADP malic enzyme (EC 1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49) or NAD malic enzyme (EC 1.1 .1.39). The occurrence of high NADP malic enzyme activity was always associated with higher NADP malate dehydrogenase activity, while those species distinguished by high activities of either of the other two decarboxylases invariably contained high aspartate aminotransferase and alanine amino- transferase activities. Each of these decarboxylating enzymes was located in bundle sheath cells. NAD malic enzyme, but not phosphoenolpyruvate carboxykinase, was associated with mitochondria. Light and electron micrographs revealed differences between these groups with respect to the intracellular location of chloroplasts and mitochondria in bundle sheath cells, and the content and ultrastructure of mitochondria. The trend was for species with high NAD malic enzyme to contain the most mitochondria in the bundle sheath cells with apparently the most extensively developed cristae membrane systems. However, mitochondrial respiratory enzyme activities were similar for the three groups of species. The basic similarities and differences between the three groups of C4 plants distinguished by their differing C4 acid decarboxylating systems are discussed, and schemes for the probable photosynthetic reactions in bundle sheath cells are presented. A nomenclature to distinguish between these groups is proposed.