beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form β-carotene from NaH¹⁴CO₃ or [U-¹⁴C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-¹⁴C]-3-PGA or 3 to 6 millimolar NaH¹⁴CO₃ was fully sufficient to supply β-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles ¹⁴C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the β-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO₂ to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute β-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.     

Analysis of 1-deoxy-d-xylulose 5-phosphate synthase activity in Grey poplar leaves using isotope ratio mass spectrometry

Deoxy-xylulose phosphate synthase (DXS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway and it might regulate the metabolic flux in plastidic isoprenoid biosynthesis. We developed a sensitive assay suitable for plant extracts that is based on the decarboxylation of labeled pyruvate (1-¹³C)-PYR and detection of ¹³CO₂ by isotope ratio mass spectrometry. We tested our method investigating the DXS activity in poplar leaves. Apparent DXS activity showed Michaelis constants of 111 and 158 μM for glyceraldehyde phosphate and pyruvate, respectively; pH and temperature optima were found at pH 8.6 and 45 °C. DXS activity was inhibited when the competitive inhibitor β-fluoropyruvate was added to the reaction mixture. DXS activity strongly depended on leaf development with higher activity in young leaves and correlated fairly well with leaf isoprene emission potential. In mature poplar leaves, isoprene emission is the main metabolic sink of plastidic isoprenoid intermediates. Consequently, we found lower DXS activity in non-isoprene-emitting lines of poplar than in emitting plants as indicator of a lower demand of metabolic flux within the MEP pathway.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana

Isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. In this study, we investigated the cellular effects of depletion of IspE, a protein involved in the MEP pathway, using virus-induced gene silencing (VIGS). The IspE gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. The GFP fusion protein of IspE was targeted to chloroplasts. Reduction of IspE expression by VIGS resulted in a severe leaf yellowing phenotype. At the cellular level, depletion of IspE severely affected chloroplast development, dramatically reducing both the number and size of chloroplasts. Interestingly, mitochondrial development was also impaired, suggesting a possibility that the plastidic MEP pathway contributes to mitochondrial isoprenoid biosynthesis in leaves. A deficiency in IspE activity decreased cellular levels of the metabolites produced by the MEP pathway, such as chlorophylls and carotenoids, and stimulated expression of some of the downstream MEP pathway genes, particularly IspF and IspG. Interestingly, the IspE VIGS lines had significantly increased numbers of cells of reduced size in all leaf layers, compared with TRV control and other VIGS lines for the MEP pathway genes. The increased cell division in the IspE VIGS lines was particularly pronounced in the abaxial epidermal layer, in which the over-proliferated cells bulged out of the plane, making the surface uneven. In addition, trichome numbers dramatically increased and the stomata size varied in the affected tissues. Our results show that IspE deficiency causes novel developmental phenotypes distinct from the phenotypes of other MEP pathway mutants, indicating that IspE may have an additional role in plant development besides its role in isoprenoid biosynthesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Genbank accession number for IspE: ABO87658.     

Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway

Isopentenyl diphosphate (IPP) is the biological C₅ precursor of isoprenoids. By labeling experiments using [1-¹³C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (β-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.     

Biosynthesis,accumulation and emission of carotenoids, α-tocopherol,plastoquinone, and isoprene in leaves under high photosynthetic irradiance

The localization of isoprenoid lipids in chloroplasts, the accumulation of particular isoprenoids under high irradiance conditions, and channelling of photosynthetically fixed carbon into plastidic thylakoid isoprenoids, volatile isoprenoids, and cytosolic sterols are reviewed. During leaf and chloroplast development in spring plastidic isoprenoid biosynthesis provides primarily thylakoid carotenoids, the phytyl side-chain of chlorophylls and the electron carriers phylloquinone K1, alpha-tocoquinone and alpha-tocopherol, as well as the nona-prenyl side-chain of plastoquinone-9. Under high irradiance, plants develop sun leaves and high light (HL) leaves with sun-type chloroplasts that possess, besides higher photosynthetic CO2 assimilation rates, different quantitative levels of pigments and prenylquinones as compared to shade leaves and low light (LL) leaves. After completion of chloroplast thylakoid synthesis plastidic isoprenoid biosynthesis continues at high irradiance conditions, constantly accumulating alpha-tocopherol (alpha-T) and the reduced form of plastoquinone-9 (PQ-9H2) deposited in the steadily enlarging osmiophilic plastoglobuli, the lipid reservoir of the chloroplast stroma. In sun leaves of beech (Fagus) and in 3-year-old sunlit Ficus leaves the level of alpha-T and PQ-9 can exceed that of chlorophyll b. Most plants respond to HL conditions (sun leaves, leaves suddenly lit by the sun) with a 1.4-2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin), an enhanced operation of the xanthophyll cycle and an increase of beta-carotene levels. This is documented by significantly lower values for the weight ratio chlorophylls to carotenoids (range: 3.6-4.6) as compared to shade and LL leaves (range: 4.8-7.0). Many plant leaves emit under HL and high temperature conditions at high rates the volatile compounds isoprene (broadleaf trees) or methylbutenol (American ponderosa pines), both of which are formed via the plastidic 1-deoxy-D: -xylulose-phosphate/2-C-methylerythritol 5-phosphate (DOXP/MEP) pathway. Other plants by contrast, accumulate particular mono- and diterpenes. Under adequate photosynthetic conditions the chloroplastidic DOXP/MEP isoprenoid pathway essentially contributes, with its C5 isoprenoid precusors, to cytosolic sterol biosynthesis. The possible cross-talk between the two cellular isoprenoid pathways, the acetate/MVA and the DOXP/MEP pathways, that preferentially proceeds in a plastid-to-cytosol direction, is shortly discussed.     

The plastidic 3-phosphoglycerate → acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids,plastidic isoprenoids and fatty acids during chloroplast development

Earlier studies on the synthesis of C₃-derived amino acids, plastidic isoprenoids and fatty acids from CO₂ by isolated chloroplasts in the light indicate the presence of a complete, but low-capacity, chloroplast (chlp) 3-phosphoglycerate acetyl-CoA pathway which is predominantely active in immature (developing) chloroplasts (A. Heintze et al., 1990, Plant Physiol. 93, 1121–1127). In this paper, we demonstrate the activity of the enzymes involved i.e. chlp phosphoglycerate mutase, chlp enolase, chlp pyruvate kinase and chlp pyruvate-dehydrogenase complex (PDC), in the stroma of purified barley (Hordeum sativum L.) chloroplasts of different developmental stages. The chlp phosphoglycerate mutase was partially purified for the first time. The activities of the enzymes of this chlp pathway (except PDC) were about a magnitude lower than those of the cytosolic enzymes. The chlp PDC of barley was more active than that of spinach. The apparent K _m values of the enzymes of this pathway were about 100 M or lower except for the chlp phosphoglycerate mutase which had a K _m of 1.6–1.8 mM for 3-phospho-d-glycerate. Interestingly, no appreciable change in the activity of these enzymes was observed during maturation of the chloroplasts. In contrast, the activity of the reversible NADP⁺-glyceraldehyde 3-phosphate dehydrogenase increased about five times (from 140 to 590 nkat per g leaf dry weight). The following hypothesis is put forward to explain the regulation of carbon metabolism during chloroplast development: 3-phospho-d-glycerate is withdrawn from a common pool by the actions of 3-phosphoglycerate kinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase, the activity of which increases considerably during maturation of chloroplasts. This leads to an insufficient supply of 3-phospho-glycerate for the chlp phosphoglycerate mutase, which has a low affinity for its substrate.Abbreviations C₃ C₂₅ pathway 3-phospho-d-glycerate acetyl-CoA pathway - Chl chlorophyll - chlp chloroplast(ic) - GAP d-glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PDC pyruvate dehydrogenase complex - PEP phosphoenolpyruvate - 2- and 3-PGA 2- and 3-phospho-d-glycerate - U unit - mmol·min^t-1 (=16.67 nkat) This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG and Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie e. V., Frankfurt/Main, FRG, (scholarship to P.H.). The authors thank Dr. K.P. Heise (Institut für Biochemie der Pflanzen, Universität Göttingen, FRG) for the gas-liquid chromatography measurements, Gabriele Böl, Dietmar Budde, Daniel Gruber, Andreas Haaf, and Antje Wassmann (all Zentrum Biochemie, Medizinische Hochschule Hannover, FRG) and Kerstin Meereis, Martin Preiss, Uwe Schwanke (all Botanisches Institut, Tierärztliche Hochschule Hannover, FRG) for detailed and skillful work, Dr. Indra Willms-Hoff, Carola Leuschner and Dr. Christian L. Schmidt for constructive criticism, and Mrs. Saime Aydogdu for technical assistance.     

Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60

The reductive carboxylic acid cycle, the autotrophic pathway of CO₂ assimilation in prokaryotes (photosynthetic and nonphotosynthetic autotrophic bacteria), was investigated in Chlamydomonas reinhardtii F-60, an algal mutant lacking a complete photosynthetic carbon reduction pathway (C₃) due to a deficiency in phosphoribulokinase. Evidence was obtained consistent with the presence of the reductive carboxylic acid cycle in F-60. This conclusion is based on the fact that: (a) acetate approximately doubled CO₂ fixation in whole cells (4 micromoles per milligram chlorophyll per hour) and in chloroplasts (32 nanomoles per milligram chlorophyll per hour); and (b) pyruvate synthase, α-ketoglutarate synthase, and ATP-citrate lyase, three indicators of the cycle, were found in cell-free extracts.     

Differential positioning of chloroplasts in C4 mesophyll and bundle sheath cells

Chloroplast photorelocation movement is extensively studied in C₃ but not C₄ plants. C₄ plants have two types of photosynthetic cells: mesophyll and bundle sheath cells. Mesophyll chloroplasts are randomly distributed along cell walls, whereas bundle sheath chloroplasts are located close to the vascular tissues or mesophyll cells depending on the plant species. The cell-specific C₄ chloroplast arrangement is established during cell maturation, and is maintained throughout the life of the cell. However, only mesophyll chloroplasts can change their positions in response to environmental stresses. The migration pattern is unique to C₄ plants and differs from that of C₃ chloroplasts. in this mini-review, we highlight the cell-specific disposition of chloroplasts in C₄ plants and discuss the possible physiological significances.Key words: abscisic acid, aggregative movement, avoidance movement, blue light, bundle sheath cell, C₄ plant, chloroplast, cytoskeleton, environmental stress, mesophyll cellChloroplasts can change their intracellular positions to optimize photosynthetic activity and/or reduce photodamage occurring in response to light irradiation. On treating with high-intensity light, the chloroplasts move away from the light to minimize photodamage (avoidance response). Meanwhile, on irradiating with low-intensity light, they move toward the light source to maximize photosynthesis (accumulation response). These chloroplast-photorelocation movements are observed in a wide variety of plant species from green algae to seed plants,^1–3 although little attention has been paid to C₄ plants. There is a report stating that monocotyledonous C₄ plants showed changes in the light transmission of leaves in response to blue light,⁴ although the direction of migration of the chloroplasts is not described.C₄ plants have two types of photosynthetic cells: mesophyll (M) cells and bundle sheath (BS) cells, which have numerous well-developed chloroplasts. BS cells surround the vascular tissues, while M cells encircle the cylinders of the BS cells (Fig. 1). The C₄ dicarboxylate cycle of photosynthetic carbon assimilation is distributed between the two cell types, and acts as a CO₂ pump to concentrate CO₂ in the BS chloroplasts.^5,6 C₄ plants are divided into three subtypes on the basis of decarboxylating enzymes: NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvate carboxykinase. Although the M chloroplasts of all C₄ species are randomly distributed along the cell walls, BS chloroplasts are located either in a centripetal (close to the vascular tissue) or in a centrifugal (close to M cells) position, depending on the species (Fig. 1A).⁷ Thus, C₄ M and BS cells have different systems for chloroplast positioning: an M cell-specific system for dispersing chloroplasts and a BS cell-specific system for holding chloroplasts in a centripetal or centrifugal disposition.Open in a separate windowFigure 1The intracellular arrangement of chloroplasts in finger millet (Eleusine coracana), an NAD-ME-type C₄ plant. (A) Light micrograph of a transverse section of a leaf blade from a control plant. Bundle sheath (BS) cells surround the vascular tissues, while mesophyll (M) cells encircle the cylinders of the BS cells. BS chloroplasts are well developed, and are located in a centripetal position, whereas M chloroplasts are randomly distributed along the cell walls. B, bundle sheath cell; M, mesophyll cell; V, vascular bundle. (B) Transverse section of a leaf blade from a drought-stressed plant. Most M chloroplasts are aggregatively distributed toward the BS side, while the centripetal arrangement of BS chloroplasts is unchanged. (C and D) Transverse sections of leaf segments irradiated with blue light of intensity 500 µmol m⁻² s⁻¹ with or without 30 µM ABA for 8 h (C and D, respectively). The adaxial side of each leaf section (upper side in the photograph) was illuminated. In the absence of ABA, M chloroplasts exhibited avoidance movement on the illuminated side and aggregative movement on the opposite side. In the presence of ABA, aggregative movement was observed on both sides. Scale bars = 50 µm.     

Helper component‐proteinase enhances the activity of 1‐deoxy‐D‐xylulose‐5‐phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y‐infected tobacco

Virus‐infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus‐infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)‐infected tobacco. Using yeast two‐hybrid assays, we demonstrated an interaction between virus protein PVY helper component‐proteinase (HC‐Pro) and tobacco chloroplast protein 1‐deoxy‐D‐xylulose‐5‐phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull‐down assays. The Transket_pyr domain (residues 394–561) of NtDXS was required for interaction with HC‐Pro, while the N‐terminal region of HC‐Pro (residues 1–97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC‐Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC‐Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC‐Pro. Our study reveals a new role of HC‐Pro in the host plant metabolic system and will contribute to the study of host–virus relationships.     

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

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

Photosynthesis and Other Traits in Relation to Chloroplast Number during Soybean Leaf Senescence

Soon after attaining full expansion, soybean (Glycine max [L.] Merr.) leaves enter a senescence phase marked by decline in photosynthetic rate and the progressive loss of chloroplast activity and composition. Our primary goal was to determine if this loss could be accounted for by sequential degradation of whole chloroplasts or by simultaneous degeneration of all chloroplasts. Total photosynthesis (TPs) measured as ¹⁴CO₂ uptake, chloroplast number, ribulose 1,5-bisphosphate carboxylase activity, uncoupled photosynthetic electron transport activity, soluble protein content, and chlorophyll content declined progressively during the 37 days after full leaf expansion. During this period, chloroplast number per unit leaf area was constant for all genotypes studied. We conclude that leaf senescence may be a two-stage process wherein the first stage chloroplast activity and composition declines, but chloroplast numbers do not change. During a brief terminal stage (11 days in our experiment), whole chloroplasts may be lost as well. As a second objective we wished to determine if variation in single-leaf total photosynthetic rate among soybean cultivars is related to corresponding variation in chloroplast number and/or chloroplast activity/composition. By comparing the means for three cultivars known to have rapid leaf TPs and for the three known to have slow TPs, we found the former group to be superior to the latter for all the previously mentioned leaf physiological traits. This superiority was related primarily to differences in chloroplast number and only secondarily to differences in activity and composition per chloroplast.     

Effects of salt-tolerant rootstock grafting on ultrastructure,photosynthetic capacity,and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-scavenging system in chloroplasts of cucumber seedlings under NaCl stress

Plant growth, photosynthetic parameters, chloroplast ultrastructure, and the ascorbate-glutathione cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves were investigated. Grafted plants were grown hydroponically and were exposed to 0, 50, and 100 mM NaCl concentrations for 10 days. Under NaCl stress, the hydrogen peroxide (H₂O₂) content in cucumber chloroplasts increased, the chloroplast ultrastructure was damaged, and the gas stomatal conductance, intercellular CO₂ concentration, as well as shoot dry weight, plant height, stem diameter, leaf area, and leaf relative water content were inhibited, whereas these changes were less severe in rootstock-grafted plants. The activities of ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and dehydroascorbate reductase (DHAR EC 1.8.5.1) were higher in the chloroplasts of rootstock-grafted plants compared with those of self-grafted plants under 50 and 100 mM NaCl. Similar trends were shown in leaf net CO₂ assimilation rate and transpiration rate, as well as reduced glutathione content under 100 mM NaCl. Results suggest that rootstock grafting enhances the H₂O₂-scavenging capacity of the ascorbate–glutathione cycle in cucumber chloroplasts under NaCl stress, thereby protecting the chloroplast structure and improving the photosynthetic performance of cucumber leaves. As a result, cucumber growth is promoted.     

Chloroplast osmotic adjustment and water stress effects on photosynthesis

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψ_w) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψ_w reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψ_w may involve stromal volume maintenance.     

CHLOROPLAST SPECIALIZATION IN DICOTYLEDONS POSSESSING THE C4-DICARBOXYLIC ACID PATHWAY OF PHOTOSYNTHETIC CO2 FIXATION

The C₄-dicarboxylic-acid pathway of photosynthetic CO₂ fixation found in tropical grasses has recently been demonstrated in members of the Amaranthaceae and Chenopodiaceae. In the tropical grasses this CO₂-fixation pathway is correlated with specialized leaf anatomy and chloroplast structure. This investigation was undertaken to determine if leaf cells of some representatives of these other families had structural features similar to those of tropical grasses. The leaf anatomy of Amaranthus edulis and a variety of Atriplex species is very similar and it resembles that of grasses such as sugar cane. Prominent bundle sheaths are surrounded by a layer of palisade cells. Bundle-sheath cells of Am. edulis have large chloroplasts containing much starch, but the chloroplasts have grana. The palisade cells have much smaller chloroplasts containing very little starch. The bundle-sheath cell chloroplasts of At. lentiformis have grana, their profiles tend to be ovoid, and they contain abundant starch grains. The palisade cell chloroplasts contain little starch and their profiles are discoid. The bundle-sheath cells of both species contain mitochondria which are much larger than those in the palisade cells. The chloroplasts in both types of cells in both species have a highly developed peripheral reticulum. This reticulum is composed of anastomosing tubules which are contiguous with the inner plastid membrane. The leaf anatomy and cell ultrastructure of these dicots are similar to those of the tropical grasses possessing this new photosynthetic carbon-fixation pathway. These morphological features are interpreted as adaptations for the rapid transport of precursors and end products of photosynthesis. A hypothesis is presented stating that the unique morphological and biochemical characters of these plants represent adaptations for efficient and rapid carbon fixation in environments where water stress frequently limits photosynthesis.     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

Two major metabolic factors for an efficient NADP-malic enzyme type C4 photosynthesis

Compared to the large number of studies focused on the factors controlling C₃ photosynthesis efficiency, there are relatively fewer studies of the factors controlling photosynthetic efficiency in C₄ leaves. Here, we used a dynamic systems model of C₄ photosynthesis based on maize (Zea mays) to identify features associated with high photosynthetic efficiency in NADP-malic enzyme (NADP-ME) type C₄ photosynthesis. We found that two additional factors related to coordination between C₄ shuttle metabolism and C₃ metabolism are required for efficient C₄ photosynthesis: (1) accumulating a high concentration of phosphoenolpyruvate through maintaining a large PGA concentration in the mesophyll cell chloroplast and (2) maintaining a suitable oxidized status in bundle sheath cell chloroplasts. These identified mechanisms are in line with the current cellular location of enzymes/proteins involved in the starch synthesis, the Calvin–Benson cycle and photosystem II of NADP-ME type C₄ photosynthesis. These findings suggested potential strategies for improving C₄ photosynthesis and engineering C₄ rice.

High levels of PGA and PEP in mesophyll cell chloroplasts and a suitable oxidation state in bundle sheath cell chloroplasts are the requirements for efficient C₄ photosynthesis.     

Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis

The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 μmoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD⁺, and coenzyme-A and the products as NADH, CO₂, and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.     

Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO₃⁻. When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C₄ species (79-98% inhibition for a range of seven C₄ species), but only moderately in C₃ species (12-46% for four C₃ species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C₄) net photosynthesis to intercellular pCO₂ showed that carboxylation efficiency, as well as the CO₂ saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C₄ species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO₂ over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO₂ by the bundle sheath cells makes any significant contribution to photosynthetic CO₂ assimilation in C₄ species. The results support the concept that C₄-selective herbicides may be developed based on inhibitors of C₄ pathway reactions.     

Acclimation of photosynthesis to low leaf water potentials

Photosynthesis is reduced at low leaf water potentials (Ψ_l) but repeated water deficits can decrease this reduction, resulting in photosynthetic acclimation. The contribution of the stomata and the chloroplasts to this acclimation is unknown. We evaluated stomatal and chloroplast contributions when soil-grown sunflower (Helianthus annuus L.) plants were subjected to water deficit pretreatments for 2 weeks. The relationship between photosynthesis and Ψ_l, determined from gas-exchange and isopiestic thermocouple psychometry, was shifted 3 to 4 bars towards lower Ψ_l, in pretreated plants. Leaf diffusive resistance was similarly affected. Chloroplast activity, demonstrated in situ with measurements of quantum yield and the capacity to fix CO₂ at all partial pressures of CO₂, and in vitro by photosystem II activity of isolated organelles, was inhibited at low Ψ_l but less in pretreated plants than in control plants. The magnitude of this inhibition indicated that decreases in chloroplast activity contributed more than closure of stomata both to losses in photosynthesis and to the acclimation of photosynthesis to low Ψ_l.