Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C₄ leaves is a key requirement for C₄ photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C₄ species using our PD quantification method, which combines three‐dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP‐ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 μmol m^?2 s^?1), and high light (1,000 μmol m^?2 s^?1), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C₄ leaf PD density depended on plant age and species. The high light treatment resulted in two to four‐fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M‐BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C₄ leaf PD formation to growth irradiance.     

Coleataenia prionitis,a C4-like species in the Poaceae

C₄-like plants represent the penultimate stage of evolution from C₃ to C₄ plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C₄ plant, its leaf anatomy and gas exchange traits suggest that it may be a C₄-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C₄ Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C₄-like intermediate plants rather than C₄ plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO₂. These data demonstrate that C. prionitis is not a true C₄ plant but should be considered as a C₄-like plant.

     

Mechanism of c(4) photosynthesis: the size and composition of the inorganic carbon pool in bundle sheath cells

We sought to characterize the inorganic carbon pool (CO₂ plus HCO₃⁻) formed in the leaves of C₄ plants when C₄ acids derived from CO₂ assimilation in mesophyll cells are decarboxylated in bundle sheath cells. The size and kinetics of labeling of this pool was determined in six species representative of the three metabolic subgroups of C₄ plants. The kinetics of labeling of the inorganic carbon pool of leaves photosynthesizing under steady state conditions in ¹⁴CO₂ closely paralleled those for the C-4 carboxyl of C₄ acids for all species tested. The inorganic carbon pool size, determined from its ¹⁴C content at radioactivity saturation, ranged between 15 and 97 nanomoles per milligram of leaf chlorophyll, giving estimated concentrations in bundle sheath cells of between 160 and 990 micromolar. The size of the pool decreased, together with photosynthesis, as light was reduced from 900 to 95 microeinsteins per square meter per second or as external CO₂ was reduced from 400 to 98 microliters per liter. A model is developed which suggests that the inorganic carbon pool existing in the bundle sheath cells of C₄ plants during steady state photosynthesis will comprise largely of CO₂; that is, CO₂ will only partially equlibrate with bicarbonate. This predominance of CO₂ is believed to be vital for the proper functioning of the C₄ pathway.     

C4 plants use fluctuating light less efficiently than do C3 plants: a study of growth,photosynthesis and carbon isotope discrimination

Plants in the field are commonly exposed to fluctuating light intensity, caused by variable cloud cover, self‐shading of leaves in the canopy and/or leaf movement due to turbulence. In contrast to C₃ plant species, only little is known about the effects of dynamic light (DL) on photosynthesis and growth in C₄ plants. Two C₄ and two C₃ monocot and eudicot species were grown under steady light or DL conditions with equal sum of daily incident photon flux. We measured leaf gas exchange, plant growth and dry matter carbon isotope discrimination to infer CO₂ bundle sheath leakiness in C₄ plants. The growth of all species was reduced by DL, despite only small changes in steady‐state gas exchange characteristics, and this effect was more pronounced in C₄ than C₃ species due to lower assimilation at light transitions. This was partially attributed to increased bundle sheath leakiness in C₄ plants under the simulated lightfleck conditions. We hypothesize that DL leads to imbalances in the coordination of C₄ and C₃ cycles and increasing leakiness, thereby decreasing the quantum efficiency of photosynthesis. In addition to their other constraints, the inability of C₄ plants to efficiently utilize fluctuating light likely contributes to their absence in such environments as forest understoreys.     

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.     

Characteristics of photosynthesis in maize leaves (С<Subscript>4</Subscript> plants) upon changes in the level of illuminance and nitrate nutrition

We studied assimilation of ¹⁴СО₂ and distribution of ¹⁴С among the products of 3-min-long photosynthesis of maize (Zea mays L.) leaves. The day before the experiment, half of the plants were fertilized with Ca(NO₃)₂ (1 g/L of water) at a rate of 6 L/m². Five days before the experiment, some plants were shaded for adaptation (illuminance was reduced by 50%). On the day of the experiment (before the application of ¹⁴СО₂), several shaded plants were exposed to direct sunlight for 3 min, and some plants grown at full light (light plants) were shaded for 3 min (illuminance of 50%). Unfertilized plants adapted for 5 days to shading showed photosynthesis of 75.9% of control level (full light). If light plants were transferred to shading for 3 min, their photosynthesis decreased to 42.1%. In plants shaded for 5 days and then transferred to full light, photosynthesis in 3 min was 96.3% of control level. At full light, fertilization with nitrate boosted photosynthesis to 132.6% as compared with control material, but photosynthesis decreased to 43.5 and 65.4% of control level in plants shaded for 5 days and those shaded for 3 min, respectively. At the same time, the plants shaded for 5 days and then exposed for 3 min to full light restored photosynthesis to almost control level (95.5%). Analysis of ¹⁴С distribution among the products of 3-min-long photosynthesis showed that, the same as in C₃ plants, a decrease in illuminance (especially a sudden one) in maize reduced the ratio between labeled sucrose and hexoses and elevates incorporation of ¹⁴С into malate, which indicated that its consumption in bundle sheath cells was suppressed. A decrease in the ratio between labeled sucrose and hexoses became more pronounced under the influence of nitrates with this effect also occurring in transport products of photosynthesis (20 cm below ¹⁴С-providing leaf area). In plants fertilized with nitrates, radioactivity of sucrose (% of radioactivity of soluble compounds) decreased in all the types of illumination. When illuminance was suddenly reduced for 3 min, incorporation of ¹⁴С into sucrose was 21.5 against 51.2% in light plants, and radioactivity of aspartate and malate sharply rose to 13.7 and 26.1% (against 2.1 and 8.9% in control material). Incorporation of ¹⁴С into compounds of glycolate pathway was low (less than 2.5%), but it was somewhat greater in nitrate plants. We concluded that the same mechanism of interaction between stomatal apparatus of leaf epidermis, invertase of mesophyll apoplast, and photosynthetic metabolism of carbon with electron flux via electron transport chain in chloroplasts of bundle sheath cells, which governs the rate of photosynthesis and assimilate export from the leaf but is triggered by the extent of consumption in the bundle sheath cells of C₄ acids produced in the mesophyll operates in C₄ plants (the same as in C₃ plants).     

C4 photosynthetic carbon metabolism in the leaves of aromatic tropical grasses — I. Leaf anatomy,CO2 compensation point and CO2 assimilation

A few species of Cymbopogon and Vetiveria are potentially important tropical grasses producing essential oils. In the present study, we report on the leaf anatomy and photosynthetic carbon assimilation in five species of Cymbopogon and Vetiveria zizanioides. Kranz-type leaf anatomy with a centrifugal distribution of chloroplasts and exclusive localization of starch in the bundle sheath cells were common among the test plants. Besides the Kranz leaf anatomy, these grasses displayed other typical C₄ characteristics including a low (0–5 µl/l) CO₂ compensation point, lack of light saturation of CO₂ uptake at high photon flux densities, high temperature (35°C) optimum of net photosynthesis, high rates of net photosynthesis (55–67 mg CO₂ dm^-2 leaf area h^-1), little or no response of net photosynthesis to atmospheric levels of O₂ and high leaf ¹³C/¹²C ratios. The biochemical studies with ¹⁴CO₂ indicated that the leaves of the above plant species synthesize predominantly malate during short term (5 s) photosynthesis. In pulse-chase experiments it was shown that the synthesis of 3-phosphoglycerate proceeds at the expense of malate, the major first formed product of photosynthesis in these plant species.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

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

Images 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.     

Metabolic regulation of carbon flux during C4 photosynthesis

The potential for glycolate and glycine metabolism and the mechanism of refixation of photorespiratory CO₂ in leaves of C₄ plants were studied by parallel inhibitor experiments with thin leaf slices, different leaf cell types and isolated mitochondria of C₃ and C₄ Panicum species. CO₂ evolution by leaf slices of P. bisulcatum, a C₃ species, fed glycolate or glycine was light-independent and O₂-sensitive. The C₄ P. maximum and P. miliaceum leaf slices fed glycolate or glycine evolved CO₂ in the dark but not in the light. In C₄ species, dark CO₂ evolution was abolished by the addition of phosphoenolpyruvate (PEP)⁴. The addition of maleate, a PEP carboxylase inhibitor, resulted in photorespiratory CO₂ efflux by C₄ leaf slices in the light also. However, PEP and maleate had no effect on either glycolate-dependent O₂ uptake by the C₄ leaf slices or on glycolate and glycine metabolism in C₃ leaf slices. The rate of photorespiratory CO₂ evolution in the C₃ Panicum species was 3 times higher than that observed with the C₄ species. The ratio of glycolate-dependent CO₂ evolution to O₂ uptake in both groups was 1:2. Isolated C₄ mesophyll protoplasts or their mitochondria did not metabolize glycolate or glycine. However, both C₃ mesophyll protoplasts and C₄ bundle sheath strands readily metabolized glycolate and glycine in a light-independent, O₂-sensitive manner, and the addition of PEP or maleate had no effect. C₄ bundle sheath- and C₃-mitochondria were capable of oxidizing glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. C₄ bundle sheath- and C₃-mitochondrial glycine decarboxylation was stimulated by oxaloacetate and NAD had no effect. In marked contrast, mitochondria isolated from C₄ mesophyll cells were incapable of oxidizing or decarboxylating added glycine. The results suggest that in leaves of C₄ plants bundle sheath cells are the primary site of O₂-sensitive photorespiratory CO₂ evolution and the PEP carboxylase present in the mesophyll cells has the Potential for efficiently refixing CO₂ before it escapes out of the leaf. The relative role of the PEP carboxylase mediated CO₂ pump and reassimilation of photorespiratory CO₂ are discussed in relation to the apparent lack of photorespiration in leaves of C₄ species.Abbreviations BSA bovine serum albumin - Chl chlorophyll - PEP phosphoenolpyruvate - Rbu-P ₂ ribulose 1,5-bisphosphate - Rib-5-P ribose-5-phosphate - Ru-5-P ribuluse-5-phosphate - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone Journal Series Paper, New Jersey Agricultural Experiment Station     

C4 photosynthesis in C3 rice: a theoretical analysis of biochemical and anatomical factors

Engineering C₄ photosynthesis into rice has been considered a promising strategy to increase photosynthesis and yield. A question that remains to be answered is whether expressing a C₄ metabolic cycle into a C₃ leaf structure and without removing the C₃ background metabolism improves photosynthetic efficiency. To explore this question, we developed a 3D reaction diffusion model of bundle‐sheath and connected mesophyll cells in a C₃ rice leaf. Our results show that integrating a C₄ metabolic pathway into rice leaves with a C₃ metabolism and mesophyll structure may lead to an improved photosynthesis under current ambient CO₂ concentration. We analysed a number of physiological factors that influence the CO₂ uptake rate, which include the chloroplast surface area exposed to intercellular air space, bundle‐sheath cell wall thickness, bundle‐sheath chloroplast envelope permeability, Rubisco concentration and the energy partitioning between C₃ and C₄ cycles. Among these, partitioning of energy between C₃ and C₄ photosynthesis and the partitioning of Rubisco between mesophyll and bundle‐sheath cells are decisive factors controlling photosynthetic efficiency in an engineered C₃–C₄ leaf. The implications of the results for the sequence of C₄ evolution are also discussed.     

Photosynthetic Characteristics of Cassava (Manihot esculenta Crantz), a C3 Species with Chlorenchymatous Bundle Sheath Cells

Cultivars of cassava, Manihot esculenta Crantz, were studiedto determine the mechanism of photosynthetic carbon assimilationin this species. The results, contrary to recent reports, indicatethat cassava is a C₃ plant based on a number of physiologicaland biochemical photosynthetic characteristics. The CO₂ compensationpoints among 10 cassava cultivars ranged from 55 to 62 µlliter^–1, which was typical for C₃ plants including castorbean, a member of the same family (Euphorbiaceae). The initialproducts of photosynthesis in cassava are C₃-like; the activitiesof several key C₄ enzymes in cassava are low and similar tothose of C₃ plants. Data on the rates of photosynthesis perunit of leaf area and the photosynthetic response of cassavato CO₂ is also consistent with C₃ photosynthesis. Cassava hasa distinctive chlorenchymatous vascular bundle sheath locatedbelow a single layer of palisade cells. Unlike C₃-C₄ intermediatesand C₄ species, the bundle sheaths of cassava are not surroundedby mesophyll cells. The bundle sheath cells which occur at highfrequency in cassava may function in both photosynthesis andtransport of photosynthates in the leaf. (Received July 31, 1990; Accepted September 25, 1990)     

The efficiency of C4 photosynthesis under low light conditions in Zea mays,Miscanthus x giganteus and Flaveria bidentis

The efficiency of C₄ photosynthesis in Zea mays, Miscanthus x giganteus and Flaveria bidentis in response to light was determined using measurements of gas exchange, ¹³CO₂ photosynthetic discrimination, metabolite pools and spectroscopic assays, with models of C₄ photosynthesis and leaf ¹³CO₂ discrimination. Spectroscopic and metabolite assays suggested constant energy partitioning between the C₄ and C₃ cycles across photosynthetically active radiation (PAR). Leakiness (φ), modelled using C₄ light‐limited photosynthesis equations (φ_mod), matched values from the isotope method without simplifications (φ_is) and increased slightly from high to low PAR in all species. However, simplifications of bundle‐sheath [CO₂] and respiratory fractionation lead to large overestimations of φ at low PAR with the isotope method. These species used different strategies to maintain similar φ. For example, Z. mays had large rates of the C₄ cycle and low bundle‐sheath cells CO₂ conductance (g_bs). While F. bidentis had larger g_bs but lower respiration rates and M. giganteus had less C₄ cycle capacity but low g_bs, which resulted in similar φ. This demonstrates that low g_bs is important for efficient C₄ photosynthesis but it is not the only factor determining φ. Additionally, these C₄ species are able to optimize photosynthesis and minimize φ over a range of PARs, including low light.     

The efficiency of the CO2-concentrating mechanism during single-cell C4 photosynthesis

The photosynthetic efficiency of the CO₂‐concentrating mechanism in two forms of single‐cell C₄ photosynthesis in the family Chenopodiaceae was characterized. The Bienertioid‐type single‐cell C₄ uses peripheral and central cytoplasmic compartments (Bienertia sinuspersici), while the Borszczowioid single‐cell C₄ uses distal and proximal compartments of the cell (Suaeda aralocaspica). C₄ photosynthesis within a single‐cell raises questions about the efficiency of this type of CO₂‐concentrating mechanism compared with the Kranz‐type. We used measurements of leaf CO₂ isotope exchange (Δ¹³C) to compare the efficiency of the single‐cell and Kranz‐type forms of C₄ photosynthesis under various temperature and light conditions. Comparisons were made between the single‐cell C₄ and a sister Kranz form, S. eltonica[NAD malic enzyme (NAD ME) type], and with Flaveria bidentis[NADP malic enzyme (NADP‐ME) type with Kranz Atriplicoid anatomy]. There were similar levels of Δ¹³C discrimination and CO₂ leakiness (?) in the single‐cell species compared with the Kranz‐type. Increasing leaf temperature (25 to 30 °C) and light intensity caused a decrease in Δ¹³C and ? across all C₄ types. Notably, B. sinuspersici had higher Δ¹³C and ? than S. aralocaspica under lower light. These results demonstrate that rates of photosynthesis and efficiency of the CO₂‐concentrating mechanisms in single‐cell C₄ plants are similar to those in Kranz‐type.     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

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.     

Localisation of sucrose-phosphate synthase and starch in leaves of C4 plants

The activity and intercellular distribution of sucrose-phosphate synthase (SPS; EC 2.4.1.14) were determined in fully expanded leaves from a range of C₄ plants. In Zea mays L. and Atriplex spongiosa F. Muell., SPS was located almost exclusively in the mesophyll cells. In other species, SPS was found in both cell types, with the activity in the bundle sheath cells ranging from 5% of the total leaf activity in Echinochloa crus-galli (L.) Beauv. to 35% in Sorghum bicolor Moench. At the end of the light period, starch was found only in the bundle sheath cells in all of the species examined. There appears to be little correlation between C₄-acid decarboxylation type and the location of sucrose and starch synthesis in the leaves of C₄ plants. Received: 18 October 1996 / Accepted: 20 November 1996     

The influence of light quality on C4 photosynthesis under steady-state conditions in Zea mays and Miscanthus×giganteus: changes in rates of photosynthesis but not the efficiency of the CO2 concentrating mechanism

Differences in light quality penetration within a leaf and absorption by the photosystems alter rates of CO₂ assimilation in C₃ plants. It is also expected that light quality will have a profound impact on C₄ photosynthesis due to disrupted coordination of the C₄ and C₃ cycles. To test this hypothesis, we measured leaf gas exchange, ¹³CO₂ discrimination (Δ¹³C), photosynthetic metabolite pools and Rubisco activation state in Zea mays and Miscanthus × giganteus under steady‐state red, green, blue and white light. Photosynthetic rates, quantum yield of CO₂ assimilation, and maximum phosphoenolpyruvate carboxylase activity were significantly lower under blue light than white, red and green light in both species. However, similar leakiness under all light treatments suggests the C₄ and C₃ cycles were coordinated to maintain the photosynthetic efficiency. Measurements of photosynthetic metabolite pools also suggest coordination of C₄ and C₃ cycles across light treatments. The energy limitation under blue light affected both C₄ and C₃ cycles, as we observed a reduction in C₄ pumping of CO₂ into bundle‐sheath cells and a limitation in the conversion of C₃ metabolite phosphoglycerate to triose phosphate. Overall, light quality affects rates of CO₂ assimilation, but not the efficiency of CO₂ concentrating mechanism.     

Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO₂ compensation point and the inhibition of photosynthesis by 21% O₂ were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C₄ cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO₂ compensation point and the degree of O₂ inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO₂ between Rubisco of the C₃ pathway and phosphoenolpyruvate carboxylase of the C₄ cycle was determined by exposing leaves to ¹⁴CO₂ for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ~94% of the CO₂ was fixed by the C₄ cycle in high light grown plants, versus ~78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C₄ photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C₄-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.     

Photosynthetic pathway alters xylem structure and hydraulic function in herbaceous plants

Plants using the C₄ photosynthetic pathway have greater water use efficiency (WUE) than C₃ plants of similar ecological function. Consequently, for equivalent rates of photosynthesis in identical climates, C₄ plants do not need to acquire and transport as much water as C₃ species. Because the structure of xylem tissue reflects hydraulic demand by the leaf canopy, a reduction in water transport requirements due to C₄ photosynthesis should affect the evolution of xylem characteristics in C₄ plants. In a comparison of stem hydraulic conductivity and vascular anatomy between eight C₃ and eight C₄ herbaceous species, C₄ plants had lower hydraulic conductivity per unit leaf area (K_L) than C₃ species of similar life form. When averages from all the species were pooled together, the mean K_L for the C₄ species was 1.60 × 10^?4 kg m^?1 s^?1 MPa^?1, which was only one‐third of the mean K_L of 4.65 × 10^?4 kg m^?1 s^?1 MPa^?1 determined for the C₃ species. The differences in K_L between C₃ and C₄ species corresponded to the two‐ to three‐fold differences in WUE observed between C₃ and C₄ plants. In the C₄ species from arid regions, the difference in K_L was associated with a lower hydraulic conductivity per xylem area, smaller and shorter vessels, and less vulnerable xylem to cavitation, indicating the C₄ species had evolved safer xylem than the C₃ species. In the plants from resource‐rich areas, such as the C₄ weed Amaranthus retroflexus, hydraulic conductivity per xylem area and xylem anatomy were similar to that of the C₃ species, but the C₄ plants had greater leaf area per xylem area. The results indicate the WUE advantage of C₄ photosynthesis allows for greater flexibility in hydraulic design and potential fitness. In resource‐rich environments in which competition is high, an existing hydraulic design can support greater leaf area, allowing for higher carbon gain, growth and competitive potential. In arid regions, C₄ plants evolved safer xylem, which can increase survival and performance during drought events.