Light-dependent development of single cell C4 photosynthesis in cotyledons of Borszczowia aralocaspica (Chenopodiaceae) during transformation from a storage to a photosynthetic organ

BACKGROUND AND AIMS: Previous work has shown that Borszczowia aralocaspica (Chenopodiaceae) accomplishes C4 photosynthesis in a unique, polarized single-cell system in leaves. Mature cotyledons have the same structure as leaves, with chlorenchyma cells having biochemical polarization of dimorphic chloroplasts and C4 functions at opposite ends of the cell. KEY RESULTS: Development of the single-celled C4 syndrome in cotyledons was characterized. In mature seeds, all cell layers are already present in the cotyledons, which contain mostly lipids and little starch. The incipient chlorenchyma cells have a few plastids towards the centre of the cell. Eight days after germination and growth in the dark, small plastids are evenly distributed around the periphery of the expanding cells. Immunolocalization studies show slight labelling of Rubisco in plastids in seeds, including chlorenchyma, hypodermal and water storage, but not epidermal, cells. After imbibition and 8 d of growth in the dark labelling for Rubisco progressively increased, being most prominent in chlorenchyma cells. There was no immunolabelling for the plastid C4 enzyme pyruvate, Pi dikinase under these conditions. Cotyledons developing in light show formation of chlorenchyma tissue, induction of the cytosolic enzyme phosphoenolpyruvate carboxylase and development of dimorphic chloroplasts at opposite ends of the cells. Proximal chloroplasts have well-developed grana, store starch and contain Rubisco; those located distally have reduced grana, lack starch and contain pyruvate, Pi dikinase. CONCLUSIONS: The results show cotyledons developing in the dark have a single structural plastid type which expresses Rubisco, while light induces formation of dimorphic chloroplasts from the single plastid pool, synthesis of C4 enzymes, and biochemical and structural polarization leading to the single-cell C4 syndrome.     

Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae)

Kranz anatomy, with its separation of elements of the C4 pathway between two cells, has been an accepted criterion for function of C4 photosynthesis in terrestrial plants. However, Bienertia cycloptera (Chenopodiaceae), which grows in salty depressions of Central Asian semi-deserts, has unusual chlorenchyma, lacks Kranz anatomy, but has photosynthetic features of C4 plants. Its photosynthetic response to varying CO2 and O2 is typical of C4 plants having Kranz anatomy. Lack of night-time CO2 fixation indicates it is not acquiring carbon by Crassulacean acid metabolism. This species exhibits an independent, novel solution to function of the C4 mechanism through spatial compartmentation of dimorphic chloroplasts, other organelles and photosynthetic enzymes in distinct positions within a single chlorenchyma cell. The chlorenchyma cells have a large, spherical central cytoplasmic compartment interconnected by cytoplasmic channels through the vacuole to the peripheral cytoplasm. This compartment is filled with mitochondria and granal chloroplasts, while the peripheral cytoplasm apparently lacks mitochondria and has grana-deficient chloroplasts. Immunolocalization studies show enzymes compartmentalized selectively in the CC compartment, including Rubisco in chloroplasts, and NAD-malic enzyme and glycine decarboxylase in mitochondria, whereas pyruvate, Pi dikinase of the C4 cycle is localized selectively in peripheral chloroplasts. Phosphoenolpyruvate carboxylase, a cytosolic C4 cycle enzyme, is enriched in the peripheral cytoplasm. Our results show Bienertia utilizes strict compartmentation of organelles and enzymes within a single cell to effectively mimic the spatial separation of Kranz anatomy, allowing it to function as a C4 plant having suppressed photorespiration; this raises interesting questions about evolution of C4 mechanisms.     

Differentiation of cellular and biochemical features of the single-cell C4 syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae)

The terrestrial plant Bienertia cycloptera has been shown to accomplish C(4) photosynthesis within individual chlorenchyma cells by spatially separating the phases of carbon assimilation into distinct peripheral and central compartments. In this study, anatomical, physiological, and biochemical techniques were used to determine how this unique compartmentation develops. Western blots show ribulose-1,5-bisphosphate carboxylase (Rubisco) (chloroplastic) is present in the youngest leaves and increases during development, while levels of C(4) enzymes-pyruvate,Pi dikinase (chloroplastic), phosphoenolpyruvate carboxylase (PEPC) (cytosol), and NAD-malic enzyme (mitochondrial)-increase later in development. Immunolocalization confirmed this for Rubisco and PEPC. The youngest chlorenchyma cells have a central nucleus surrounded by monomorphic granal chloroplasts containing Rubisco. Later stages show progressive development of a central cytoplasmic compartment enriched with chloroplasts and mitochondria and of a peripheral cytoplasm with chloroplasts. A complex reticulum of connections between the compartments also developed and was characterized. δ(13)C isotope analyses show mature leaves have distinct C(4)-type isotope composition, while the composition in younger leaves is "C(4)-like." Based on the results, this form of single-cell C(4) photosynthesis develops from a common pool of organelles through partitioning to separate compartments, and the development of biochemically and ultrastructurally dimorphic chloroplasts.     

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.     

Development of biochemical specialization and organelle partitioning in the single-cell C4 system in leaves of Borszczowia aralocaspica (Chenopodiaceae)

The terrestrial plant Borszczowia aralocaspica (Chenopodiaceae) has recently been shown to contain the entire C(4) photosynthesis mechanism within individual, structurally and biochemically polarized chlorenchyma cells rather than in a dual cell system, as has been the paradigm for this type of carbon fixation (Nature 414: 543-546, 2001). Analysis of carbon isotope composition and (14)CO(2) fixation shows that photosynthesis and growth of B. aralocaspica occurs through carbon acquired by C(4) photosynthesis. The development of this unique single-cell C(4) system in chlorenchyma cells was studied by analysis of young (0.2-0.3 cm length), intermediate (ca. 0.5-0.6 cm length), and mature leaves (ca. 3 cm length). The length of chlorenchyma cells approximately doubles from young to intermediate and again from intermediate to the mature leaf stage. In young chlorenchyma cells, there is a single type of chloroplast; the chloroplasts are evenly distributed throughout the cytosol, and all contain starch and rubisco. During leaf development, the activities of phosphoenolpyruvate carboxylase (PEPC; which is cytosolic), rubisco, and pyruvate,Pi dikinase (PPDK) increase on a chlorophyll basis. As leaves mature, chloroplasts differentiate into two distinct structural and biochemical types that are spatially separated into the proximal and distal parts of the cell (the proximal end being closest to the center of the leaf). The early stages of this polarization are observed in intermediate leaves, and the polarization is fully developed in mature leaves. The chloroplasts in the distal ends of the cell have reduced grana and little starch, while those at the proximal ends have well-developed grana and abundant starch. In mature leaves, PPDK is expressed in chloroplasts at the distal end of the cells, while rubisco and adenosine diphosphate glucose (ADPG) pyrophosphorylase are selectively expressed in chloroplasts at the proximal end of the cell. Mitochondrial polarization also occurs during development as nicotinamide-adenine dinucleotide phosphate-malic enzyme (NAD-ME) and the photorespiratory enzyme glycine decarboxylase are expressed in mature but not young leaves and are localized in mitochondria at the proximal end of the cells. The data show that single-cell C(4) develops from a single pool of identical organelles that develop differential biochemical functions and spatial partitioning in the cell during maturation.     

Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays)

Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C₄ plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C₄ photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP⁺-malate dehydrogenase proceeded more rapidly than photosynthesis.     

Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici

Bienertia sinuspersici is a land plant known to perform C(4) photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C(4) cycle. Western-blot analyses indicated that the CC has Rubisco from the C(3) cycle, the C(4) decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C(3) cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C(4) and C(3) cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C(4) species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C(4) species.     

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30°C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C₄ plants.

Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

     

CO(2) Assimilation and Activities of Photosynthetic Enzymes in High Chlorophyll Fluorescence Mutants of Maize Having Low Levels of Ribulose 1,5-Bisphosphate Carboxylase

Photosynthetic properties were examined in several hcf (high chlorophyll fluorescence 11, 21, 42 and 45) nuclear recessive mutants of maize which were previously found to have normal photochemistry and low CO₂ fixation. Mutants usually either died after depletion of seed reserves (about 18 days after planting), or survived with slow growth up to 7 or 8 weeks. Both the activity and quantity of ribulose 1,5-bisphosphate carboxylase (Rubisco) were low in the mutants (5-25% of the normal siblings on a leaf area basis) and the loss of Rubisco tended to parallel the reduction in photosynthetic capacity. The Rubisco content in the mutants was often marginal for photosynthetic carbon gain, with some leaves and positions along a leaf having no net photosynthesis, while other leaves had a low carbon gain. Conversely, the activities of C₄ cycle enzymes, phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malate dehydrogenase, and NADP-malic enzyme, were the same or only slightly reduced compared to the normal siblings. The mutants had about half as much chlorophyll content per leaf area as the normal green plants. However, the Rubisco activity in the mutants was low on both a leaf area and chlorophyll basis. Low Rubisco activity and lower chlorophyll content may both contribute to the low rates of photosynthesis in the mutants on a leaf area basis.     

Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate,Pi dikinase

Pyruvate,Pi dikinase regulatory protein (PDRP) has been highly purified from maize leaves, and its role in catalyzing both ADP-mediated inactivation (due to phosphorylation of a threonine residue) and Pi-mediated activation (due to dephosphorylation by phosphorolysis) of pyruvate,Pi dikinase has been confirmed. These reactions account for the dark/light-mediated regulation of pyruvate,Pi dikinase observed in the leaves of C4 plants. During purification to apparent homogeneity the ratio of these two activities remained constant. The molecular weight of the native PDRP was about 180,000 at pH 8.3 and 90,000 at pH 7.5. Its monomeric molecular weight was 45,000. It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation. Michaelis constants for orthophosphate and the above form of active pyruvate,Pi dikinase were determined, as well as the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi. For the inactivation reaction, Km values were 1.2 microM for the active pyruvate,Pi dikinase and 52 microM for ADP. CDP and GDP but not UDP could substitute for ADP. The inactivation reaction is inhibited by inactive pyruvate,Pi dikinase competitively with respect to both active pyruvate,Pi dikinase and ADP. Both the activation and inactivation reactions catalyzed by PDRP have a broad pH optimum between 7.8 and 8.3. The results are discussed in terms of the likely mechanism of dark/light regulation of pyruvate,Pi dikinase in vivo.     

Cloning of cDNA for pyruvate, Pi dikinase from maize leaves.

To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E. coli, strain RR1. Colonies were screened by sequential hybridization with 32P-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)+RNA fractionated by sucrose density centrifugation. Among the clones bearing cDNA homologous to high molecular weight poly(A)+RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf. Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits. Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.     

Comparison of leaf structure and photosynthetic characteristics of C3 and C4 Alloteropsis semialata subspecies

Alloteropsis semialata (R. Br.) Hitchcock includes both C3 and C4 subspecies: the C3 subspecies eckloniana and the C4 subspecies semialata. We examined the leaf structural and photosynthetic characteristics of these plants. A. semialata ssp. semialata showed high activities of photosynthetic enzymes involved in phosphoenolpyruvate carboxykinase-type C4 photosynthesis and an anomalous Kranz anatomy. Phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase and glycine decarboxylase (GDC) were compartmentalized between the mesophyll (M) and inner bundle sheath cells, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in both cells. A. semialata ssp. eckloniana also showed an anomalous non-Kranz anatomy, in which the mestome sheath cells included abundant chloroplasts and mitochondria. Rubisco and GDC accumulated densely in the M and mestome sheath cells, whereas the levels of C4 enzymes were low. The activity levels of photo-respiratory enzymes in both subspecies were intermediate between those in typical C3 and C4 plants. The values of CO2 compensation points in A. semialata ssp. semialata were within the C4 range, whereas those in A. semialata ssp. eckloniana were somewhat lower than the C3 range. These data suggest that the plants are C3-like and C4-like but not typical C3 and C4, and when integrated with previous findings, point to important variability in the expression of C4 physiology in this species complex. A. semialata is therefore an intriguing grass species with which to study the evolutionary linkage between C3 and C4 plants.     

Towards a dynamic photosynthesis model to guide yield improvement in C4 crops

The most productive C4 food and biofuel crops, such as Saccharum officinarum (sugarcane), Sorghum bicolor (sorghum) and Zea mays (maize), all use NADP-ME-type C4 photosynthesis. Despite high productivities, these crops fall well short of the theoretical maximum solar conversion efficiency of 6%. Understanding the basis of these inefficiencies is key for bioengineering and breeding strategies to increase the sustainable productivity of these major C4 crops. Photosynthesis is studied predominantly at steady state in saturating light. In field stands of these crops light is continually changing, and often with rapid fluctuations. Although light may change in a second, the adjustment of photosynthesis may take many minutes, leading to inefficiencies. We measured the rates of CO₂ uptake and stomatal conductance of maize, sorghum and sugarcane under fluctuating light regimes. The gas exchange results were combined with a new dynamic photosynthesis model to infer the limiting factors under non-steady-state conditions. The dynamic photosynthesis model was developed from an existing C4 metabolic model for maize and extended to include: (i) post-translational regulation of key photosynthetic enzymes and their temperature responses; (ii) dynamic stomatal conductance; and (iii) leaf energy balance. Testing the model outputs against measured rates of leaf CO₂ uptake and stomatal conductance in the three C4 crops indicated that Rubisco activase, the pyruvate phosphate dikinase regulatory protein and stomatal conductance are the major limitations to the efficiency of NADP-ME-type C4 photosynthesis during dark-to-high light transitions. We propose that the level of influence of these limiting factors make them targets for bioengineering the improved photosynthetic efficiency of these key crops.     

Development of structural and biochemical characteristics of C(4) photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae)

Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and biochemical development of C(4) occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygenase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C(4) biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the (13)C/(12)C isotope composition of leaves to values characteristic of C(4) photosynthesis. The findings indicate that these two forms of NAD-ME type C(4) photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes.     

The effects of salinity on photosynthesis and growth of the single-cell C<Subscript>4</Subscript> species <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis> (Chenopodiaceae)

Recent research on the photosynthetic mechanisms of plant species in the Chenopodiaceae family revealed that three species, including Bienertia sinuspersici, can carry out C₄ photosynthesis within individual photosynthetic cells, through the development of two cytoplasmic domains having dimorphic chloroplasts. These unusual single-cell C₄ species grow in semi-arid saline conditions and have semi-terete succulent leaves. The effects of salinity on growth and photosynthesis of B. sinuspersici were studied. The results show that NaCl is not required for development of the single-cell C₄ system. There is a large enhancement of growth in culture with 50–200 mM NaCl, while there is severe inhibition at 400 mM NaCl. With increasing salinity, the carbon isotope values (δ¹³C) of leaves increased from −17.3^o/_oo (C₄-like) without NaCl to −14.6^o/_oo (C₄) with 200 mM NaCl, possibly due to increased capture of CO₂ from the C₄ cycle by Rubisco and reduced leakiness. Compared to growth without NaCl, leaves of plants grown under saline conditions were much larger (~2 fold) and more succulent, and the leaf solute levels increased up to ~2000 mmol kg solvent⁻¹. Photosynthesis on an incident leaf area basis (CO₂ saturated rates, and carboxylation efficiency under limiting CO₂) and stomatal conductance declined with increasing salinity. On a leaf area basis, there was some decline in Rubisco content with increasing salinity up to 200 mM NaCl, but there was a marked increase in the levels of pyruvate, Pi dikinase, and phosphoenolpyruvate carboxylase (possibly in response to sensitivity of these enzymes and C₄ cycle function to increasing salinity). The decline in photosynthesis on a leaf area basis was compensated for on a per leaf basis, up to 200 mM NaCl, by the increase in leaf size. The influence of salinity on plant development and the C₄ system in Bienertia is discussed.     

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.     

Sensitivity of photosynthesis in a C4 plant,maize, to heat stress

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38 degrees C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30 degrees C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO(2) assimilation, the maximum quantum yield of photosystem II (F(v)/F(m)) was relatively insensitive to leaf temperatures up to 45 degrees C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40 degrees C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45 degrees C. The activation state of Rubisco decreased at temperatures exceeding 32.5 degrees C, with nearly complete inactivation at 45 degrees C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30 degrees C.     

Light induction of cell type differentiation and cell-type-specific gene expression in cotyledons of a C(4) plant, Flaveria trinervia

In Flaveria trinervia (Asteraceae) seedlings, light-induced signals are required for differentiation of cotyledon bundle sheath cells and mesophyll cells and for cell-type-specific expression of Rubisco small subunit genes (bundle sheath cell specific) and the genes that encode pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase (mesophyll cell specific). Both cell type differentiation and cell-type-specific gene expression were complete by d 7 in light-grown seedlings, but were arrested beyond d 4 in dark-grown seedlings. Our results contrast with those found for another C(4) dicot, Amaranthus hypochondriacus, in which light was not required for either process. The differences between the two C(4) dicot species in cotyledon cell differentiation may arise from differences in embryonic and post-embryonic cotyledon development. Our results illustrate that a common C(4) photosynthetic mechanism can be established through different developmental pathways in different species, and provide evidence for independent evolutionary origins of C(4) photosynthetic mechanisms within dicotyledonous plants.     

Photosynthetic carbon metabolism of the cool-temperate C4 grass Spartina anglica Hubb.

The aim of this work was to describe the photosynthetic carbon metabolism of the cooltemperate C₄ grass Spartina anglica. With the exception of pyruvate, phosphate dikinase and pyruvate kinase, the maximum catalytic activities in leaves of putative enzymes of the C₄ cycle of a phosphoenolpyruvate-carboxykinase C₄ plant were considerably in excess of the observed, steady-state rate of photosynthesis, and were comparable with the maximum catalytic activities of key enzymes of the reductive pentose-phosphate pathway. Radioactive carbon from ¹⁴CO₂ supplied to attached leaves during steady-state photosynthesis appeared first in malate and aspartate from which it moved to intermediates of the reductive pentose-phosphate pathway, and then to sucrose. These experiments show that photosynthetic carbon metabolism in this cool-temperate C₄ plant is similar to that of C₄ plants of hotter climates.