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This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Portulaca grandiflora (Lind.) is a succulent species with C4 photosynthesis and crassulacean acid metabolism (CAM) cycling in leaves, and CAM-idling type photosynthesis in stems. We investigated the level and localization of carbon fixation enzymes and photosynthetic activity of leaves and stems of P. grandiflora under well-watered and drought conditions. As CAM activity increased during water stress, the leaf water-storage tissue collapsed, presumably transferring water to the bundle sheath and mesophyll cells, and so maintaining the C4 photosynthetic pathway. Tissue prints indicated an increase in phosphoenolpyruvate carboxylase (PEPC) in the water-storage tissue of leaves and the cortex of stems. Immunoblot analyses after 10 d of water stress showed that leaves had a slight decrease in the proteins of the C4-CAM pathway, while at the same time a new isoform of NADP-malic enzyme (NADP-ME) appeared. In contrast, the stem showed increases in proteins of the CAM pathway when water stressed. Under water stress, diurnal fluctuation in acidity in leaves was not accompanied by a net gain or loss of CO2 at night, and there was sustained, but decreased, fixation of CO2 during the day, characteristic of CAM cycling. High gross rates of O2 evolution were maintained during the day under water stress, suggesting induction of alternative electron sinks. With induced diurnal fluctuations in acidity in stems, there was no net carbon gain during the day or night. These results demonstrate, for the first time, that the stem of P. grandiflora is an inducible CAM-idling tissue. Our results also indicate that the C4 and CAM pathways operate independently of one another in P. grandiflora.

Background Hydrogen cyanamide (HC) and pruning (P) have frequently been used to break dormancy in grapevine floral buds. However, the exact underlying mechanism remains elusive. This study aimed to address the early mode of action of these treatments on accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and expression of related genes in the dormancy breaking buds of grapevine in the summer. Results The budbreak rates induced by pruning (P), hydrogen cyanamide (HC), pruning plus hydrogen cyanamide (PHC) and water (control) after 8 days were 33, 53, 95, and 0 %, respectively. Clearly, HC was more effective in stimulating grapevine budbreak and P further enhanced its potency. In situ staining of longitudinal bud sections after 12 h of treatments detected high levels of ROS and nitric oxide (NO) accumulated in the buds treated with PHC, compared with HC or P alone. The amounts of ROS and NO accumulated were highly correlated with the rates of budbreak among these treatments, highlighting the importance of a rapid, transient accumulation of sublethal levels of ROS and RNS in dormancy breaking. Microarray analysis revealed specific alterations in gene expression in dormancy breaking buds induced by P, HC and PHC after 24 h of treatment. Relative to control, PHC altered the expression of the largest number of genes, while P affected the expression of the least number of genes. PHC also exerted a greater intensity in transcriptional activation of these genes. Gene ontology (GO) analysis suggests that alteration in expression of ROS related genes is the major factor responsible for budbreak. qRT-PCR analysis revealed the transient expression dynamics of 12 specific genes related to ROS generation and scavenge during the 48 h treatment with PHC. Conclusion Our results suggest that rapid accumulation of ROS and NO at early stage is important for dormancy release in grapevine in the summer, and the identification of the commonly expressed specific genes among the treatments allowed the construction of the signal transduction pathway related to ROS/RNS metabolism during dormancy release. The rapid accumulation of a sublethal level of ROS/RNS subsequently induces cell wall loosening and expansion for bud sprouting. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0889-y) contains supplementary material, which is available to authorized users.

 Premise of the study: Chloroplast development and structure are highly conserved in vascular plants, but the bizonoplast of Selaginella is a notable exception. In the shade plant S. erythropus , each dorsal epidermal cell contains one bizonoplast, while other cells have normal chloroplasts. Our quest was to (1) determine the origin of bizonoplasts, (2) explore developmental plasticity, and (3) correlate developmental changes with photosynthetic activity to provide insights unavailable in other green plants with more constrained development.  Methods: Bizonoplast development was studied in juvenile prostrate and older erect shoots of S. erythropus. Plastid plasticity was studied in plants cultivated under different light conditions. Chlorophyll fl uorescence was measured and correlated with photosynthetic activity.  Key results: The bizonoplast originates from a proplastid, forming a distinctive upper zone rapidly after exposure to low light. In the prostrate shoots, the proplastid develops through early stages only. When the shoot becomes erect, the proplastid soon develops into a mature bizonoplast. Erect shoots have signifi cantly higher photosynthetic effi ciency than prostrate shoots. No bizonoplasts were found in the plants growing in high light, where 2–4 spheroidal chloroplasts formed, or with light from below.  Conclusions: The upper zone develops above a normal-looking chloroplast structure to produce a bizonoplast. Bizonoplast developmental plasticity suggests that regular lamellar structure and monoplastidy are adaptations to deep shade environments. Such novel variation in S. erythropus is in stark contrast to known plastid development in other vascular plants, possibly refl ecting retention of developmental fl exibility in the basal clade, Lycophyta, to which it belongs.

The C4-Pdk gene encoding the C4 enzyme pyruvate, orthophosphate dikinase (PPDK) of maize (Zea mays cv Golden Cross Bantam) was introduced into the C3 plant, rice (Oryza sativa cv Kitaake). When the intact maize C4-Pdk gene, containing its own promoter and terminator sequences and exon/intron structure, was introduced, the PPDK activity in the leaves of some transgenic lines was greatly increased, in one line reaching 40-fold over that of wild-type plants. In a homozygous line, the PPDK protein accounted for 35% of total leaf-soluble protein or 16% of total leaf nitrogen. In contrast, introduction of a chimeric gene containing the full-length cDNA of the maize PPDK fused to the maize C4-Pdk promoter or the rice Cab promoter only increased PPDK activity and protein level slightly. These observations suggest that the intron(s) or the terminator sequence of the maize gene, or a combination of both, is necessary for high-level expression. In maize and transgenic rice plants carrying the intact maize gene, the level of transcript in the leaves per copy of the maize C4-Pdk gene was comparable, and the maize gene was expressed in a similar organ-specific manner. These results suggest that the maize C4-Pdk gene behaves in a quantitatively and qualitatively similar way in maize and transgenic rice plants. The activity of the maize PPDK protein expressed in rice leaves was light/dark regulated as it is in maize. This is the first reported evidence for the presence of an endogenous PPDK regulatory protein in a C3 plant.

The combined effects of O2 on net rates of photosynthesis, photosystem II activity, steady-state pool size of key metabolites of photosynthetic metabolism in the C4 pathway, C3 pathway and C2 photorespiratory cycle and on growth were evaluated in the C4 species Amaranthus edulis and the C3 species Flaveria pringlei. Increasing O2 reduced net CO2 assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O2 up to 5‐10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O2 dependent photorespiration, O2 is required for maximizing C4 cycle activity to concentrate CO2 in bundle sheath cells. Therefore, the response of net photosynthesis to O2 in C4 plants may result from the balance of these two opposing effects. Under 21 versus 5% O2, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C4 photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C3 and C4 pathways, 14C labeling of primary photosynthesis products and 13C/12C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C4 type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C3 and C4 types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C4 type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C4 type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C3 type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C4 photosynthesis in the family Chenopodiaceae.

Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

A C3 monocot, Hordeum vulgare and C3 dicot, Vicia faba, were studied to evaluate the mechanism of inhibition of photosynthesis due to water stress. The net rate of CO2 fixation (A) and transpiration (E) were measured by gas exchange, while the true rate of O2 evolution (J O2) was calculated from chlorophyll fluorescence analysis through the stress cycle (10 to 11 days). With the development of water stress, the decrease in A was more pronounced than the decrease in J O2 resulting in an increased ratio of Photosystem II activity per CO2 fixed which is indicative of an increase in photorespiration due to a decrease in supply of CO2 to Rubisco. Analyses of changes in the J O2 A ratios versus that of CO2 limited photosynthesis in well watered plants, and RuBP pool/RuBP binding sites on Rubisco and RuBP activity, indicate a decreased supply of CO2 to Rubisco under both mild and severe stress is primarily responsible for the decrease in CO2 fixation. In the early stages of stress, the decrease in C i (intercellular CO2) due to stomatal closure can account for the decrease in photosynthesis. Under more severe stress, CO2 supply to Rubisco, calculated from analysis of electron flow and CO2 exchange, continued to decrease. However, C i, calculated from analysis of transpiration and CO2 exchange, either remained constant or increased which may be due to either a decrease in mesophyll conductance or an overestimation of C i by this method due to patchiness in conductance of CO2 to the intercellular space. When plants were rewatered after photosynthesis had dropped to 10-30% of the original rate, both species showed near full recovery within two to four days.

The effect of O2 on photosynthesis was determined in maize (Zea mays) leaves at different developmental stages. The optimum level of O2 for maximum photosynthetic rates was lower in young and senescing tissues (2-5 kPa) than in mature tissue (9 kPa). Inhibition of photosynthesis by suboptimal levels of O2 may be due to a requirement for functional mitochondria or to cyclic/pseudocyclic photophosphorylation in chloroplasts; inhibition by supraoptimal levels of O2 is considered to be due to photorespiration. Analysis of a range of developmental stages (along the leaf blade and at different leaf ages and positions) showed that the degree of inhibition of photosynthesis by supraoptimal levels of O2 increased rapidly once the ribulose-1,5-bisphosphate carboxylase/oxygenase and chlorophyll contents were below a critical level and was similar to that of C3 plants. Tissue having a high sensitivity of photosynthesis to O2 may be less effective in concentrating CO2 in the bundle sheath cells due either to limited function of the C4 cycle or to higher bundle sheath conductance to CO2. An analysis based on the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase was used to predict the maximum CO2 level concentrated in bundle sheath cells at a given degree of inhibition of photosynthesis by supraoptimal levels of O2.

Study of the unique leaf anatomy and chloroplast structure in shade-adapted plants will aid our understanding of how plants use light efficiently in low light environments. Unusual chloroplasts in terms of size and thylakoid membrane stacking have been described previously in several deep-shade plants. In this study, a single giant cup-shaped chloroplast, termed a bizonoplast, was found in the abaxial epidermal cells of the dorsal microphylls and the adaxial epidermal cells of the ventral microphylls in the deep-shade spike moss Selaginella erythropus. Bizonoplasts are dimorphic in ultrastructure: the upper zone is occupied by numerous layers of 2–4 stacked thylakoid membranes while the lower zone contains both unstacked stromal thylakoids and thylakoid lamellae stacked in normal grana structure oriented in different directions. In contrast, other cell types in the microphylls contain chloroplasts with typical structure. This unique chloroplast has not been reported from any other species. The enlargement of epidermal cells into funnel-shaped, photosynthetic cells coupled with specific localization of a large bizonoplast in the lower part of the cells and differential modification in ultrastructure within the chloroplast may allow the plant to better adapt to low light. Further experiments are required to determine whether this shade-adapted organism derives any evolutionary or ecophysiological fitness from these unique chloroplasts.

Castor bean (Ricinus communis L.) has a high photosynthetic capacity under high humidity and a pronounced sensitivity of photosynthesis to high water vapor pressure deficit (VPD). The sensitivity of photosynthesis to varying VPD was analyzed by measuring CO2 assimilation, stomatal conductance (gs), quantum yield of photosystem II (φII), and nonphotochemical quenching of chlorophyll fluorescence (qN) under different VPD. Under both medium (1000) and high (1800 micromoles quanta per square meter per second) light intensities, CO2 assimilation decreased as the VPD between the leaf and the air around the leaf increased. The gs initially dropped rapidly with increasing VPD and then showed a slower decrease above a VPD of 10 to 20 millibars. Over a temperature range from 20 to 40°C, CO2 assimilation and gs were inhibited by high VPD (20 millibars). However, the rate of transpiration increased with increasing temperature at either low or high VPD due to an increase in gs. The relative inhibition of photosynthesis under photorespiring (atmospheric levels of CO2 and O2) versus nonphotorespiring (700 microbars CO2 and 2% O2) conditions was greater under high VPD (30 millibars) than under low VPD (3 millibars). Also, with increasing light intensity the relative inhibition of photosynthesis by O2 increased under high VPD, but decreased under low VPD. The effect of high VPD on photosynthesis under various conditions could not be totally accounted for by the decrease in the intercellular CO2 in the leaf (Ci) where Ci was estimated from gas exchange measurements. However, estimates of Ci from measurements of φII and qN suggest that the decrease in photosynthesis and increase in photorespiration under high VPD can be totally accounted for by stomatal closure and a decrease in Ci. The results also suggest that nonuniform closure of stomata may occur in well-watered plants under high VPD, causing overestimates in the calculation of Ci from gas exchange measurements. Under low VPD, 30°C, high light, and saturating CO2, castor bean (C3 tropical shrub) has a rate of photosynthesis (61 micromoles CO2 per square meter per second) that is about 50% higher than that of tobacco (C3) or maize (C4) under the same conditions. The chlorophyll content, total soluble protein, and ribulose-1,5-bisphosphate carboxylase/oxygenase level on a leaf area basis were much higher in castor bean than in maize or tobacco, which accounts for its high rates of photosynthesis under low VPD.

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C(3), C(3)-C(4), C(4)-like, and C(4) plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C(3)-C(4) intermediates showed an inverse continuum in level of photorespiration and development of the C(4) syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C(4) photosynthesis to F. Among the intermediates, the photosynthetic CO(2) compensation points at 30 degrees C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C(4)-like species varied from 3 to 6 microbars, similar to those measured for the C(4) species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C(3) to C(3)-C(4) to C(4)-like and C(4) species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C(3) species, but generally lower activities compared to C(4)-like and C(4) species. The levels of these C(4) enzymes are correlated with the degree of C(4) photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C(4) syndrome in C(3)-C(4)Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C(4) photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C(4) pathway. In the progression from C(3) to C(4) species in Flaveria, the CO(2) compensation point decreased more rapidly than did the decrease in O(2) inhibition of photosynthesis or the increase in the degree of C(4) photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO(2) and prior to substantive reduction in O(2) inhibition and development of the C(4) syndrome. However, further reduction in O(2) inhibition in some intermediates and C(4)-like species is considered primarily due to the development of the C(4) syndrome. Thus, the evolution of C(3)-C(4) intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO(2) concentration first via refixation of photorespired CO(2), followed by development of the C(4) syndrome.

PhosphoEnolPyruvate Carboxykinase (Pck) Is A C4-Acid Decarboxylation Enzyme Located In The Cytosol Of Bundle Sheath Cells Of Pck Subtype C4 Plants. In This Study, We Have Introduced The Maize Pck Gene Into Rice Genome And Targeted The Enzyme To Mesophyll Chloroplast As A First Step In An Attempt To Raise Co2 Concentration In The Chloroplast Of Rice Via Decarboxylation Of Oxaloacetate. Molecular Analyses Confirmed The Integration Of The Maize Gene Into Rice Genome And Its Expression At Transcript Level In Several Transgenic Lines. Although Expression Of The Maize Pck Gene In Transgenic Rice Plants Was Driven By The Tomato RbcS3C Promoter The Protein Was Expressed In An Organ-Specific Manner: High In Leaf, Stem And Floret, But Not In Root. The Protein Amounts In The Leaf Were 4–6 Fold Higher In Transgenic Than In Wild Type Plants. The Photosynthetic Rates Of Three Greenhouse Grown Transgenic Lines Were Comparable Or Slightly Higher Than That Of Untransformed Wild Type Plants. In A Preliminary Field Trial, Transgenic Rice Plants Exhibited Enhanced Growth (20–50%) And Grain Yields (10–50%) Than Did Wild Type. Since Pck Is Only One Of The Biochemical Steps In The Entire C4 Pathway Other Affiliated Mechanisms, Such As Effective Co2 Acquisition And Rapid Carboxylation By Pep Carboxylase, Will Have To Be In Place In Order To Have A Functional “C4 Rice”.

Under Low Co2 Or Alkaline Water Conditions, Cyanobacteria Use Bicarbonate Transporters To Pump In Bicarbonate As A Major Carbon Source. This Adaptive Co2 Concentrating Mechanism Allows Cyanobacteria To Survive Unfavorable Growth Conditions. In This Study, We Have Constitutively Expressed The High Affinity Bicarbonate Transporter Gene, IctB, From Cyanobacterium In Rice. All Four Transgenic Rice Lines Expressing The Transporter Exhibited Enhanced Photosynthetic Capacity, Growth And Grain Yield. Relative To Untransformed Wild Type Plants, The Transgenic Plants Had 10–30% Higher Photosynthesis Rates, 15–20% Higher Carboxylation Efficiencies, And Lower Photosynthetic Co2 Compensation Points. Activities Of Ribulose 1,5-Bisphosphate Carboxylase And Pep Carboxylase Were Also Higher In These Transgenic Lines. Consistently, The Transgenic Plants Produced 10–120% More Tillers Or Panicles Per Plant And 10–70% More Grains, Relative To The Wild Type. The Enhancements In Growth And Grain Yield Are Closely Related With The Increased Photosynthetic Capacity Among The Transgenic Lines. Yield Increases Were Also Confirmed In A Preliminary Field Trial. This Study Demonstrates That The Simple Co2 Concentrating Mechanism From Cyanobacterium Can Largely Improve The Photosynthetic Efficiency, Growth And Productivity Of C3 Crops.

The paraveinal mesophyll (PVM) is a spatially, morphologically and ultrastructurally specialized tissue in soybean leaves. It accounts for about 18% of the mesophyll volume as determined by stereological measurements and represents a significant cellular compartment, equal in size to that of the spongy mesophyll. Mesophyll cells (MC), their protoplasts (MP) and PVM protoplasts (PVMP) were isolated from leaves of non-nodulated plants and their physical characteristics and relative activity in some aspects of carbon metabolism were determined. PVMP were about twice the diameter and seven times the volume of MP: they had smaller and 2 3 fewer chloroplasts, and about one third the chlorophyll content of MP. Ribulose 1,5-biphosphate carboxylase (RuBPCase) activity and 14CO2 fixation rates of PVMP were 40% and 30%, respectively, of the values measured in MP on a protoplast basis. PVMP showed net oxygen consumption (240 nmol O2 (106 protoplasts)−1 h−1) in the light with 10 mM bicarbonate, while MP showed the expected photosynthetic oxygen evolution (89 nmol O2 (106 protoplasts)−1 h−1) under similar conditions. The dark respiration rate of PVMP was about six times greater on a chlorophyll basis, three times greater on a protoplast basis and one point seven times greater on a cytoplasmic volume basis than the rate measured in MP. The data show that the PVM plays a minor role in carbon fixation in soybean leaves and supports the hypothesis that one of its primary roles is as a cellular pathway for metabolite transport within the leaf.

Transgenic rice plants expressing the maize phosphoeno/pyruvate carboxylase (PEPC) and pyruvate, orthophosphate dikinase (PPDK) exhibit a higher photosynthetic capacity (up to 35%) than untransformed plants. The increased photosynthetic capacity in these plants is mainly associated with an enhanced stomatal conductance and a higher internal CO2 concentration. Plants simultaneously expressing high levels of both enzymes also have a higher photosynthetic capacity. The results suggest that both PEPC and PPDK play a key role in organic acid metabolism in the guard cells to regulate stomatal opening. Under photoinhibitory and photooxidative conditions, PEPC transgenic rice plants are capable of maintaining a higher photosynthetic rate, a higher photosynthetic quantum yield by PSII and a higher capacity to dissipate excess energy photochemically and non-photochemically than untransformed plants. Preliminary data from field trials show that relative to untransformed plants, the grain yield is about 10-20% higher in selected PEPC and 30-35% higher in PPDK transgenic rice plants, due to increased tiller number. Taken together, these results suggest that introduction of C4 photosynthesis enzymes into rice has a good potential to enhance its tolerance to stress, photosynthetic capacity and yield.

The induction of Crassulacean acid metabolism in M:esembryanthemum crystallinum was investigated in response to foliar application of gibberellic acid (GA). After 5 weeks of treatment, GA-treated plants showed 1.7- to almost a 4-fold increase of phosphoenolpyruvate carboxylase (PEPcase) activity with a concomitant increase in acid metabolism when compared to control plants. Immunoblot analysis indicated an increase in the PEPcase protein similar to that of salt treatment while Rubisco did not show a similar rise. The results indicate that exogenously applied GA accelerates plant developmental expression of PEPcase and Crassulacean acid metabolism in M: crystallinum.

The chloroplastic NADP-malic enzyme (NADP-ME) is a key enzyme of the C4 photosynthesis pathway in NADPME type C4 plants such as maize. To express the chloroplastic NADP-ME in leaves of a C 3 plant, rice, full-length cDNAs encoding the rice C 3 -specific isoform and the maize C4-specific isoform of the enzyme were expressed under the control of the rice Cab promoter. Transformants carrying the rice cDNA showed the NADP-ME activities in the leaves less than several-fold that of non-transformants, while those carrying the maize cDNA showed activities up to 30-fold that of non-transformants or about 60% of the NADP-ME activity of maize leaves. These results indicate that expression of the rice C3-specific NADP-ME is suppressed at co- and/or post-transcriptional levels by some regulation mechanisms intrinsic to rice, while that of the foreign C 4 -specific isoform can escape from such suppression. The accumulation of the maize C 4 -specific NADP-ME led to bleaching of leaf color and growth hindrance in rice plants under natural light. These deteriorative effects resulted from enhanced photoinhibition of photosynthesis due to an increase in the level of NADPH inside the chloroplast by the action of the maize enzyme.

Salsola arbusculiformis is identified as a C 3 ‐C 4 intermediate species based on anatomical, biochemical and physiological characteristics. This is the first report of a naturally occurring intermediate species in the Chenopodiaceae, the family with the largest number of C4 species amongst the dicots. In the genus Salsola, most species have Salsoloid anatomy with Kranz type bundle sheath cells and C4 photosynthesis, while a few species have Sympegmoid anatomy and were found to have non-Kranz type bundle sheath cells and C3 photosynthesis. In the cylindrical leaves of C4 Salsola with Salsoloid type anatomy, there is a continuous layer of distinct, chlorenchymatous Kranz type bundle sheath cells surrounded by a single layer of mesophyll cells; whereas species with Sympegmoid type anatomy have an indistinct bundle sheath with few chloroplasts and multiple layers of chlorenchymatous mesophyll cells. However, S. arbusculiformis has intermediate anatomical features. While it has two-to-three layers of mesophyll cells, characteristic of Sympegmoid anatomy, it has distinctive, Kranz-like bundle sheath cells with numerous chloroplasts and mitochondria. Measurements of its CO2 compensation point and CO2 response of photosynthesis show S. arbusculiformis functions as an intermediate species with reduced levels of photorespiration. The primary means of reducing photorespiration is suggested to be by refixing photorespired CO2 in bundle sheath cells, since analysis of photosynthetic enzymes (activity and immunolocalization) and 14 CO2 labelling of initial fixation products suggests minimal operation of a C4 cycle. # 2001 Annals of Botany Company

Transgenic rice plants overexpressing maize C 4 -specific phospho enol pyruvate carboxylase (PEPC) exhibit a higher photosynthetic rate (up to 30%) and a more reduced O 2 inhibition of photosynthesis than untransformed plants. There is a small increase in the amount of atmospheric CO 2 being directly fixed by PEPC. Similarly, transgenic rice plants overexpressing the maize chloroplastic pyruvate, orthophosphate dikinase (PPDK), also have higher photosynthetic rates (up to 35%) than untransformed plants. This increased photosynthetic capacity is at least in part due to an enhanced stomatal conductance and a higher internal CO 2 concentration. Using conventional hybridization, we have integrated maize PEPC and PPDK genes into the same transgenic rice plants. In the segregating population, the photosynthetic rates of plants with high levels of both maize enzymes are up to 35% higher than those of untransformed plants. Under full-sunlight conditions, the photosynthetic capacity of field-grown PEPC transgenic rice plants is twice as high as that of untransformed plants. PEPC transgenic plants consistently have a higher photosynthetic quantum yield by photosystem II and a higher capacity to dissipate excess energy photochemically and nonphotochemically. Preliminary data from field tests show that the grain yield is about 10–30% higher in PEPC and 30–35% higher in PPDK transgenic rice plants relative to untransformed plants. Taken together, these results suggest that introduction of C 4 photosynthesis enzymes into rice has a good potential for enhancing the crop’s photosynthetic capacity and yield.

The effects of elevated CO(2) concentrations on the photochemistry, biochemistry and physiology of C(4) photosynthesis were studied in maize (Zea mays L.). Plants were grown at ambient (350mgr;L L(-1)) or ca. 3 times ambient (1100mgr;L L(-1)) CO(2) levels under high light conditions in a greenhouse for 30 d. Relative to plants grown at ambient CO(2) levels, plants grown under elevated CO(2) accumulated ca. 20% more biomass and 23% more leaf area. When measured at the CO(2) concentration of growth, mature leaves of high-CO(2)-grown plants had higher light-saturated rates of photosynthesis (ca. 15%), lower stomatal conductance (71%), higher water-use efficiency (225%) and higher dark respiration rates (100%). High-CO(2)-grown plants had lower carboxylation efficiencies (23%), measured under limiting CO(2), and lower leaf protein contents (22%). Activities of a number of C(3) and C(4) cycle enzymes decreased on a leaf-area basis in the high-CO(2)-grown plants by 5-30%, with NADP-malate dehydrogenase exhibiting the greatest decrease. In contrast, activities of fructose 1,6-bisphosphatase and ADP-glucose pyrophosphorylase increased significantly under elevated CO(2) condition (8% and 36%, respectively). These data show that the C(4) plant maize may benefit from elevated CO(2) through acclimation in the capacities of certain photosynthetic enzymes. The increased capacity to synthesize sucrose and starch, and to utilize these end-products of photosynthesis to produce extra energy by respiration, may contribute to the enhanced growth of maize under elevated CO(2).

The Chenopodiaceae contains species with C3, C4 and possibly C3-C4 intermediate photosynthesis. This family has a second place in world flora in abundance of plants with C4 photosynthesis (about 350-400 species) and it displays a large diversity of biochemical mechanisms of CO2 fixation and Kranz-anatomy in mature leaves. C4 Chenopodiaceae species dominate in arid desert regions of Africa, Asia and Europe, and are adapted to extreme environment. Previous studies were concentrated on investigation of C4 photosynthesis in basic assimilative organs (leaves and assimilatory shoots) and demonstrated a large structural and biochemical diversity of C4 photosynthesis (1). At the same time, leaf-like organs of plants such as cotyledons were, studied poor, but give evidence of occurrence both C3 and C4 photosynthetic types in these organs (2–3). The purpose of this study was a comparative structural and biochemical investigation of photosynthetic types in leaves and leaf-like organs in Chenopodiaceae species differed in taxonomy, evolution position, structural and biochemical features of C4 photosynthesis.

Because photosynthetic rates in C4 plants are the same at normal levels of O2 (c. 20 kPa) and at c. 2 kPa O2 (a conventional test for evaluating photorespiration in C3 plants) it has been thought that C4 photosynthesis is O2 insensitive. However, we have found a dual effect of O2 on the net rate of CO2 assimilation among species representing all three C4 subtypes from both monocots and dicots. The optimum O2 partial pressure for C4 photosynthesis at 30 °C, atmospheric CO2 level, and half full sunlight (1000 μmol quanta m-2 s-1) was about 5-10 kPa. Photosynthesis was inhibited by O2 below or above the optimum partial pressure. Decreasing CO2 levels from ambient levels (32.6 Pa) to 9.3 Pa caused a substantial increase in the degree of inhibition of photosynthesis by supra-optimum levels of O2 and a large decrease in the ratio of quantum yield of CO2 fixation/quantum yield of photosystem II (PSII) measured by chlorophyll α fluorescence. Photosystem II activity, measured from chlorophyll α fluorescence analysis, was not inhibited at levels of O2 that were above the optimum for CO2 assimilation, which is consistent with a compensating, alternative electron flow as net CO2 assimilation is inhibited. At suboptimurn levels of O2, however, the inhibition of photosynthesis was paralleled by an inhibition of PSII quantum yield, increased state of reduction of quinone A, and decreased efficiency of open PSII centres. These results with different C4 types suggest that inhibition of net CO2assimilation with increasing O2 partial pressure above the optimum is associated with photorespiration, and that inhibition below the optimum O2 may be caused by a reduced supply of ATP to the C4 cylcle as a result of inhibition of its production photochemically.

Some succulent plants initially perform C3 photosynthesis but subsequently shift to CAM after developmental or environmental changes (e.g. photoperiod, water stress, day/night temperature). Species which have a strong dependency on the environment for expression of CAM are facultative, in contrast to obligate CAM plants which tend to function in the CAM mode under all conditions (Cockburn 1985; Winter 1985). Associated with this transition in facultative species is nocturnal opening of stomata with concomitant CO2 uptake, and diurnal fluctuations of tissue acidity and malate content. Facultative CAM plants may maximize their growth by assimilating carbon via the C3 pathway when environmental conditions are less stressful, and utilize the CAM mode when environmental conditions cause more potential for water stress.

Two major indicators were used to access the degree of photorespiration in various photosynthetic types of Flaveria species (C3, C3-C4, C4-like, and C4): the O2 inhibition of photosynthesis measured above the O2 partial pressure which gives a maximum rate, and O2- and light-dependent whole-chain electron flow measured at the CO2 compensation point (Γ). The optimum level of O2 for maximum photosynthetic rates under atmospheric levels of CO2 (34 Pa) was lower in C3 and C3-C4 species (ca. 2 kPa) than in C4-like and C4 species (ca. 9 kPa). Increasing O2 partial pressures from the optimum for photosynthesis up to normal atmospheric levels (ca. 20 kPa) caused an inhibition of photosynthesis which was more severe under lower CO2. This inhibition was calculated as the O2 inhibition index (ΘA, the percentage inhibition of photosynthesis per kPa increase in O2). From measurements of 18 Flaveria species at atmospheric CO2, the ΘA values decreased from C3 (1.9–2.1) to C3-C4 (1.2–1.6), C4-like (0.6–0.8) and C4 species (0.3–0.4), indicating a progressive decrease in apparent photorespiration in this series. With increasing irradiance at Γ under atmospheric levels of O2, and increasing O2 partial pressure at 300 μmol quanta·m−2·s−1, there was a similar increase in the rate of O2 evolution associated with whole-chain electron flow (Jo 2, calculated from chlorophyll fluorescence analysis) in the C3 and C3-C4 species compared to a much lower rate in the C4-like and C4 species. The results indicate that there is substantial O2-dependent electron flow in C3 and C3-C4 species, reflecting a high level of photorespiration compared to that in C4-like and C4 species. Consistent with these results, there was a significant decrease in Γ from C3 (6–6.2 Pa) to C3-C4 (1.0–3.0 Pa), to C4-like and C4 species (0.3–0.8 Pa), indicating a progressive decrease in apparent photorespiration. However, C3 and C3-C4 species examined had high intrinsic levels of photorespiration with the latter maintaining low apparent rates of photorespiration and lower Γ values, primarily by refixing photorespired CO2. The C4-like and C4 Flaveria species had low, but measurable, levels of photorespiration via selective localization of ribulose-1,5-bisphosphate carboxylase in bundle sheath cells and operation of a CO2 pump via the C4 pathway.

Despite previous reports of no apparent photorespiration in C4 plants based on measurements of gas exchange under 2 versus 21% O2 at varying [CO2], photosynthesis in maize (Zea mays) shows a dual response to varying [O2]. The maximum rate of photosynthesis in maize is dependent on O2 (approximately 10%). This O2 dependence is not related to stomatal conductance, because measurements were made at constant intercellular CO2 concentration (Ci); it may be linked to respiration or pseudocyclic electron flow. At a given Ci, increasing [O2] above 10% inhibits both the rate of photosynthesis, measured under high light, and the maximum quantum yield, measured under limiting light ([phi]CO2). The dual effect of O2 is masked if measurements are made under only 2 versus 21% O2. The inhibition of both photosynthesis and [phi]CO2 by O2 (measured above 10% O2) with decreasing Ci increases in a very similar manner, characteristically of O2 inhibition due to photorespiration. There is a sharp increase in O2 inhibition when the Ci decreases below 50 [mu]bar of CO2. Also, increasing temperature, which favors photorespiration, causes a decrease in [phi]CO2 under limiting CO2 and 40% O2. By comparing the degree of inhibition of photosynthesis in maize with that in the C3 species wheat (Triticum aestivum) at varying Ci, the effectiveness of C4 photosynthesis in concentrating CO2 in the leaf was evaluated. Under high light, 30[deg]C, and atmospheric levels of CO2 (340 [mu]bar), where there is little inhibition of photosynthesis in maize by O2, the estimated level of CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the bundle sheath compartment was 900 [mu]bar, which is about 3 times higher than the value around Rubisco in mesophyll cells of wheat. A high [CO2] is maintained in the bundle sheath compartment in maize until Ci decreases below approximately 100 [mu]bar. The results from these gas exchange measurements indicate that photorespiration occurs in maize but that the rate is low unless the intercellular [CO2] is severely limited by stress.

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C(3) pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.

Dans le but de determiner si le mecanisme d'assimilation photosynthetique du carbone chez le manioc est de type C 3 ou C 4 , les caracteristiques photosynthetiques physiologiques et biochimiques de cette plante sont etudiees. Le taux de photosynthese, le point de compensation en CO 2 et l'activite des enzymes de la voie C 4 sont mesures. Les produits primaires de la fixation du CO 2 sont identifies. L'anatomie des feuilles est observee

Eleocharis contains many amphibious species, and displays diversity of photosynthetic mechanism (C3, C4 or C3-C4 intermediates). A unique feature of Eleocharis is the plasticity in the photosynthetic mechanism of some species in response to the environment. In this study, we have examined the culm anatomy and photosynthetic property of several Eleocharis species grown terrestrially and the changes in the newly produced culms over a short period time frame after switching from terrestrial to submerged condition. Eleocharis baldwinii (Torrey) Chapman is C4-like in terrestrial habitat, exhibiting O2 inhibition of photosynthesis with Rubisco expressed in both mesophyll and bundle sheath cells and PEPC strictly in the mesophyll cells, but switches to C3-C4 intermediacy when submerged. In addition to Eleocharis vivipara Link type 1 (which switches from C4-like to C3), two other photosynthetic types examined in this study were shown to have different responses to growth in either terrestrial or submerged conditions. E. vivipara type 2 is a typical C4 plant in the terrestrial habitat, but becomes a C3-C4 intermediate under submerged conditions. Further, terrestrially, E. vivipara type 3 is a C3-C4 intermediate, but when submerged the δ13C value increases to –6.7‰, indicating its use of bicarbonate as a major carbon source. The submerged form of this plant exhibited about three times higher photosynthetic O2 evolution rate, compared to the C3 species Eleocharis erythropoda Steudel. These Eleocharis species possess different molecular switches for regulating C4 gene expression in response to environmental stimuli both between different species, and in E. vivipara among different populations. The apparent expression of a bicarbonate transport system by E. vivipara type 3 while submerged represents a unique adaptation to low CO2 availability.

One hundred and one species (in 36 families) of weeds on cultivated land in Taiwan were investigated for the occurrence of Kranz leaf anatomy and activities of key enzymes of C4 photosynthesis to determine their photosynthetic mechanisms. Based on the anatomical and biochemical analyses, 75 species were found to possess the C3 and 26 species the C4 pathway of photosynthetic CO2 fixation. Among the 26 C4 species, 15 species are in Gramineae, 6 in Cyperaceae, 2 each in Euphorbiaceae and Amaranthaceae, and 1 in Portulacaceae. Two C4 species in the Gramineae, namely Digitaria radicosa (Presl) Miq. and Sporobolus fertilis (Steud.) Clayton, were recorded as C4 plants for the first time. The biochemical subdivisions of these C4 weeds were also determined. As in the natural C4 populations, the NADP-malic enzyme subtype of C4 photosynthesis dominates the list of C4 weeds on this island (62%), while the PEP carboxykinase subtype is relatively rare (12%). NAD-malic enzyme subtype has an intermediate representation (26%). The high proportion of weeds in Taiwan being C3 plants is noteworthy, and it may be accounted for by the high precipitation in this subtropical island.

The quantum yields of electron transport from photosystem II (PSII) (Φe, determined from chlorophyll a fluorescence), and CO2 assimilation (ΦCO2, photosynthetic rate/light intensity) were measured simultaneously in vivo with representative species of Flaveria which show a progression in development between C3 and C4 photosynthesis and in reduction of photorespiration. These were F. pringlei (C3), F. sonorensis (C3-C4, but lacking a C4 cycle), F. floridana (C3-C4, with partially functional C4 cycle), F. brownii (C4-like) and F. bidentis (C4). The level of PSII activity with varying CI under 210 mbar O2 was very similar in all species. However, the progressive development of C4 characteristics among the species produced an increased efficiency in utilisation of PSII derived energy for CO2 assimilation under 210 mbar O2, due to reduced photorespiratory losses at low CO2 levels. In all species, when photorespiration was limited by low O2 (20 mbar), there was a linear or near linear relationship between the quantum yield of PSII v. the quantum yield of CO2 fixation with varying intercellular levels of CO2 (Ci) indicating that CO2 fixation in this case is linked to PSII activity. When switching from 20 to 210 mbar O2 at atmosphere levels of CO2, there was a similar decrease in the efficiency in utilising PSII activity for CO2 assimilation at different light intensities, but the degree of sensitivity to O2 progressively decreased among the species concomitant with the development of C4 photosynthesis. These results may help explain why there is an advantage to evolution of C4 photosynthesis in environments where Ci becomes limiting.

The initial products of photosynthesis by the C3 species Flaveria cronquistii, the C4 species F. trinervia, and the C3-C4 intermediate species F. ramosissima were determined using a pulse-chase technique with 14CO2-12CO2. The intermediate species F. ramosissima incorporated at least 42% of the total soluble 14C fixed into malate and aspartate after 10 seconds of photosynthesis in 14CO2, as compared with 90% for the C4 species F. trinervia and 5% for the C3 species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in 12CO2, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C4 pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C3 and C4 plants.

Mesophyll protoplasts and bundle-sheath cells of Pennisetum purpureum Schum., a C4 plant with low phenol-oxidase activity, were enzymatically separated according to methods recently developed with sugarcane (Saccharum officinarum L.), maize (Zea mays L.), and sorghum (Sorghum bicolor L.). The phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase of the C4 pathway were found to be localized in the mesophyll protoplasts while ribulose-1,5-diphosphate (RuDP) carboxylase, phosphoribulokinase and NADP-malic enzyme were localized in the bundle-sheath cells. The levels of these enzyme activities in the leaf extracts and in certain cellular preparations of P. purpureum are sufficient to account for the rate of photosynthesis in the leaf. These results on the activities and distribution of photosynthetic enzymes with P. purpureum preparations are consistent with our previous evidence for cellular separation of the C4 and the reductive pentose-phosphate pathways in C4 species.With chlorogenic acid as the substrate, P. purpureum, Setaria lutescens (Weigel) Hubb. and Panicum texanum Buckl. have relatively low phenol-oxidase activity, similar to that found in spinach (Spinacia oleracea L.); while sorghum, sugarcane, maize, Panicum capillare L. and P. miliaceum L. have relatively high phenoloxidase activity, similar to that in tobacco (Nicotiana tabacum L.). C4 species having high phenol-oxidase activity have substantial activity of the enzyme in both mesophyll and bundle-sheath extracts. Since phenol oxidase is found in both cell types it is not logical to expect preferential inhibition of RuDP carboxylase or other photosynthetic enzymes through phenol oxidation in mesophyll extracts, as has been previously suggested. When dithiothreitol and polyvinylpyrrolidone were included in the enzyme extraction medium, the activity of RuDP carboxylase increased 10% in P. purpureum and 59% in sugarcane leaf extracts.

Individual leaves of potato (Solanum tuberosum L. W729R), a C(3) plant, were subjected to various irradiances (400-700 nm), CO(2) levels, and temperatures in a controlled-environment chamber. As irradiance increased, stomatal and mesophyll resistance exerted a strong and some-what paralleled regulation of photosynthesis as both showed a similar decrease reaching a minimum at about 85 neinsteins.cm(-2).sec(-1) (about (1/2) of full sunlight). Also, there was a proportional hyperbolic increase in transpiration and photosynthesis with increasing irradiance up to 85 neinsteins.cm(-2).sec(-1). These results contrast with many C(3) plants that have a near full opening of stomata at much less light than is required for saturation of photosynthesis.Inhibition of photosynthesis by 21% O(2) was nearly overcome by a 2-fold increase in atmospheric levels of CO(2) (about 1,200 ng.cm(-3)). Photosynthesis at 25 C, high irradiance, 2.5% O(2) and atmospheric levels of CO(2) was about 80% of the CO(2)-saturated rate, suggesting that CO(2) can be rate-limiting even without O(2) inhibition of photosynthesis. With increasing CO(2) concentration, mesophyll resistance decreased slightly while stomatal resistance increased markedly above 550 ng.cm(-3) which resulted in a significant reduction in transpiration.Although potato is a very productive C(3) crop, there is substantial O(2) inhibition of photosynthesis. The level of O(2) inhibition was maximum around 25 C but the percentage inhibition of photosynthesis by O(2) increased steadily from 38% at 16 C to 56% at 36 C. Photosynthesis and transpiration showed broad temperature optima (16-25 C). At higher temperatures, both the increased percentage inhibition of photosynthesis by O(2) and the increased stomatal resistance limit photosynthesis, while increased stomatal resistance limits transpiration. Water use efficiency, when considered at a constant vapor pressure gradient, increased with increasing irradiance, CO(2) concentration, and temperature.

Four species of the genus Flaveria, namely F. anomala, F. linearis, F. pubescens, and F. ramosissima, were identified as intermediate C(3)-C(4) plants based on leaf anatomy, photosynthetic CO(2) compensation point, O(2) inhibition of photosynthesis, and activities of C(4) enzymes. F. anomala and F. ramosissima exhibit a distinct Kranz-like leaf anatomy, similar to that of the C(4) species F. trinervia, while the other C(3)-C(4) intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO(2) compensation points of these intermediates at 30 degrees C were very low relative to those of C(3) plants, ranging from 7 to 14 microliters per liter. In contrast to C(3) plants, net photosynthesis by the intermediates was not sensitive to O(2) concentrations below 5% and decreased relatively slowly with increasing O(2) concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% O(2) varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentum, a typical C(3) species. The inhibition of carboxylation efficiency by 21% O(2) varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C(4) (2% for F. trinervia) and C(3) (53% for L. esculentum) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C(4) enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C(4) photosynthesis. It appears that these Flaveria species may be true biochemical C(3)-C(4) intermediates.

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO(2) compensation concentration (20-25 microliters per liter at 30 degrees C and 21% O(2)) and by a reduced sensitivity of net photosynthesis to 21% O(2). In contrast, the related C(3) species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO(2) compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O(2). Furthermore, in P. hysterophorus the CO(2) compensation concentration is relatively less sensitive to changes in O(2) concentrations and shows a biphasic response to changing O(2), with a transition point at about 11% O(2). Based on these results, P. hysterophorus is classified as a C(3)-C(4) intermediate. The activities of diagnostic enzymes of C(4) photosynthesis in P. hysterophorus were very low, comparable to those observed in the C(3) species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to (14)CO(2) (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial (14)C products (77-84%), with/=4% of the (14)C-label in malate plus aspartate. These results indicate that in the C(3)-C(4) intermediate P. hysterophorus, the reduction in leaf photorespiration cannot be attributed to C(4) photosynthesis.

Among higher plants, C3 species have high levels of photorespiration, which limit the rate of net carbon dioxide assimilation. C4 plants have evolved a mechanism that overcomes photorespiration. Some species have photorespiration levels intermediate to C3 and C4 species, as well as other intermediate anatomical, physiological, and biochemical characteristics. These C3-C4 intermediates provide excellent opportunities for studying how photorespiration is reduced, as well as the genetics and evolution of C4 photosynthesis. (Accepted for publication 11 April 1984)

Leaves of Flaveria brownii exhibited slightly higher amounts of oxygen inhibition of photosynthesis than the C4 species, Flaveria trinervia, but considerably less than the C3 species, Flaveria cronquistii. The photosynthetic responses to intercellular CO2, light and leaf temperature were much more C4-like than C3-like, although 21% oxygen inhibited the photosynthetic rate, depending on conditions, up to 17% of the photosynthesis rate observed in 2% O2. The quantum yield for CO2 uptake in F. brownii was slightly higher than that for the C4 species F. trinervia in 2% O2, but not significantly different in 21% O2. The quantum yield was inhibited 10% in the presence of 21% O2 in F. brownii, yet no significant inhibition was observed in F. trinervia. An inhibition of 27% was observed for the quantum yield of F. cronquistii in the presence of 21% O2. The photosynthetic response to very low intercellular CO2 partial pressures exhibited a unique pattern in F. brownii, with a break in the linear slope observed at intercellular CO2 partial pressure values between 15 and 20 μbar when analyzed in 21% O2. No significant break was observed when analyzed in 2% O2. When taken collectively, the gas-exchange results reported here are consistent with previous biochemical studies that report incomplete intercellular compartmentation of the C3 and C4 enzymes in this species, and suggest that F. brownii is an advanced, C4-like C3-C4 intermediate.

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.

Carbon-isotope ratios were examined as δ(13)C values in several C3, C4, and C3-C4 Flaveria species, and compared to predicted δ(13)C, values generated from theoretical models. The measured δ(13)C values were within 4‰ of those predicted from the models. The models were used to identify factors that contribute to C3-like δ(13)C values in C3-C4 species that exhibit considerable C4-cycle activity. Two of the factors contributing to C3-like δ(13)C values are high CO2 leakiness from the C4 pathway and pi/pa values that were higher than C4 congeners. A marked break occurred in the relationship between the percentage of atmospheric CO2 assimilated through the C4 cycle and the δ(13)C value. Below 50% C4-cycle assimialtion there was no significant relationship between the variables, but above 50% the δ(13)C values became less negative. These results demonstrate that the level of C4-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C4 to the C3 cycle. As expression increaces above 50%, however, increased integration of C3- and C4-cycle co-function occurs.

The magnitude of the percentage inhibition of photosynthesis by atmospheric levels of O2 in the C3 species Solanum tuberosum L., Medicago sativa L., Phaseolus vulgaris L., Glycine max L., and Triticum aestivum L. increases in a similar manner with an increase in the apparent solubility ratio of O2/CO2 in the leaf over a range of solubility ratios from 25 to 45. The solubility ratio is based on calculated levels of O2 and CO2 in the intercellular spaces of leaves as derived from whole leaf measurements of photosynthesis and transpiration. The solubility ratio of O2/CO2 can be increased by increased leaf temperature under constant atmospheric levels of O2 and CO2 (since O2 is relatively more soluble than CO2 with increasing temperature); by increasing the relative levels of O2/CO2 in the atmosphere at a given leaf temperature, or by increased stomatal resistance. If the solubility ratio of O2/CO2 is kept constant, as leaf temperature is increased, by varying the levels of O2 or CO2 in the atmosphere, then the percentage inhibition of photosynthesis by O2 is similar. The decreased solubility of CO2 relative to O2 (decreased CO2/O2 ratio) may be partly responsible for the increased percentage inhibition of photosynthesis by O2 under atmospheric conditions with increasing temperature.

The potential for C4 photosynthesis was investigated in five C3-C4 intermediate species, one C3 species, and one C4 species in the genus Flaveria, using (14)CO2 pulse-(12)CO2 chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating (14)CO2 into the C4 acids malate and aspartate, following an 8-s pulse. The proportion of (14)C label in these C4 products ranged from 50-55% to 20-26% in the C3-C4 intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, (14)CO2 into aspartate as into malate. Generally, about 5-15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C4 photosynthesis among the intermediate species based on the apparent rate of conversion of (14)C label from the C4 cycle to the C3 cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C4-cycle products and an increase in percentage label in C3-cycle products during chase periods with (12)CO2, although the rate of change was slower than in the C4 species, F. palmeri. In these C3-C4 intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C3-C4 intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C4-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C4 and C3 cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C3 species, 7-18% of the initial label was in malate+aspartate. However, only 40-50% of this label was in the C-4 position, indicating C4-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O2, the absorbed quantum yields for CO2 uptake (in mol CO2·[mol quanta](-1)) averaged 0.053 in F. cronquistii (C3), 0.051 in F. trinervia (Spreng.) Mohr (C4), 0.052 in F. ramosissima (C3-C4), 0.051 in F. anomala (C3-C4), 0.050 in F. linearis (C3-C4), 0.046 in F. floridana (C3-C4), and 0.044 in F. pubescens (C3-C4). In 2% O2 an enhancement of the quantum yield was observed in all of the C3-C4 intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O2 were intermediate in value to the C3 and C4 species, indicating a co-function of the C3 and C4 cycles in CO2 assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O2 presumably reflect an ineffcient transfer of carbon from the C4 to the C3 cycle. The response of the quantum yield to four increasing O2 concentrations (2-35%) showed lower levels of O2 inhibition in the C3-C4 intermediate F. ramosissima, relative to the C3 species. This indicates that the co-function of the C3 and C4 cycles in this intermediate species leads to an increased CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O2.

Etude de la photosynthese chez cinq des sept especes de Flaveria presumees de type C 4 afin de determiner si ce sont des especes de type C 4 ou de type C 4 -like

The effect of environmental factors on the post-illumination burst of CO2 (PIB) and O2 inhibition of apparent photosynthesis (APS) in wheat (Triticum aestivum L.) was studied in an open gas exchange system utilizing the mathematics of non-steady-state systems. Two components of inhibition by O2 are suggested: one is caused by photorespiration as measured from the maximum rate of the PIB, and the second is direct inhibition as taken as APS2%O2— (APSx%O2+ PIBx%O2) where X is the oxygen concentration. A primary PIB which occurred from 16–28 s after the darkening of the foliage was attributed to photorespiration. No primary PIB was observed at 2% O2. At a CO2 concentration of 100 μ/1 in the atmosphere (about 2.5 μM based on leaf intercellular concentration) and at 30°C and 145 nE/cm2 nE/cm2·s, APS decreased curve-linearly with increasing O2 and reached an O2 compensation point of 560 μM (48% by volume), above which there was a net loss of CO2 in the light. The PIB increased with increasing O2 and became saturated at about 500 μM O2 but decreased above 900 μM O2. Direct inhibition of photosynthesis by O2 increased with increasing O2 concentration. Decreasing CO2 concentration had an effect on the magnitude of the PIB similar to that of increasing O2. At 30°C and 21% O2, the PIB increased with decreasing CO2 down to the CO2 compensation point (I) of 1.4 μM (47 μM/l). Below Γ, both PIB and CO2 evolution into the air in the light (at 21% O2) increased and then decreased at CO2 below 0.8 μM. The ratio of the PIB to APS2% o O2 increased linearly with increasing O2/CO2 ratio where O2 was held constant at 21% and CO2 was varied from 1.4 to 8.5 μM, while direct inhibition of photosynthesis expressed as a proportion of APS2%O2 remained constant over this range. At low CO2 concentration photorespiration as estimated by the PIB is the major part of O2 photosynthesis, while at atmospheric CO2 levels, direct inhibition is the major component. The PIB and APS at 2% and 21% O2 increased hyperbolically with increasing irradiance and all became light-saturated at about 65 nE/cm2 s. The percentage total O2 inhibition of photosynthesis remained constant with increasing irradiance as did the relative contribution of direct O2 inhibition or photorespiration (PIB) to total O2 inhibition. The PIB and APS at 21% O2 had similar temperature optima of 30°C when experimental conditions were adjusted to provide a constant internal O2/CO2 solubility ratio at varying temperatures. However, with a constant external CO2 concentration, the temperature optimum for the PIB shifted upward to 35°C while that for APS at 21% O2 remained at 30°C, which may be due to an increased O2/CO2 concentration in the leaf with increasing temperature.

Paraveinal mesophyll (PVM) is a specialized soybean ( Glycine max Merr.) leaf tissue which represents a significant biochemical compartment. Stereological measurements showed that PVM makes up 23% of the mesophyll volume in nodulated soybean. To get an indication of the extent of involvement of PVM in ureide metabolism, physical characteristics, distribution of allantoinase activity and ureide content were determined in isolated PVM protoplasts (PVMP) and mesophyll protoplasts (MP). PVMP were larger and contained less chlorophyll and protein than MP. PVMP had twice as much allantoinase activity per protoplast but only half as much allantoinase activity when expressed on a volume basis as compared to MP. Total leaf ureide concentration was high and nearly equally distributed between MP and PVMP. PVMP had a higher ureide content per protoplast, a higher concentration of allantoic acid and a lower ratio of allantoin to allantoic acid. These results suggest that both tissues have the capacity to assimilate allantoin in vivo. The data are discussed with reference to the relative access of the two mesophyll tissues to incoming ureides.