Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in <Emphasis Type="Italic">Mammillaria gracilis</Emphasis> Tissues

In vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation and hyperhydricity in in vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H₂O₂) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS. The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed. Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation and hyperhydricity and linked to the loss of tissue organization in M. gracilis. B. Balen and M. Tkalec contributed equally to this work.     

Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C₄ plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C₄ and CAM plants, respectively.     

Nitrogen metabolism-related enzymes in <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> after <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

We compared C₃ and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2^nd leaf pair and non-inoculated 3^rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C₃ plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C₃ and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C₃ plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3^rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C₃- and CAM-specific.     

Limiting Factors in Photosynthesis: II. IRON STRESS DIMINISHES PHOTOCHEMICAL CAPACITY BY REDUCING THE NUMBER OF PHOTOSYNTHETIC UNITS

It has been proposed that Fe stress may be used in the study of limiting factors in photosynthesis as an experimental means of varying photochemical capacity in vivo (Plant Physiol 1980 65: 114-120). In this paper the effect of Fe stress on photosynthetic unit number, size, and composition was investigated by measuring P₇₀₀, cytochrome (Cyt) f, chlorophyll (Chl) a, and Chl b in sugar beet leaves. The results show that when Fe stress reduced Chl per unit area by 80% (from 60 to 12 micrograms per square centimeter), it decreased the number of P₇₀₀ molecules per unit area by 88% and Cyt f per unit area by 86%; over the same range the Chl to P₇₀₀ ratio increased by 37% but there was no significant change in the Chl to Cyt f ratio. These data suggest that Fe stress decreases photochemical capacity and Chl per unit area by diminishing the number of photosynthetic units per unit leaf area.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO₂ into organic acids for subsequent C₄ or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC₄PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.     

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H₂O₂ from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.     

Morphology and ultrastructure of Mammillaria gracillis (Cactaceae) in in vitro culture

Morphogenetic status of cactus Mammillaria gracillis Pfeiff. tissue culture was studied by light and electron microscopy. In vitro propagated shoots spontaneously developed callus. This callus regenerated normal and hyperhydric shoots without exogenous hormones. Tumour tissue induced by wild or rooty strains of Agrobacterium tumefaciens never expressed any morphogenetic potential. Light microscopy showed cellular characteristics of morphologically different tissues. Ultrastructural studies revealed changes in plastids: secondary dedifferentiation of mature chloroplasts, thylakoid swelling and disruption, phytoferritin accumulation, plastid elongation and increase in size. Changes in chlorophyll and carotenoid content were in accordance with degradation of the thylakoid system. Plastids were confirmed as very sensitive organelles to an artificial hyperhydric environment as well as to Agrobacteria-mediated cell transformation.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH₂) through the cytochrome b₆f complex (Cyt b₆f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700⁺ and PC⁺ was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC⁺ and P700⁺ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b₆f to the PSI donors. A significant down-regulation of Cyt b₆f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b₆f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e⁻ transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

DAC Is Involved in the Accumulation of the Cytochrome b6/f Complex in Arabidopsis

The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b₆/f complex, and provide evidence suggesting that the efficiency of cytochrome b₆/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b₆/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b₆/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b₆ protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b₆/f complex, possibly through interaction with the PetD protein.The cytochrome b₆/f (Cyt b6/f) complex is a multisubunit complex that resides in the thylakoid membrane and functions in linear and cyclic electron transport. In the linear process, the complex receives electrons from PSII and transfers them to PSI, a process that is accompanied by the generation of a proton gradient, which is essential for ATP synthesis (Mitchell, 1961; Saraste, 1999). The native form of this complex is present as a dimer with a mass of 310 kD that can be converted into a 140-kD monomer with increasing detergent concentrations (Huang et al., 1994; Breyton et al., 1997; Mosser et al., 1997; Baniulis et al., 2009). In higher plants, the Cyt b6/f monomer contains at least eight subunits: Cyt f, Cyt b₆, PetC, PetD, PetM, PetL, PetG, and PetN (Wollman, 2004). PetC and PetM are encoded by nuclear genes, whereas the others are encoded by plastid genes. It has been shown that PetG and PetN are necessary for complex stability in tobacco (Nicotiana tabacum; Schwenkert et al., 2007). By contrast, PetL is not required for the accumulation of other subunits of the Cyt b6/f complex, even though it is involved in the stability and formation of the functional dimer (Bendall et al., 1986; Schwenkert et al., 2007). Inactivation of PetC in Arabidopsis (Arabidopsis thaliana) resulted in significantly reduced amounts of Cyt b6/f subunits and completely blocked linear electron transport, indicating that PetC participates in the formation of the functionally assembled Cyt b6/f complex (Maiwald et al., 2003). In Synechocystis sp. PCC 6803, the PetM subunit has no essential role in Cyt b6/f complex electron transfer or accumulation; however, the absence of this subunit apparently affects the levels of other protein complexes involved in energy transduction (Schneider et al., 2001). In addition to the other proteins, FNR was identified as a subunit of the Cyt b6/f complex isolated from spinach (Spinacia oleracea) thylakoid membranes (Zhang et al., 2001).Previous research has revealed how the Cyt b6/f complex assembles into a functional dimer (Bendall et al., 1986; Lemaire et al., 1986; Kuras and Wollman, 1994). In the Cyt b6/f complex, Cyt b₆ and PetD form a mildly protease-resistant subcomplex that serves as a template for the assembly of Cyt f and PetG, producing a protease-resistant cytochrome moiety (Wollman, 2004). The PetC and PetL proteins then participate in the assembly of the functional dimer (Schwenkert et al., 2007). PetD becomes more unstable in the absence of Cyt b₆, and the synthesis of Cyt f is greatly reduced when either Cyt b₆ or PetD is inactivated, indicating that both Cyt b₆ and PetD are prerequisite for the synthesis of Cyt f (Kuras and Wollman, 1994). The reduced synthesis of Cyt f can be explained by the so-called CES (for controlled by epistasy of synthesis) mechanism. It is suggested that, in this mechanism, the synthesis rate of some chloroplast-encoded subunits of photosynthetic protein complexes is regulated by the availability of their assembly partners from the same complexes (Choquet et al., 2001). The mechanism of CES for Cyt f has been studied in detail in Chlamydomonas reinhardtii (Choquet et al., 1998; Choquet and Vallon, 2000). In it, the unassembled Cyt f inhibits its own translation through a negative feedback mechanism, and MCA1 and TCA1 have been demonstrated to be involved in the regulation of Cyt f synthesis (Boulouis et al., 2011).Many studies have focused on understanding the conversion of apocytochrome to holocytochrome via the covalent binding of heme in Cyt f and Cyt b₆ during the assembly of Cyt b6/f through the CCS and CCB pathways (Nakamoto et al., 2000; Wollman, 2004; de Vitry, 2011). The CCS pathway was originally discovered in the green alga C. reinhardtii through genetic studies of ccs mutants (for cytochrome c synthesis) that display a specific defect in membrane-bound Cyt f and soluble Cyt c₆, two thylakoid lumen-resident c-type cytochromes functioning in photosynthesis (Xie and Merchant, 1998). In the CCS pathway, six loci that include plastid ccsA and nuclear CCS1 to CCS5 have been found in C. reinhardtii (Xie and Merchant, 1998). In these mutants, the apocytochrome is normally synthesized, targeted, and processed, but heme attachment is perturbed. The CCB pathway is involved in the covalent attachment of heme c(i) to Cyt b₆ on the stromal side of the thylakoid membranes (Kuras et al., 2007). The ccb mutants show defects in the accumulation of subunits of the Cyt b6/f complex and covalent binding of heme to Cyt b₆ (Lyska et al., 2007; Lezhneva et al., 2008). However, heme binding is not a prerequisite for the assembly of Cyt b₆ into the Cyt b6/f complex, although the fully formed Cyt b6/f showed an increased sensitivity to protease (Saint-Marcoux et al., 2009).The assembly of the Cyt b6/f complex is a multistep process, and current studies have shown that the covalent binding of heme to Cyt f and Cyt b₆ is highly regulated. Thus, it is reasonable to speculate that, similar to the other photosynthetic protein complexes (Mulo et al., 2008; Nixon et al., 2010; Rochaix, 2011), the assembly of the Cyt b6/f complex is also assisted by many nucleus-encoded factors. In this study, we characterized an Arabidopsis protein, DAC (for defective accumulation of Cyt b6/f complex), that seems to be involved in the assembly of the Cyt b6/f complex. In addition, we provide evidence that DAC interacts directly with PetD before it assembles within the Cyt b6/f complex.     

Variability in the synthesis of cytochromes f and b-563 during the cell cycle of Euglena gracilis

The study of the successive formation of the photosynthetic apparatus in Euglena gracilis (Brandt, P. and Von Kessel, B. (1983) Plant Physiol. 72, 616–619) was extended to the determination of the stage-specific synthesis of cytochrome $\text{b}\text{f}$ complex during the cell cycle of this alga. Most of the cytochrome f (33 kDa) has properties of an intrinsic membrane protein, but part of it is soluble. Cytochrome b-563 (18 kDa) is only intrinsic. The intensity of binding the intrinsic cytochromes in the thylakoids depends on the developmental stage of the organism. The light-independent synthesis of cytochrome f takes place prior to the assembly of the chlorophyll-protein complex I (CP I). Immediately after this assembly of CP I, cytochrome b-563 is synthesized in the light. Hence, the ratio cytochrome b-563/cytochrome f changes during the cell cycle of E. gracilis. The physiological implication of presumably non-complexed cytochrome f and of complex-bound cytochromes f and b-563 on the stage-specific efficiency of photosynthesis of E. gracilis is discussed.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Aiming at understanding the odd case of CAM expression by a C₄ plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxylyase, phosphorylating) were comparatively studied in leaves of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and under water-stress. CAM induction in leaves of these plants (designated as CAM) is indicated by the nocturnal acidification and by the clear diurnal oscillation pattern and amplitude of acidity, malic acid, and PEPC activity characteristic of CAM plants. Treatment of the other plant group (designated as C₄) by growth under a 16-h photoperiod and well-watered conditions did not induce expression of the tested criteria of CAM in plants. In these C₄ plants, the mentioned CAM criteria were undetectable. PEPC from CAM and C₄ Portulaca responded differently to any of the studied assay conditions or effectors. For example, extent and timing of sensitivity of PEPC to pH change, inhibition by malate, activation by glucose-6-phosphate or inorganic phosphate, and the enzyme affinity to the substrate PEP were reversed with induction of CAM from the C₄-P. oleracea. These contrasting responses indicate distinct kinetic and regulatory properties of PEPC of the two modes. Thus by shifting to CAM in the C₄ Portulaca a new PEPC isoform may be synthesised to meet CAM requirements. Simultaneous occurrence of both C₄ and CAM is suggested in P. oleracea when challenged with growth under stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photocontrol of sorghum leaf phosphoenolpyruvate carboxylase : characterization of messenger RNA and of photoreceptor

The mechanism underlying the light effect on phosphoenolpyruvate carboxylase (PEPC) from the C₄ plant sorghum (Sorghum vulgare Pers., var Tamaran) leaves was investigated. Following exposure to light a new isozyme of PEPC, specific for the green leaf and responsible for primary CO₂ fixation in photosynthesis, was established. Northern blot experiments revealed the presence of PEPC mRNA showing a molecular weight of 3.4 kilobases. During the greening process, concomitant to enzyme activity, PEPC protein and PEPC messenger RNA amounts increased considerably. This photoresponse was shown to be under phytochrome control.     

Different CO<Subscript>2</Subscript> acclimation strategies in juvenile and mature leaves of <Emphasis Type="Italic">Ottelia alismoides</Emphasis>

The freshwater macrophyte, Ottelia alismoides, is a bicarbonate user performing C4 photosynthesis in the light, and crassulacean acid metabolism (CAM) when acclimated to low CO₂. The regulation of the three mechanisms by CO₂ concentration was studied in juvenile and mature leaves. For mature leaves, the ratios of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) are in the range of that of C4 plants regardless of CO₂ concentration (1.5–2.5 at low CO₂, 1.8–3.4 at high CO₂). In contrast, results for juvenile leaves suggest that C4 is facultative and only present under low CO₂. pH-drift experiments showed that both juvenile and mature leaves can use bicarbonate irrespective of CO₂ concentration, but mature leaves have a significantly greater carbon-extracting ability than juvenile leaves at low CO₂. At high CO₂, neither juvenile nor mature leaves perform CAM as indicated by lack of diurnal acid fluctuation. However, CAM was present at low CO₂, though the fluctuation of titratable acidity in juvenile leaves (15–17 µequiv g^?1 FW) was slightly but significantly lower than in mature leaves (19–25 µequiv g^?1 FW), implying that the capacity to perform CAM increases as leaves mature. The increased CAM activity is associated with elevated PEPC activity and large diel changes in starch content. These results show that in O. alismoides, carbon-dioxide concentrating mechanisms are more effective in mature compared to juvenile leaves, and C4 is facultative in juvenile leaves but constitutive in mature leaves.     

Freezing of Nonwoody Plant Tissue: II. Cell Damage and the Fine Structure of Freezing Curves

Increasing pretreatment day temperatures of 20, 30, and 40 C resulted in decreased net photosynthesis in Agropyron smithii (C₃) while in Bouteloua gracilis (C₄) net photosynthesis was increased. The effect on photosynthesis of increasing analysis temperatures was the same as observed by increasing pretreatment temperatures. Resistance of the stomata and boundary layer were less affected by pretreatment temperatures than were the remaining resistances of a physical and chemical nature. Resistances for A. smithii were increased and those for B. gracilis were decreased by increasing pretreatment temperatures. Phenology of the species in the shortgrass prairie is such that A. smithii has its greatest growth activity during the cool portion of the growth season, whereas B. gracilis is most active in the warm portion. Thus, photosynthetic adaptation to temperature is strongly suggested as a strategy for ecosystem utilization by reduction of interspecific competition.     

Over-expression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of CO₂ in C₄ plants. Under the control of the rice Rubisco small subunit promoter, cDNA of a C₄ SiPPC gene cloned from Seteria italica was introduced into Japonica rice by Agrobacterium-mediated transformation. Integration of the gene was confirmed by PCR analysis. RT-PCR showed expression of the gene at the RNA level in transgenic plants, and enzyme activity measurements confirmed the increase in PEPC protein. The transformants showed improvements in both photosynthesis rate and yield only under upland field cultivation. The possible function of PEPC in rice stress tolerance is discussed.     

Photoperiodism and Crassulacean acid metabolism

Measurements of net CO₂ exchange, malate accumulation, properties and capacity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaves of different ages of two short-day dependent Crassulacean acid metabolism (CAM) plants (Kalanchoe blossfeldiana v. Poelln. Tom thumb and K. velutina Welw.) show that, in both species: a) young leaves from plants grown under long days display a CO₂ exchange pattern typical of C₃ plants; b) leaf aging promotes CAM under long-day conditions; c) short-day treatment induces CAM in young leaves to a higher degree than aging under long days; d) at least in K. blossfeldiana, the PEPC form developed with leaf aging under long days and the enzyme form synthetized de novo in young leaves grown under short days were shown to have similar properties. Short days also promote CAM in older leaves though at a lesser extent than in young leaves: The result is that this photoperiodic treatment increases the general level of CAM performance by the whole plant. The physiological meaning of the control of PEPC capacity by photoperiodism could be to afford a precisely timed seasonal increase in CAM potentiality, enabling the plant to immediately optimize its response to the onset of drought periods.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC phosphoenolpyruvate carboxylase (EC 4.1.1.31) - LD long day - SD short day