Xerosicyos danguyi H. Humb. (Cucurbitaceae) is a Crassulacean acid metabolism (CAM) species native to Madagascar. Previously, it was shown that when grown under good water conditions, it is a typical CAM plant, but when water stressed, it shifts to a dampened form of CAM, termed CAM-idling, in which stomata are closed day and night but with a continued, low diurnal organic acid fluctuation. We have now studied the kinetics of some metabolic features of the shift from CAM to CAM-idling under severe water stress and the recovery upon rewatering. When water is withheld, there is a steady decrease in relative water content (RWC), reaching about 50%, at which point the water potential decreases precipitously from about -2 or -3 bars to -12 bars.Abscisic acid (ABA) increases sharply at about 75% RWC. Stomata close, which limits COz uptake, and there is a dampened diurnal organic acid fluctuation typical of CAM-idling. Throughout an extended stress period to 50% RWC, there is no change in chlorophyll, protein, and ribulose bisphosphate carboxylase activity compared with the well-watered plants. Despite the fact that the tissue was already in CAM, the stress is accompanied by an increase in phosphoenolpyruvate carboxylase (PEPc) mRNA, extractable PEPc activity, and PEPc protein (such that the specific activity remained approximately constant) and a decrease in the apparent KJPEP). It is not known if the changes in KJPEP) in response to drought are related to or are separate from the increases in PEPc protein and mRNA. The changes in Km(PEP) could be in response to the decreased endogenous levels of organic acids, but evidently are not an assay artifact. The increases in PEPc protein and mRNA appear to be related to the water-stress treatment and may result from the increased concentration of ABA or the decreased levels of endogenous organic acids. When rewatered, the metabolism quickly returns to the well-watered control typical of CAM.
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,X,K,(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 40C. 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 waterstress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, L-malate, decreased the V.., and increased the K,,xpEp) 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,,,p) for all treatments. At saturafing 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 I 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.PEPC' is the initial C02-fixing enzyme in C4 plants (in the light) and CAM plants (in the dark). In both C4 and CAM plants, the amount of PEPC protein does not change between '
During the development of Peperomia camptotricha leaves, metabolism changes from C3‐photosynthesis to Crassulacean acid metabolism (CAM). The youngest leaves showed no diurnal fluctuation of organic acids or P‐enolpyruvate carboxylase (PEPc) activity. There was little evidence for PEPc protein using PEPc antibodies prepared from the CAM form of PEPc, nor was there evidence for PEPc mRNA when tested using a cDNA probe made from CAM P. scandens. As leaves matured, there was a parallel increase in titratable acidity, PEPc activity, PEPc protein, and PEPc mRNA. In leaf whorls 1 through 6, there was a significant linear correlation between the diurnal fluctuation of organic acids and PEPc activity indicating a functional relationship. The specific activity of PEPc increased as leaves matured and the Km (PEP) decreased indicating that the enzyme was becoming more active. The ratio of PEPc protein to PEPc mRNA decreased as leaves matured. During the expression of CAM, the spongy mesophyll where most of the CAM activity occurs increased in thickness and per cent air space, whereas the palisade mesophyll where most of the C3 activity occurs did not increase in size dramatically. The diurnal fluctuation of organic acids and the expression of PEPc activity, protein, and mRNA increased as the thickness of the spongy mesophyll increased. During the expression of CAM in Peperomia camptotricha, there appears to be coordinated expression of PEPc mRNA, protein, and activity, the commencement of diurnal organic acid fluctuation, and the development of the CAM‐like spongy mesophyll. Thus the evidence suggests that CAM in this species is expressed during normal development and not in response to environmental signals.
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