J. Brulfert

Photoperiodism and crassulacean acid metabolism : I. Immunological and kinetic evidences for different patterns of phosphoenolpyruvate carboxylase isoforms in photoperiodically inducible and non-inducible Crassulacean acid metabolism plants.

Plants of Kalanchoe blossfeldiana v. Poelln. Tom Thumb and Sedum morganianum E. Walth. were grown under controlled photoperiodic conditions under either short or long days. Gaz exchange measurements confirmed that in K. blossfeldiana Crassulacean acid metabolism (CAM) was photoperiodically inducible and that S. morganianum performed CAM independently of photoperiod. With K. blossfeldiana, a comparison of catalytic and regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from short-day and long-day grown plants showed differences, but not with S. morganianum. Ouchterlony double diffusion tests and immunotitration experiments (using a S. morganianum PEPC antibody) established that CAM is induced in K. blossfeldiana—but not in S. morganianum—through the synthesis of a new PEPC isoform; this form shows an immunological behavior different from that prevailing under non-inductive conditions and can be considered as specific for CAM performance.

Effect of NaCl on Photosynthesis of Two Wheat Species (Triticum durum and T. aestivum) Differing in their Sensitivity to Salt Stress

Summary Two wheat species, Triticum aestivum , cv. Tanit and T. durum , cv. Ben Bachir, were cultivated hydroponically and subjected to salt stress (50 or 100 mmol/L NaCl) up to 21 days. NaCl treatment resulted in growth reduction for both varieties (but less for Ben Bachir than for Tanit), equivalent Na + and Cl − absorption by the whole plants, and the capacity for Ben Bachir to transport ions into the leaves where they accumulated. This accumulation process accounts for the maintainance of the water status in Ben Bachir leaves. As a whole, Ben Bachir shows characteristics of a NaCl-tolerant species. Net CO 2 uptake by the non-tolerant Tanit variety was markedly reduced at PPFD higher than 500 µmol m −2 s −1 , mostly because of stomatal closure, as shown by the decrease in the C i /C a ratio, the effect of saturating CO 2 concentrations (5 %) and the increase in the δ 13 C in the salt-treated leaf tissues. No effect of salt stress could be found either on the initial slopes of the net CO 2 assimilation curve, on the F v /F m ratio of chlorophyll fluorescence, or on the carboxylative capacities in the leaves. As a whole, these results favour the hypothesis of an exclusive effect of salt stress on the stomatal behaviour of the treated leaves.

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants.

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C4, Crassulacean acid metabolism (CAM) and C3 types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C4 type), kalanchoe (CAM type), french bean, and spinach (C3 type). In the green leaves of C4 plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C3 plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.

Effect of drought stress on net CO2 uptake by Zea leaves

The net CO2 assimilation by leaves of maize (Zea mays L. cv. Adonis) plants subjected to slow or rapid dehydration decreased without changes in the total extractable activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (ME). The phosphorylation state of PEPC extracted from leaves after 2–3 h of exposure to light was not affected by water deficit, either. Moreover, when plants which had been slowly dehydrated to a leaf relative water content of about 60% were rehydrated, the net CO2 assimilation by leaves increased very rapidly without any changes in the activities of MDH, ME and PEPC or phosphorylation state of PEPC. The net CO2-dependent O2 evolution of a non-wilted leaf measured with an oxygen electrode decreased as CO2 concentration increased and was totally inhibited when the CO2 concentration was about 10%. Nevertheless, high CO2 concentrations (5–10%) counteracted most of the inhibitory effect of water deficit that developed during a slow dehydration but only counteracted a little of the inhibitory effect that developed during a rapid dehydration. In contrast to what could be observed during a rapidly developing water deficit, inhibition of leaf photosynthesis by cis-abscisic acid could be alleviated by high CO2 concentrations. These results indicate that the inhibition of leaf net CO2 uptake brought about by water deficit is mainly due to stomatal closure when a maize plant is dehydrated slowly while it is mainly due to inhibition of non-stomatal processes when a plant is rapidly dehydrated. The photosynthetic apparatus of maize leaves appears to be as resistant to drought as that of C3 plants. The non-stomatal inhibition observed in rapidly dehydrated leaves might be the result of either a down-regulation of the photosynthetic enzymes by changes in metabolite pool sizes or restricted plasmodesmatal transport between mesophyll and bundle-sheath cells.

Interaction of Photoperiod and Drought as CAM Inducing Factors in Kalanchoë blossfeldiana Poelln., cv. Tom Thumb.

Summary Long-day (C3-performing) or short-day (CAM-performing) treated plants of Kalanchoe blossfeldiana Poelln. cv. Tom Thumb, were subjected to drought (no watering, 30 % relative humidity in the air) under long days; in the C3-performing leaves, CAM was induced after a lag of 2 days, with progressive development of typical CO2 exchange patterns, diurnal oscillation of malic acid content and increasing capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) through enzyme neosynthesis. From day 7 of drought, typical CAM shifted to a mode of operation in which nocturnal CO2 uptake was maintained, day CO2 exchange suppressed, malic acid pool fully depleted and PEPC capacity lowered. In contrast, in the already CAM-performing (short-day-grown) plants, the effect of drought was immediate (no lag time), resulting in a very steep increase in PEPC capacity; the level of dark CO2-fixation was maintained, day CO2 exchange was suppressed and the malic acid pool decreased to attain a lowered but stabilized level of day-night oscillation. The mechanism involved in CAM induction by drought showed some similarity to that of CAM induction by short days. This would suggest that the probably different messengers triggered by phytochrome modification and by water stress affect the same target as a first step of the induction process.

Identification of multiple PEPC isogenes in leaves of the facultative Crassulacean Acid Metabolism (CAM) plant Kalanchoe blossfeldiana Poelln. cv. Tom Thumb

In the facultative Crassulacean Acid Metabolism (CAM) plant Kalanchoe blossfeldiana cv. Tom Thumb, CAM can be induced by short‐day treatment or water deficiency stress. From young leaves of well‐watered and water‐stressed individuals of this plant, cDNA clones coding for a partial sequence of the key enzyme of CAM, phosphoenolpyruvate carboxylase, were isolated after transcription of mRNA. cDNA polymorphism was established by enzyme restriction profiles and sequencing data. Four PEPC isogenes could be shown to exist in K. blossfeldiana forming two gene pairs, with 95%–98% homology inside and only 75% between the pairs. One cDNA sequence pair having a length of 1113 bp and an open reading frame of 371 AA was identified as PEPC isoform specific for the C3 state, whereas the pair having a length of 1116 bp and an open reading frame of 372 AA could be attributed to the CAM state. These results were confirmed by Southern Blot hybridization. (EMBL, accession numbers X 87818, X 87819, X 87820, X 87821.)

Ecophysiological Studies on the Vegetation of Madagascar: A δ13C and δD Survey for Incidence of Crassulacean Acid Metabolism (Cam) Among Orchids From Montane Forests and Succulents from the Xerophytic Thorn-Bush

Abstract The incidence of Crassulacean acid metabolism (CAM) in plants collected at various habitats in Madagascar was investigated by survey of carbon and hydrogen isotope ratios ((δ13C and δD values). In about 50% of the epiphytic orchids from evergreen higher and lower montane forests the δ13C values were indicative for CAM. The remainder of the species are presumably C3 plants. In all samples of malagasy epiphytic leafless orchids comprising 9 species, the δ13C values suggest extreme CAM with CO2 uptake proceeding entirely during the night. All terrestrial orchids collected in the lower montane forests obviously acquire external carbon by C3-photosynthesis, whereas Lissochilus decaryi, a terrestrial orchid from the semi-arid south of Madagascar and various other species of this genus are CAM plants. This is the first report of CAM occurrence in sympodial terrestrial orchids. Judged by the δ13C values, all succulents (mainly Didiereaceae, Euphorbiaceae, Crassulaceae and Asclepiadaceae) collected at the...

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism.

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using 14C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by 14CO2 fumigations and NaH14CO3 injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO2 in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.

Daily rhythm of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants : Immunological evidence for the absence of a rhythm in protein synthesis.

Immunotitration of phosphoenolpyruvate carboxylase (EC 4.1.1.31) extracted from leaves of Kalanchoe blossfeldiana v. Poelln. cv. Tom Thumb. It was established that at different times of the day-night cycle the daily rhythm of enzyme capacity does not result from a rhythm in protein synthesis, but rather from changes in the specific activity of the enzyme.

Ecophysiological studies in Kalanchoë porphyrocalyx (Baker) and K. miniata (Hils et Bojer), two species performing highly flexible CAM

Preceding results, based on the determination of stable carbon isotope composition (δ13C) of leaf tissues from various Kalanchoë species, suggested a close coincidence between the photosynthetic flexibility of the species and their habitat, life form and taxonomic position within the genus. The ability to shift from C3-to Crassulacean Acid Metabolism (CAM)-type of photosynthesis seemed to concern in particular the more ancestral species in the genus and to be linked to epiphytism and changing climatic situations. For deeper insights into these interrelationships, physiological studies in controlled conditions were carried out on K. miniata and K. porphyrocalyx. These two species differ by their habitat preference and life form. Measurements were conducted on CO2 exchange patterns, day/night fluctuation of malate content in the leaves and ‘capacity’ of phosphoenolpyruvate carboxylase (PEPC). The results show that the 2 species can be considered as ‘facultative’ CAM plants, with very high flexibility in their photosynthetic behaviour. The decrease in water availability seems to be a major factor triggering the shift from C3 to the CAM mode. In K. miniata, 21 days of drought depressed CO2 uptake to the level of CAM idling whereas in K. porphyrocalyx, CO2 exchange was considerably more resistant. At least for K. miniata, short-day treatment was found to be a further CAM-inducing factor. The results are discussed in terms of their ecophysiological significance under the environmental conditions of the sites where the investigated species naturally grow.