CAM and carbon isotope composition: Origin of the constitutive carbon in young leaves of Bryophyllum daigremontianum Berger

Abstract. The export of assimilates from mature leaves towards the young leaves was investigated: 100% and 65% of constitutive matter of leaves of rank 0 (the youngest leaves at the top of the plant) and of rank 1, respectively, originated from other parts of the plant. Photosynthesis of a particular leaf covers the total carbon requirement of that leaf only when it reaches about two-thirds of its mature size. When pairs of mature leaves were excised, the young leaves increased their own autotrophic growth while the level of assimilates exported by the remaining leaves remained unchanged. The existence of permanent pools in the leaves that export the assimilates was demonstrated; about 50% of the carbon, both in the soluble and insoluble fractions of mature and senescent leaves (ranks 5 to 8 from the apex), was not renewed by turnover. It is shown that the ¹³C-enrichment of the components of the starch-malate sequence in young leaves results, at least in part, from the incorporation of imported carbon chains. The significance of the δ¹³C diagnosis in CAM determination is discussed in relation to the origin of the constitutive carbon of the leaves.     

Diurnal changes in sucrose phosphate synthase activity in leaves

Studies were conducted to identify and compare diurnal changes in sucrose phosphate synthase (EC 2.4.1.14) activity in leaves of different species, and the effect of nitrogen nutrition on the rhythm in soybean [ Glycine max (L). Merr] leaves. In recently expanded corn ( Zea mays L.) leaves, a single peak of enzyme activity was observed at the beginning of the photoperiod. A similar pattern was observed in older corn leaves, but activities (leaf fresh weight basis) were lower. In recently expanded pea ( Pisum sativum L.) and soybean leaves, two peaks of sucrose phosphate synthase activity were observed over a 24-h light:dark period, one at the beginning and one at the end of the photoperiod. A similar pattern was observed in older soybean leaves, but activities were generally lower and the amplitude of the changes was reduced. In a separate experiment, soybean plants were grown in the greenhouse with either 2 or 10 m M nitrate. The high-N plants had higher rates of photosynthesis and translocation, and greater activities of sucrose phosphate synthase in leaf extracts, compared to low-N plants. Over both experiments with soybeans, changes in sucrose phosphate synthase activity during the photoperiod were closely aligned with changes in translocation rate.     

The leaf as the site of gibberellin action in flower formation in Bryophyllum daigremontianum

Summary The long-short-day plant Bryophyllum daigremontianum can be induced to flower by transfer from long to short days (LDSD), or by gibberellin (GA) application under SD. Application of GA to mature leaves of intact or partially defoliated plants induces flowering more effectively than when applications are made to the youngest leaf pair and the shoot tip.Mature leaves on de-budded plants in SD are induced to produce floral stimulus by GA application, as demonstrated by grafting LD receptor scions onto the debudded plants, or by grafting SD leaves treated with GA onto receptor stocks in LD. This shows that GA applied to Bryophyllum in SD exerts its flower-promoting effect in the leaves.The minimal number of SD necessary for flower formation in Bryophyllum is approximately 15, both in case of photoinduction by the shift LDSD, and after GA treatment in SD. It is concluded that the LD part of photinduction establishes a high level of endogenous GAs in the leaves which is a prerequisite for production of floral stimulus under subsequent SD.Work supported by the United States Atomic Energy Commission, Contract No. AT(11-1) 1338.     

Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum berger,a plant with crassulacean acid metabolism: Some physiological aspects related to CO2 dark fixation

Isotope analysis of the biochemical fractions isolated quantitatively from young and mature leaves of Bryophyllum daigremontianum Berger have been carried out before and after a dark period of accumulation of organic acids. The mature leaf is enriched in ¹³C compared to the young leaf. The ¹³C values of the different leaf constituents vary between the ¹³C values of C₄ plants (-11) and those of C₃ plants (-27). During the dark period, the two types of leaves store organic acids with ¹³C values of -15 and lose insoluble sugars, including starch with a ¹³C value of -12. Furthermore, young leaves store phosphorylated compounds with ¹³C values of -11 and lose weakly polymerised sugars with ¹³C values of -18. These results led to the formulation of a hypothesis of the origin of the two substrates of -carboxylation: phosphoenolpyruvate arises from the glycolytic breakdown of the insoluble sugars rich in ¹³C, and the major portion of the CO₂ is the result of the complete breakdown (respiration) of the soluble sugars rich in ¹²C. The existence of two independent sugar pools leads to the assumption that there are two separate glycolytic pathways. The ¹³C enrichment of the stored products of the young leaves in the day seems to be the result of a weak discrimination for ¹³C by ribulose diphosphate carboxylase, which reassimilates to a great extent the CO₂ released from malate accumulated in the night.Abbreviations CAM crassulacean acid metabolism - C₃ metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway - C₄ metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway - ¹³C() (R_sample-R_PDB) 10³/R_PDB where PDB=Pee Dee belemnite (belemite from the Pee Dee formation South Carolina) and R=¹³C/¹²C - NAD-MDH(EC1.1.1.37) NAD-malate dehydrogenase - NADP-ME (EC1.1.1.40) NADP-malic enzyme - PEP phosphoenolpyruvate - PEPC (EC4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK(EC2.7.9.1) pyruvate, Pi-dikinase - RuDP ribulose diphosphate - RuDPC (EC4.1.1.39) RuDP carboxylase     

Effect of leaf age on light and dark 14CO2 fixation in a CAM plant, Bryophyllum calycinum

Leaves of different ages from B. calycinum were exposed to ¹⁴CO₂in light during day and night. The labelling pattern on thechromatogram differed with leaf age. Young leaves had similarpatterns to those of C3 plants during both day and night. Matureleaves showed high incorporation of ¹⁴C into C4 acids, especiallyat night. In contrast, no significant difference with leaf agewas observed in the pattern of dark ¹⁴CO₂ fixation products.Study of the enzyme activity and the content of titratable acidat each leaf age suggested that high incorporation of ¹⁴C inC4 acids during the night was due to the simultaneous absorptionof CO₂ by both enzymes RuDPcarboxylase and PEPcarboxylase. (Received November 24, 1977; )     

Diurnal changes in photosystem II photochemistry, photoprotective compounds and stress-related phytohormones in the CAM plant, Aptenia cordifolia

Acclimation of photosynthetic light reactions to daily changes in solar radiation requires adjustments in photosystem II photochemistry and may be affected by environmental stresses, such as drought. In this study, we examined the effects of a short-term, severe water deficit on diurnal variations in photosystem II photochemistry, photoprotective compounds (tocopherols and carotenoids, including the xanthophyll cycle) and stress-related phytohormones (abscisic acid and salicylic acid) in the CAM plant, Aptenia cordifolia L. f. Schwantes. Violaxanthin was rapidly converted to zeaxanthin under high light, the de-epoxidation state of the xanthophyll cycle reaching maximum levels of 0.95 at midday in irrigated plants. Under a higher photoprotective demand caused by water deficit, plants showed significant increases in abscisic acid and γ-tocopherol levels, which were followed by decreases in β-carotene and the F_v/F_m ratio at later stages of stress. Decreases in this ratio below 0.70 correlated with sustained increases in the de-epoxidation state of the xanthophyll cycle, which kept above 0.90 at night after 15 days of water deficit. In contrast to abscisic acid, salicylic acid levels kept constant under water deficit and showed a sharp decrease during the day both under irrigated and water stress conditions. We conclude that the CAM plant, A. cordifolia showed several strategies of acclimation to short-term water deficit, including abscisic acid and γ-tocopherol accumulation, as well as sustained increases in the de-epoxidation state of the xanthophyll cycle, which was tightly coupled to daily variations in photosystem II photochemistry. The differential accumulation of tocopherol homologues under water deficit and the diurnal fluctuations of salicylic acid levels in this CAM plant will also be discussed.     

Effects of photoperiod and ageing on the carbon isotope composition of Bryophyllum daigremontianum Berger

Abstract. Changes in crassulacean acid metabolism (CAM) in the leaves of Bryophyllum daigremontianum were studied comparatively under closely controlled (phytotron) and semi-natural (greenhouse) conditions utilizing measurements of natural carbon isotope composition (δ¹³C) of the total leaf matter. Abrupt transfer of plants from long days to short days resulted in an overall increase of CAM in mature leaves (no. 3 from the apex and older) and thus in a higher CAM level for the whole plant. Study during the course of a year in semi-natural conditions showed that a progressive increase in length of day and day night temperature differences (February ← June) activated CAM but only a passage to short days (June ← October) produced the maximum CAM. Under the experimental conditions employed, the maximum shift from the C₃-type to the C₄-type of metabolism was observed in plants subjected to semi-natural progressive variations in the environment, i.e. the δ¹³C values indicated that for plants in the greenhouse the total carbon flow entering the cells was mediated by phosphoenolpyruvate carboxylase activity.     

Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM plant Bryophyllum fedtschenkoi

Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg²⁺ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg²⁺ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg²⁺ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg²⁺ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg²⁺ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO₂ exchange. However, in contrast to the rhythm in CO₂ exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.     

Frerea indica,a stem succulent CAM plant with deciduous C3 leaves

Summary During the winter in greenhouse culture, Frerea indica(Asclepiadaceae) is a leafless stem succulent resembling the other members of the Stapelieae subfamily. However, in spring it produces leaves which persist during the summer period. CO₂ exchange measurements were carried out with Frerea indica at its different seasonal states of development. The leafless stems in winter as well as the defoliated ones in summer show all characteristics of Crassulacean acid metabolism. The leaves, on the other hand, fix CO₂ with high rates according to the C₃ pathway; no diurnal malate fluctuations are detectable. This feature can be interpreted as an ecological adaptation where a seasonal plant dimorphism enables a functional change in the prevailing mode of CO₂ metabolism. In the leafless state, which probably represents the appearance of the plant during the dry period in nature, it is saving water by pursuing CAM. The season with enough water available can be used for high photosynthetic gain by C₃ leaves.     

Diurnal changes in chlorophylla fluorescence and carotenoid composition inOpuntia ficus-indica,a CAM plant,and in three C3 species in Portugal during summer

Summary Diurnal changes in chlorophylla fluorescence were determined in four species, differing in life form, in Portugal during the summer of 1989. These includedOpuntia ficus-indica, a CAM plant, andHelianthus annuus, Ficus carica andArbutus unedo, three C₃ species. Steady state fluorescence yield,F _S, and maximum fluorescence yield,F _M′, were determined at different times of the day. Using the model of Genty et al. (1989), the photon use efficiency of photosystem II electron transport,φ _e, was calculated from (F _M′−F _S)/F _M′. Diurnal changes in relative rate of non-cyclic electron transport through photosystem II,J _e, were derived by multiplyingφ _e by the incident photon flux density (PFD). WhenJ _e, determined for each species for various points in time throughout the day, was plotted against corresponding values of PFD, the light response curves obtained showed thatJ _e was linearly dependent on PFD in low light and approached saturation in high light. The highest values ofJ _e were observed inHelianthus annuus, followed byOpuntia ficus-indica, Ficus carica andArbutus unedo. The proportion of the xanthophyll zeaxanthin to total carotenoids, determined around noon, was inversely related to maximum rates ofJ _e.     

Gibberellin-like substances in Bryophyllum daigremontianum and the distribution and persistence of applied gibberellin A3

Summary Acidic extracts of the long-short-day plant Bryophyllum daigremontianum contain two gibberellin (GA)-like substances called fractions I and II. In plants under permanent short-day (SD) conditions the levels of both I and II are very low. In continuous long days (LD) the total GA content is approximately 20 times higher than in SD, mainly due to an increased level of II. Extracts of plants induced to flower by the shift LDSD show a further increase in the level of II. Application of GA₃ to plants in SD causes normal flower formation, but the level of fraction II remains as low as in vegetative plants in permanent SD.Approximately 10% of the GA₃ applied could still be recovered from leaves and inflorescences after 45 days, indicating that GA₃ is very stable in Bryophyllum, Most of the GA₃ recovered was still associated with the treated leaves, but small amounts could be detected in other leaves and in inflorescences. Results of grafting experiments indicate that these low levels of GA₃ are adequate to induce production of the floral stimulus.     

Diurnal fluctuation of light and dark CO2 fixation in peeled and unpeeled leaves of Bryophyllum daigremontiana

Diurnal fluctuation of light and dark CO₂ fixation in peeledand unpeeled leaves of Bryophyllum daigremontiana was examined.A distinct difference in light CO₂ fixation was observed inunpeeled leaves but not in peeled ones. No measurable differencein dark CO₂ fixation was observed in either type. These resultsindicate that the leaves of CAM plants have a high capacityfor CO₂ fixation in the daytime, but it is suppressed by theclosing of the stomata. Also, the rapid depression of CO₂ uptakewhen the illumination was directed at on dark acidified leavescould be prevented by peeling off the epidermis. The net photosyntheticCO₂ uptake in peeled leaves was 77 µmoles/mg chllrophyll/hrin the 3rd leaf and 62 in the 4th leaf. (Received August 7, 1978; )     

Light-induced changes in the period of the circadian rhythm of carbon dioxide output in Bryophyllum leaves

Summary The period of the rhythm of carbon dioxide output from leaves of Bryophyllum fedtschenkoi R. Hamet et Perrier at 15°C was shorter in continuous white light than in darkness. The period was monitored in leaves exposed to narrow spectral bands of monochromatic radiation at an incident quantum flux density of 4.7×10^-11 einsteins cm^-2s^-1. Bands centred on 660, 600, 730 and 530 nm significantly shortened the period, the greatest effect being achieved at 660 nm and the smallest at 530 nm; those centred on 760 and 450 nm were without effect. None of the bands tested significantly lengthened the period. The period of the rhythm in leaves exposed continuously to monochromatic radiation at 660 nm decreased with increasing quantum flux density. The extent to which a quantum flux density of 4.7×10^-11 einsteins cm^-2s^-1 at wavelength 660 nm shortened the period depended on the ambient temperature. At 15°C a significant reduction of 4.4 h occurred as compared with the dark control, while at 30°C no significant reduction was observed. The transient (the time from the initiation of the rhythm to the first peak) showed a greater dependence on temperature than did the steady-state period. No such difference could be detected in relation to the intensity or quality of irradiation. The reduction of the transient by the various irradiation treatments was, in general, proportional to the reduction of the period.     

Diurnal variation of the acid phosphatase activity in the leaves of Cowpea

Summary The activity of acid phosphatases (glycerophosphatase, phenylphosphatase and pyrophosphatase) in Cowpea (Vigna catjang) leaves has been studied at different hours of the day and a marked diurnal variation has been observed. The phosphatase activity in leaves collected between 10 A.M. to 2 P.M. was always higher than in leaves collected earlier in the morning and late at evening. Pyrophosphatase activity exhibited greater variation than phenylphosphatase and glycerophosphatase activity. The significance of the increased phosphatase activity is discussed in relation to the production of carbohydrates from organic acids in light.     

Internal and external photoprotection in developing leaves of the CAM plant Cotyledon orbiculata

Leaves of the CAM plant Cotyledon orbiculata produced a dense epidermal wax which decreased the absorption of light, possibly functioning as an external photoprotective mechanism (Robinson et al. 1993). However, developing leaves did not accumulate wax until after 21 d with full wax coating not achieved until at least 35 d. In addition, young leaves had lower rates of electron transport than mature leaves. Leaf development therefore occurs at higher incident PFD than that experienced by the mature leaves, and, for young leaves, can lead to an increase in the proportion of light energy which is excess to requirements and must be dissipated non-photochemically. Changes in the photosynthetic capacity, PSII efficiency, rate of energy dissipation, and the content of chlorophyll (Chi), carotenoids, wax and anthocyanins were followed in developing leaves of C. orbiculata in an attempt to elucidate the relative importance of the various photoprotective mechanisms during leaf ontogeny. The largest pools of xanthophyll cycle pigments (on a Chi basis) were found in the waxless, young leaves and were correlated with greater levels of energy dissipation activity. The importance of xanthophyll cycle-dependent energy dissipation in young C. orbiculata leaves prior to development of a reflective wax covering, and full photosynthetic capacity which for CAM plants includes appreciable nocturnal acid accumulation, is discussed. Also, we consider the possibility that anthocyanin pigments in the upper and lower epidermis may increase reflectivity and act as external photoprotectants.     

Gibberellin A20 content of Bryophyllum daigremontianum under different photoperiodic conditions as determined by gas-liquid chromatography

Summary The gibberellin A₂₀ content of the long-short-day plant Bryophyllum daigremontianum (R. Hamet and Perr.) Berg. under different photoperiodic conditions was determined by gas-liquid chromatography. Purified extracts from ten plants were adequate for quantitative analysis by this method. The level of GA₂₀ increased following transfer from long-day (LD) to short-day (SD) conditions until after 38 SD it was three times higher on a dry weight basis than in comparable plants under continuous LD. No GA₂₀ could be detected in extracts of plants under permanent SD. These results are in agreement with earlier data obtained by assaying Bryophyllum extracts with the d-5 mutant of corn.     

Diurnal changes in the regulatory properties of PEP-carboxylase in Crassulacean Acid Metabolism (CAM)

Abstract. The CAM plants Kalanchoe tubiflora and K. blossfeldiana were grown under photoperiodically controlled conditions (short days). In these plants, phos-phoenolpyruvate carboxylase capacity and the sensitivity of the enzyme to the effectors L-malate (inhibitor) and glucose-6-phosphate (activator) were measured throughout the diurnal CAM cycle. In K. tubiflora , enzyme capacity was higher if measured at pH 7.0 than at pH 8.0 and displayed a rhythmical behavior with highest values at the end of the light period. As reported earlier, in K. blossfeldiana PEP-C capacity was higher during the night. It was more pronounced when plants were kept in CO₂-free air during the dark period. In both plants, the sensitivity of the enzyme to the effectors showed very clear diurnal changes: inhibition by malate and activation by glucose-6-phosphate were strikingly higher during the day than during the night; the effect depended on PEP concentration. The changing activation of the enzyme by glucose-6-phos-phate reflects diurnal changes of the Km for PEP which was found to be higher during the day than during the night. Manipulations of malate accumulation by nocturnal application of CO₂-free air did not influence these effects. The results are discussed in context with the metabolic control of CAM.     

Kinetic changes with temperature of phosphoenolpyruvate carboxylase from a CAM plant

Abstract. Purified and crude phosphoenolpyruvate carboxylase from the CAM plant Kalanchoë daigremontiana Hamet et Perrier ( Bryophyllum diagremontianum ) was assayed at temperatures between 10 and 45° C. The optimum temperature of the enzyme activity changed with substrate availability and effector concentration in the assay. l -malate inhibited the enzyme activity and lowered the optimum temperature. Glucose-6-phosphate raised the optimum temperature to 43°C. K _m values for phosphoenolpyruvate increased with assay temperature from 0.12 mol m^-3 at 15° C to 0.36 mol^m−3 at 35° C. Inhibition by malate increased with temperature and acidity of the assay. In the crude enzyme 50% of control activity was inhibited by 1.65 mol m^-3 malate at 15° C and by 0.5 mol m^-3 at 35° C (at pH 7.0). With purification malate sensitivity was lost ( K _i values for malate at least 10 times higher). The shift in optimum temperatures for PEP-carboxylase activity thus results from changes in the kinetic parameters with temperature and allosteric effectors. The often low optimum temperatures for CO₂ fixation observed in nature may thus be the result of substrate and effector concentrations in the cytoplasm and the antagonistic effect of temperature on substrate affinity and effector efficiency on phosphoenolpyruvate carboxylase.