Orlando Queiroz

Photoperiodism and enzyme rhythms: Kinetic characteristics of the photoperiodic induction of Crassulacean acid metabolism.

SummaryThe effect of photoperiod on Crassulacean acid metabolism (CAM) in Kalanchoe blossfeldiana Poellniz, cv. Tom Thumb, has characteristics similar to its effect on flowering in this plant (although these two phenomena are not causally related). The photoperiodic control of CAM is based on (a) dependance on phytochrome, (b) an endogenous circadian rhythm of sensitivity to photoperiodic signals, (c) a balance between specific positive (increase in enzyme capacity) and negative (inhibitory substances) effects of the photoperiod. Variations in malate content, capacity of phosphoenolpyruvate (PEP) carboxylase, and capacity of CAM inhibitors in young leaves were measured under photoperiodic conditions noninductive for CAM and after transfer into photoperiodic conditions inductive for CAM. Essential characteristics of the photoperiodic induction of CAM are: 1) lag time for malate accumulation; 2) after-effect of the inductive photoperiod on the malate accumulation, on the increase in PEP carboxylase capacity, and on the decrease in the level of long-day produced inhibitors; final levels of malate, enzyme capacity and inhibitor are proportional to the number of inductive day-night cycles; 3) cireadian rhythm in PEP carboxylase capacity with a fixed phase under noninductive photoperiods and a continuously shifting phase under inductive photoperiods, after complex advancing and delaying transients. Kinetic similarities indicate that photoperiodic control of different physiological functions, namely, CAM and flowering, may be achieved through similar mechanisms. Preliminary results with species of Bryophyllum and Sedum support this hypothesis. Phase relationships suggest different degrees of coupling between endogenous enzymic rhythm and photoperiod, depending on whether the plants are under long days or short days.

Enzymic and metabolic changes in bean leaves during continuous pollution by subnecrotic levels of SO2

Abstract A rapid increase in enzyme capacity appears to be the primary response to subnecrotic (0·1 ppm, 300μg m −3), continuous pollution by SO2 in the leaves of Phaseolus vulgaris. The process is not restricted to a specific metabolic function but affects the key-enzymes of the main central metabolic pathways, and also peroxidases. Changes in the pools of organic acids, amino acids and polyamines are concomitant, and consistent, with the enzymic changes. In contrast, concentration in sulphur-containing amino acids is not modified. The results suggest a co-ordinated increase in the metabolic potentiality of the cell affording a temporarily increased capacity of resisting pollution by a faster metabolisation of SO2 and a stabilisation of internal pH (postulated role of the variation in polyamides). The temporary (about 2–3 weeks) character of this readjustment and its physiological cost are discussed in connection with recent work.

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.

Photoperiodism and Crassulacean acid metabolism : II. Relations between leaf aging and photoperiod in Crassulacean acid metabolism induction.

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.

Changes in the isozymic pattern of phosphoenolpyruvate : An early step in photoperiodic control of crassulacean acid metabolism level.

Two major isofunctional forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) have been separated from the leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb by acrylamide gel electrophoresis and diethylaminoethyl cellulose techniques: one of the forms prevails under long-day treatment (low crassulacean acid metabolism level), the other develops under short-day treatment (high Crassulacean acid metabolism level). Molecular weights are significantly different: 175·103 and 186·103, respectively. These results indicate that two populations of phosphoenolyruvate carboxylase are present in the plant, one of which is responsible for Crassulacean acid metabolism activity under the control of photoperiod.The Crassulacean acid metabolism appears to depend on the same endogenous clock that governs other photoperiodically controlled events (e.g. flowering). The metabolic and energetic significance of this feature is discussed. It is suggested that modification in isozymic composition could be an early step in the response to photoperiodism at the metabolic level.

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.

Photoperiodic control of phenolic metabolism in Kalanchoe blossfeldiana

Abstract When grown in conditions of long day length, the leaves of Kalanchoe blossfeldiana contain high levels of soluble phenolic compounds, mainly present as tannins. A decreasing concentration gradient is observed in the leaves from the apex to the base. When transferred to short day conditions, the ability of leaves of the same physiological age to accumulate phenolics decreases with time. The very low phenolic content after 25 short days indicates pronounced changes in the metabolism of the plant induced by new photoperiodic conditions. Moreover, during development in short days the amount of tannins per leaf reaches a maximum then decreases suggesting an over-polymerization or even a degradation of the substances. A similar lag time is required for the depressing effect of short days on phenolies and for their stimulating effect on CAM.

Malate Dehydrogenase Forms a Complex with and Activates Phosphoenolpyruvate Carboxylase from Crassulacean Acid Metabolism Plants

Summary Gel chromatography and kinetic techniques give evidence for the association of malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) extracted from leaves of the Crassulacean Acid Metabolism plant Kalanchoe blossfeldiana . PEPC and one form of MDH co-eluted in the same peak from Ultrogel AcA 54, Ultrogel AcA 34 or Sephacryl 200. The complex was disrupted by gradient acrylamide gel electrophoresis and reconstituted in vitro by recovering the purified PEPC from the gel in the presence of MDH. This effect of MDH was not obtained with other proteins or enzymes at the same protein concentration. The molecular masses observed for the postulated complex are consistent with the association of one MDH dimer with one PEPC tetrameric form, or under higher ionic strength, one PEPC dimer. The PEPC-MDH interaction was shown to stimulate PEPC activity several times, or to depress it when MDH was in excess, which supports specificity of the effect. The inhibitory action of malate on PEPC activity was affected by the PEPC-MDH association.

Multiple Levels in the Control of Rhythms in Enzyme Synthesis and Activity By Circadian Clocks: Recent Trends

It is now well established that circadian clocks can control rhythmicity at different stages of the sequence of events leading from gene activity to a functional enzyme molecule. Conceptual and experimental distinction of the effective control targets is emphasized, with particular attention to recent results obtained on circadian clock control of transcription and to data indicating that proteins can behave as conformational oscillators. Thus circadian rhythmicity both in gene expression and in the dynamics of the enzyme molecule would contribute to the temporal compartmentation of processes such as metabolic co-ordination and channeling necessary for the adaptive efficiency of physiological programmes.

Enzyme circadian rhythms and conformational oscillators survey and prospects

Abstract An unconventional hypothesis is proposed combining for the first time two different features which are basic for cell metabolism but have been as yet investigated separately: (a) circadian rhythms in enzyme activity, which play a fundamental role in adaptation to the 24h cyclities of environmental cues, and (b) the dynamic character of protein structure which results in continuous shifting between conformational states. Recent data indicate that large time‐scale (hours, circadian) periodicities can thus arise spontaneously in the catalytic characteristics of enzymes in solution, alternating phases of high and low sensitivity to substrate and effectors. The question raises whether conformational oscillators would provide a molecular component of the processes which govern rhythmicity of enzyme activity. This hypothesis also opens a new approach to the study of the temporal organization of enzyme‐enzyme interactions and metabolic channeling, particularly in photoperiodism.