Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Period-2 cycles and 2:1 phase locking in a biological clock driven by temperature pulses

Crassulacean acid metabolism (CAM) serves as a botanical model system for the investigation of circadian rhythmicity. In a new set of experiments with the obligatory CAM plant Kalancho? daigremontiana the response to periodic stimulations with temperature pulses has been studied. On the basis of an experimental phase-response curve of net CO(2)-gas exchange the effect of periodic stimulation has been simulated using a finite-difference equation. These simulations revealed the locations of two period-2 cycles in the CO(2) uptake of the CAM plant. In subsequent experiments based upon the simulated bifurcation diagram the position and amplitude of one of these cycles were confirmed, while experimental evidence for the second cycle could be found. Possible roles of such dynamics for the functioning of the biological clock are discussed.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

Circadian Stomatal Rhythms in Epidermal Peels from Vicia faba

Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 30°C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population.     

Evidence of multiple circadian oscillators in bean plants.

Circadian rhythms in stomatal opening and photosynthesis had shorter free-running periods than circadian rhythms in leaflet movement in bean plants (Phaseolus vulgaris L.) transferred from 12-hr photoperiods to constant conditions. The rhythm in leaflet movement had a period close to 27 hr, whereas the rhythm in stomatal opening, measured as conductance to water vapor, had a period close to 24 hr. Photosynthesis, measured as net assimilation of CO2, also oscillated with a period close to 24 hr. The periods of these rhythms did not vary with increasing temperature, demonstrating temperature compensation of the controlling oscillators. The difference in free-running periods displayed by these rhythms is evidence that multiple oscillators with different intrinsic frequencies operate in bean plants.     

Priority of light/dark entrainment over temperature in setting the circadian rhythms of the prokaryote Synechococcus RF-1

Light/dark (L/D) and temperature are two major factors in the entrainment of circadian rhythms. The input pathways of these two environmental factors for the entrainment of circadian rhythms in Synechococcus RF-1 are different since the overt rhythms in mutant CR-1, one of the circadian-rhythm mutants of Synechococcus RF-1, could be established by temperature cycles but not by L/D. Therefore, it was of interest to investigate the phases of Synechococcus RF-1 cells entrained simultaneously by L/D and temperature. The circadian rhythms of nitrogenase activity and protein synthesis in RF-1 cells entrained by L/D, and by lowered or raised temperatures differed in their peaks of activity. Comparison of the phases of RF-1 cells entrained by L/D and temperature independently, and by L/D and temperature simultaneously indicated that L/D entrainment has priority over the temperature effect. Received: 8 February 1999 / Accepted: 1 April 1999     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

铁皮石斛叶片光合作用的碳代谢途径

 利用LI-6400光合测定系统测定了不同天气条件下铁皮石斛（Dendrobium officinale）叶片24h CO2吸收的动态以及CO2吸收对光强和温度的响应。晴天的白天和夜间铁皮石斛都能吸收CO2,中午CO2吸收速率为负值, CO2的交换方式具景天酸代谢途径(CAM)的特点。阴雨天,只有白天吸收CO2,夜间表现为暗呼吸,光合作用碳代谢的途径为C3途径。在多云的天气条件下,白天吸收CO2,并持续至日落后。夜间21∶00仍有CO2吸收,23∶00以后至次日凌晨处于暗呼吸状态。在500 μmol·m-2·s-1光照件下,20℃出现最大CO2吸收值。在夜间,25℃时CO2的吸收速率最高。有光和无光条件下,低温或高温引起CO2吸收速率下降均为非气孔因素所致。晴天上午,铁皮石斛叶片的表观量子产额为0.035,光合补偿点为2.9μmol·m-2·s-1,饱和光强为500μmol·m-2·s-1,强光下出现光抑制现象。叶片受到强光预先照射后,即使光照减弱光抑制效应仍保持一段时间,致使光合补偿点升高,表观量子产额下降,相同光强下的CO2吸收效率降低。结果表明：铁皮石斛为兼性CAM植物,随着环境条件的变化,其光合作用在景天酸代谢途径(CAM)与C3途径间变化。     

The circadian timekeeping system of Drosophila

Daily rhythms in behavior, physiology and metabolism are controlled by endogenous circadian clocks. At the heart of these clocks is a circadian oscillator that keeps circadian time, is entrained by environmental cues such as light and activates rhythmic outputs at the appropriate time of day. Genetic and molecular analyses in Drosophila have revealed important insights into the molecules and mechanisms underlying circadian oscillator function in all organisms. In this review I will describe the intracellular feedback loops that form the core of the Drosophila circadian oscillator and consider how they are entrained by environmental light cycles, where they operate within the fly and how they are thought to control overt rhythms in physiology and behavior. I will also discuss where work remains to be done to give a comprehensive picture of the circadian clock in Drosophila and likely many other organisms.     

Young daughter cladodes affect CO2 uptake by mother cladodes of Opuntia ficus-indica

BACKGROUND AND AIMS: Drought damages cultivated C3, C4 and CAM plants in the semi-arid lands of central Mexico. Drought damage to Opuntia is common when mother cladodes, planted during the dry spring season, develop young daughter cladodes that behave like C3 plants, with daytime stomatal opening and water loss. In contrast, wild Opuntia are less affected because daughter cladodes do not develop on them under extreme drought conditions. The main objective of this work is to evaluate the effects of the number of daughter cladodes on gas exchange parameters of mother cladodes of Opuntia ficus-indica exposed to varying soil water contents. METHODS: Rates of net CO2 uptake, stomatal conductance, intercellular CO2 concentration, chlorophyll content and relative water content were measured in mature mother cladodes with a variable number of daughter cladodes growing in spring under dry and wet conditions. KEY RESULTS: Daily carbon gain by mother cladodes was reduced as the number of daughter cladodes increased to eight, especially during drought. This was accompanied by decreased mother cladode relative water content, suggesting movement of water from mother to daughter cladodes. CO2 assimilation was most affected in phase IV of CAM (late afternoon net CO2 uptake) by the combined effects of daughter cladodes and drought. Rainfall raised the soil water content, decreasing the effects of daughter cladodes on net CO2 uptake by mother cladodes. CONCLUSIONS: Daughter cladodes significantly hasten the effects of drought on mother cladodes by competition for the water supply and thus decrease daily carbon gain by mother cladodes, mainly by inhibiting phase IV of CAM.     

Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 °C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.     

Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoë daigremontiana

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalancho? daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.     

Relationships between leaf conductance to CO2 diffusion and photosynthesis in micropropagated grapevine plants, before and after ex vitro acclimatization

In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO(2) diffusion, which often represents an important constraint to CO(2) assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO(2) diffusion were assessed in in vitro-cultured plants of the grapevine rootstock '41B' (Vitis vinifera 'Chasselas'xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m(-2) s(-1) in acclimatized plants, which ensured a higher CO(2) concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m(-2) s(-1), which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.     

Temperature Cycles as Zeitgeber for the Circadian Clock of Two Burrowing Rodents, the Normothermic Antelope Ground Squirrel and the Heterothermic Syrian Hamster

The circadian systems of two burrowing rodents, the normothermic diurnal antelope ground squirrel (Ammospermophilus leucurus) and the heterothermic nocturnal Syrian hamster (Mesocricetus auratus) were compared with respect to entrainment by temperature cycles. Both species were subjected to the same ambient temperature (Ta) cycles with amplitudes between 4 and 12ºC at constant illuminations (100 and 0.05 lux in squirrels; 1.0 lux in hamsters). Wheel running activity was continuously measured. There was considerable interindividual variation in the daily pattern of wheel-running activity and in the ability to entrain to Ta cycles of the same amplitude in both species. The activity rhythms of about 33 to 67% of the animals of the two species entrained to Ta cycles with amplitudes of 6 to 12ºC. One of six squirrels and one of nine hamsters even entrained to Ta cycles of 4ºC. In the antelope ground squirrels, activity occurred predominantly in the cooler phase of the Ta cycle, whereas hamsters were mainly active during the warmer phase. In some squirrels, the activity rhythms were split in two main components which were both entrained to the cooler fraction of the Ta cycle, sometimes with additional (masking) activity during the warmer fraction (above 30ºC). The results do not support the earlier view that temperature cycles affect the circadian systems of heterothermic mammals, including hibernators, more strongly than those of normothermic species. It is suggested that behavioral and physiological adjustments to the environmental conditions play an important role for mammalian circadian systems to respond to temperature changes as a zeitgeber.     

Temperature Cycles as Zeitgeber for the Circadian Clock of Two Burrowing Rodents,the Normothermic Antelope Ground Squirrel and the Heterothermic Syrian Hamster

The circadian systems of two burrowing rodents, the normothermic diurnal antelope ground squirrel (Ammospermophilus leucurus) and the heterothermic nocturnal Syrian hamster (Mesocricetus auratus) were compared with respect to entrainment by temperature cycles. Both species were subjected to the same ambient temperature (Ta) cycles with amplitudes between 4 and 12ºC at constant illuminations (100 and 0.05 lux in squirrels; 1.0 lux in hamsters). Wheel running activity was continuously measured. There was considerable interindividual variation in the daily pattern of wheel-running activity and in the ability to entrain to Ta cycles of the same amplitude in both species. The activity rhythms of about 33 to 67% of the animals of the two species entrained to Ta cycles with amplitudes of 6 to 12ºC. One of six squirrels and one of nine hamsters even entrained to Ta cycles of 4ºC. In the antelope ground squirrels, activity occurred predominantly in the cooler phase of the Ta cycle, whereas hamsters were mainly active during the warmer phase. In some squirrels, the activity rhythms were split in two main components which were both entrained to the cooler fraction of the Ta cycle, sometimes with additional (masking) activity during the warmer fraction (above 30ºC). The results do not support the earlier view that temperature cycles affect the circadian systems of heterothermic mammals, including hibernators, more strongly than those of normothermic species. It is suggested that behavioral and physiological adjustments to the environmental conditions play an important role for mammalian circadian systems to respond to temperature changes as a zeitgeber.     

Circadian Rhythms in Photosynthesis : Oscillations in Carbon Assimilation and Stomatal Conductance under Constant Conditions

Net carbon assimilation and stomatal conductance to water vapor oscillated repeatedly in red kidney bean, Phaseolus vulgaris L., plants transferred from a natural photoperiod to constant light. In a gas exchange system with automatic regulation of selected environmental and physiological variables, assimilation and conductance oscillated with a free-running period of approximately 24.5 hours. The rhythms in carbon assimilation and stomatal conductance were closely coupled and persisted for more than a week under constant conditions. A rhythm in assimilation occurred when either ambient or intercellular CO₂ partial pressure was held constant, demonstrating that the rhythm in assimilation was not entirely the result of stomatal effects on CO₂ diffusion. Rhythms in assimilation and conductance were not expressed in plants grown under constant light at a constant temperature, demonstrating that the rhythms did not occur spontaneously but were induced by an external stimulus. In plants grown under constant light with a temperature cycle, a rhythm was entrained in stomatal conductance but not in carbon assimilation, indicating that the oscillators driving the rhythms differed in their sensitivity to environmental stimuli.     

Some effects of air temperature and humidity on crop and leaf photosynthesis, transpiration and resistance to gas transfer

Measurements of carbon dioxide exchange and transpiration were made, at various air temperatures, on wheat and barley using a field enclosure system. From these were derived the stomatal and mesophyll resistances to carbon dioxide transfer. Optimum temperatures for net CO₂ uptake were about 24°C for wheat and barley. Above these optima, as temperature increased so net CO₂ uptake rates decreased, because of increasing stomatal and mesophyll resistances; transpiration rates decreased in wheat but were constant in barley. In laboratory growth cabinets, wheat plants were subjected to different regimes of temperature and humidity. Optimum temperature for net CO₂ uptake of individual leaves was 25°C. At constant humidity, a decline in net uptake rates above 25°C was associated with large increases in mesophyll resistance. At a constant 25°C, as the vapour pressure deficit (v.p.d. was increased above 1 k Pa (10 mb) v.p.d. the net uptake declined, with an increase in mesophyll resistance and a small increase in stomatal resistance. When the v.p.d. exceeded 1 k Pa at a temperature of 30°C, conditions that are experienced by field plants, then there were large increases in both mesophyll and stomatal resistances and the net uptake rates declined. Photo-respiration, as measured by CO₂ uptake in oxygen-free air, was independent of temperature, but both dark respiration and CO₂ compensation concentration increased with temperature.