Identification of rhythmic subsystems in the circadian cycle of crassulacean acid metabolism under thermoperiodic perturbations

Leaves of the Crassulacean acid metabolism (CAM) plant Kalancho? daigremontiana Hamet et Perrier de la Bathie show overt circadian rhythms in net CO2 uptake, leaf conductance to water and intercellular CO2 concentration, which are entrained by periodic temperature cycles. To probe their sensitivity to thermoperiodic perturbations, intact leaves were exposed to continuous light intensity and temperature cycles with a period of 16 h, applying a set of different baseline temperatures and thermodriver amplitudes. All three overt rhythms were analyzed with respect to their frequency spectra and their phase relations with the thermodriver. For most stimulation protocols, stomatal conductance and net CO2 change were fully or partially entrained by the temperature pulses, while the internal CO2 concentration remained dominated by oscillations in the circadian range. Prolonged time series recorded for up to 22 d in continuous light underline the robustness of these circadian oscillations. This suggests that the overt circadian rhythm of net CO2 uptake in CAM results from the interaction of two coupled original systems: (i) an endogenous cycle of CO2 fixation in the mesophyll, showing very robust periodic activity, and (ii) stomatal movements that respond to environmental stimuli independently of rhythmic processes in the mesophyll, and thus modulate the gas exchange amplitude.     

The "Kluge-Lüttge Kammer": a preliminary evaluation of an enclosed, Crassulacean acid metabolism (CAM) Mesocosm that allows separation of synchronized and desynchronized contributions of plants to whole system gas exchange

Crassulacean acid metabolism (CAM) is recognized as a photosynthetic adaptation of plants to arid habitats. This paper presents a proof-of-concept evaluation of partitioning net CO2 exchanges for soil and plants in an arid, exclusively CAM mesocosm, with soil depth and succulent plant biomass approximating that of natural Sonoran Desert ecosystems. We present the first evidence that an enclosed CAM-dominated soil and plant community exposed to a substantial day/night temperature difference (30/20 degrees C), exhibits a diel gas exchange pattern consisting of four consecutive phases with a distinct nocturnal CO2 uptake. These phases were modulated by plant assimilation and soil respiration processes. Day-time stomatal closure of the CAM cycle during phase III was used to eliminate aboveground photosynthetic assimilation and respiration and thereby to estimate belowground plant plus soil respiration. Rapid changes in temperature appeared to synchronize single plant gas exchange but individual plant gas exchange patterns were desynchronized at constant day/night temperatures (25 degrees C), masking the distinct mesocosm pattern. Overall, the mean carbon budget of this CAM model Sonoran Desert system was negative, releasing an average of 22.5 mmol CO2 m-2 d-1. The capacity for nocturnal CO2 assimilation in this exclusively CAM mesocosm was inadequate to recycle CO2 released by plant and soil respiration.     

高浓度二氧化碳对植物影响的研究进展

工业革命后全球大气CO2浓度持续上升,不仅对全球气候的变迁产生重大影响,而且对植物的形态、水分利用、蛋白质合成、光合、抗性、生长及生物量等都有不同程度的影响。高浓度CO2促进植物根、幼苗的生长,叶片增厚,降低气孔密度、气孔导度及蒸腾速率,增加水分利用效率、作物的产量及生物量,促进乙烯生物合成,增强植物的抗氧化能力。不同光合途径(C3、C4及CAM)及不同植被类型的植物对高浓度CO2的响应不同。长期和短期的高浓度CO2处理,植物响应方式有很大的差异,如短期高CO2处理使光合能力增强,而长期处理则使光合能力下调。     

Environment or development? Lifetime net CO2 exchange and control of the expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

The relative influence of plant age and environmental stress signals in triggering a shift from C(3) photosynthesis to Crassulacean acid metabolism (CAM) in the annual halophytic C(3)-CAM species Mesembryanthemum crystallinum was explored by continuously monitoring net CO(2) exchange of whole shoots from the seedling stage until seed set. Plants exposed to high salinity (400 mm NaCl) in hydroponic culture solution or grown in saline-droughted soil acquired between 11% and 24% of their carbon via net dark CO(2) uptake involving CAM. In contrast, plants grown under nonsaline, well-watered conditions were capable of completing their life cycle by operating in the C(3) mode without ever exhibiting net CO(2) uptake at night. These observations are not consistent with the widely expressed view that the induction of CAM by high salinity in M. crystallinum represents an acceleration of preprogrammed developmental processes. Rather, our study demonstrates that the induction of the CAM pathway for carbon acquisition in M. crystallinum is under environmental control.     

Digital Computer Simulation of Respiratory Response to Cerebrospinal Fluid PCO2 in the Cat

The respiratory control system is treated as linear with a transmission delay between ventilation and sensing points (chemoreceptors). To the accepted variables involving body gas stores, ventilatory effects, transmission effects, and steady state pH, P(CO2), P(O2) chemoreceptor response, certain detailed analysis of the central receptors have been added. By construction of a model for medullary CO(2) receptor utilizing expected values of CNS (central nervous system) circulation, CO(2) production, and tissue-buffering effects, results of experimental observation of the effects of alteration of CSF were simulated. The inclusion of CSF effects also allowed simulation of the response to alteration in inspired CO(2), hyperventilation, and the periodic breathing with prolongation of circulation time.     

大气CO2浓度升高对植物的直接影响-国外十余年来模拟实验研究主要手段及基本结论

 本文针对国外近十几年来在CO2浓度升高对植物的直接影响方面所开展的生理生态学研究方法、动态、基本结论、存在问题等内容做了简要的介绍。大气CO2浓度在过去200年内已增加了80μmol·mol-1,生长在高CO2环境下的植物,其生理生态、形态及化学成分等方面将会发生相应的变化。表现在光合作用速率出现不同程度的提高;呼吸作用受抑制;气孔密度减少,水分利用效率增加;生物量及产量增加;一些关键蛋白质及酶、非结构性碳水化合物含量增加;组织中的氮、硫等元素含量降低;根系及花的发育也随CO2浓度的升高而提前等。不同光合途径(C3、C4及CAM)及不同植被类型(自然植被、栽培植被)的植物随CO2浓度发生的上述指标的变化在长期反应与短期反应方面具有很大的差异。另外,实验控制条件如温度、光照、水分、养分甚至实验装置(如花盆)的大小对预测结果也有很大的影响。     

Plant CO2 responses: an issue of definition, time and resource supply

In this review I am drawing attention to some constraints and biases in CO2 enrichment experiments and the analysis of data in the literature. Conclusions drawn from experimental works differ when the data are grouped in a way such that the relative frequency of test conditions does not determine the emerging trends, for instance unrealistically strong CO2-'fertilization' effects, which are in conflict with some basic ecological principles. I suggest separating three test conditions: uncoupled systems (plants not depending in a natural nutrient cycle) (I); expanding systems, in which plants are given ample space and time to explore otherwise limited resources (II); and fully coupled systems in which the natural nutrient cycling governs growth at steady-state leaf area index (LAI) and fine root renewal (III). Data for 10 type III experiments yield rather moderate effects of elevated CO2 on plant biomass production, if any. In steady-state grassland, the effects are water-related; in closed tree stands, initial effects decline rapidly with time. Plant-soil coupling (soil conditions) deserves far greater attention than plant-atmosphere coupling (CO2 enrichment technology).     

CO(2) homeostasis during periodic breathing in obstructive sleep apnea.

The contribution of apnea to chronic hypercapnia in obstructive sleep apnea (OSA) has not been clarified. Using a model (D. M. Rapoport, R. G. Norman, and R. M. Goldring. J. Appl. Physiol. 75: 2302-2309, 1993), we previously illustrated failure of CO(2) homeostasis during periodic breathing resulting from temporal dissociation between ventilation and perfusion ("temporal V/Q mismatch"). This study measures acute kinetics of CO(2) during periodic breathing and addresses interapnea ventilatory compensation for maintenance of CO(2) homeostasis in 11 patients with OSA during daytime sleep (37-171 min). Ventilation and expiratory CO(2) and O(2) fractions were measured on a breath-by-breath basis by means of a tight-fitting full facemask. Calculations included CO(2) excretion, metabolic CO(2) production, and CO(2) balance (metabolic CO(2) production - exhaled CO(2)). CO(2) balance was tabulated for each apnea/hypopnea event-interevent cycle and as a cumulative value during sleep. Cumulative CO(2) balance varied (-3,570 to +1,388 ml). Positive cumulative CO(2) balance occurred in the absence of overall hypoventilation during sleep. For each cycle, positive CO(2) balance occurred despite increased interevent ventilation to rates as high as 45 l/min. This failure of CO(2) homeostasis was dependent on the event-to-interevent duration ratio. The results demonstrate that 1) periodic breathing provides a mechanism for acute hypercapnia in OSA, 2) acute hypercapnia during periodic breathing may occur without a decrease in average minute ventilation, supporting the presence of temporal V/Q mismatch, as predicted from our model, and 3) compensation for CO(2) accumulation during apnea/hypopnea may be limited by the duration of the interevent interval. The relationship of this acute hypercapnia to sustained chronic hypercapnia in OSA remains to be further explored.     

Crassulacean acid metabolism in the ZZ plant, Zamioculcas zamiifolia (Araceae)

Zamioculcas zamiifolia (Araceae), a terrestrial East African aroid, with two defining attributes of crassulacean acid metabolism (CAM) (net CO(2) uptake in the dark and diel fluctuations of titratable acidity) is the only CAM plant described within the Araceae, a mainly tropical taxon that contains the second largest number of epiphytes of any vascular plant family. Within the Alismatales, the order to which the Araceae belong, Z. zamiifolia is the only documented nonaquatic CAM species. Zamioculcas zamiifolia has weak CAM that is upregulated in response to water stress. In well-watered plants, day-night fluctuations in titratable acidity were 2.5 μmol H(+)·(g fresh mass)(-1), and net CO(2) uptake in the dark contributed less than 1% to daily carbon gain. Following 10 d of water stress, net CO(2) uptake in the light fell 94% and net CO(2) uptake in the dark increased 7.5-fold, such that its contribution increased to 19% of daily carbon gain. Following rewatering, dark CO(2) uptake returned to within 5% of prestressed levels. We postulate that CAM assists survival of Z. zamiifolia by reducing water loss and maintaining carbon gain during seasonal droughts characteristic of its natural habitat.     

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.     

On the nature of facultative and constitutive CAM: environmental and developmental control of CAM expression during early growth of Clusia, Kalanchöe, and Opuntia

The capacity to induce crassulacean acid metabolism developmentally (constitutive CAM) and to up-regulate CAM expression in response to drought stress (facultative CAM) was studied in whole shoots of seven species by measuring net CO(2) gas exchange for up to 120 day-night cycles during early growth. In Clusia rosea, CAM was largely induced developmentally. Well-watered seedlings began their life cycle as C(3) plants and developed net dark CO(2) fixation indicative of CAM after the initiation of the fourth leaf pair following the cotyledons. Thereafter, CAM activity increased progressively and drought stress led to only small additional, reversible increases in dark CO(2) fixation. In contrast, CAM expression was overwhelmingly under environmental control in seedlings and mature plants of Clusia pratensis. C(3)-type CO(2) exchange was maintained under well-watered conditions, but upon drought stress, CO(2) exchange shifted, in a fully reversible manner, to a CAM-type pattern. Clusia minor showed CO(2) exchange reponses intermediate to those of C. rosea and C. pratensis. Clusia cretosa operated in the C(3) mode at all times. Notably, reversible stress-induced increases of dark CO(2) fixation were also observed during the developmental progression to pronounced CAM in young Kalancho? daigremontiana and Kalancho? pinnata, two species considered constitutive CAM species. Drought-induced up-regulation of CAM was even detected in young cladodes of a cactus, Opuntia ficus-indica, an archetypal constitutive CAM species. Evidently, the defining characteristics of constitutive and facultative CAM are shared, to variable degrees, by all CAM species.     

Sensitivity of the Plant Vacuolar Malate Channel to pH, Ca2+ and Anion-Channel Blockers

The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in which large fluxes of malate occur during the day/night cycle. By applying the patch-clamp technique, in the whole-vacuole configuration, to isolated vacuoles from leaf mesophyll cells of the CAM plant Kalancho? daigremontiana, we studied the regulation of the vacuolar malate channel by pH and Ca2+, as well as its sensitivity to anion-channel blockers. Malate currents were found to be insensitive to Ca2+ on the cytoplasmic side of the membrane over a range from approximately 10(-8) M to 10(-4) M. In contrast, decreasing cytoplasmic pH below 7.5 had a significant modulatory effect on channel activity, reducing malate currents by 40%, whereas increasing cytoplasmic pH above 7.5 resulted in no change in current. Several known Cl?-channel blockers inhibited the vacuolar malate currents: niflumic acid and indanoyloxyacetic acid (IAA-94) proved to be the most effective inhibitors, exerting half-maximal effects at concentrations of approximately 20 mM, suggesting that the plant vacuolar malate channel may share certain similarities with other classes of known anion channels.     

CO2浓度升高对沉水克隆植物生长速率及营养元素积累的影响

 研究在不同CO2浓度下水生克隆植物刺苦草（Vallisneria spinulosa）整个生活周期中生长的动态变化及对营养元素积累情况。在不损伤植物体的前提下,采用刺苦草形态学指标组合史估计了植株生物量的动态变化。结果表明：刺苦草鳞茎的萌发不受CO2浓度变化的影响。在高浓度CO2即(1000±50) μmol·mol-1下,刺苦草源株地上部分生长速率在整个生长前期和中期都远远高于低浓度CO2即(400±20) μmol·mol-1,而在后期则出现相反的现象,其中一个原因是因为高浓度CO2下后期光合物质向地下大量转移形成鳞茎引起地上部分生长减慢。但高浓度CO2下克隆株中的初级和次级分株生长速率均高于低浓度CO2。在两种CO2浓度下相同克隆植株构件中的总碳含量没有明显差异;除鳞茎外,根、叶、匍匐茎中的总磷含量随CO2浓度升高显著增加;由于各构件生物量增加有明显差异,导致叶和鳞茎因为生物稀释作用而使其含氮量降低了12%～14%,但根和茎中含量基本保持不变。在高浓度CO2中植株总生物量显著升高,所以总碳、总氮和总磷吸收量均显著大于在低浓度CO2中的吸收量。研究结果揭示,大气CO2浓度升高对沉水克隆植物生长的促进,有利于提高水生克隆植物在群落中的竞争能力;水生植物克隆生长将增加水生生态系统中碳的沉积;水环境中N、P含量将直接影响到水生克隆植物生长。     

大气CO2浓度升高对植物的直接影响—国外十余年来模拟实验研究主要手段及基本结论

 本文针对国外近十几年来在CO2浓度升高对植物的直接影响方面所开展的生理生态学研究方法、动态、基本结论、存在问题等内容做了简要的介绍。大气CO2浓度在过去200年内已增加了80μmol·mol-1,生长在高CO2环境下的植物,其生理生态、形态及化学成分等方面将会发生相应的变化。表现在光合作用速率出现不同程度的提高;呼吸作用受抑制;气孔密度减少,水分利用效率增加;生物量及产量增加;一些关键蛋白质及酶、非结构性碳水化合物含量增加;组织中的氮、硫等元素含量降低;根系及花的发育也随CO2浓度的升高而提前等。不同光合途径(C3、C4及CAM)及不同植被类型(自然植被、栽培植被)的植物随CO2浓度发生的上述指标的变化在长期反应与短期反应方面具有很大的差异。另外,实验控制条件如温度、光照、水分、养分甚至实验装置(如花盆)的大小对预测结果也有很大的影响。     

Curiosity and context revisited: crassulacean acid metabolism in the Anthropocene

Having gained some understanding of the consequences of the CO(2)-concentrating mechanisms in crassulacean acid metabolism (CAM) that internalize the photosynthetic environment of the Cretaceous on a daily basis, it may be time to consider potential long-term effects of the planetary CO(2)-concentrating mechanism on growth and ecology of these plants in the Anthropocene. This paper emphasizes our limited understanding of the carbohydrate economy of CAM in relation to growth processes and briefly reviews recent studies of the diel cycles of growth in these plants. An inadvertent long-term, regional-scale experiment from the past is revisited in which an Opuntia monoculture grew to occupy >25 million hectares of farmland in central eastern Australia, producing a total biomass of about 1.5 billion tonnes in about 80 years. Although at the time it does not seem to have been recognized that this invasion involved CAM, a botanist from the University of Melbourne, Jean White-Haney emerges as a heroic pioneer in the control of the invader by poison and pioneered its biological control. The Opuntia population was expanding at 10-100 ha h(-1) when it was brought to a halt within a decade by the voracious appetite of Cactoblastis cactorum larvae. It is now known that the female parent moth of this predator detects CAM in O. stricta prior to oviposition by deploying the most sensitive CO(2) detector system yet found in the Lepidoptera. The O. stricta invasion is a dramatic demonstration of the capacity of CAM plants to attain and sustain high biomass; to sequester and retain atmospheric CO(2). In conclusion, experiments are reviewed that show stimulation of CO(2) assimilation, growth, and biomass of CAM plants by elevated atmospheric [CO(2)], and the proposition that these plants may have a role in atmospheric CO(2) sequestration is re-examined. This role may be compromised by predators such as Cactoblastis. However the moth CO(2) sensors are adapted to pre-industrial atmospheric [CO(2)] and FACE (free-air CO(2) enrichment) experiments show this exquisite system of biological control is also compromised by rising global [CO(2)] in the Anthropocene.     

CO(2)-concentrating: consequences in crassulacean acid metabolism

The consequences of CO(2)-concentrating in leaf air-spaces of CAM plants during daytime organic acid decarboxylation in Phase III of CAM (crassulacean acid metabolism) are explored. There are mechanistic consequences of internal CO(2) partial pressures, p(i)(CO(2)). These are (i) effects on stomata, i.e. high p(i)(CO(2)) eliciting stomatal closure in Phase III, (ii) regulation of malic acid remobilization from the vacuole, malate decarboxylation and refixation of CO(2) via Rubisco (ribulose bisphosphate carboxylase/oxygenase), and (iii) internal signalling functions during the transitions between Phases II and III and III and IV, respectively, in the natural day/night cycle and in synchronizing the circadian clocks of individual leaf cells or leaf patches in the free-running endogenous rhythmicity of CAM. There are ecophysiological consequences. Obvious beneficial ecophysiological consequences are (i) CO(2)-acquisition, (ii) increased water-use- efficiency, (iii) suppressed photorespiration, and (iv) reduced oxidative stress by over-energization of the photosynthetic apparatus. However, the general potency of these beneficial effects may be questioned. There are also adverse ecophysiological consequences. These are (i) energetics, (ii) pH effects and (iii) Phase III oxidative stress. A major consequence of CO(2)-concentrating in Phase III is O(2)-concentrating, increased p(i)(CO(2)) is accompanied by increased p(i)(O(2)). Do reversible shifts of C(3)/CAM-intermediate plants between the C(3)-CAM-C(3) modes of photosynthesis indicate that C(3)-photosynthesis provides better protection from irradiance stress? There are many open questions and CAM remains a curiosity.     

Effects of elevated CO2 on the tolerance of photosynthesis to acute heat stress in C3, C4, and CAM species

Determining the effect of elevated CO(2) on the tolerance of photosynthesis to acute heat stress (AHS) is necessary for predicting plant responses to global warming because photosynthesis is heat sensitive and AHS and atmospheric CO(2) will increase in the future. Few studies have examined this effect, and past results were variable, which may be related to methodological variation among studies. In this study, we grew 11 species that included cool and warm season and C(3), C(4), and CAM species at current or elevated (370 or 700 ppm) CO(2) and at species-specific optimal growth temperatures and at 30°C (if optimal ≠ 30°C). We then assessed thermotolerance of net photosynthesis (P(n)), stomatal conductance (g(st)), leaf internal [CO(2)], and photosystem II (PSII) and post-PSII electron transport during AHS. Thermotolerance of P(n) in elevated (vs. ambient) CO(2) increased in C(3), but decreased in C(4) (especially) and CAM (high growth temperature only), species. In contrast, elevated CO(2) decreased electron transport in 10 of 11 species. High CO(2) decreased g(st) in five of nine species, but stomatal limitations to P(n) increased during AHS in only two cool-season C(3) species. Thus, benefits of elevated CO(2) to photosynthesis at normal temperatures may be partly offset by negative effects during AHS, especially for C(4) species, so effects of elevated CO(2) on acute heat tolerance may contribute to future changes in plant productivity, distribution, and diversity.     

High light-induced switch from C(3)-photosynthesis to Crassulacean acid metabolism is mediated by UV-A/blue light

The high light-induced switch in Clusia minor from C(3)-photosynthesis to Crassulacean acid metabolism (CAM) is fast (within a few days) and reversible. Although this C(3)/CAM transition has been studied intensively, the nature of the photoreceptor at the beginning of the CAM-induction signal chain is still unknown. Using optical filters that only transmit selected wavelengths, the CAM light induction of single leaves was tested. As controls the opposite leaf of the same leaf pair was studied in which CAM was induced by high unfiltered radiation (c. 2100 micromol m(-2) s(-1)). To evaluate the C(3)-photosynthesis/CAM transition, nocturnal CO(2) uptake, daytime stomatal closure and organic acid levels were monitored. Light at wavelengths longer than 530 nm was not effective for the induction of the C(3)/CAM switch in C. minor. In this case CAM was present in the control leaf while the opposite leaf continued performing C(3)-photosynthesis, indicating that CAM induction triggered by high light conditions is wavelength-dependent and a leaf internal process. Leaves subjected to wavelengths in the range of 345-530 nm performed nocturnal CO(2) uptake, (partial) stomatal closure during the day (CAM-phase III), and decarboxylation of citric acid within the first 2 d after the switch to high light conditions. Based on these experiments and evidence from the literature, it is suggested that a UV-A/blue light receptor mediates the light-induced C(3)-photosynthesis/CAM switch in C. minor.     

CO2 and O2 dependence of PS II activity in C4 plants having genetically produced deficiencies in the C3 or C4 cycle

The CO2 dependence of rates of CO2 fixation (A) and photochemistry of PS II at 5, 15 and 30% O2 were analyzed in the C4 plant Amaranthus edulis having a C4 cycle deficiency [phosphoenolpyruvate carboxylase (PEPC) mutants], and in the C4 plant Flaveria bidentis having a C3 cycle deficiency [Rubisco small subunit antisense (SSU)]. In the wild type (WT) A. edulis and its heterozygous mutant having less than 50% WT PEPC activity there was a similar dependence of A and PS II photochemistry on varying CO2, although the CO2 saturated rates were 25% lower in heterozygous plants. The homozygous plants having less than 2% PEPC of the WT had significant levels of photorespiration at ambient levels of CO2 and required about 30 times ambient levels for maximum rates of A. Despite variation in the capacity of the C4 cycle, more than 91% of PS II activity was linearly associated with A under varying CO2 at 5, 15 and 30% O2. However, the WT plant had a higher PS II activity per CO2 fixed under saturating CO2 than the homozygous mutant, which is suggested to be due to elimination of the C4 cycle and its associated requirement for ATP from a Mehler reaction. In the SSU F. bidentis plants, a decreased rate of A (35%) and PS II activity (33%) accompanied a decrease in Rubisco capacity. There was some increase in alternative electron sinks at high CO2 when the C3 cycle was constrained, which may be due to increased flux through the C4 cycle via an ATP generating Mehler reaction. Nevertheless, even with constraints on the function of the C4 or C3 cycle by genetic modifications, analyses of CO2 response curves under varying levels of O2 indicate that CO2 assimilation is the main determinant of PS II activity in C4 plants.