The Azolla-Anabaena azollae Relationship : XI. PHYCOBILIPROTEINS IN THE ACTION SPECTRUM FOR NITROGENASE-CATALYZED ACETYLENE REDUCTION

Visible absorption spectra are presented for the Azolla caroliniana Willd.-Anabaena azollae Strass. association and the individual partners. Although absorption by the phycobiliproteins of the endophytic cyanobacterium clearly complements the absorption by the fern pigments, their contribution to the absorption spectrum of the association is effectively concealed by the preponderance of the Azolla pigments. Action spectra for nitrogenase-catalyzed C₂H₂ reduction in both the Azolla-Anabaena association and the endophytic Anabaena demonstrate that quanta absorbed by the phycobiliproteins is as effective as that absorbed by chlorophyll a in driving this photosystem I-linked process. Under anaerobic conditions, the inhibition of photosystem II activity by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron did not selectively decrease the relative quantum yields in the region of phycobiliprotein absorption. At the well-below saturating light intensities used for the action spectra studies, the absolute rates of C₂H₂ reduction were increased uniformly via respiratory-linked processes under aerobic conditions. The occurrence of phycobiliproteins in heterocysts of the endophytic Anabaena was demonstrated using fluorescence microscopy of intact filaments. Fluorescence micrographs of Anabaena cylindrica filaments are presented for comparison.     

Exogenous expression of the wheat chloroplastic fructose-1,6-bisphosphatase gene enhances photosynthesis in the transgenic cyanobacterium, <Emphasis Type="BoldItalic">Anabaena</Emphasis> PCC7120

The paper reports a study on the genetic regulation of photosynthesis by introducing the gene encoding wheat chloroplastic fructose-1,6-bisphosphatase (FBPase) into the cyanobacterium Anabaena PCC7120. The gene was RT-PCR amplified from wheat and modified by replacement of the 5′-terminal encoding sequence with optimal and A/T-rich codons to promote prokaryotic expression. The resultant FBPase gene was ligated downstream of the strong promoter, PpsbA of expression vector pRL-439, then inserted into of shuttle vector pDC-08. The resulting shuttle expression vector (pDC-fbp) was transferred into the filamentous, heterocystour cyanobacterium, Anabaena PCC7120, by the tri-parental conjugation transfer method. Protein expression of FBPase in the transgenic Anabaena was 126.5% higher than in wild type cells, and the enzyme activity of transgenic cells was 1.41-fold higher than that of wild type cells. Under atmospheric conditions of 360 μmol mol⁻¹ CO₂, Anabaena cells overexpressing the FBPase gene further showed increases in net photosynthesis (117.2%) and true photosynthesis (122.5%) as compared to wild type cells. In addition, transgenic Anabaena grew faster and contained more Chl a than did wild type cells. Together, these results indicate that introduction of the wheat chloroplastic FBPase gene into Anabaena increase photosynthesis and cell growth; furthermore, these trends were more evident under stress condition (higher CO₂ concentration). This is the first report of enhanced photosynthesis in cyanobacteria expressing genes from higher plants.     

TheAzolla-Anabaena association: Historical perspective,symbiosis and energy metabolism

The heterosporous water-fern genusAzolla is one of the few symbioses with a cyanobacterium in the genusAnabaena. TheAzolla-Anabaena association includes six extant speciesof Azolla, which are widely distributed in relatively placid tropical and/or temperate freshwater environments. The earliest mention of the plant seems to be in an ancient Chinese dictionary that appeared about 2000 years ago.Azolla was used in about the 11th century in Vietnam. By 1980 renewed interest in this symbiotic association was shown by the demand for a less fossil energy-dependent agricultural technology. The importation of a variety ofA. filiculoides may have been a most significant breakthrough for the improvementof Azolla cultivation in China. The history of research may be divided into three periods and a new biotechnological stageof Azolla research has recently begun. Each mature dorsal leaf lobe has an ellipsoid cavity which containsAnabaena azollae throughout its development. HeterocystousA. azollae from sixAzolla species share identical and highly specific antigens.Azolla and its endophyte exhibit a coordinated pattern of differentiation and development. Epidermal hair cells of the host are probably interactive with the symbiont. The interior surface of a mature leaf cavity is lined with an envelope and covered by a mucilaginous layer.A. azollae shares the cavity with small populations of the bacteriaPseudomonas andAzotobacter. Endophyte-freeAzolla may rarely occur in nature and can be generated by aseptic techniques.Anabaena azollae can be isolated fromAzolla fronds by gentle pressure and by enzymatic digestion. The free living cultures derived from theAnabaena so obtained differ in some respects, however, from the freshly extracted symbiont, and might better be called the presumptive isolate. BothAzolla andAnabaena contain specific photosynthetic pigments. The optimum conditions for photosynthesis have been measured.Azolla is a C₃ plant and has high net photosynthesis. PSII activity in the symbiont is low. Nitrogenase is localized in the heterocysts of the symbiont and has some advantages compared with free-living cyanobacteria. SymbioticA. azollae has a high frequency of heterocysts. Unidirectional hydrogenase occurs in the symbiont and recycles electrons and ATP. Simultaneous measurements of N₂ fixation and photosynthesis show the dependence of nitrogenase on photosynthetically captured radiation for energy by an indirect dependence on CO₂ fixation. The host contains most of the total GS and GDH activities, and the symbiont excretes a substantial portion of its newly fixed nitrogen as ammonium. The two partners in the association exhibit a comparable developmental gradient and a mechanism of cooperative integration for their energy metabolism, thus improving the efficiency of solar energy conversion and presenting a unique model for biotechnology.     

Simultaneous Transport of CO(2) and HCO(3) by the Cyanobacterium Synechococcus UTEX 625

A mass spectrometer was used to simultaneously follow the time course of photosynthetic O₂ evolution and CO₂ depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO₂ and HCO₃⁻ were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO₃⁻ transport by Na⁺ addition had no effect on ongoing CO₂ transport. This result is interpreted to indicate that the CO₂ and HCO₃⁻ transport systems are separate and distinctly different transport systems. Measurement of CO₂-dependent photosynthesis indicated that CO₂ uptake involved active transport and that diffusion played only a minor role in CO₂ acquisition in cyanobacteria.     

Photosynthesis: action spectra for leaves in normal and low oxygen

The action spectrum of apparent photosynthesis for attached radish (Raphanus sativus L. var. Early Scarlet Globe) and corn (Zea mays L. var. Pride V.) leaves was measured at 300 μl/l CO₂ and both 21% and 2% O₂. The spectra were measured at light intensities where apparent photosynthesis was proportional to intensity. For radish, a high compensation point plant, oxygen had an inhibiting effect on photosynthesis at all wavelengths from 402 to 694 mμ. If a constant rate of photosynthesis at 21% O₂ for the different wavelengths was chosen, then the percent increase in net CO₂ fixation at 2% O₂ was constant. For corn, a low compensation point plant, no inhibitory effect of oxygen concentration from 2% to 21% O₂ was found over the visible spectrum. The CO₂ compensation point for light intensities greater than the light compensation point was found to be constant and independent of wavelength for both radish and corn leaves. For radish, the lowering of the oxygen concentration from 21% to 2% at these intensities was found to reduce the CO₂ compensation point by the same amount for the wavelengths studied.     

Effect of Boron Deficiency on Photosynthesis and Reductant Sources and Their Relationship with Nitrogenase Activity in Anabaena PCC 7119

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O₂ evolution and in CO₂ fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N₂ fixation is independent of its effects on photosynthesis and reductant supply.     

The photosynthetic action spectrum of the bean plant

The photosynthetic action spectrum of the bean plant leaf, Phaseolus vulgaris L. (variety Red Kidney), has been determined with a diffraction grating illuminated by a 6500-watt xenon arc. An infrared CO₂ analyzer was used to determine the gross photosynthetic rate of the terminal leaflet of the first trifoliate leaf. The rate was measured as a function of the light intensity at steps of 12.5 nanometers which approximates the length of the leaflet used. Twenty-five curves between 400 and 700 nanometers were used to establish the action spectrum. All light curves were some linear function of the incident intensity, and all were extrapolated to zero. The action spectrum shows the following features. (a) there are two peaks (i.e., at about 670 and 630 nanometers) and a shoulder between 600 and 612 nanometers in the red region where the highest rate of photosynthesis is found. Lower peaks in descending order are found in the blue (at about 437 nanometers) and the green (at about 500 nanometers) regions. (b) There are two small minima at about 650 nanometers and between 470 and 480 nanometers, and a broad minimum is found between 540 and 530 nanometers. (c) The photosynthetic rate declines rapidly above 680 nanometers, reaching the lowest value at 700 nanometers. (d) At wave lengths below the blue maximum, the rate decreases progressively to 400 nanometers.     

CO(2) and O(2) Exchange in Two Mosses, Hypnum cupressiforme and Dicranum scoparium

Photosynthetic CO₂ and O₂ exchange was studied in two moss species, Hypnum cupressiforme Hedw. and Dicranum scoparium Hedw. Most experiments were made during steady state of photosynthesis, using ¹⁸O₂ to trace O₂ uptake. In standard experimental conditions (photoperiod 12 h, 135 micromoles photons per square meter per second, 18°C, 330 microliters per liter CO₂, 21% O₂) the net photosynthetic rate was around 40 micromoles CO₂ per gram dry weight per hour in H. cupressiforme and 50 micromoles CO₂ per gram dry weight per hour in D. scoparium. The CO₂ compensation point lay between 45 and 55 microliters per liter CO₂ and the enhancement of net photosynthesis by 3% O₂versus 21% O₂ was 40 to 45%. The ratio of O₂ uptake to net photosynthesis was 0.8 to 0.9 irrespective of the light intensity. The response of net photosynthesis to CO₂ showed a high apparent K_m (CO₂) even in nonsaturating light. On the other hand, O₂ uptake in standard conditions was not far from saturation. It could be enhanced by only 25% by increasing the O₂ concentration (saturating level as low as 30% O₂), and by 65% by decreasing the CO₂ concentration to the compensation point. Although O₂ is a competitive inhibitor of CO₂ uptake it could not replace CO₂ completely as an electron acceptor, and electron flow, expressed as gross O₂ production, was inhibited by both high O₂ and low CO₂ levels. At high CO₂, O₂ uptake was 70% lower than the maximum at the CO₂ compensation point. The remaining activity (30%) can be attributed to dark respiration and the Mehler reaction.     

Introduction of a Synthetic CO2-fixing Photorespiratory Bypass into a Cyanobacterium

Global photosynthetic productivity is limited by the enzymatic assimilation of CO₂ into organic carbon compounds. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the carboxylating enzyme of the Calvin-Benson cycle, poorly discriminates between CO₂ and O₂, leading to photorespiration and the loss of fixed carbon and nitrogen. With the advent of synthetic biology, it is now feasible to design, synthesize, and introduce biochemical pathways in vivo. We engineered a synthetic photorespiratory bypass based on the 3-hydroxypropionate bi-cycle into the model cyanobacterium, Synechococcus elongatus sp. PCC 7942. The heterologously expressed cycle is designed to function as both a photorespiratory bypass and an additional CO₂-fixing pathway, supplementing the Calvin-Benson cycle. We demonstrate the function of all six introduced enzymes and identify bottlenecks to be targeted in subsequent bioengineering. These results have implications for efforts to improve photosynthesis and for the “green” production of high value products of biotechnological interest.     

The Sequence of Change within the Photosynthetic Apparatus of Wheat following Short-Term Exposure to Ozone

The basis of inhibition of photosynthesis by single acute O₃ exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O₃ for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO₂ uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO₂ uptake and the internal CO₂ concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O₃; no significant increase occurred for any of the other treatments. Analysis of the response of CO₂ uptake to the internal CO₂ concentration implied that the predominant factor responsible for the reduction in light-saturated CO₂ uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O₃, respectively. At saturating concentrations of CO₂, photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O₃. Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO₂ uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O₃, requiring 8 hours at 400 nanomoles per mole O₃ before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO₂ uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O₃ fumigation.     

Genetically engineering cyanobacteria to convert CO2, water,and light into the long-chain hydrocarbon farnesene

Genetically engineered cyanobacteria offer a shortcut to convert CO₂ and H₂O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C₁₅H₂₄), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO₂, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1?±?1.8 μg·L^?1·O.D.^?1·d^?1. Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.     

Sodium Requirement for Photosynthesis and Its Relationship with Dinitrogen Fixation and the External CO(2) Concentration in Cyanobacteria

Cells of Anabaena PCC 7119 and of a mutant strain of Nostoc muscorum unable to fix dinitrogen, grown at pH 8 and under low CO₂ tension (air), showed a reduced capacity for photosynthesis when cultured in the absence of sodium, this inhibition being followed by symptoms of photooxidation, such as chlorosis, oxygen consumption in the light, and decrease of superoxide dismutase activity. The impairment of photosynthesis preceded that of nitrogenase activity, indicating that the requirement for sodium in photosynthesis was independent of its effects on nitrogen metabolism. However, when cyanobacteria were grown at pH 6.3 or under high CO₂ tensions, sodium was not required for photosynthesis and no symptoms of photooxidation were observed.     

O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation

Leaves of C₃ plants which exhibit a normal O₂ inhibition of CO₂ fixation at less than saturating light intensity were found to exhibit O₂-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O₂-insensitive photosynthesis has been reported in nine other C₃ species and usually occurred when the intercellular CO₂ pressure was about double the normal pressure. A lack of O₂ inhibition of photosynthesis was always accompanied by a failure of increased CO₂ pressure to stimulate photosynthesis to the expected degree. O₂-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O₂ and CO₂ insensitive at physiological CO₂ pressures. Postillumination CO₂ exchange kinetics showed that O₂ and CO₂ insensitivity was not the result of elimination of photorespiration.

It is proposed that O₂ and CO₂ insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO₂ assimilation.

     

Photoinhibition of photosynthesis in the cyanobacterium Microcystis aeruginosa

We have examined characteristics of the photoinhibition of photosynthesis which occur in the unicellular cyanobacterium Microcystis aeruginosa, following exposure to photon fluence rates in excess of those required for growth. Photoinhibition occurs following exposure of cells to a photon fluence rate of 1,000 μmol m^-2 s^-1, which is manifested as a decrease in either light-limited CO₂ fixation or light-saturated CO₂-dependent O₂ evolution. The extent and rapidity of this photoinhibition is greatly enhanced under CO₂-depleted conditions. Experiments in which cultures were sparged with different gases indicate that photoinhibition is not an obvious consequence of elevated O₂ tensions, unlike the photooxidative bleaching of photosynthetic pigments. Comparative studies on the photoinactivation of CO₂-dependent O₂ evolution and of the methyl viologen-dependent Mehler reaction, in whole cells, indicate that a primary site of light damage is within the photosynthetic electron-transport reactions and that carbon fixation is initially unaffected.     

Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells

The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O₂-dependent CO₂ compensation concentration, a nonlinear time course for photosynthetic ¹⁴CO₂ uptake at low CO₂ and high O₂ concentrations, and an O₂ stimulation of glycine and serine synthesis which was reversed by high CO₂ concentration. The compensation concentration was a linear function of O₂ concentration and increased as temperature increased. At atmospheric CO₂ concentration, 21% O₂ inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO₂ and increased with increasing temperature. The Km (CO₂) of photosynthesis was also temperature-dependent, increasing from 12 μm CO₂ at 15 C to 38 μm at 35 C. In contrast, the Ki (O₂) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O₂, where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O₂ inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO₂ and O₂ concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO₂ and O₂.     

Oxygen Inhibition of Photosynthesis: I. Temperature Dependence and Relation to O(2)/CO(2) Solubility Ratio

The magnitude of the percentage inhibition of photosynthesis by atmospheric levels of O₂ in the C₃ species Solanum tuberosum L., Medicago sativa L., Phaseolus vulgaris L., Glycine max L., and Triticum aestivum L. increases in a similar manner with an increase in the apparent solubility ratio of O₂/CO₂ in the leaf over a range of solubility ratios from 25 to 45. The solubility ratio is based on calculated levels of O₂ and CO₂ in the intercellular spaces of leaves as derived from whole leaf measurements of photosynthesis and transpiration. The solubility ratio of O₂/CO₂ can be increased by increased leaf temperature under constant atmospheric levels of O₂ and CO₂ (since O₂ is relatively more soluble than CO₂ with increasing temperature); by increasing the relative levels of O₂/CO₂ in the atmosphere at a given leaf temperature, or by increased stomatal resistance. If the solubility ratio of O₂/CO₂ is kept constant, as leaf temperature is increased, by varying the levels of O₂ or CO₂ in the atmosphere, then the percentage inhibition of photosynthesis by O₂ is similar. The decreased solubility of CO₂ relative to O₂ (decreased CO₂/O₂ ratio) may be partly responsible for the increased percentage inhibition of photosynthesis by O₂ under atmospheric conditions with increasing temperature.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

Azolla-Anabaena azollae Relationship: V. N(2) Fixation, Acetylene Reduction, and H(2) Production

In order to characterize the reactions catalyzed by nitrogenase in the Azolla-Anabaena association, ¹⁵N₂ fixation, C₂H₂ reduction, and ATP-dependent H₂ production were measured in both the Azolla-Anabaena complex and in the alga isolated from the complex.     

Some observations on the carbon dioxide burst in chlorella and chlamydomonas

In the course of mass spectrometer studies with the algae Chlorella and Chlamydomonas, data were obtained which indicate that the CO₂ burst and gulp are sensitive to oxygen. Furthermore, the CO₂ burst was found to be strongly suppressed when wave lengths shorter than 460 nanometers were blocked at intensities adequate to saturate photosynthesis. Under appropriate conditions at 30°, the CO₂ burst was interrupted by a brief CO₂ gulp and the post illumination gulp by a brief burst of CO₂. The post illumination gulp of CO₂ could be induced during illumination by interposition of a filter blocking wave lengths shorter than 460 nanometers. These data are discussed in relation to earlier reports of the phenomenon and briefly as they affect the detection of photorespiration.     

Action Spectra for Guard Cell Rb Uptake and Stomatal Opening in Vivia faba

Abaxial epidermal strips, containing guard cells as the only viable cells, were prepared from leaves of Vicia faba following a period in darkness, and floated, under CO₂-free air, on 2 mm RbCl + 0.1 mm CaCl₂ labeled with ⁸⁶Rb⁺. Under white light (high pressure mercury vapor lamp), stomatal opening in these strips approached its maximum at less than 0.02 calorie per square centimeter per minute. Under light of different wavelengths, 20 nanometers apart, and at a low quantum flux density of 7 × 10¹⁴ quanta per square centimeter per second, Rb⁺ uptake and stomatal opening were activated only in the blue and long ultraviolet regions, with a peak at 420 to 460 nanometers. The action spectrum suggests that the underlying process is not photosynthesis. At higher quantum flux density (38 × 10¹⁴ quanta per square centimeter per second), uptake and opening also responded to red (600-680 nanometers) and somewhat to green light, with a minimum at 540 to 560 nanometers, indicating a possible involvement of the photosynthetic process. This light-induced opening appeared not to be mediated by a lowering of CO₂ concentration, since CO₂-free air was used in all treatments and controls. Stomatal opening paralleled Rb⁺ uptake in all cases. This constitutes further evidence for the potassium transport hypothesis of stomatal movement.