Interaction between Green and Far-Red Light on the Low Fluence Rate Chloroplast Orientation in Mougeotia

Continuous irradiation of Mougeotia with linearly polarized green light (550 nanometers, 0.2 watt per square meter) induces a change in the orientation of its chloroplast from profile to face position, if the electrical vector of the green light is vibrating normal to the cell axis. This change is complete within 25 minutes of the onset of irradiation. In contrast, if the electrical vector of the green light is parallel to the cell axis, no chloroplast reorientation is induced, even with a fluence rate as high as 3 watts per square meter. Furthermore, unpolarized far-red light (727 nanometers, 2 watts per square meter) given alone has no effect on chloroplast reorientation. Simultaneous and continuous irradiation with polarized green light, regardless of its plane of polarization, together with unpolarized far-red light, however, does lead to chloroplast reorientation. These data indicate that, in addition to the red-absorbing form of phytochrome, there exists in Mougeotia another sensory pigment absorbing green light.     

A Circadian Rhythm in the Number of Daughter Cells in Synchronous Chlorella fusca var vacuolata

Chlorella fusca var vacuolata cells were transferred to continuous darkness or weak light (0.07 watts per square meter) (both were called waiting time, WT) after a 12-hour light and 12-hour dark schedule. A daily dilution is performed at the end of the light/dark schedule, resulting in always the same average production of 18 autospores per mother cell. After 12 and 24 hours of WT in darkness, the production of autospores in a subsequent light/dark schedule was 50 and 100%, respectively. If the WT was performed in weak light (0.07 watts per square meter) the lowest production was obtained, independently of the length of WT. However, an interruption of this weak light by dark pulses (3 hours) increased the autospore production by an amount that depends upon the phase of the circadian rhythm, and varied up to 70% of the control (WT in permanent darkness). If the WT (total darkness) was interrupted by light pulses of 0.5 hour (white, same as used for growth), a phase response curve of productivity resulted. Pulses between the 12th and 18th hour of WT in darkness gave a 3-hour delay of maximum; later on pulses shifted the maximum autospore production 3 hours ahead.     

Influence of the Irradiance on Carbohydrate Content and Rooting of Cuttings of Pine Seedlings (Pinus sylvestris L.)

Seedlings of Pinus sylvestris L. were grown for 6 weeks at an irradiance of either 8 or 40 watts per square meter in a controlled environment room. Cuttings from these plants were rooted in tap water for 75 days at either 8 or 40 watts per square meter. The photoperiod was 17 hours.

During the first 30 days of the rooting period quantitative changes in carbohydrates were recorded in cuttings from the different treatments. The carbohydrate contents of the cuttings were mainly regulated by the irradiance during the stock plant stage and generally a higher carbohydrate level was found in cuttings from stock plants grown at 40 watts per square meter.

The irradiance during the rooting period had only minor effects on the time course of root formation, whereas the irradiance during the stock plant stage did influence the subsequent root formation. Cuttings from stock plants grown at 8 watts per square meter rooted faster and with higher frequency than those from stock plants grown at 40 watts per square meter. These results are discussed in relation to the mentioned irradiance effects on carbohydrate content.

     

Effect of the Intensity and Duration of Light at Various Temperatures on the Germination of Oldenlandia corymbosa L. Seeds

At temperatures below 35 to 40°C, fairly intense continuous white light (13 watts per square meter) inhibits germination of Oldenlandia corymbosa L. seeds, and the lower the temperature, the greater the inhibition. However, such lighting may enable seeds to germinate later in the dark; their degree of germinability depends both on the duration of lighting and on the temperature during lighting and after transfer to the dark.     

Participation of Photosynthesis in Floral Induction of the Long Day Plant Anagallis arvensis L

The saturating photon flux density (400 to 700 nanometers) for induction of flowering of the long day plant Anagallis arvensis L. was 1,900 micromoles per square meter per second (6,000 foot-candles) when an 8-hour daylength was extended to 24 hours by a single period of supplementary irradiation. The saturating photon flux density for photosynthetic CO₂ uptake during the same single supplementary light period was lower, at about 1,000 to 650 micromoles per square meter per second (3,000 to 2,000 foot-candles).

The per cent flowering and mean number of floral buds per plant were significantly reduced when the light extension treatment was given in CO₂-free air, and glucose (10 kilograms per cubic meter in water) relieved this effect. Glucose solution also significantly increased flowering of plants given supplementary light treatment in atmospheric air under a photon flux density of 80 micromoles per square meter per second. Increasing the CO₂ concentration to 1.27 grams per cubic meter of CO₂ in air during the supplementary light period did not increase flowering.

It is concluded that high photon flux densities promote flowering of Anagallis through both increased photosynthesis and the photomorphogenic action of high irradiance.

     

Sugar Concentrations in Guard Cells of Vicia faba Illuminated with Red or Blue Light : Analysis by High Performance Liquid Chromatography

Concentrations of soluble sugars in guard cells in detached, sonicated epidermis from Vicia faba leaves were analyzed quantitatively by high performance liquid chromatography to determine the extent to which sugars could contribute to changes in the osmotic potentials of guard cells during stomatal opening. Stomata were illuminated over a period of 4 hours with saturating levels of red or blue light, or a combination of red and blue light. When stomata were irradiated for 3 hours with red light (50 micromoles per square meter per second) in a solution of 5 millimolar KCl and 0.1 millimolar CaCl₂, stomatal apertures increased a net maximum of 6.7 micrometers and the concentration of total soluble sugar was 289 femtomoles per guard cell (70% sucrose, 30% fructose). In an identical solution, 2.5 hours of irradiation with 25 micromoles per square meter per second of blue light caused a maximum net increase of 7.1 micrometers in stomatal aperture and the total soluble sugar concentration was 550 femtomoles per guard cell (91% sucrose, 9% fructose). Illumination with blue light at 25 micromoles per square meter per second in a solution lacking KCl caused a maximum net increase in stomatal aperture of 3.5 micrometers and the sugar concentration was 382 femtomoles per guard cell (82% sucrose, 18% fructose). In dual beam experiments, stomata irradiated with 50 micromoles per square meter per second of red light opened steadily with a concomitant increase in sugar production. Addition of 25 micromoles per square meter per second of blue light caused a further net gain of 3.7 micrometers in stomatal aperture and, after 2 hours, sugar concentrations had increased by an additional 138 femtomoles per guard cell. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were performed with epidermis illuminated with 50 micromoles per square meter per second of red light or with 25 micromoles per square meter per second of blue light in solutions containing or lacking KCl. DCMU completely inhibited sugar production under red light, had no effect on guard cell sugar production under blue light when KCl was present, and inhibited sugar production by about 50% when guard cells were illuminated with blue light in solutions lacking KCl. We conclude that soluble sugars can contribute significantly to the osmoregulation of guard cells in detached leaf epidermis of V. faba. These results are consistent with the operation of two different sugar-producing pathways in guard cells: a photosynthetic carbon reduction pathway and a pathway of blue light-induced starch degradation.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Growth and Tuberization of Potato (Solanum tuberosum L.) under Continuous Light

The growth and tuberization of potatoes (Solanum tuberosum L.) maintained for 6 weeks under four different regimes of continuous irradiance were compared to plants given 12 hours light and 12 hours dark. Treatments included: (a) continuous photosynthetic photon flux of 200 micromoles per square meter per second cool-white fluorescent (CWF); (b) continuous 400 micromoles per square meter per second CWF; (c) 12 hours 400 micromoles per square meter per second CWF plus 12 hours dim CWF at 5 micromoles per square meter per second; (d) 12 hours micromoles per square meter per second CWF plus 12 hours dim incandescent (INC) at 5 micromoles per square meter per second and a control treatment of 12 hours light at 400 micromoles per square meter per second CWF and 12 hours dark. The study included five cultivars ranging from early- to late-season types: `Norland,' `Superior,' `Norchip,' `Russet Burbank,' and `Kennebec.' Tuber development progressed well under continuous irradiation at 400 micromoles per square meter per second and under 12 hours irradiance and 12 hours dark, while tuber development was suppressed in all other light treatments. Continuous irradiation at 200 or 400 micromoles per square meter per second resulted in severe stunting and leaf malformation on `Superior' and `Kennebec' plants, but little or no injury and vigorous shoot growth in the other cultivars. No injury or stunting were apparent under 12-dim light or 12-dark treatments. Plants given 12 hours dim INC showed significantly greater stem elongation but less total biomass than plants in other treatments. The continuous light encouraged shoot growth over tuber growth but this trend was overridden by providing a high irradiance level. The variation among cultivars for tolerance to continuous lighting indicates that potato may be a useful species for photoinhibition studies.     

Enhancement of the Stomatal Response to Blue Light by Red Light, Reduced Intercellular Concentrations of CO(2), and Low Vapor Pressure Differences

The effects of environmental parameters on the blue light response of stomata were studied by quantifying transient increases in stomatal conductance in Commelina communis following 15 seconds by 0.100 millimole per square meter per second pulses of blue light. Because conductance increases were not observed following red light pulses of the same or greater (30 seconds by 0.200 millimole per square meter per second) fluences, the responses observed could be reliably attributed to the specific blue light response of the guard cells, rather than to guard cell chlorophyll. In both Paphiopedilum harrisianum, which lacks guard cell chloroplasts, and Commelina, the blue light response was enhanced by 0.263 millimole per square meter per second continuous background red light. Thus, the blue light response and its enhancement do not require energy derived from red-light-driven photophosphorylation by the guard cell chloroplasts. In Commelina, reduction of the intercellular concentration of CO₂ by manipulation of ambient CO₂ concentrations resulted in an enhanced blue light response. In both Commelina and Paphiopedilum, the blue light response was decreased by an increased vapor pressure difference. The magnitude of blue-light-specific stomatal opening thus appears to be sensitive to environmental conditions that affect the carbon and water status of the plant.     

Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica

The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H⁺ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C₃ photosynthesis. The total nocturnal H⁺ accumulation and CO₂ uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H⁺ accumulation and 4 moles per square meter per day for CO₂ uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H⁺ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.

A minimum ratio of H⁺ accumulated to CO₂ taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H⁺-to-CO₂ ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H⁺ accumulation and CO₂ uptake decreased, but the H⁺-to-CO₂ ratio increased. A ratio of two H⁺ per CO₂ is consistent with the H⁺ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO₂ uptake is strongly favored over respiratory activity.

     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Blue-Light Regulation of Epicotyl Elongation in Pisum sativum

Blue light is known to induce suppression of stem elongation. To avoid the complication of blue-light-induced transformation of phytochrome we have adapted the procedure of measuring blue-light-induced suppression of stem elongation in Pisum sativum L. var Alaska grown under continuous red light. The resulting fluence-response curve for suppression of epicotyl elongation measured twenty-four hours after a blue-light treatment is bell-shaped, with the peak of suppression between 10⁰ and 10¹ micromoles per square meter, and no suppression at 10⁴ micromoles per square meter. Suppression is first observed 5 and 11 hours after the blue-light treatment for the fourth and third internodes, respectively. No significant differences in elongation rates were noted for the 10⁴ micromoles per square meter treated seedlings throughout the 24 hour period. Reciprocity holds for both third and fourth internodes in response to 10¹ and 10⁴ micromoles per square meter of blue light over the range of irradiation times tested (10⁰ to 10⁴ seconds, 10¹ micromoles per square meter; 10⁰ to 10³ seconds, 10⁴ micromoles per square meter). In contrast to the bell-shaped fluence-response obtained for epicotyl elongation, measurements of chlorophyll and carotenoid accumulation indicate increasing accumulation with increasing fluence.     

Phototropism in Hypocotyls of Radish : III. Influence of Unilateral or Bilateral Illumination of Various Light Intensities on Phototropism and Distribution of cis- and trans-Raphanusanins and Raphanusamide

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light at 0.05, 0.1, or 1 watt per square meter, the suppression of the growth rate on the lighted side depended on the light intensity. The growth rate at the shaded side was only a little affected by the illumination at 0.05 and 0.1 watt per square meter but considerably suppressed by that at 1 watt per square meter. Upon a continuous unequal bilateral illumination, the growth rate was more strongly suppressed on the side of the higher intensity than on the side of the lower one, resulting in phototropic curvature toward the light source of the higher intensity. It was calculated from correlation analysis of light intensity and growth rate that, on an average, 6.9% of the irradiation applied to one side reached the opposite side. The amounts of cis- and trans-raphanusanins and raphanusamide in hypocotyls subjected to unilateral or unequal bilateral illumination increased much more at the side of the lighted or the higher intensity than at the opposite side. The present study demonstrates that phototropism in radish hypocotyl is correlated with and we conclude caused by a gradient of growth inhibition in the hypocotyl, depending on irradiation-induced amounts of cis- and trans-raphanusanins and raphanusamide.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: I. Cells in Darkness

We have developed protocols for phase shifting the circadian rhythm of Chlamydomonas reinhardtii by light pulses. This paper describes the photobiology of phase-resetting the Chlamydomonas clock by brief (3 seconds to 15 minutes) light pulses administered during a 24 hour dark period. Its action spectrum exhibited two prominent peaks, at 520 and 660 nanometers. The fluence at 520 nanometers required to elicit a 4 hour phase shift was 0.2 millimole photon per square meter, but the pigment that is participating in resetting the clock under these conditions is unknown. The fluence needed at 660 nanomoles to induce a 4 hour phase shift was 0.1 millimole photon per square meter, which is comparable with that needed to induce the typical low fluence rate response of phytochrome in higher plants. However, the phase shift by red light (660 nanometers) was not diminished by subsequent administration of far-red light (730 nanometers), even if the red light pulse was as short as 0.1 second. This constitutes the first report of a regulatory action by red light in Chlamydomonas.     

Water Relations, Diurnal Acidity Changes, and Productivity of a Cultivated Cactus, Opuntia ficus-indica

Physiological responses of the Crassulacean acid metabolism (CAM) plant Opuntia ficus-indica (Cactaceae) were studied on a commercial plantation in central Chile. Young cladodes (flattened stems) and flower buds exhibited daytime stomatal opening, whereas mature cladodes and fruit exhibited the nocturnal stomatal opening characteristic of CAM plants. Severe water stress suppressed the nocturnal stomatal opening by mature cladodes, but their high water vapor conductance occurring near dawn was not affected. Nocturnal acidity increases were not as sensitive to water stress as was the nocturnal stomatal opening. The magnitude of the nocturnal acidity increases depended on the total daily photosynthetically active radiation (PAR), being 90% PAR-saturated at 27 moles per square meter per day for a mean nighttime air temperature of 5°C and at 20 moles per square meter per day for 18°C. Inasmuch as the PAR received on unshaded vertical surfaces averaged about 21 moles per square meter per day, nocturnal acidity increases by the cladodes were on the verge of being PAR-limited in the field. The net assimilation rate, which was positive throughout the year, annually averaged 3.4 grams per square meter per day for 1.0- and 2.0-year-old plants. Plants that were 5.4 years old had 7.2 square meters of cladode surface area (both sides) and an annual dry weight productivity of 13 megagrams (metric tons) per hectare per year when their ground cover was 32%. This substantial productivity for a CAM plant was accompanied by the highest nocturnal acidity increase so far observed in the field, 0.78 mole H⁺ per square meter.     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Light Intensity and CO(2) in the C(3) Annuals Chenopodium album L. and Phaseolus vulgaris L

The light and CO₂ response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (k_cat) of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C₃ annuals Chenopodium album and Phaseolus vulgaris at 25°C. The initial slope of the photosynthetic CO₂ response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO₂ response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO₂ partial pressures (C₁) between the CO₂ compensation point and 500 microbars. Above a C₁ of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C₁, decreasing as the C₁ was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C₁ only under conditions when the activation state of rubisco was dependent on C₁. Otherwise, RuBP pool sizes increased as C₁ was reduced. ATP pools in C. album tended to increase as C₁ was reduced. In P. vulgaris, decreasing C₁ at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k_cat. These results support modelled simulations of the rubisco response to light and CO₂, where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.     

Effect of N-(Phosphonomethyl)glycine on Carbon Assimilation and Metabolism during a Simulated Natural Day

The effects of N-(phosphonomethyl)glycine (glyphosate) on the regulation of carbon assimilation, metabolism, and translocation were studied in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under a light regimen that began with gradually increasing irradiance as generally occurs on a natural day. Soon after application, glyphosate caused a marked increase in ribulose bisphosphate and a decrease in phosphoglyceric acid. The response is most simply explained by direct inhibition of ribulose bisphosphate carboxylase activity. The extent of inhibition was small, and the carbon assimilation rate did not decrease. As predicted, photosynthesis declined within an hour after glyphosate was applied to leaves under gradually increasing light. Inhibition resulted from a decrease in ribulose bisphosphate due to depletion of carbon from the photosynthetic carbon reduction cycle. Photoinhibition, a light-dependent limitation of photosynthetic capacity, appeared to be necessary for marked glyphosate-induced inhibition of photosynthesis. As a result, photosynthesis rate increased with irradiance until it exceeded 400 micromoles per square meter per second but then declined as the light level increased beyond 500 micromoles per square meter per second. Glyphosate changed the allocation of newly fixed carbon between starch and sucrose for export. Changes in the levels of ribulose bisphosphate and phosphoglyceric acid produced important effects on the regulation of carbon assimilation and metabolism.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.