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.     

Biphasic fluence response curves for induction of seed germination

Fluence-response curves for the induction of seed germination after 24 hours pretreatment at 35°C of Rumex obtusifolius and Arabidopsis thaliana show two phases of response: (a) a very low fluence-response (10⁻⁴ - 10⁻¹ micromoles per square meter) and (b) a low fluence-response (1 - 10³ micromoles per square meter).     

A Common Fluence Threshold for First Positive and Second Positive Phototropism in Arabidopsis thaliana

The relationship between the amount of light and the amount of response for any photobiological process can be based on the number of incident quanta per unit time (fluence rate-response) or on the number of incident quanta during a given period of irradiation (fluence-response). Fluence-response and fluence rate-response relationships have been measured for second positive phototropism by seedlings of Arabidopsis thaliana. The fluence-response relationships exhibit a single limiting threshold at about 0.01 micromole per square meter when measured at fluence rates from 2.4 × 10⁻⁵ to 6.5 × 10⁻³ micromoles per square meter per second. The threshold values in the fluence rateresponse curves decrease with increasing time of irradiation, but show a common fluence threshold at about 0.01 micromole per square meter. These thresholds are the same as the threshold of about 0.01 micromole per square meter measured for first positive phototropism. Based on these data, it is suggested that second positive curvature has a threshold in time of about 10 minutes. Moreover, if the times of irradiation exceed the time threshold, there is a single limiting fluence threshold at about 0.01 micromole per square meter. Thus, the limiting fluence threshold for second positive phototropism is the same as the fluence threshold for first positive phototropism. Based on these data, we suggest that this common fluence threshold for first positive and second positive phototropism is set by a single photoreceptor pigment system.     

Two distinct blue-light responses regulate the levels of transcripts of specific nuclear-coded genes in pea

Fluence-response characteristics for bluelight(BL)-mediated changes in the steady-state levels ofCab-, pEA215 and pEA207-RNA in red-light(RL) grown pea (Pisum sativum L. cv. Alaska) seedlings indicate the existence of two BL responses: a blue-lowfluence (BLF) response, causing an increase inCab- and pEA215-RNA, and a blue-high-fluence (BHF) response, causing a return to control levels forCab- and pEA215-RNA and a decrease in pEA207-RNA levels (Warpeha and Kaufman, 1989, Plant Physiol.,91, 1030–1035). We now show that under dark growth conditions, only the BLF response is apparent;Cab- and pEA215-RNA increase at all fluences tested, whereas pEA207-RNA levels are unaltered over the range of BL fluence tested. The treatment of dark-grown seedlings with RL immediately prior to BHF irradiation does not elicit the BHF response forCab-, pEA215 and pEA207-RNA, indicating that the role of growth in RL is to enable the seedling to reach a particular developmental state, rather than ensuring the presence of active phytochrome at the time of BL-irradiation. The apical bud of RL-grown seedlings has only the BLF response;Cab-RNA levels increase while pEA207-RNA exhibits no change at any of the fluences tested. The developing leaves of the fourth node show the BHF response; bothCab- and pEA207-RNA decrease following treatment with high-fluence BL. These data also indicate the necessity for reaching a specific developmental state before the BHF response can be activated. This research was supported by U.S. Department of Agriculture, Competitive Grants Office, grant No. 86CRCR12228 to L.S.K. K.M.F.W. gratefully acknowledges a University of Illinois at Chicago Laboratory for Molecular Biology Fellowship.     

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.     

Acclimation of Ribulose Bisphosphate Carboxylase and mRNAs to Changing Irradiance in Adult Tobacco Leaves: Differential Expression in LSU And SSU mRNA

The transfer of Nicotiana tabacum plants grown in low light (60 micromoles quanta per square meter per second) to higher light (360 micromoles quanta per square meter per second) was previously shown to induce adaptive stimulation of photosynthetic capacities. The variations of ribulose bisphosphate carboxylase/oxygenase (RubisCo) expression in mature leaves was examined as a result of this acclimation. Maximum or initial activities increased markedly after low- to high-light transfer with a maximum effect after 2 to 3 days. The higher activity is mainly explained by RubisCo protein synthesis as shown by immunorocket technique. Small subunits of RubisCo (SSU) mRNA relative content determined by hybridization of total RNA with DNA probe by Dot-blot method, followed the same pattern as RubisCo quantity. The magnitude of this response was amplified when more contrasting light conditions (25 versus 360 micromoles per square meter per second) were established on the same leaf: RubisCo activity, RubisCo protein, and SSU mRNA contents decreased in the shaded zone and increased in the high-light zone within 1 day. After 2 days the shade/light ratio was 1 to 3 for RubisCo protein and 1 to 4 for SSU-RNA, whereas the ratios remained equal to one in controls. Hybridization of the same RNA extracts with large subunits of RubisCo (LSU) probe showed no variation in LSU-RNA content. So in green adult leaves, the expression of SSU and LSU genes is regulated differently. The observed white light quantitative effect on RubisCo expression was not dependent on the photosynthetic rate or assimilate content since low CO₂ concentration around the leaf after the light shift did not modify the response.     

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.     

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.

     

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.     

Photosynthetic dynamics in chrysanthemum in response to single step increases and decreases in photon flux density

The time-course of CO₂ assimilation rate and stomatal conductance to step changes in photosynthetic photon flux density (PPFD) was observed in Chrysanthemum × morifolium Ramat. `Fiesta'. When PPFD was increased from 200 to 600 micromoles per square meter per second, the rate of photosynthetic CO<sub>2</sub> assimilation showed an initial rapid increase over the first minute followed by a slower increase over the next 12 to 38 minutes, with a faster response in low-light-grown plants. Leaves exposed to small step increases (100 micromoles per square meter per second) reached the new steady-state assimilation rate within a minute. Both stomatal and biochemical limitations played a role during photosynthetic induction, but carboxylation limitations seemed to predominate during the first 5 to 10 minutes. Stomatal control during the slow phase of induction was less important in low-light compared to high-light-grown plants. In response to step decreases in PPFD, photosynthetic rate decreased rapidly and a depression in CO<sub>2</sub> assimilation prior to steady-state was observed. This CO<sub>2</sub> assimilation `dip' was considerably larger for the large step (400 micromoles per square meter per second) than for the small step. The rapid photosynthetic response seems to be controlled by biochemical processes. High- and low-light-grown plants did not differ in their photosynthetic response to PPFD step decreases.     

Novel light-regulated chloroplast thylakoid membrane protein

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.     

Endogenous Abscisic Acid Content Correlates with Photon Fluence Rate and Induced Leaf Morphology in Hippuris vulgaris

This research focused on studying how light and endogenous abscisic acid regulate leaf development in Hippuris vulgaris, a species of heterophyllic aquatic plant. Amounts of photosynthetically active radiation greater than 300 micromoles per square meter per second caused submerged H. vulgaris shoots to produce aerial-type leaves. Abscisic acid was not detected in shoots grown under noninducing light quantities (100 micromoles per square meter per second), but was present at 13.4 nanograms per gram fresh weight in shoot tips after plants were exposed to 1 photoperiod of inducing light (500 micromoles per square meter per second). This supports a role for abscisic acid in the high light-induced heterophylly in H. vulgaris, and provides additional support for the general hypothesis that abscisic acid regulates leaf development in heterophyllic aquatic plants. No relationship was observed here between postphotoperiodic light treatments of various red/far red ratios and heterophylly in H. vulgaris.     

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.     

Inhibition of stem elongation in cucumis seedlings by blue light requires calcium

The effects of blue light and calcium on elongation of hypocotyl segments of Cucumber (Cucumis sativa L. cv Burpee's Pickler) were studied. Cucumber seedlings grown in dim red light showed a rapid decline in the rate of hypocotyl elongation when irradiated with high intensity (100 micromoles per square meter per second) blue light. In intact, 4-day-old seedlings the inhibition began within 2 minutes after the onset of blue-light irradiation and reached a maximum of approximately 55% within 4 minutes. Hypocotyl segments cut from 4-day-old seedlings also showed an inhibition of elongation in response to blue light when segments were floated on aqueous buffer and exposed to blue light for 3 hours. In the presence of 2 micromolar indole-3-acetic acid, blue light caused a 50% inhibition of elongation. Buffering free calcium in the incubation medium with 0.1 millimolar ethylene glycol bis(-aminoethyl ether)- N,N,N′,N′-tetraacetic acid eliminated the blue-light inhibition of segment elongation. Several experiments confirmed a specific requirement for calcium for the blue-light-induced inhibition of segment elongation. Treating segments with 0.2 micromolar fusicoccin abolished the inhibition of elongation by blue light as did buffering the medium at pH 4. Adding 1 millimolar ascorbate to incubation medium also eliminated the inhibition of segment elongation caused by blue light. Several compounds implicated in cell-wall redox reactions alter the magnitude of the blue-light-induced inhibition. The activity of peroxidase isolated from the cell-wall free space of cucumber hypocotyls was inhibited by ascorbate and low pH. The results are consistent with the hypothesis that blue light inhibits elongation by inducing an increase in cell-wall peroxidase activity and implicate calcium ions in the response to blue light.     

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.

     

Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy

The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of ¹⁴CO₂ fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged −18.7 micromoles per square meter per second for 6 days after anthesis and declined to −2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO₂ was maximal at −158 micromoles CO₂ per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled ¹⁴CO₂ injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of ¹⁴CO₂ in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO₂ loss, do serve a vital role in the recycling of internal CO₂ and therein, function as important sources of assimilate for reproductive development.     

Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO(2) Exchange

The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO₂ assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light-mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed.     

Characterization of a Rapid, Blue Light-Mediated Change in Detectable Phosphorylation of a Plasma Membrane Protein from Etiolated Pea (Pisum sativum L.) Seedlings

When crude microsomal membranes from apical stem segments of etiolated Pisum sativum L. cv Alaska are mixed in vitro with γ-[³²P]ATP, a phosphorylated band of apparent molecular mass 120 kilodaltons can be detected on autoradiographs of sodium dodecyl sulfate electrophoresis gels. If the stem sections are exposed to blue light immediately prior to membrane isolation, this band is not evident. The response is observed most strongly in membranes from the growing region of the stem, but no 120 kilodalton radiolabeled band is detected in membranes from the developing buds. Fluence-response curves for the reaction show that the system responds to blue light above about 0.3 micromole per square meter, and the visible phosphorylation completely disappears above 200 micromoles per square meter. Reciprocity is valid for the system, because varying illumination time or fluence rate give similar results. If the stem segments are left in the dark following a saturating blue irradiation, the radio-labeled band begins to return after about 10 minutes and is as intense as that from the dark controls within 45 to 60 minutes. A protein that comigrates with the phosphorylated protein on polyacrylamide gels is also undetectable after saturating blue light irradiations. The fluence range in which the protein band disappears is the same as that for the disappearance of the phosphorylation band. Its dark recovery kinetics and tissue distribution also parallel those for the phosphorylation. In vitro irradiation of the isolated membranes also results in a phosphorylation change at that molecular mass, but in the opposite direction. Comparisons of the kinetics, tissue distribution, and dark recovery of the phosphorylation response with those published for blue light-mediated phototropism or rapid growth inhibition indicate that the phosphorylation could be linked to one or both of those reactions. However, the fluence-response relationships for the change in detectable phosphorylation match quite closely those reported for phototropism but not those for growth inhibition. Blue light has also been found to regulate the capacity for in vitro phosphorylation of a second protein. It has an apparent molecular mass of 84 kilodaltons and is localized primarily in basal stem sections.     

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.