Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Combined Effects of CO2 and O3 on Antioxidative and Photoprotective Defense Systems in Needles of Ponderosa Pine

Ponderosa pine (Pinus ponderosa Dougl. ex Laws.) seedlings were exposed to near ambient or elevated CO₂ (average concentrations during the last growing season 446 versus 699 mol mol^–1), combined with low or elevated O₃ for three seasons. Ozone exposure during the last growing season (accumulated dose above threshold 0.06 mol mol^–1) was 0.05 versus 26.13 mol mol^–1 h. Needles of the youngest age class were harvested after the dormancy period. Ozone exposure decreased needle contents of chlorophyll a, chlorophyll b, and ascorbate, and resulted in a more oxidized total ascorbate and a more de-epoxidized xanthophyll cycle pool irrespective of the CO₂ level. Trees under elevated CO₂ had a more oxidized glutathione pool and lower chlorophyll a content. Contents of glutathione, tocopherol, and carotenoids were not affected by the CO₂ or O₃ treatments. There were no interactive effects between elevated CO₂ and elevated O₃ on any of the parameters measured. The results suggest that elevated atmospheric CO₂ concentration does not compensate for ozone stress by increasing antioxidative capacity in ponderosa pine.     

Growth and tissue senescence inPrunus avium shoots grownin vitro at different CO3/O2 ratios

Summary The rate of metabolism and biosynthetic processes makein vitro cultures very sensitive to environmental changes, and therefore subject to physiological and morphological alterations leading to senescence in the short term. The effect of three different calibrated atmospheric compositions were studied duringin vitro culture ofPrunus avium shoots. At 0.034% CO₂-21% O₂ (vol/vol), which stimulate the natural atmosphere, the highest growth rate and chlorophyll content were recorded. When grown at 0.09% CO₂-8% O₂ (vol/vol), a favorable condition for photosynthesis and growth, cultures showed a higher percentage of dry matter and elevated ethylene production, but total chlorophyll was lower. These shoots were also highly lignified and fibrous with red pigmentation along the leaves and stems. At 0% CO₂-21% O₂ (vol/vol), in contrast, growth and ethylene formation were inhibited; chlorophyll content was lowest in comparison with the other two environmental conditions, but regreening of tissues was observed after the first half of the culture period. Senescence symptoms, as indicated by decreased chlorophyll, appeared after about 18 d of culture for tissues grown in CO₂-containing atmospheres. These experiments provided evidence that in CO₂-enriched cultures biomass production steadily increased even when chlorophyll decreased. A possible role of CO₂ in promoting tissue-senescence through activation of photooxidative events and ethylene synthesis is discussed.     

Changes in Lipid Metabolism during Adaptation of the Dunaliella salina Photosynthetic Apparatus to High CO2 Concentration

The effects of CO₂ on the content and composition of lipid fatty acids (FA) and on the photosynthetic characteristics of unicellular halophilic green alga Dunaliella salina (known to be susceptible to CO₂ stress) were investigated. It was shown that even one-day-long increase in the CO₂ concentration (from 2 to 10%) provoked an increase in the total amount of FA on the dry weight basis by 30%. After 7-day-long growth at 10% CO₂, this value was 2.7-fold higher than that at 2% CO₂. The difference in the FA content and composition indicated the activation of FA synthesis de novo and inhibition of their elongation and desaturation, as well as the increase in the relative content of saturated FA at 10% CO₂. It was demonstrated that, after one-day-long CO₂ stress, the MGDG/DGDG ratio increased fourfold without change in the sum of their FA, which indicates the increase in the proportion of lipids predisposed to micellar (hexagonal phase) but not lamellar structure formation. Under short-term CO₂ stress, the ratio of 3/6 FA increased and the content of E-16:113 FA in phosphatidylglycerols increased sharply. The drop in protein content especially in the photosystem I (PSI) preparations, as well as diminishing the ratio of F ₇₀₀-to-F ₆₈₆ nm fluorescence (F ₇₀₀/F ₆₈₆) under short-term CO₂ stress argued for the significant damage to PSI. The reversibility of these changes at more prolonged treatment (7 and 10 days) demonstrated that D. salina cells could restore the functional activity of PSI. The lower level of F ₇₀₀/F ₆₈₆, chlorophyll a (Chla)/Chlb, and 3/6 FA ratio in line with the higher level of E-16:113 in the cells growing for a long time at the high CO₂ concentration is characteristic for the new structural and functional state of the photosynthetic apparatus providing for the effective photosynthesis of D. salina under these conditions.     

Light and dark CO2 fixation in Clusia uvitana and the effects of plant water status and CO2 availability

Summary In well-watered plants of Clusia uvitana, a species capable of carbon fixation by crassulacean acid metabolism (CAM), recently expanded leaves gained 5 to 13-fold more carbon during 12 h light than during 12 h dark periods. When water was withheld from the plants, daytime net CO₂ uptake strongly decreased over a period of several days, whereas there was a marked increase in nocturnal carbon gain. Photosynthetic rates in the chloroplasts were hardly affected by the water stress treatment, as demonstrated by measurements of chlorophyll a fluorescence of intact leaves, indicating efficient decarboxylation of organic acids and refixation of carbon in the light. Within a few days after rewatering, plants reverted to the original gas exchange pattern with net CO₂ uptake predominantly occurring during daytime. The reversible increase in dark CO₂ fixation was paralleled by a reversible increase in the content of phosphoenolpyruvate (PEP) carboxylase protein. In wellwatered plants, short-term changes in the degree of dark CO₂ fixation were induced by alterations in CO₂ partial pressure during light periods: a decrease from 350 to 170 bar CO₂ caused nocturnal carbon gain, measured in normal air (350 bar), to increase, whereas an increase to 700 bar CO₂, during the day, caused net dark CO₂ fixation to cease. The increased CAM activity in response to water shortage may, at least to some extent, be directly related to the reduced carbon gain during daytime.     

Isolation and characterization of three types of membranes from the cyanobacterium (blue-green alga) Synechocystis PCC 6714

Three types of membranes were separated from the cyanobacterium Synechocystis PCC 6714 by mechanical disruption and density gradient centrifugation. Orange-colored membranes contained xanthophylls but little -carotene or chlorophyll a, green-colored membranes contained chlorophyll a, -carotene and xanthophylls, and another type of orange-colored membranes contained unknown xanthophylls. These membrane preparations were similar to those from Anacystis nidulans in pigmentation and buoyant density and were identified as purified preparations of the cytoplasmic membranes, thylakoid membranes and cell walls of Synechocystis PCC 6714, respectively.Abbreviations SDS sodium dodecyl sulfate - Tes N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - TLC thin-layer chromatography     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.     

Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K _m(PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing and antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO₂ assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO₂ release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO₂ assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO₂ assimilation determined in low CO₂ was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO₂ compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.Abbreviations CO₂ compensation point - CO₂ quantum efficiency of CO₂ assimilation - _PSII quantum efficiency of photosystem II electron transport - A CO₂ assimilation rate - C_i intercellular CO₂ concentration; e, electron - PFD photon flux density - Q_A primary quinone electron acceptor of photosystem II - Q_N non-photochemical chlorophyll a fluorescence quenching - q_P photochemical chlorophyll a fluorescence quenching     

Regreening of Zantedeschia aethiopica Spreng. Spathe induced by reapplied cytokinins

The excision of the inflorescence of Zantedeschia aethiopica Spreng. and its substitution by the cytokinin 6-(o-hydroxybenzylamino)-purine and its riboside 6-(o-hydroxybenzylamino)-9--D-ibofuranosylpurine, previously isolated from Z. aethiopica fruits showed that these cytokinins present in fruits are correlated with the self-inhibition of senescence.The determination of chlorophyll levels revealed that both base and riboside promoted regreening, 241–300% over 30 days compared with 341% after normal fructification, the riboside being the most effective (at 10^–8 M).Electron microscopic observations on palisade parenchyma cells showed that the reapplied endogenous cytokinins produced an increase in the cytoplasmic and chloroplastidial ribosomes. At the same time that the stroma was restructured, osmiophilic material and starch diminished. The formation of thylakoids begins in osmiophilic dense regions. The sequence of these events is responsible for the regreening and complete restructuring of spathe palisade cells where well structured and functional chloroplasts are evident.This study has been supported by grant INIC-BL₁     

Photosynthesis in the basal growing zone of barley leaves

Cell proliferation, elongation, determination and differentiation mainly take place in the basal 5 mm of a barley leaf, the so-called basiplast. A considerable portion of cDNAs randomly selected from a basiplast cDNA library represented photosynthetic genes such as CP29, RUBISCO-SSU and type I-LHCP II. Therefore, we became interested in the role of the basiplast in establishing photosynthesis. (1) Northern blot analysis revealed expression of photosynthetic genes in the basiplast, although at a low level. Analysis of basiplasts at different developmental stages of the leaves revealed maximal expression of photosynthetic genes during early leaf development. The activity of these genes shows that plastid differentiation involves the development of the photosynthetic apparatus even at this early state of leaf cell expansion. (2) This conclusion was supported by the fact that chlorophylls and carotenoids are synthesized in the basiplast. The qualitative pattern of pigment composition was largely similar to that of fully differentiated green leaves. (3) The transition from proplastids to chloroplasts progressed in the basal 5 mm of the leaf, so that the number of grana lamellae per thylakoid stack increased with distance from the meristem from zero to about five. (4) Photosynthetic function was studied by chlorophyll a-fluorescence measurements. In dark-adapted 8-day-old primary leaves, the fluorescence ratio (F_P-F_o)/F_P was little decreased in basiplasts as compared to leaf blades. During steady state photosynthesis, the ratio (F_M-F_o)/F_M was high in leaf blade (0.5), but low in the sheath (0.25) and in the basiplast (0.18), indicating the existence of functional, albeit low light-adapted chloroplasts in the basiplast. (5) Further on, chlorophyll a fluorescence analysis in relation to seedling age revealed efficient photosynthetic performance in the basiplast of 3- to 6-day-old seedlings which later-on differentiates into leaf blade as compared to the basiplast of 7- to 12-day-old seedlings which develops into leaf sheath and finally ceases to grow. The leaf age dependent changes in basiplast photosynthesis were reflected by changes in pigment contents and LHCP II expression both of which also revealed a maximum in the basiplast of 4-day-old seedlings.Abbreviations bas 1 basiplast-associated gene 1 encoding a peroxide reductase - cab chlorophyll a/b binding protein - CP 29 29 kDa chlorophyll binding protein - DIG digoxigenin - EMIP epidermal major intrinsic protein - LHCP II light harvesting complex of Photosystem II - LSU large subunit of Rubisco - NPQ non photochemical chlorophyll a fluorescence quenching - PSI/PS II Photosystem I/II - PQ photochemical chlorophyll a fluorescence quenching - Rubisco Ribulose-1,5-bisphosphate carboxylase - SSU small subunit of Rubisco     

The relationship between carbon and water transport in single cells ofChara corallina

Summary The hydraulic resistance of the plasma membrane was measured on single internodal cells ofChara corallina using the method of transcellular osmosis. The hydraulic resistance of the plasma membrane of high CO₂-grown cells was significantly higher than the hydraulic resistance of the plasma membrane in low CO₂-grown cells. Therefore we tested the possibility that the bicarbonate transport system, postulated to be present in low CO₂-grown cells, serves as a water channel that lowers the hydraulic resistance of the plasma membrane. We were unable to find any correlation between agents that inhibited the bicarbonate transport system and agents that increased the hydraulic resistance of low CO₂-grown cells. We did, however, find a correlation between the permeability of the cell to water and CO₂. We propose that the reduced hydraulic resistance of the plasma membrane of the low CO₂-grown cells is a function of a change in either the structural properties of the lipid bilayer or the activity of a CO₂ transport protein so that under conditions of reduced inorganic carbon, the plasma membrane becomes more permeable to CO₂, and consequently to other small molecules, including H₂O, methanol and ethanol.Dedicated to our teacher, Professor Masashi Tazawa, on the occasion of his 65th birthday     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.     

Indication of transthylakoid proton-fluxes in Aegopodium podagraria L. by light-induced changes of plasmalemma potential,chlorophyll fluorescence and light-scattering

The time course of the responses of chlorophyll fluorescence in leaves of Aegopodium podagraria to changes in irradiance does not necessarily show the time constant of thylakoid energization at energy fluence rates below 10–25 W·m^-2. In addition, other measures of thylakoid energization, such as lightscattering at 532 nm and the responses to saturating flashes, show that the related component disappears from these signals at low fluence rates, but not necessarily all together at the same fluence rate. However, this time constant still appears in the light-induced responses of the plasmalemma potential. This implies that the effect on the electrogenic proton pump in the plasmalemma is the most sensitive indicator of proton fluxes into the inner thylakoid space. These results are a further indication that energy-quenching is coupled ther indication that energy-quenching is coupled to transthylakoid proton fluxes via an intermediate, which is not active in Aegopodium podagraria at low irradiances.Abbreviations and symbols _i time constant - F chlorophyll fluorescence - I constant component of irradiance - I _v variable component of irradiance - S light-scattering - q _E high-energy state quenching of chlorophyll fluorescence - T transmittance at 532 nm - V plasmalemma potential     

Recycling of respiratory CO2 during Crassulacean acid metabolism: alleviation of photoinhibition in Pyrrosia piloselloides

The regulation of Crassulacean acid metabolism (CAM) in the fern Pyrrosia piloselloides (L.) Price was investigated in Singapore on two epiphytic populations acclimated to sun and shade conditions. The shade fronds were less succulent and had a higher chlorophyll content although the chlorophyll a:b ratio was lower and light compensation points and dark-respiration rates were reduced. Dawn-dusk variations in titratable acidity and carbohydrate pools were two to three times greater in fronds acclimated to high photosynthetically active radiation (PAR), although water deficits were also higher than in shade fronds. External and internal CO₂ supply to attached fronds of the fern was varied so as to regulate the magnitude of CAM activity. A significant proportion of titratable acidity was derived from the refixation of respiratory CO₂ (27% and 35% recycling for sun and shade populations, respectively), as measured directly under CO₂-free conditions. Starch was shown to be the storage carbodydrate for CAM in Pyrrosia, with a stoichiometric reduction of C₃-skeleton units in proportion to malic-acid accumulation. Measurements of photosynthetic O₂ evolution under saturating CO₂ were used to compare the light responses of sun and shade fronds for each CO₂ supply regime, and also following the imposition of a photoinhibitory PAR treatment (1600 mol·m^-2·s^-1 for 3 h). Apparent quantum yield declined following the high-PAR treatment for sun- and shade-adapted plants, although for sun fronds CAM activity derived from respiratory CO₂ prevented any further reduction in photosynthetic efficiency. Recycling of respiratory CO₂ by shade plants could only partly prevent photoinhibitory damage. These observations provide experimental evidence that respiratory CO₂ recycling, ubiquitous in CAM plants, may have developed so as to alleviate photoinhibition.Abbreviations and symbols CAM Crassulacean acid metabolism - F_M maximal photosystem II fluorescence - F_T terminal steady-state fluorescence - PAR photosynthetically active radiation, 400–700 nm - H⁺ (dawn-dusk) variation in titratable acidity     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 mol photons · m^–2 · s^–1, 350 bar CO₂, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO₂ concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50–20% of the wildtype level) resulted in a strong and proportional inhibition of CO₂ assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF₁)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this excess Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO₂ concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.Abbreviations C_i CO₂ concentration in the air spaces within the leaf - CF₁ coupling factor 1 - Chl chlorophyll Fru1 - 6bisP fructose-1,6-bisphosphate - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground-level of fluorescence obtained using a weak non-actinic modulated beam in the dark - PGA glycerate-3-phosphate - rbcS gene for the nuclear-encoded small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Ru1, 5bisP ribulose-1,5-bisphosphate     

CHARACTERIZATION OF BLEACHING IN CHLORELLA INDUCED BY SUBOPTIMAL GROWTH TEMPERATURES1

Incubation of cultures of a high-temperature strain of chlorella at 10 C stopped growth and bleached all chlorophyll in the cells in 24 hr. Optimal conditions of light (3.0 mw/cm²), gas (1% CO₂-in-air), and inorganic medium for maximal growth at 39 C were maintained in the transfer from 39 to 12, 10, or 5 C. The bleaching process at 10 C is characterized by a lag period for the first few hours followed by a linear decrease in chlorophyll content of cultures. The amounts of time required to bleach half of the chlorophyll initially present (effective half-times) at 10 C were 14 hr for chlorophyll a and 17 hr for chlorophyll b. Effective half-times of bleaching for total chlorophyll were 47 hr at 12 C and 6 hr at 5 C. Addition of glucose to inorganic medium delayed but did not prevent bleaching. Use of argon gas instead, of 1% CO₂-in-air prevented cells from bleaching in both inorganic and glucose media, and indicated an oxygen requirement for bleaching. Incubation of 6 additional strains of Chlorella at 10 C resulted in responses ranging from bleaching to no growth to growth.     

Inhibition of photosynthesis,acidification and stimulation of zeaxanthin formation in leaves by sulfur dioxide and reversal of these effects

Leaves of Pelargonium zonale L. and Spinacia oleracea L. were fumigated with high concentrations of SO₂ for very short periods of time with the aim of first producing acute symptoms of damage and then observing repair. The response of different photosynthetic parameters to SO₂ was monitored during and after fumigation. The following results were obtained: (1) Inhibition of CO₂ assimilation in the light was accompanied by increased reduction of the quinone acceptor, Q_A, of photosystem II and by increased oxidation of the electrondonor pigment P₇₀₀ of photosystem I. Increased control of photosystem II activity in the SO₂-inhibited state was also indicated by increased light scattering and by increased non-photochemical quenching of chlorophyll fluorescence. Both are indicators of chloroplast energization. Apparently, SO₂ did not decrease but rather increased energization of the chloroplast thylakoid system by light. (2) Accumulation of dihydroxyacetone phosphate, fructose-1,6-phosphate and ribulose-1,5-phosphate and a decrease of 3-phosphoglycerate and hexosephosphate indicated that SO₂ inhibited enzymes of the Calvin cycle. (3) Stimulated postillumination CO₂ evolution suggested that when photosynthesis declined respiration increased to provide energy for repair reactions. (4) Increased leaf absorbance at 505 nm indicated increased stimulation of zeaxanthin formation in thylakoid membranes under the influence of SO₂. A similar increase in 505-nm absorbance could be induced by high concentrations of CO₂. In darkened leaves, SO₂ did not produce changes in 505-nm absorbance. (5) While zeaxanthin formation was stimulated, changes in the fluorescence of the pH-indicating dye pyranine, which had been fed to the leaves, indicated acidification of the cytoplasm of leaf cells by SO₂. Maximum acid production by SO₂ required light. In contrast, cytoplasmic acidification of leaf cells by CO₂ was similar in the light and in the dark. (6) Since zeaxanthin formation is known to depend on the acidification of the thylakoid lumen, SO₂-dependent zeaxanthin formation indicated SO₂-dependent acidification of the thylakoid lumen as the indirect result of cytoplasmic acidification by SO₂. (7) Inhibition of photosynthesis and other effects of SO₂ were fully reversible in the light. Detoxification of SO₂ and reactivation of the photosynthetic apparatus were slow or absent in the dark. Light had a dual effect on the action of SO₂. Transiently, it first increased the extent of inhibition of assimilation, but, finally, it reversed inhibition. Sulfur dioxide was inhibitory as a consequence of the chemical reactivity of its hydration products rather than as a result of cellular acidification by the produced acid. The initial acidification was followed by an appreciable alkalisation demonstrating the action of the pH-stat mechanism. (8) The data are discussed in relation to SO₂ toxicity under field conditions when plants are chronically exposed to polluted air.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - F6P fructoce-6-phosphate - F, Fm, Fm, Fo, Fo chlorophyll fluorescence levels - PGA 3-phosphoglycerate - P₇₀₀ primary donor of photosystem I - Q_A primary quinone acceptor of photosystem II - q_p photochemical quenching of chlorophyll fluorescence - NPQ non-photochemical quenching of chlorophyll fluorescence - RuBP ribulose-1,5-bisphosphate Dedicated to Professor O.L. Lange on the occasion of his 65th birthdayOn leave from the Centre for Multidisciplinary Sciences, University of Belgrade, YugoslaviaThis work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 251 of the University of Würzburg. S. V.-J. acknowledges support by the Leibniz program of the Deutsche Forschungsgemeinschaft and by the Fonds for Science of the Republic of Serbia (contract no. 8604). We are grateful to Drs. Z.-H. Yin, U. Takahama and K.-J. Dietz (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, FRG) for cooperation and helpful discussions.     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid