Accumulation of Plastoquinone A during Low Temperature Growth of Winter Rye

Chloroplasts isolated from rye (Secale cereale L. cv Puma) grown at 5°C (RH) accumulated 260% more plastoquinone A (PQA) per plastid than chloroplasts isolated from rye grown at 20°C (RNH). The number of plastoglobuli increased by 270% in RH chloroplasts compared with RNH plastids. When RH plastids were lysed and washed, the number of plastoglobuli associated with thylakoid membranes decreased significantly, yet the PQA levels remained high. Room temperature fluorescence induction indicated that (a) there is no change in the size of the PQA pool immediately available for photochemistry in RNH and RH thylakoids and (b) there is a pool of oxidized PQA present in RNH and RH thylakoids which is not available for photochemistry. The accumulated PQA in RH thylakoids may reflect an increased nonphotochemical function such as regulation of thylakoid protein phosphorylation or protection against photoinhibition.     

Low Temperature Development Induces a Specific Decrease in trans-Delta-Hexadecenoic Acid Content which Influences LHCII Organization

Lipid and fatty acid analyses were performed on whole leaf extracts and isolated thylakoids from winter rye (Secale cereale L. cv Puma) grown at 5°C cold-hardened rye (RH) and 20°C nonhardened rye (RNH). Although no significant change in total lipid content was observed, growth at low, cold-hardening temperature resulted in a specific 67% (thylakoids) to 74% (whole leaves) decrease in the trans-Δ³-hexadecenoic acid (trans-16:1) level associated with phosphatidyldiacylglycerol (PG). Electron spin resonance and differential scanning calorimetry (DSC) indicated no significant difference in the fluidity of RH and RNH thylakoids. Separation of chlorophyll-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the ratio of oligomeric light harvesting complex:monomeric light harvesting complex (LHCII₁:LHCII₃) was 2-fold higher in RNH than RH thylakoids. The ratio of CP1a:CP1 was also 1.5-fold higher in RNH than RH thylakoids. Analyses of winter rye grown at 20, 15, 10, and 5°C indicated that both, the trans-16:1 acid levels in PG and the LHCII₁:LHCII₃ decreased concomitantly with a decrease in growth temperature. Above 40°C, differential scanning calorimetry of RNH thylakoids indicated the presence of five major endotherms (47, 60, 67, 73, and 86°C). Although the general features of the temperature transitions observed above 40°C in RH thylakoids were similar to those observed for RNH thylakoids, the transitions at 60 and 73°C were resolved as inflections only and RH thylakoids exhibited transitions at 45 and 84°C which were 2°C lower than those observed in RNH thylakoids. Since polypeptide and lipid compositions of RH and RNH thylakoids were very similar, we suggest that these differences reflect alterations in thylakoid membrane organization. Specifically, it is suggested that low developmental temperature modulates LHCII organization such that oligomeric LHCII predominates in RNH thylakoids whereas a monomeric or an intermediate form of LHCII predominates in RH thylakoids. Furthermore, we conclude that low developmental temperature modulates LHCII organization by specifically altering the fatty composition of thylakoid PG.     

Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes

Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the ³²Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.     

Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua

Thermal acclimation by Saxifraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 20°C and 10°C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10°C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20°C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20°C- and 10°C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10°C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20°C-acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10°C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem II to photosystem II reaction centers. The relative amounts of chlorophyll-protein complexes and thylakoid polypeptides separated electro-phoretically were similar for both 20°C- and 10°C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition.     

Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications

The effects of chilling in the light (4 days at 5°C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40°C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze-fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light-harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light-harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Δ³-hexadecenoic acid in the phosphatidyldiacylglycerol fraction.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5°C or 20°C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol → methylviologen) after dark preincubation at temperatures between 0 and 60°C. Thylakoids isolated in the presence or absence of Na⁺/Mg²⁺ from 20°C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60°C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20°C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20°C grown rye, thylakoids isolated from 5°C grown rye in the presence of Na⁺/Mg²⁺ exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60°C. This was not due to damage to PSI electron transport in thylakoids isolated from 5°C grown plants since light saturated PSI activity was 60% higher in 5°C thylakoids than 20°C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5°C thylakoids could be observed after dark preincubation only when 5°C thylakoids were initially isolated in the absence of Na⁺/Mg²⁺. We suggest that 5°C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20°C thylakoids and hence cannot be increased further by dark preincubation.     

Photosystem electron-transport capacity and light-harvesting antenna size in maize chloroplasts

Spectrophotometric and kinetic measurements were applied to yield photosystem (PS) stoichiometries and the functional antenna size of PSI, PSII_α, and PSII_β in Zea mays chloroplasts in situ. Concentrations of PSII and PSI reaction centers were determined from the amplitude of the light-induced absorbance change at 320 and 700 nm, which reflect the photoreduction of the primary electron acceptor Q of PSII and the photooxidation of the reaction center P700 of PSI, respectively. Determination of the functional chlorophyll antenna size (N) for each photosystem was obtained from the measurement of the rate of light absorption by the respective reaction center. Under the experimental conditions employed, the rate of light absorption by each reaction center was directly proportional to the number of light-harvesting chlorophyll molecules associated with the respective photosystem. We determined N_P700 = 195, N_α = 230, N_β = 50 for the number of chlorophyll molecules in the light-harvesting antenna of PSI, PSII_α, and PSII_β, respectively. The above values were used to estimate the PSII/PSI electron-transport capacity ratio (C) in maize chloroplasts. In mesophyll chloroplasts C > 1.4, indicating that, under green actinic excitation when Chl a and Chl b molecules absorb nearly equal amounts of excitation, PSII has a capacity to turn over electrons faster than PSI. In bundle sheath chloroplasts C < 1, suggesting that such chloroplasts are not optimally poised for linear electron transport and reductant generation.     

Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll-Protein Complexes and Heme-Binding Proteins

Three chlorophyll-protein complexes (CP I, CP III, CP IV) were electrophoretically separated from thylakoids of the eukaryotic red alga Porphyridium cruentum. CP I contained the primary photochemical reaction center of photosystem I as judged by its light-induced reversible absorbance change at 700 nanometers, by its fluorescence emission maximum at 720 nanometers (−196°C), and by the molecular weight of its apoprotein (68,000 daltons). CP III and CP IV appeared to belong with photosystem II as suggested by the absence of light-reversible absorbance at 700 nanometers, by their fluorescence maximum at 690 nanometers (−196°C), and by the presence of a chlorophyll-binding polypeptide with a molecular weight of about 52,000 daltons. CP IV when completely denatured had two additional polypeptides of about 40,000 and 48,000 daltons. All three chlorophyll-protein complexes contained carotenoids: the chlorophyll/carotenoid molar ratio of 15:1 for CP I, and 20:1 for CP III and CP IV. The thylakoid membranes of P. cruentum contained four cytochromes, detected by heme-dependent peroxidase activity, but there was no observed association with the electrophoretically separated chlorophyll-protein complexes.     

Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress

The response of the photosynthetic apparatus in the green alga Dunaliella salina, to irradiance stress was investigated. Cells were grown under physiological conditions at 500 millimoles per square meter per second (control) and under irradiance-stress conditions at 1700 millimoles per square meter per second incident intensity (high light, HL). In control cells, the light-harvesting antenna of photosystem I (PSI) contained 210 chlorophyll a/b molecules. It was reduced to 105 chlorophyll a/b in HL-grown cells. In control cells, the dominant form of photosystem II (PSII) was PSII_α(about 63% of the total PSII) containing >250 chlorophyll a/b molecules. The smaller antenna size PSII_β centers (about 37% of PSII) contained 135 ± 10 chlorophyll a/b molecules. In sharp contrast, the dominant form of PSII in HL-grown cells accounted for about 95% of all PSII centers and had an antenna size of only about 60 chlorophyll a molecules. This newly identified PSII unit is termed PSII_γ. The HL-grown cells showed a substantially elevated PSII/PSI stoichiometry ratio in their thylakoid membranes (PSII/PSI = 3.0/1.0) compared to that of control cells (PSII/PSI = 1.4/1.0). The steady state irradiance stress created a chronic photoinhibition condition in which D. salina thylakoids accumulate an excess of photochemically inactive PSII units. These PSII units contain both the reaction center proteins and the core chlorophyll-protein antenna complex but cannot perform a photochemical charge separation. The results are discussed in terms of regulatory mechanism(s) in the plant cell whose function is to alleviate the adverse effect of irradiance stress.     

The Role of Acyl Lipids in Reconstitution of Lipid-Depleted Light-Harvesting Complex II from Cold-Hardened and Nonhardened Rye

The role of acyl lipids in the in vitro stabilization of the oligomeric form of light-harvesting complex II of winter rye (Secale cereale L. cv Muskateer) grown at 5 or 20°C was investigated. Purified light-harvesting complex II was enzymically delipidated to various extents by treatment with the following lipolytic enzymes: phospholipase C, phospholipase A₂, and galactolipase. Complete removal of phosphatidylcholine had no effect on the stability of the oligomeric form, whereas the removal of phosphatidylcholine plus phosphatidylglycerol caused a decrease in the ratio of oligomeric:monomeric forms from 1.86 ± 0.17 to 0.85 ± 0.17 and 3.51 ± 0.82 to 0.81 ± 0.29 for purified cold-hardened and nonhardened light-harvesting complex II, respectively, with no change in free pigment content. Incubation of delipidated cold-hardened or nonhardened light-harvesting complex with purified thylakoid phosphatidylglycerol containing trans-Δ³-hexadecenoic acid resulted in 48% reconstitution of the oligomeric form on a total chlorophyll basis with an oligomer:monomer of about 1.90. Incubation in the presence of di- 16:0 or di- 18:1 phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglyceride, or digalactosyldiacylglyceride caused no oligomerization, but rather a further destabilization of the monomeric form. These lipid-dependent structural changes were correlated with significant changes in the 77K fluorescence emission spectra for purified light-harvesting complex II. We conclude that the stabilization of the supramolecular organization of light-harvesting complex II from rye is specifically dependent upon molecular species of phosphatidylglycerol containing trans-Δ³-hexadecenoic acid.     

Photoacclimation in the Red Alga Porphyridium cruentum: Changes in Photosynthetic Enzymes, Electron Carriers, and Light-Saturated Rate of Photosynthesis as a Function of Irradiance and Spectral Quality

Acclimation of the photosynthetic apparatus to changes in the light environment was studied in the unicellular red alga Porphyridium cruentum (American Type Culture Collection No. 50161). Absolute or relative amounts of four photosynthetic enzymes and electron carriers were measured, and the data were compared with earlier observations on light-harvesting components (F.X. Cunningham, Jr., R.J. Dennenberg, L. Mustárdy, P.A. Jursinic, E. Gantt [1989] Plant Physiol 91: 1179-1187; F.X. Cunningham, Jr., R.J. Dennenberg, P.A. Jursinic, E. Gantt [1990] Plant Physiol 93: 888-895) and with measurements of photosynthetic capacity. P_max, the light-saturated rate of photosynthesis on a chlorophyll (Chl) basis, increased more than 4-fold with increase in growth irradiance from 6 to 280 μeinsteins·m⁻²·s⁻¹. Amounts of ferredoxin-NADP⁺ reductase, ribulose-1,5-bisphosphate carboxylase, and cytochrome f increased in parallel with P_max, whereas numbers of the light-harvesting complexes (photosystem [PS] I, PSII, and phycobilisomes) changed little, and ATP synthase increased 7-fold relative to Chl. The calculated minimal turnover time for PSII under the highest irradiance, 5 ms, was thus about 4-fold faster than that calculated for cultures grown under the lowest irradiance (19 ms). A change in the spectral composition of the growth light (irradiance kept constant at 15 μeinsteins·m⁻²·s⁻¹) from green (absorbed predominantly by the phycobilisome antenna of PSII) to red (absorbed primarily by the Chl antenna of PSI) had little effect on the amounts of ribulose-1,5-bisphosphate carboxylase, ATP synthase, and phycobilisomes on a Chl, protein, or thylakoid area basis. However, the number of PSI centers declined by 40%, cytochrome f increased by 40%, and both PSII and ferredoxin-NADP⁺ reductase increased approximately 3-fold on a thylakoid area basis. The substantial increase in ferredoxin-NADP⁺ reductase under PSI light is inconsistent with a PSI-mediated reduction of NADP as the sole function of this enzyme. Our results demonstrate a high degree of plasticity in content and composition of thylakoid membranes of P. cruentum.     

Development at Cold-Hardening Temperatures : The Structure and Composition of Purified Rye Light Harvesting Complex II

Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mm KCl and 10 mm MgCl₂ resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-Δ³-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII₁:LHCII₃) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII₁:LHCII₃ of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LCHII observed in situ and in vitro.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

The Nature of Light-Induced Inhibition of Photosystem II in Pumpkin (Cucurbita pepo L.) Leaves Depends on Temperature

Attached leaves of pumpkin (Cucurbita pepo L.) were treated in high or moderate light at room temperature or a 1°C. The symptoms of photoinhibition appearing during light treatments at room temperature could be attributed to a decrease in the primary activity of PSII. However, when the light treatment was given at 1°C, the quantum yield of photosynthetic oxygen evolution decreased much more than would be expected from the decrease in the ratio of variable to maximum fluorescence at 77°K. Also, light treatment at 1°C lowered the chloroplast wholechain electron transfer capacity much more than it affected PSII electron transport (H₂O to paraphenylbenzoquinone). Light treatments at both room temperature and 1°C led to an increase in B_max, which indicates an increase in the proportion of PSII_β centers. PSI was not affected by the light treatments, and the treatments in the dark at 1°C caused only minor changes in the measured properties of the leaves. We conclude that high light always inhibits the primary activity of PSII, but at low temperature there is greater inhibition of electron transfer from primary electron accepting plastoquinone of PSII to the plastoquinone pool, which leads to a drastic decrease in the quantum yield of oxygen evolution in the chilling-sensitive pumpkin.     

Rearrangement of the chloroplast thylakoid at chilling temperature in the light

The leaves of chilling-sensitive pumpkin (Cucurbita pepo L.) showed symptoms reminiscent of photoinhibition when kept for 4 days at 5°C in moderate light. A decrease was observed in the variable part of chlorophyll α fluorescence, apparent quantum yield, and maximum rate of O₂ evolution. Chloroplast whole-chain electron transport activity measured from chloroplast thylakoids had decreased to 51% of the control value. Photosystem II (PSII) activity decreased by only 9%, suggesting that photoinhibition was not responsible for the loss of electron transport activity. An increase in the proportion of PSII_β (measured as a β_max value) was observed after the chilling treatment. Fractionation of thylakoid membranes showed a 42% increase in PSII activity in the nonappressed region while that in the appressed region decreased slightly. This was accompanied by a decrease in the ratio of the length of appressed to nonappressed thylakoid membranes. Leaf photosynthesis largely recovered within 24 hours of returning to the original growth conditions. We suggest that the increase in the proportion of PSII_β during chilling in light plays a role in protecting PSII from photoinhibitory damage.     

Photosynthetic electron transport in isolated maize bundle sheath cells

Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (μeq/hr · mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 μeq/hr · mg Chl. This was increased to 200 to 350 μeq/hr · mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 μeq/hr · mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 μeq/hr · mg Chl).     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Low growth temperature-induced increase in light saturated photosystem I electron transport is cation dependent

Thylakoid membranes isolated from cold tolerant, herbaceous monocots and dicots grown at 5°C exhibit a 1.5-fold to 2.7-fold increase in light saturated rates of photosystem I (PSI) electron transport compared to thylakoids isolated from the same plant species grown at 20°C. This was observed only when either water or reduced dichlorophenolindophenol was used as an electron donor. The apparent quantum yield for PSI electron transport was not affected by growth temperature. The higher light saturated rates of PSI electron transport in 5°C thylakoids had an absolute requirement for the presence of Na⁺ and Mg⁺². The accessibility of reduced dichlorophenolindophenol to the donor site was not affected by growth temperature since 5°C and 20°C thylakoids exhibited no significant difference in the concentration of this electron donor required for half-maximal PSI activity. The cation dependent higher rates of light saturated PSI activity were also observed when rye thylakoids were developed under intermittent light conditions at 5°C. Thus, this cation effect on PSI activity appeared to be independent of light harvesting complex I and II. The extent of the in vitro reversibility of this cation effect appeared to be limited by an inherent decay process for PSI electron transport. The rate of decay for PSI activity was greatest when thylakoids were isolated in the absence of NaCl and MgCl₂. We conclude that exposure of plants to low growth temperatures induces a reorganization of thylakoid membranes which increases the light saturated rates of PSI electron transport with no change in the apparent quantum efficiency for this reaction. Cations are required to stabilize this reorganization.     

Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl-3(2H)Pyridazinone)

When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H₂O → methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30°C. Below 30°C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6-dichlorophenol indophenol-reduced → methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30°C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide → methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H₂O → methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1°C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55°C; this decrease was biphasic in the control and monophasic in the treated plants.