Light-dependent development of thermoluminescence, delayed emission and fluorescence variation in dark-grown spruce leaves

Thermoluminescence profiles of spruce leaves grown under various light or dark conditions were measured after excitation at a low temperature (−70 to −20 °C) by 1-min illumination with red light, and the following results were obtained. Mature spruce leaves showed five thermoluminescence bands at −30, −5, +20, +40 (or +35) and +70 °C (denoted as Z_v, A, B₁, B₂ and C bands, respectively), but dark-grown spruce leaves with a similar chlorophyll content showed only two bands, at −30 and +70 °C (the Z_v and C bands) and were devoid of the three other bands (the A, B₁ and B₂ bands). On exposure of the dark-grown leaves to continuous red light, the A, B₁ and B₂ bands were rapidly developed, and the development was accompanied by enhancement of delayed emission, fluorescence variation and the Hill activity (photoreduction of 2,6-dichlorophenolindophenol with water as electron donor). It was demonstrated that the dark-grown spruce leaves are devoid of the water-splitting system in Photosystem II, and that the latent water-splitting activity is rapidly photoactivated by exposure of the leaves to continuous red light. These results on the gymnosperm spruce leaves, in which greening proceeds in complete darkness, being independent of the development of the water-splitting system in light, were discussed in relation to previous observations on angiosperm leaves, in which both greening and the activity generation proceed in the light.     

Development of photosynthetic activities in dark-grown spruce seedlings

The chloroplasts in dark-grown, 5-day-old seedlings of Piceaabies contained doubly stacked primary thylakoid membranes connectedwith prolamellar bodies. The photo-system II reaction centerand photosystem II-associated electron transfer were assembledin the primary thylakoid membranes, but the oxygen-evolvingsystem remained latent unless the seedlings were exposed tolight. The photoactivation of this system was strongly dependenton temperature during pre-illumination. This implies that somethermal process, in addition to the photoprocess, is involvedin the photoactivation of the oxygen-evolving system. ¹Laboratory of Plant Physiology, The Institute of Physical andChemical Research, Wako-shi, Saitama 351, Japan. ²Department of Biology, Fukuoka Dental College, Fukuoka 814,Japan. (Received June 22, 1977; )     

Protochlorophyllide resynthesis in dark-grown bean leaves

The protochlorophyllide content of dark-grown bean leaves was determined at various ages. It was detectable the second day after germination and reached a maximum on about the tenth day.     

Photosynthetic CO2-fixing activity in dark-grown spruce seedlings

Bicarbonate-dependent O₂-evolving activity in dark-grown cotyledonsof Picea abies was measured with an oxygen electrode with differentpreillumination times. The activity showed a slight linear increasewith increasing preillumination time. On the other hand, O₂-evolvingactivity (Hill activity) of chloroplasts prepared from preilluminateddark-grown cotyledons exhibited a characteristic change of asteep rise followed by a gradual increase with increasing preilluminationtime. The results obtained were discussed in connection withthe light activation of the latent, inactive O₂-evolving centerin dark-grown cotyledons. (Received December 8, 1978; )     

Tissue localization of phytochrome in dark-grown barley leaves

Phytochrome was spectrophotometrically determined to be differentially concentrated among separated tissues of dark-grown, norflurazon-treated barley l     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the watersplitting activity in photosynthesis

Plant materials (intact leaves, chloroplasts or subchloroplast particles) preilluminated at a low temperature (e.g. −60°C) were rapidly cooled to −196°C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as Z_v, A, B₁, B₂ and C bands. The A, B₁, B₂ and C bands appeared at constant temperatures, −10, +25, +40 and +55°C, respectively, being independent of the illumination temperature, but the Z_v band appeared at a variable temperature slightly higher than the illumination temperature. The B₁ and B₂ bands were absent in the thermoluminescence profiles of samples devoid of the oxygenevolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water-splitting activity in photosynthesis.

Plant materials (intact leaves, chloroplasts or subchloroplast particles) pre-illuminated at a low temperature (e.g. -60 degrees C) were rapidly cooled to -196 degrees C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as ZV, A, B1, B2 and C bands. The A, B1, B2 and C bands appeared at constant temperatures, -10, +25, +40 and +55 degrees C, respectively, being independent of the illumination temperature, but the ZV band appeared at a variable temperature slightly higher than the illumination temperature. The B1 and B2 bands were absent in the thermoluminescence profiles of samples devoid of the oxygen-evolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

Lipid metabolism in ageing leaves of dark-grown barley seedlings

The rapid senescence of the etiolated leaves of dark-grown barley seedlings in the dark is accompanied by the loss of those lipids associated with the plastids. The linolenate content of the plastid glycerolipids rapidly decreased whereas it tended to increase in the extraplastidic phospholipids. Kinetin treatment slowed down the loss of the plastid lipids and their constituent fatty acids. The hormone treatment brought about increased linolenate, particularly in phosphatidylcholine and monogalactosyldiacylglycerol. The senescing leaf attempts to adapt to ageing by increased membrane synthesis and/or membrane repair. Kinetin appears to control the sequential desaturation of oleate to linolenate.     

Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2, 6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10 s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light → A₀-light → A₁-dark → A₂-light → A₃ in which P is a precursor convertible to A₀, the first intermediate with a longer lifetime of $\text{t}\text{1}\text{2}\text{≈ 100}\text{s}$ and A₃ is the final activated compound or state converted by short-interval flashes from A₀ through A₁ and A₂, two other intermediates with shorter lifetimes of $\text{t}\text{1}\text{2}\text{≈ 0.4}\text{s}$ and 5 s, respectively.     

Photochlorophyllide (P650) turnover in dark-grown barley leaves

Laevulinate (LA) induced an increase in protochlorophyllide (P650) in dark-grown ageing barley leaves. The increase was due to a suppression of a P650 breakdown mechanism. The LA inhibition of P650 destruction allowed an estimate to be made of turnover of P650 in ageing etiolated leaves. The rate constant for P650 destruction in 8-day-old dark-grown leaves was 139 pmol/nmol/hr with a half life of 5 hr.     

Ultrastructure and lipid composition of etioplasts in developing dark-grown wheat leaves

Changes in the ultrastructure and lipid composition of etioplasts have been evaluated in three regions from the base to the tip of 8-day-old darkgrown wheat leaves and in the upper-2/3 region of etiolated leaves of different ages. In developing darkgrown tissues, the main morphological changes that etioplasts undergo consist of an increase in the amount of thylakoïds which, in the most mature etioplasts, align in parallel arrays. Concomitantly, galactolipids and sulfolipid form an increasing proportion of the total lipids. Trans-3-hexadecenoic acid was not detectable in phosphatidylglycerol (PG) of etioplasts showing appressed thylakoïds isolated from 5-day-old leaves, but was present in significant amounts in etioplasts in the basal part of 8-day-ols leaves in which membrane appression was barely visible. The proportions of this acid increase as etioplasts develop, reaching 25% of the PG fatty acids (1.2% of the total fatty acids) in the most differentiated etioplasts. In wheat etioplasts, it appears that trans-3-hexadecenoic acid may accumulate in considerable amounts in darkgrown tissues and that its accumulation is not directly involved in membrane appression.Abbreviations AP phosphatidic acid - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission.

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2,6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light leads to A0-light leads to A1-dark leads to A2-light leads to A3 in which P is a precursor convertible to A0, the first intermediate with a longer lifetime of t 1/2 approximately 100s and A3 is the final activated compound or state converted by short-interval flashes from A0 through A1 and A2, two other intermediates with shorter lifetimes of t 1/2 approximately 0.4s and 5s, respectively.     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

Stimulatory effect of calcium on photoactivation of the water oxidation system in dark-grown spruce chloroplasts

Treatment of platelets or red cells with small amounts of phospholipase C from Clostridium welchii enables both cells, prior to the onset of lysis, to stimulate prothrombin conversion by coagulation factor Xa and Va in the presence of calcium. Phospholipase C treatment of both cells also exposes significant amounts of phosphatidylserine at the outer surface. The level of phosphatidic acid formed from diglycerides produced by phospholipase C action, is similar to that formed in activated platelets upon triggering the phosphatidylinositol cycle. A possible involvement of this cycle to activate platelets to become more procoagulant is discussed.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

The association of protein synthesis with protochlorophyllide holochrome regeneration in dark-grown barley leaves

A light-stimulated increase in incorporation of radioactive amino acids into protein associated with protochlorophyllide holochrome occurs concomitantly with the regeneration of phototransformable protochlorophyllide in dark-grown barley leaves. This increase in radioactivity and the protochlorophyllide regeneration process are both abolished by incubation of the leaves with inhibitors of cytoplasmic protein synthesis. Prelimiary data implicate protein in the molecular weight range of 45,000–60,000 daltons in this process.     

Cytokinins induce photomorphogenic development in dark-grown gametophytes of Ceratopteris richardii

The cytokinins benzylaminopurine, kinetin and isopentenyladenine induce photomorphogenesis in dark-grown gametophytes of the fern Ceratopteris richardii. At sub-nanomolar concentrations each altered the rate and pattern of cell division, elongation and differentiation, mimicking aspects of the light-mediated transition from filamentous to prothallial growth. Untreated dark-grown gametophytes grow as narrow, elongate, asexual filaments with an apical meristem. Cytokinin treatments as low as 10(-12) M reduced the length-to-width ratio through decreased cell elongation, increased periclinal cell division and induced the formation of rhizoid initials in the cells immediately below the apical meristem. Higher concentrations (10(-9)-10(-8) M) induced conversion of the meristem from apical to notch morphology. Cytokinins induced both red- and blue-light-mediated photomorphogenic events, suggesting stimulation of both phytochrome and cryptochrome signaling; however, cytokinin treatment only partially substituted for light in that it did not induce hermaphroditic sexual development or spore germination in the dark. Additionally, cytokinins did not increase chlorophyll synthesis in dark-grown gametophytes, which unlike angiosperms are able to produce mature chloroplasts in the dark. Cytokinin treatment had only slight effects on light-grown gametophytes. These results suggest evolutionary conservation between angiosperms and pteridophytes in the role of cytokinins in regulating photomorphogenesis.     

Functional development of photosystems I and II in dark-grown pine seedlings

Green plastids prepared from seedlings of Pinus silvestris harvestedafter three weeks of growth in the dark, without exposure tolight, catalyzed photoreductions of methyl viologen and nicotinamide-adeninedinucleotide phosphate, and cyclic photophosphory-lation withN-methylphenazonium methosulfate, but could not catalyze thephotoreduction of 2,4-dichlorophenol indophenol in the absenceand presence of diphenylcarbazide. In dark-grown seedlings ofPinus silvestris, functional photosystem I developed with noexposure to light, but no photosystem II activity was abserved. (Received August 22, 1973; )     

The mechanism of the ozone-induced changes in thermoluminescence glow curves of barley leaves

The changes in thermoluminescence (TL) signals induced by short-term ozone exposure of leaves are characterized by a down-shift of the peak-temperature of the TLB-band and an increase of a TL band at 55°C. We investigated the relationship of these changes to photosystem 2 (PS2) photochemistry. The changes were not only detectable in the presence of ozone, but also after irradiation of dark-adapted leaves and after aging of irradiated detached leaf segments. The opposite effect on TL, an up-shift of the peak-temperature of the B-band and the decrease of the intensity of the band at 55°C were found after infiltration of leaves with nigericin, antimycin A, and diphenyleneiodonium chloride (DPI). Propyl gallate down-shifted the peak-temperature of the B-band. 2,5-dimethyl-1,4-benzoquinone up-shifted the peak-temperature of the B-band and decreased the intensity of the 55°C band. The intensity of the 55°C band did not change significantly in the presence of oxygen in comparison to that in nitrogen atmosphere. It decreased with time of dark adaptation (50% intensity was observed after 3 h of dark adaptation at room temperature), however, it was reactivated to its initial value (at 5 min of dark adaptation) after 1 single-turnover flash. The 55°C band was not significantly changed in the presence of DCMU. Thus the ozone-induced band at 55°C is assigned to charge recombination in PS2. Changes in the electron transport chain at the acceptor side of PS2, probably related to the cyclic electron transport around photosystem 1 and/or chlororespiration, could play an important role in the increase of the 55°C band and the down-shift of the B-band. The changes at the acceptor side indicated by TL can be an ex pression of a physiological regulatory mechanism functional under stress conditions.     

Photoactivation of the latent O2-evolving center in chloroplasts isolated from dark-grown spruce seedlings

The latent O₂-evolving center in chloroplasts isolated from spruce [Picea abies (L.) Karst.] seedlings grown in the dark was readily activated by pre-illuminating the chloroplast suspension with weak white light. The photoactivation depended on pH with the optimum at pH 7–8, and was strongly stimulated by ascorbic acid. The optimal stimulation was also obtained at pH 7–8. The temperature dependence of the photoactivation suggested the involvement of some dark reaction in the activation process.