Photochemical Activity of Chloroplasts Isolated from Etiolated Plants

The rates of phosphorylation, ferricyanide, and dye reductionwere determined with chloroplasts isolated from Linum usitatissimumgrown in darkness and subjected to periods of light of differentduration. With an increased period of illumination, the chlorophyllcontent increased as did also the rate of the three processesmeasured, but no correlation between these two factors was observed.Neither was there any correlation between the rate of any photochemicalreaction and the plastoquinone content. It was concluded thatsome unspecified factor, possibly structural, which developsduring illumination must control the rate of the photochemicalreactions.     

Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

Changes in Sterol Composition during Greening of Etiolated Barley Shoots

The following sterols were identified in barley shoots: stigmasterol, β-sitosterol, campesterol, and cholesterol. The total sterol content of green and etiolated tissue was 2.84 and 3.20 milligrams per gram dry weight, respectively. The free sterols accounted for most of the difference in total sterol content. The sterol ester, sterol glycoside, and acylated sterol glycoside contents of green and etiolated barley shoots were essentially the same. Etiolated tissue had twice as much total β-sitosterol as stigmasterol, while green tissue had equal amounts of these two sterols. The campesterol and cholesterol content was the same in green and etiolated tissue. This same sterol composition pattern held true for the free, glycosidic, and acylated glycosidic sterols; however, the sterol ester fraction had a completely different composition pattern. The esterified stigmasterol content was quite low in green and etiolated tissue, and campesterol was the second largest esterfied sterol component in etiolated tissue. Etiolated barley seedlings exposed to light had a shift in the ratio of free stigmasterol to β-sitosterol in favor of stigmasterol; however, no correlation was observed between chlorophyll synthesis and shift in sterol composition.     

Changes in Lipid Composition during Greening of Etiolated Pea Seedlings

After 7 days of germination in the dark, the three sections of pea seedlings studied (cotyledons, stems, and young leaves) are rich in linoleic acid; after illumination of the seedlings a very significant increase in linolenic acid is observed in the young leaves section, whereas only small variations are noted in the fatty acid composition of the other sections. The increase in linolenic acid results from the increase in galactolipid content of the young leaves; these already linolenic acid-rich galactolipids are present but only in small amounts in the etiolated seedlings (10% of total lipid).     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves

Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O₂ evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P₇₀₀, the photoreduction of C-550, and the photo-oxidation of cytochrome b₅₅₉ in intact leaves frozen to −196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at −196 C.     

The Photochemical Activity of Chloroplasts and Subchloroplast Particles Isolated from Etiolated Bean Leaves Exposed to Continuous Light

The photochemical activity of chloroplasts and subchloroplastparticles isolated from primary bean leaves between the 4thand 24th hour of illumination of etiolated seedlings is thesubject of this paper. The photosystem I activity (oxygen uptakein the presence of MV, DCIP, ascorbate and DCMU), expressedon a unit chlorophyll basis, decreased approximately 10-foldbetween 4 and 8 h of greening. At the same time the photosystemII activity (DCIP photoreduction in the presence of DPC) wasreduced to a half. The photosystem I activity also decreasedin all hitherto investigated fractions which were isolated fromthe digitonin-treated chloroplasts. However, at the initialphase of greening this decrease was the most significant inthe fraction containing heavy particles. After 24 h of greening DCMU, at concentrations higher than 10^–10M, limited the rate of ferricyanide photoreduction by isolatedchloroplasts, whereas after 6 h of greening this effect wasobservable even in the presence of 10^–12 M DCMU. The resultsobtained demonstrated that under those conditions both photosystemswere active after 4 h of greening and PS I activity developedmore rapidly than that of PS II. It also follows from the presenteddata that the water splitting reactions were delayed in developmentas compared to the other reactions investigated, and that PSII units may limit the electron flow in chloroplasts at earlierstages of leaf greening.     

Relationship between Increased NADP-linked Glyceraldehyde-3-phosphate Dehydrogenase Activity and Protein Synthesis during the Greening of Etiolated Pea Seedlings

Photoinduction of NADP-linked glyceraldehyde-3-phosphate dehydrogenase activity in etiolated pea seedlings was investigated in the presence of various concentrations of four inhibitors of protein synthesis (cycloheximide, actinomycin D, chloramphenicol and puromycin) and one photosynthesis inhibitor (DCMU), and compared with increase in chlorophyll and total protein contents. The enzymatic activity and chlorophyll showed similar responses to the action of the antibiotics, whereas they were not significantly affected by the presence of DCMU.     

逆境胁迫对菜豆黄化苗转绿过程中交替呼吸途径与叶绿素荧光特征的影响

以菜豆黄化幼苗作为试验材料,探讨了铅(Pb)或PEG(聚乙二醇)胁迫下交替呼吸途径在植物转绿过程中对叶绿素含量以及叶绿素荧光特性的影响,以阐明逆境胁迫下植物交替呼吸途径的生理学作用。结果显示：(1)与菜豆黄化幼苗正常转绿过程(对照)相比,Pb或PEG胁迫导致菜豆黄化幼苗的叶绿素含量积累延迟,使叶片PSⅡ潜在最大光化学量子效率(F_v/F_m)、光适应下最大光化学效率(F_v′/F_m′)、PSⅡ光适应下实际光化学效率(Y(Ⅱ))和光化学荧光猝灭系数(qP)显著下降,而非光化学猝灭系数(NPQ)则显著增加。(2)在菜豆黄化幼苗转绿过程中,Pb或PEG胁迫导致其交替呼吸途径容量较对照均显著上升。(3)Pb或PEG胁迫下,交替呼吸途径抑制剂［水杨基氧肟酸(SHAM,1 mmol/L)］使菜豆黄化幼苗转绿过程中叶绿素含量、F_v/F_m、F_v′/F_m′、Y(Ⅱ)和qP进一步下降, NPQ却进一步增加,说明抑制交替呼吸途径会加剧Pb或PEG胁迫对PSⅡ反应中心活性的进一步抑制,使还原力积累加剧,造成热耗散进一步增加。研究表明,Pb或PEG胁迫均显著降低了菜豆黄化幼苗PSⅡ对光能的利用率,进而阻碍了菜豆黄化幼苗转绿进程;交替呼吸途径有助于在胁迫条件下缓解PSⅡ的过度还原,可能在一定程度上缓解了Pb或PEG胁迫对其转绿进程的阻碍作用。     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

Accumulation of Pigments during the Greening of Etiolated Seedlings of Hordeum vulgare L.

The pigment changes that occur during transformation of etioplaststo fully developed chloroplasts have been studied in seedlingsof barley (Hordeum vulgare L.) by greening with white lightof low (15–25 µmol m^–2 s^–1) and medium(150 µmol m^–2 s^–1) intensity. At least 24h longer was required in the low light regime for the same concentrationof pigment to be accumulated in the seedlings. The increasein pigment content was mainly due to the synthesis of chlorophyllsa and b. Of the carotenoids present, the increases in the levelsof neoxanthin and, especially, ß-carotene were muchgreater than those observed for the other carotenoids. Levelsof lutein also increased but this change was small by comparisonto those observed for neoxanthin and ß-carotene. Inthe long-term the concentration of violaxanthin remained unalteredalthough significant transient changes were recorded. The levelsof antheraxanthin and zeaxanthin were markedly reduced duringgreening. The rate of pigment synthesis decreased with increasingcell age, i.e. from the base to the tip of the primary leaf.Overall, carotenoid levels increased by approximately 100% atthe base of the seedling but hardly at all at the tip. Key words: Hordeum vulgare, carotenoids, violaxanthin-cycle, etiolation     

Glycerolipid Synthesis in Avena Leaves during Greening of Etiolated Seedlings IV. Effect of Light on Fatty Acid Desaturation

Etiolated Avena sativa L. leaves were fed with [l-¹⁴C]acetatefor 20 h in the dark and labeled fatty acids in glycerolipidswere chased during 24 h in the light (greening condition) orin the dark, to determine the light effect on the fatty aciddesaturation. Oleate decrease in phosphatidylcholine was thesame in the light and in the dark, showing that oleate desaturationis independent of light (or greening). Linoleate desaturationin galactolipids, especially in monogalactosyl diacylglycerol,was enhanced by light and palmitate desaturation to hexadecenoatein phosphatidylglycerol was strictly light-dependent. (Received May 11, 1983; Accepted August 16, 1983)     

Variations in Protein Synthesis in Different Regions of Greening Leaves of Barley Seedlings and Effects of Imposed Water Stress

Water stress, applied to the roots of six-day-old barley seedlingsmarkedly reduced the capacity of their leaves to synthesizeproteins during greening, a process that normally involves intensesynthetic activity. Upon illumination of etiolated seedlingsprotein synthesis commenced most rapidly in the basal region,and then declined in activity. Thereafter, in turn, the middleand apical regions exhibited a similar pattern of syntheticactivity; this is indicative of a ‘wave’ of proteinsynthesis progressing from the base of the leaf to the apexas greening proceeded. The synthesis of proteins varied bothquantitatively and qualitatively in different regions of theleaf during greening. For example, there was a noticeable increasein ribulose-1, 5-bisphosphate carboxylase synthesis in the apicalregion of the leaf compared to the basal region. Water stressreduced protein synthesis in all regions of the leaf, althoughmost effectively in the oldest, apical regions. Upon returnto full water status, the basal regions recovered the most rapidlyand to the greatest extent. Similar results were obtained whenboth intact greening leaves, and isolated segments from differentregions of the leaf were used. The reduction in protein synthesiselicited by water stress was not due to a selective quantitativechange in any particular protein; one protein, of an approximatemolecular weight of 60 kD, appeared to be synthesized only understress conditions. Key words: Water stress, Protein synthesis, Barley seedlings     

黄化水稻幼苗转绿期AOX1基因家族的表达与功能分析

完全黄化的水稻幼苗叶片在持续光照下总呼吸速率、交替途径的速率以及交替途径在总呼吸中的比值均上升,但以水稻AOX1基因家族3个成员的特异性片段为探针,仅观察到其中AOX1c转录本的增加。交替途径的专一性抑制剂SHAM可以降低水稻幼苗在持续光照过程中的相对光合放氧速率与叶绿素含量。同时,水稻黄化幼苗光照前黑暗处理时间越长,在恢复光照后交替途径能力的增加越显著,表现了转绿进程与交替途径之间的相关性。推测加强交替途径可能是植物缓和能量和物质需求矛盾的一个重要调控机制。     

Development of Lipid Synthesis from CO2 in Avena Leaves during Greening of Etiolated Seedlings

The development of the lipid synthesizing system in Avena leafsections was examined in connection with carbon fixation duringthe greening of etiolated seedlings under light. During theinitial 2 h illumination there was a low level of CO₂ fixationby PEP carboxylation, but its products, malate and citrate,did not serve as a carbon source for lipid synthesis, althoughlipid synthesis from acetate had already been established. Withthe initiation of Calvin cycle activity after the initial 2h illumination, lipid synthesis began, with CO₂ fixed by RuBPcarboxylation serving exclusively as the carbon source. Fattyacid synthesis in the leaves during the initial 3 h illumination,unlike the fatty acid synthesis thereafter, was insensitiveto thiolactomycin, an inhibitor of type II fatty acid synthetasecontained in the plastids, and was not dependent on light, incontrast to light-dependent activity in greened leaves. The distribution of ¹⁴C incorporated into lipid molecules fromNaH¹⁴CO₃ showed an equal ratio of ¹⁴C in fatty acid, glyceroland choline moieties of labeled phosphatidylcholine, but a denserradioactivity in the galactose moiety than in the residual moietyof mono- and di-galactosyldiacylglycerols. This suggests a regulatedsupply of glycerol, choline and fatty acid moieties for phosphatidylcholinesynthesis, and an excess supply of galactose to diacylglycerolmoiety for galactosyldiacylglycerol synthesis in Avena leaves. (Received October 31, 1984; Accepted January 25, 1985)     

光和水分胁迫对臭柏实生幼苗光化学效率及色素组成的影响

为探明毛乌素沙地3年生臭柏(Sabina vulgaris)实生苗在不同光照和水分条件下的光抑制响应机制,研究了各处理臭柏实生苗的最大光化学效率(F_v/F_m)及叶绿素(Chla+Chlb)和叶黄素(A+V+Z)含量,分析了其叶绿素循环和叶黄素循环的变化规律。结果表明,77%透光区通过减少Chlb含量,升高Chla/Chlb,避免光能过剩;同时,增加A+V+Z及热散逸色素(A+Z)含量、提高(A+V+Z)/(Chla+Chlb)和(A+V)/(A+V+Z)值,耗散过剩光能,避免光破坏。25%透光区的叶绿素和叶黄素循环机制随着水分条件的变化迅速发生改变。10%透光区通过增加Chlb含量,降低Chla/Chlb,捕捉更多的光能,几乎不存在光抑制。毛乌素臭柏实生幼苗能够适应不同的光照和水分条件,在恶劣的沙漠中完成天然更新,形成独特的群落景观,与叶绿素循环和叶黄素循环有着密切的关系。     

Appearance of Chlorophyll-Protein Complexes in Greening Barley Seedlings

The sequential appearance of chlorophyll-protein complexes (CP)in greening barley leaves was studied by an improved methodof SDS-polyacrylamide gel electrophoresis (PAGE). Solubilizedthylakoid membranes were purified using a sucrose step gradientand CPs were separated by PAGE with low concentrations of SDSin solubilizing and reservoir buffers. At 10 min after the onsetof illumination, a chlorophyll-protein complex (CPX) was detected.It was a labile CP, its chlorophyll (Chl) being easily releasedfrom the apoprotein during electrophoresis. The P700-chlorophylla/b-protein complex (CPl) appeared after 45–60 min ofillumination together with P700 activity. Light-harvesting chlorophylla/b-protein complex (LHCP) began to accumulate at 2.5 h withthe beginning of Chl b synthesis. In some cases a small amountof CPa could be detected after 6 h of greening. The time-differencespectrum between homogenates of leaves illuminated for 30 and60 min had an absorbance maximum at 677 nm, showing that a redshift indicative of CPl formation began soon after completionof the Shibata shift. The time-difference spectrum between 3.5-hand 4.0-h illuminated leaves resembled the absolute spectrumof fully greened leaves, indicating that at this stage, spectralcomponents were being synthesized at the same ratio at whichthey exist in fully greened tissues. Both absolute and time-differencespectral data supported the SDS-PAGE results. (Received February 27, 1985; Accepted May 8, 1985)     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.