Correlated Changes in the Activity,Amount of Protein,and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.     

Characterization of protoheme levels in etiolated and greening plant tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).     

Change in Levels of Acyl Carrier Proteins in Greening Plants

The levels of acyl carrier proteins (ACP) in greening spinachcotyledons and greening oat leaves were examined by immunoblottingwith antiserum raised against spinach ACP I. Two isoforms ofACP, ACP I and ACP II, were found in spinach cotyledons, asthey were in the green leaves. The level of ACP II was higherthan that of ACP I in etiolated cotyledons. The level of ACPI increased markedly with greening. In the greened cotyledons,the major isoform was ACP I as was the case in green spinachleaves. In oat leaves, two isoforms were also identified, oatACPI (about 12kDa) and ACP II (about 17kDa), which cross-reactedwith the antiserum against spinach ACP I, but which were differentfrom spinach ACPs I and II. The levels of oat ACPs I and IIwere very low in etiolated leaves. The increase in levels ofboth ACPs corresponded to the change in the activity of fattyacid synthesis during illumination for 24 h. During furtherillumination for 24 h, the level of ACP II increased a littlein parallel with the change in the activity of fatty acid synthesis,whereas the level of ACP I increased somewhat more. The functionof oat ACPs I and II is discussed in connection with the formationof chloroplast. (Received March 27, 1989; Accepted September 18, 1989)     

Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl

Treatment with acifluorfen-methyl (AFM), methyl 5-(2-chloro-4-[tri-fluoromethyl] phenoxy)-2-nitrobenzoate, inhibited protochlorophyllide synthesis in dark-held, δ-amino levulinic acid-fed, excised cotyledons of cucumber (Cucumis sativus L.). Protochlorophyllide and protoporphyrin IX levels in AFM-treated cotyledons were inversely related and dependent on AFM concentration; as the herbicide dose increased, protoporphyrin IX levels also increased with a concomitant loss of protochlorophyllide. Significant protoporphyrin IX accumulation was induced by concentrations of AFM from the linear region of the membrane disruption dose response curve. The pattern of precursor accumulation seen in HPLC chromatograms from extracts of AFM-treated tissue indicate that Mg insertion into the tetrapyrrole ring is inhibited, suggesting interference with Mg-chelatase. An inhibitor of δ-amino levulinic acid synthesis, gabaculine (3-amino-2,3-dihydrobenzoic acid), completely blocked the membrane disruption activity of AFM in illuminated cotyledons. Protoporphyrin IX accumulating in AFM-treated tissues may serve as the primary photosensitizer for initiating lipid peroxidation.     

Inter-organ Control of Greening in Etiolated Cucumber Cotyledons

Inter-organ control of greening in etiolated cucumber (Cucumis sativus L. cv. Aonagajibae) cotyledons was investigated. Four- or six-day-old excised or intact etiolated cucumber cotyledons were illuminated under aerobic conditions. Excised cotyledons without hypocotyl hooks produced chlorophyll without a prolonged lag phase and the rate of chlorophyll formation was not depressed if they were illuminated immediately after excision. If the excised cotyledons were incubated in the dark before illumination, chlorophyll accumulation at the end of 6 h of continuous illumination was remarkably lowered as the dark period lengthened, especially in 6-day-old cotyledons. The rapid loss of chlorophyll-forming capacity of excised cotyledons during dark preincubation suggests a stimulatory effect of hypocotyls on the greening in cotyledons. The treatment of excised cotyledons with bleeding sap in the dark for 18 h resulted in the promotion of chlorophyll formation during subsequent continuous illumination. Partial fractionation of bleeding sap with organic solvents and paper chromatography indicates that the active substances showed the same behavior as cytokinins. These facts add weight to the hypothesis that cytokinins from roots flow into cotyledons and stimulate greening.     

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH₃ are discussed.     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Photoinhibition in vivo of Photosystem II reactions during developmentof the photosystems of wheat seedlings

Photoinhibition of O₂ evolution and reactions leading to millisecond-delayed light emission (ms-DLE) of chlorophyll by illumination of leaves with excess white light were investigated in wheat seedlings greened for different times in a special chamber with constant conditions (20°C; CO₂ and humidity). A sharp reduction in initial and steady state rates of O₂ evolution and in the intensity of different components of ms-DLE under excess light on the stage of lag-phase of chlorophyll biosynthesis (4–6h of greening) were observed. An increasing stability of the oxygen-evolving process and ms-DLE of chlorophyll during formation of the thylakoid membrane photosystems (12–24 h of greening) was shown. Rifampicin did not influence the stability of oxygen evolution whereas cycloheximide led to the intensification of photoinhibition of the initial and steady-state rates of oxygen evolution under the inhibitory light action. The early stages of photosystems formation during short time of greening of etiolated seedlings were more sensitive to the action of inhibitory light, possibly due to a weak interaction of the oxygen-evolving system components and connection with reaction centers of Photosystem II.     

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.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.     

Biogenesis of photosynthetic apparatus under the inhibition of energy processes in de-etiolated seedlings of barley (Hordeum vulgare L.)

Transformation of protochlorophyllide forms in etiolated barley seedlings and biogenesis of photosynthetic apparatus in greening leaves of 7-day-old etiolated barley seedlings (Hordeum vulgare L.) were studied under the inhibition of energy processes during illumination. Repression of electron transport between photosystem 2 and 1 (PS2 and PS1, respectively) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) inhibited the photochemical activity of PS2 but did not affect chlorophyll biosynthesis and ATP content in leaves compared to the control. Inhibition of mitochondrial electron transport with sodium azide increased relative content of nonphotoactive protochlorophyllide in etiolated leaves, decreased the content of ATP, chlorophylls, and carotenoids and completely suppressed the functional activity of PS 2. The inhibitor of glycolysis sodium fluoride affected all the parameters even more strongly. We observed synchronism in the accumulation of chlorophylls and carotenoids during greening for all inhibitor variants other than fluoride (correlation coefficient, r, equal to 0.98, 0.97, 0.97, and 0.47 with the significance level of 0.01; 0.015; 0.015, and 0.27 for control, diuron, azide, and sodium fluoride, respectively). The change in chlorophyll content under the influence of inhibitors positively correlated with the amount of ATP in the leaf tissue (for 24 h greening, r = 0.97 with significance level of 0.015). We suggest that sources of ATP involved in the synthesis of chlorophyll during greening of etiolated barley seedlings are mostly of non-plastid origin.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and other metalloporphyrins by isolated etioplasts and developing chloroplasts

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, δ-aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in vitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H₂O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and δ-aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own δ-aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in vitro. The metalloporphyrins synthesized in vitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.     

Impaired chlorophyll formation in etiolated cucumber cotyledons following prolonged dark incubation after red light pretreatment

Red light (R) pretreatment of etiolated cucumber seedlings ( Cucumis sativus L. var. Elem) followed by prolonged dark incubation prior to white light (WL) exposure, had an adverse effect on the greening of the cotyledons. The effect was photoreversible by far-red (FR) light. Cotyledons which were dark incubated for 24 h following the R pulse greened more rapidly when exposed to WL than did the controls, while total chlorophyll (Chl) accumulation after 24 h in the light was about the same in both. However, after 48 h post-R dark incubation greening of the treated cotyledons was delayed, and their amount of Chl which accumulated after 24 h WL was about one half of that in non-treated seedlings. As the length of the post-R dark incubation period was extended Chl production became slower, so that after 96 h post-R dark incubation the Chl level in the treated cotyledons after 24 h WL was approximately 20% of the controls. No significant differences in amounts of protochlorophyll could be detected between seedlings preilluminated with R or R followed by FR. Seedlings 4-, 5- and 6-days-old at the time of R treatment showed similar degrees of impaired Chl synthesis following prolonged post-R dark incubation.     

Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings.

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.     

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene

Background and Aims

Etiolation symptoms and the greening process are usually studied on dark-germinated seedlings and this raises the question – can these results be generalized for plants growing under field conditions? This work examines various aspects of the plastid differentiation under the covering of the achene wall, which often remains attached to the cotyledons of sunflower (Helianthus annuus) seedlings grown under light.

Methods

Cotyledons of 7- to 10-d-old sunflower seedlings grown in the dark and on light were examined. The partially covered cotyledons were sectioned into light-exposed, covered and transition zones. Pigment contents, 77 K fluorescence spectroscopy, electron microscopy and fluorescence imaging, along with fluorescence kinetic methods, were used.

Key Results

The light-exposed zone of the partially covered cotyledons was similar to cotyledons developed without achene covering. However, some of the plastids had prolamellar bodies among the granal thylakoid membranes; despite this no protochlorophyllide was detected. The fully covered, yellowish sections contained protochlorophyllide forms emitting at 633 and 655 nm and well-developed prolamellar bodies, similar to those of etiolated cotyledons. In addition, reduced amounts of chlorophyll a, chlorophyll b and stacked thylakoid membrane pairs were found in this region. The transitional sections showed a mixture of the characteristics of the covered and exposed sections. Various, but significantly different values of the photosynthetic activity parameters were found in each sector of the partially covered cotyledons.

Conclusions

The partial covering of the achene wall shades the cotyledon tissues effectively, enough to provoke the appearance of etiolation phenomena, i.e. the permanent presence of flash-photoactive protochlorophyllide complexes and prolamellar bodies (with or without protochlorophyllide), which proves that these phenomena may appear under natural illumination conditions.Key words: Cotyledon, etio-chloroplast, etioplast, etiolation, Helianthus annuus, photosynthetic activity, protochlorophyllide, prolamellar body, sunflower     

Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING

During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis.