Elimination of the lag period in chloroplast development in a chlorophyll mutant of peanuts

The mutation of a nuclear gene in peanut (Arachis hypogaea L.) plants results in a reduced light-dependent development of chloroplast fine structure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose-1, 6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and dark respiration during the 72-hour lag period of chlorophyll synthesis in dark-grown leaves exposed to continuous light. The mutation has pleiotropic affects. Kinetic analysis shows there is also a 72-hour lag period in the light-dependent development of NADP-glyceraldehyde-3-P dehydrogenase and fructose-1, 6-diP aldolase in the mutant leaves, whereas there is no lag in the development of NAD-malate dehydrogenase and dark respiration. There is minimal development of the chloroplast during the 72-hour mutationally induced lag period, but there is pronounced cytoplasmic and mitochondrial activity during this phase. There is a 24-hour lag period in the light-dependent enlargement of the mutant leaves. At the completion of leaf enlargement, chloroplast differentiation is initiated. The mutation does not result in any chloroplast deletions, it only affects the timing of the synthesis of these components.     

The effects of water stress on the development of the photosynthetic apparatus in greening leaves

The effects of low and high relative humidity and of polyethylene glycol-induced root water stress on chlorophyll accumulation, on formation of the lamellar chlorophyll-protein complexes, and on the development of photosynthetic activity during chloroplast differentiation were examined. Low relative humidity or polyethylene glycol-induced root water stress (stress conditions) resulted in a 3 to 4 hour lag in chlorophyll accumulation, retarded the rate of chlorophyll b accumulation, and reduced the rate of formation of the light-harvesting chlorophyll a/b protein. All of these effects could be overcome by high relative humidity (nonstress) conditions. Concomitant measurement of leaf water potential showed that under stress conditions greening leaves were subjected to initial water deficits of −8 bars which decreased to −5 bars after 3 to 4 hours of illumination corresponding to the end of the lag phase. Leaves greening under nonstress conditions did not experience leaf water deficits greater than about −5 bars. It seems that the attainment of a minimum leaf water potential of −5 bars may be critical in the control of early chloroplast development. These results demonstrate that the lag phase is not indicative of a programmed event in chloroplast development, but rather is attributable to environmental conditions prevailing during leaf development and greening.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Characterization of a virescent chloroplast mutant of tobacco

Virescent mutations produce plants in which young leaves have reduced chlorophyll levels but accumulate nearly normal amounts of chlorophyll as they age; they are predominantly nuclear mutations. We describe here a virescent mutation (designated Vir-c) found in a somatic hybrid line derived from Nicotiana tabacum L. and Nicotiana suaveolens Lehm. This mutation is inherited maternally. Young, half-expanded Vir-c leaves contained three to six times less chlorophyll than did control leaves, and reached maximum chlorophyll levels much later in development. Chlorophyll synthesis rates and chloroplast numbers per cell in Vir-c were similar to the control, and carotenoid content in Vir-c was sufficient to protect chlorophyll from photo-oxidation. Photosynthetic rates of Vir-c at low light intensities suggested a reduced ability to collect light. Electron micrographs of Vir-c chloroplasts from half-expanded leaves showed a significant reduction in thylakoids per granum. The decrease in granal thylakoids was strongly associated with low chlorophyll levels; mature Vir-c leaves with nearly normal chlorophyll content showed normal granal profiles. These results are discussed in relation to virescent mutants previously described.     

Regulation of synthesis of the photosystem I reaction center

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light- induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark- grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.     

High photosynthetic rate of a chlorophyll mutant of cotton

In a chlorophyll mutant (virescent) and wild-type cotton (Gossypium hirsutum L.), a number of photosynthetic parameters have been measured and compared with those published for other chlorophyll mutants. (a) The photosynthetic rates at 230 w/m² (400-700 nm) from a tungsten lamp were 36.8 mg CO₂ fixed/dm²·hr (virescent) and 39.5 mg CO₂ fixed/dm²·hr (wild-type). On a chlorphyll basis, the photosynthetic rates were 36.8 and 12.1 mg CO₂ fixed/mg chl·hr, respectively. (b) The photosynthetic rates at 13 w/m² (400-700 nm) from a tungsten source were 7.1 mg CO₂ fixed/dm²·hr (virescent) and 7.4 mg CO₂ fixed/dm²·hr (wild-type). On a chlorophyll basis, the photosynthetic rates were 6.0 and 1.4 mg CO₂ fixed/mg chl·hr, respectively. (c) The chlorophyll a/b ratios of the virescent and wild-type leaves were 3.3 and 4.1 (d) The chlorophyll/carotenoid ratios for the virescent and wild-type leaves were 3.2 and 7.3, respectively. (e) The photosynthetic carbon metabolism of the chlorophyll mutant was through the reductive pentose phosphate cycle. (f) The CO₂ compensation points for the virescent and wild-type plants were similar. (g) The mutant and wild-type leaves have the same quantum yield in the red part of the visible spectrum, but the virescent leaves have a lower quantum yield in the blue part of the spectrum. (h) Virescent and wild-type leaves contain similar levels on a protein basis of several reductive pentose phosphate cycle enzymes.     

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.     

The Effect of Phytohormones on Chlorophyll(ide), Protochlorophyll(ide) and Carotenoid Formation in Greening Dark Grown Wheat Leaves

The influence of phytohormones on chlorophyll and carotenoid formation during the greening of irradiated dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull) was studied. Leaves were floated on solutions of abscisic acid, gibberellic acid and kinetin for 24 h. The chlorophyll and carotenoid contents were determined during a subsequent period of 48 h of continuous irradiation. Leaves treated with abscisic acid showed a longer lag phase and a lower rate of accumulation of chlorophyll as compared to the control than did leaves treated with gibberellic acid and kinetin. The carotenoid content was low both in leaves treated with abscisic acid and in those treated with gibberellic acid. Treatment with abscisic acid lowered the protochlorophyllide regeneration after a saturating light flash while gibberellic acid as well as kinetin had no effect. The influence of ABA was partly dependent on an increase of the wounded part of the cut leaf segments. The accumulation of protochlorophyllide in leaves treated with δ-aminolevulinic acid was not affected by the different hormonal treatments. These results suggest that the main effect of abscisic acid is probably outside the chloroplast, i.e. on the formation or transport of δ-aminolevulinic acid.     

Control of chlorophyll production in rapidly greening bean leaves

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.

Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.

Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of δ-amino-levulinic acid partially relieves the inhibition by chloramphenicol.

It is suggested that light regulates chlorophyll synthesis by controlling the availability of δ-aminolevulinic acid, possibly by mediating the formation of an enzyme of δ-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

     

Characteristics of a virescent cotton mutant

The virescent cotton (Gossypium hirsutum) mutant described here differs from normal cultivated cotton by a single mutation in the nucleus. The mutant exhibits nuclear control of chlorophyll and carotenoid development. Young leaves are distinctly yellow and become green with age. There is no unusual photometabolism of ¹⁴CO₂ or ¹⁴C-acetate in this mutant. It is probable that the nuclear virescent mutation is in a locus concerned with making structural units. The yellow leaves do show a high photosynthetic capacity on a chlorophyll basis. At saturating light intensity the rate of CO₂ fixation is 8 fold higher than the green control leaves. Thus, impaired pigment synthesis which could be lethal is offset by a high photosynthetic capacity in the virescent leaves.     

The development of plastocyanin in greening bean leaves

The plastocyanin content of etiolated bean leaves (Phaseolus vulgaris L.) was measured, and the development of the protein in response to light was followed. Measurements were made by quantitative extraction of plastocyanin and a sensitive assay with an O₂ electrode. The electron-paramagnetic-resonance (e.p.r.) signal of oxidized plastocyanin was used as an independent check on the validity of the assay method, and on the thoroughness of extraction. After an initial lag period, the amount of plastocyanin in greening bean leaves increased to reach a maximum after 50h illumination. The chlorophyll/plastocyanin ratio reached a maximum value of 200 irrespective of the light intensity at which greening was carried out, suggesting that the synthesis of the two components is co-ordinated. Experiments involving treatment of etiolated seedlings with brief periods of light of different spectral composition indicated that phytochrome is involved in plastocyanin synthesis. The lack of inhibition of plastocyanin synthesis by specific inhibitors of chloroplast protein synthesis suggests that the protein is synthesized on cytoplasmic ribosomes. The data are discussed in relation to the development of ferredoxin in greening bean leaves.     

Inhibition of Light-Dependent Development of Chloroplasts by 5-Fluorouracil

The uracil analogue, 5-fluorouracil, inhibited the developmentof chloroplasts in Euglena gracilis, strain Z. Chlorophyll synthesiswas inhibited when dark-grown cells were illuminated in thepresence of 5-fluorouracil, but only if the 5-fluorouracil waspresent during the lag phase of chlorophyll synthesis. Ribonucleaseshowed a similar inhibition. Equimolar concentrations of uracilreleased inhibition by 5-fluorouracil, but if cells were incubatedin the light with 5-fluorouracil before addition of uracil,the ability of uracil to effect rapid reversal of 5-fluorouracilinhibition was decreased. In contrast, prior incubation with5-fluorouracil in the dark did not affect reversibility by uracil.The synthesis of a chloroplast-localized protein, cytochromec (552, Euglena), was also inhibited by 5-fluorouracil, whereasthe light-stimulated synthesis of a number of cytoplasmic enzymeswas enhanced. The results suggest that addition of 5-fluorouracilat the beginning of the illumination period preferentially interfereswith the synthesis of chloroplast protein compared with thesynthesis of cytoplasmic protein by inhibiting the formationof a ribosomal system, presumably localized in the chloroplast,that functions in the synthesis of chloroplast protein. Thedata also suggest that in uninhibited cells, the formation ofthis ribosomal system was largely completed within the first10 to 14 h of illumination and before the main period of synthesisof chloroplastproteins.     

Events Surrounding the Early Development of Euglena Chloroplasts: I. Induction by Preillumination 1

Preillumination, followed by a dark period prior to exposure of dark-grown nondividing cells of Euglena gracilis var. bacillaris to normal lighting conditions for chloroplast development, results in potentiation, or abolishment of the usual lag in chlorophyll accumulation. The degree of potentiation is a function of the length of the preillumination period, the intensity of preilluminating light, and the length of the dark period interposed before re-exposure to continuous light for development. The optimal conditions are found to be: 90 minutes of preillumination with white light at an intensity greater than 30 microwatts per square centimeter (14 foot candles) followed by a dark period of at least 12 hours. Reciprocity is not found between duration and intensity of preilluminating light. Preillumination with blue light and red light was found to be the most effective in promoting potentiation, and the ratio of effectiveness of blue to green to red is consistent with protochlorophyll-(ide) being the photoreceptor. Although red light is effective, there is no reversal by far red light, and these facts, taken together with the effectiveness of blue light, suggest that the phytochrome system is not involved. The amount of chlorophyll formed at the end of preillumination is proportional to the resulting potentiation, suggesting that the amount of protochlorophyll(ide) removed or chlorophyll(ide) formed regulates this phenomenon. Potentiated and nonpotentiated cells show comparable rates of protochlorophyll(ide) resynthesis, suggesting that this is not the limiting factor in nonpotentiated cells. Although light is required for protochlorophyll(ide) conversion in chlorophyll synthesis, a brief preillumination seems also to initiate the production of components in the subsequent dark period which, in nonpotentiated cells, are ordinarily synthesized during the lag period under continuous illumination. These components are necessary to sustain maximal rates of subsequent chlorophyll accumulation.     

Development of chlorophyll and hill activity

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding δ-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with δ-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.     

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.     

The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms. I. The stimulating effect of cold predarkening on different stages of greening under white light

Schönbohm, E., Stute, U., Thienhaus, P. and Werner, U. 1988. The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms I. The stimulating effect of cold predarkening on different stages of greening under white light. - Physiol. Plant. 72: 541–546.
The etioplast/chloroplast transformation in angiosperms is controlled by light; most of the processes are mediated by phytochrome. We have shown that in the primary leaves of etiolated seedlings of wheat ( Triticum aestivum L. cv. Kolibri), fire-bean ( Phaseolus multiflorus L. cv. Preisgewinner) and in the cotyledons of etiolated sun flower seedlings ( Helianthus annuus L. cv. macrocarpa) the chlorophyll accumulation in the phase after the end of the lag phase can be greatly stimulated by a cold predarkening period. This effect is not necessarily coupled with a red preirradiation. Furthermore the lag phase can be dramatically shortened by the cold, dark pretreatment, whereas the amount of photoconvertible protochlorophyll(ide) in the darkness remains unaffected by the cold, dark pretreatment. The stimulating effect of a cold, predarkening period on greening is fully reversible by a warm, dark phase that is placed between the cold period and the onset of the continuous white light phase. These findings cannot be generalized: We could demonstrate that in the tropical plant Momordica charantia greening under white light was not affected by different temperature pretreatments during predarkening. The stimulating effect of a cold, predarkening period on greening is assumed to have ecological relevance.     

Quantitative studies on ferredoxin in greening bean leaves

Two methods of measuring small amounts of the iron-sulphur protein ferredoxin are described. One involves measurements of the signal at g=1.96 produced by reduced ferredoxin in an e.p.r. (electron-paramagnetic-resonance) spectrometer; the other depends on the rate of ferredoxin-dependent electron transport in a chloroplast bioassay measured in an O(2) electrode. These methods of measurement were used to examine the development of ferredoxin during the greening of etiolated bean leaves. Ferredoxin is present in low concentrations in the leaves and cotyledons of 14-day-old etiolated beans (Phaseolus vulgaris L. var. Canadian Wonder), and develops in a linear manner with time when the leaves are illuminated. This synthesis appears to be independent of chlorophyll synthesis during the early stages of greening. However, the chlorophyll/ferredoxin ratio reaches a final value of approx. 360 irrespective of the light intensity, indicating the existence of a control mechanism operative in deciding the stoicheiometry of these components in the mature chloroplast. The ferredoxin synthesis appears to be light-dependent, and red light is the most effective in its promotion. The effect of red illumination is not reversed by far-red light, indicating the absence of a phytochrome control of ferredoxin synthesis. From experiments using specific inhibitors of chloroplast protein synthesis, it is concluded that ferredoxin is synthesized on cytoplasmic ribosomes.     

Studies on Moss Spores

At the beginning of germination in Polytrichum commune sporesa clear lag phase is observed in the synthesis of chlorophylls.This synthesis apparently does not start until chloroplast replicationbegins, being correlated with the increase of the number ofchloroplasts. The chlorophyll a to b ratio is 1.7 in the dryspores and it does not vary much during germination.