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.     

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.     

Protochlorophyll(ide) accumulation and degradation in the dark and photoconversion to chlorophyll in the light in pigment mutant C-2A' of Scenedesmus obliquus

Pigment mutant C-2A' of Scenedesmus obliquus accumulates only traces of chlorophyll, when grown heterotrophically in the dark. Immediately upon transfer of cells into fresh medium protochlorophyllide and protochlorophyll are formed, which accumulate to their maximum concentrations within 8 to 12 h. Subsequently, this protochlorophyll(ide) is degraded in the dark, but not transformed into chlorophyll. After 6–8 days of dark growth no protochlorophyll(ide) can be detected any more. The protochlorophyll(ide) pool of cultures, which contain reduced concentrations, can be reestablished either by addition of glucose or illumination with blue light; both increase the rate of respiration.
By low temperature spectroscopy in vivo and by absorption and fluorescence emission spectroscopy of pigment extracts it is shown that the protochlorophyllide accumulated in freshly inoculated cultures can be converted to chlorophyll in light.
From the action spectrum for chlorophyll formation after addition of glucose it can be seen that protochlorophyllide 636 and 649 are present and are photoconvertible in this mutant.     

Oak seedlings grown in different light qualities

Oak seedlings (Quercus robur L.) were germinated in darkness for 3 weeks and then given continuous long wavelength far-red light (LFR; wavelengths longer than 700 nm). A control group of seedlings was kept in darkness. After 2 additional weeks the chlorophyll formation ability in red light was examined in the different seedlings. The stability of the protochlorophyll(ide) and chlorophyll(ide) forms to high intensity red irradiation was also measured. Oak seedlings grown in darkness accumulated protochlorophyll(ide) (6 μg per g fresh matter). Absorption spectra and fluorescence spectra indicated the presence of more protochlorophyll(ide)_628–632 than protochlorophyllide_650–657. The level of protochlorophyll(ide) was higher in leaves of plants cultivated in LFR light (13 μg per g fresh matter) than in leaves of dark grown plants. 12% of the protochlorophyll(ide) was esterified in both cases. The level of protochlorophyll(ide)_628–632 in LFR grown oaks varied with the age of the leaves, being higher in the older (basal) leaves, but also in the very youngest (top-most) leaves. The ability of the leaves to form photostable chlorophyll in red light showed a similar age dependence, being low in rather young and in older leaves. A low ability to form photostable chlorophyll thus appears to be correlated with a high content of protochlorophyll(ide)_628–632. Upon irradiation only the protochlorophyllide_650–657 was transformed to chlorophyllide. After this phototransformation the chlorophyllide peak at 684 nm shifted to 671 nm within about 30 min in darkness. This shift took place without any accompanying change in photostability of the chlorophyll(ide). Upon irradiation with strong red light a similar shift took place within one minute. This indicates that the chlorophyllide after phototransformation was rather loosely bound to the photoreducing enzyme. The development towards photostable chlorophyll forms consists of three phases and is discussed.     

Structure-function correlation during the etioplast-chloroplast transition in cucumber cotyledonary leaves

We studied the development of chloroplasts from etioplasts in the cotyledonary leaves of 4-d-old dark-grown cucumber (Cucumis sativus) seedlings after irradiation (20 μmol m^-2 s^-1). Upon irradiation, the triggering of chlorophyll (Chl) synthesis and accumulation showed a relatively short lag phase. The irradiation of etiolated seedlings initiated the synthesis of apoproteins of pigment-protein complexes. While Chl-protein 2 (CP2) was detected at 6 h after irradiation, CP1 only after 29 h. The appearance and accumulation of some of the apoproteins were monitored by Western-blotting. LHC2 apoprotein was detected after a 6 h-irradiation. The amounts of D1 protein of photosystem (PS) 2 and PsaA/B protein of PS1 were quantitated by ELISA. Further, the thylakoid membrane function during this time period in terms of PS1- and PS2-mediated electron transfer activity and intersystem electron pool size were analyzed. While PS1 activity was detected after 4 h, PS2-mediated O₂ evolution was detected only after a 17 h-irradiation. F_v/F_m value of Chl a fluorescence measurements indicated that the photochemical efficiency of these leaves reached its maximum after 29 h of irradiation. The intersystem pool size of cotyledonary leaves was equivalent to that of the control cotyledonary leaves grown for 25 h under continuous irradiation. Thus etioplasts develop into fully functional chloroplasts after approximately 25 h when 4 d-dark grown cucumber seedlings are continuously moderately irradiated. The development of photosynthetic electron transport chain seems to be limited in time at the level of PS2, possibly at the donor side. This revised version was published online in September 2006 with corrections to the Cover Date.     

Phytol Changes in Dark Grown Leaves of Barley after Varying Light Treatments

Gas chromatographic determinations revealed a certain amount of free phytol in dark-grown barley leaves. When a short light impulse or continuous light is given to the leaves, the phytol pool is partly emptied due to esterification of chlorophyllide a. The regeneration is slow during the first 2–3 hours. A pretreatment with light flashes followed by a dark period accelerates the regeneration, which stops however after approximately 30 min. Some evidence points to the existence of an acceptor for excess phytol entering at this stage. Connections between phytol changes during irradiation and the lag phase of chlorophyll formation are discussed.     

The effect of photoperiod length on respiration in leaves of Saxifraga cernua L., an arctic herb

Abstract Rates of oxygen uptake were measured in leaves of Saxifraga cernua which had been exposed to an 18-h photoperiod. These rates were compared to those in plants which had been exposed to continuous light. Rates of total dark respiration and alternative pathway respiration measured at the end of the photoperiod gradually decreased over the initial 3 d of exposure to an 18-h photoperiod. Thereafter, respiratory rates were constant. Rates of total dark respiration and alternative pathway respiration decreased during the 6h dark period. Rates of normal and alternative pathway respiration are equally affected during the dark period. The respiratory rates had reached a new minimum level 3 d after the initiation of a dark period. These results suggest that respiration rates in arctic plants are high because of the long photoperiod in the arctic. The kinetics of photoperiod induced changes in respiration are slow enough to suggest the involvement of the biological clock in setting respiration rates. Indeed, total dark respiration and alternative pathway respiration show a definite circadian rhythm. Free-running experiments show that normal respiration changes much less (has a smaller amplitude of variation) than alternative pathway respiration and that alternative pathway respiration accounts for most of the rhythmicity of respiration.     

High salt stress in wheat leaves causes retardation of chlorophyll accumulation due to a limited rate of protochlorophyllide formation

When exposed to salt stress, leaves from dark-grown wheat seedlings ( Triticum aestivum , cv. Giza 168) showed reduced accumulation of chlorophyll during irradiation. To elucidate the mechanism behind salt-influenced reduction of chlorophyll biosynthesis, we have investigated the effect of salt stress on the spectral forms of Pchlide, the phototransformation of Pchlide to Chlide, the Shibata shift, the regeneration of Pchlide and the accumulation of Pchlide from 5-aminolevulinic acid (ALA). We found that the phototransformation of Pchlide to Chlide was not affected by salt stress. The blue shift (Shibata shift) of newly formed Chlide was delayed both after flash irradiation and in continuous light. The reformation of Pchlide in darkness after a flash irradiation or after a period of 3-h irradiation was retarded in the salt-treated leaves. However, after a 20-h dark period, Pchlide was reformed even in salt-treated leaves but the formation of short-wavelength Pchlide was suppressed. Compared to controls, salt treatment also reduced the amount of Pchlide accumulated in leaves floated on ALA. The increase in the low temperature fluorescence emission spectrum at 735 nm, which occurred gradually during several hours of irradiation with continuous light in control leaves, was completely suppressed in salt-treated leaves. It is concluded that salt stress inhibits chlorophyll accumulation partly by reducing the rate of porphyrin formation but, as discussed, also by a possible reduction in the formation of chlorophyll-binding proteins.     

Effects of Moderate Water Deficit (Stress) on Wheat Seedling Growth and Plastid Pigment Development

Etiolated 6-day-old wheat (Triticum aestivum L. cv. Chris) seedlings were subjected to osmotic stress by an application of polyethylene glycol 12 h prior to the exposure to a continuous 72-h light period. The water potential of the primary leaf of stressed seedlings was between –9 and –14 bars throughout the light period. Stress impaired seedling growth, leaf unfolding, and the increase in leaf area. The imposed osmotic stress reduced total chlorophyll accumulation, particularly after 9 h light, suggesting that this is the approximate time period for the depletion of the protochlorophyll(ide) pool and the pool of an essential protochlorophyll(ide) precursor. The chlorophyll a/b ratio of extracts from stressed and non-stressed plants was the same during the 72-h greening period. Water deficit stress impaired carotenoid accumulation sooner than the impairment of chlorophyll production suggesting either a smaller carotenoid pool size of precursors or that the metabolic pathway of carotenoid synthesis was more sensitive to stress. Shifts from the usual plastid pigment absorbance maxima were not observed in these studies.     

Characterization and Properties of a Protochlorophyllide Ester in Leaves of Dark Grown Barley with Geranylgeraniol as Esterifying Alcohol

The protochlorophyllide ester isolated from dark grown barley leaves was shown to contain geranylgeraniol as esterifying alcohol. No phytylester was found. The qualitative analyses were performed with combined gas chromatography-mass spec-trometry. Chromatographic separation and spectrofluorometric determination of the protochlorophyll and chlorophyll pigments before and after irradiation of the dark grown leaves with light flashes at 2°C showed that part of the protochlorophyllide ester was photoconverted to chlorophyll a.     

Accumulation of protochlorophyll and chlorophyll a as controlled by photomorphogenically effective light

Summary Data are presented which indicate that the rate of synthesis and the pool size of photoconvertible protochlorophyll(ide) in the cotyledons of the mustard seedling are controlled by the active form of phytochrome (P_fr). Inductionreversion experiments show that formation of chlorophyll a through photoconversion of the protochlorophyll(ide) by repeated red pulses (5 min each) has no effect on synthesis of carotenoids and galactolipids. Since the protochlorophyll(ide)-converting activity of the standard far-red light used in this laboratory is very low, chlorophyll-a accumulation is very slow under continuous standard far-red light. It is concluded that photosynthesis (or photosynthetic phosphorylation) does not participate in the high irradiance reaction of photomorphogenesis.     

Studies on chloroplast development in Chlamydomonas reinhardtii IV. Control of rapid chlorophyll formation in greening y-1 cells

Effects of protein synthesis inhibitors, CAP and CHI, on diegreening of Chlamydomonas reinhardtii y-1 cells, particularlyon die P-factor formation (19) in the early phase, were studied.Chlorophyll synthesis in the normal greening process, whichis divided into three phases, was strongly inhibited by bothantibiotics, although the inhibition by CAP was weaker in themiddle and late phases. The development of potential for rapidchlorophyll formation (P-factor formation) that takes placein dark-grown cells during dark incubation following brief illuminationwas completely blocked by CHI, but not by CAP. A "CHI-sensitive"period for the P-factor formation was restricted to the initial30 min during the dark incubation following brief illumination(10 min). This initial 30-min period appeared to correspondto the time of protochlorophyll(ide) formation which was inhibitedby CHI. Light-dependent conversion of protochlorophyll(ide) to chlorophylland also the subsequent protochlorophyll(ide) synthesis, whichis "CHI-sensitive" seem to be prerequisite for the inductionof P-factor synthesis. A possible control mechanism involvedin the early phase of the greening process in y-1 cells is discussed. (Received February 12, 1976; )     

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.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.     

Pigment formation in potato tubers (Solanum tuberosum) exposed to light followed by darkness

Potato tubers ( Solanum tubersoum cvs Bintje and King Edward). never exposed to light, lack chlorophyllous pigments. Continuous irradiation results in chlorophyll (Chl) formation and induces the ability for protochlorophyll (Pchl) formation when the tubers are brought back to darkness. Pigment synthesis takes place in both blue and red light, but blue light is more effective than red in starting the greening process. The pigment formation is strongest in the layers just below the periderm with a steep gradient inwards. Small amounts of Chl formed after irradiation. slowly fade away during extended darkness. However, the Chl formed after long time of irradiation is remarkably stable. Irradiated potatoes, placed in darkness, form Pchl with a fluorescence emission peak at 633 nm. A maximal level is reached after ca 7 days. Resolution of the Pchl spectrum suggests the presence of small amounts of a pigment with an emission maximum at around 642 nm. No sign of the Pchl with emission maximum at 657 nm, which dominates in etiolated leaves, is found. A faint Chl fluorescence indicates that some Pchl, probably the 642 nm form, is phototransformed into Chl in weak light. The Chl formation in the potato tuber is discussed in relation to that of roots and leaves.     

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.     

Formation of short-wavelength chlorophyll(ide) after brief irradiation is correlated with the occurrence of protochlorophyll(ide)636–642 in dark-grown epi- and hypocotyls of bean (Phaseolus vulgaris)

Chlorophyll formation capacity along the seedling of bean ( Phaseolus vulgaris L. cv. Brede zonder draad) was investigated. After 7 days of irradiation a gradient was formed, where the primary leaf contained ca 300 times more chlorophyll per gram fresh weight than the lower hypocotyl section and ca 20 times more than the epicotyl. Similar chlorophyll gradients but at lower levels were seen when the seedlings were first placed in darkness for 7 days and then irradiated for 1, 2 or 7 days. Ultrastructural investigation of seedlings grown for 7 days in darkness and then irradiated for 24 h revealed a more developed inner membrane system with grana stacks in plastids of cells in the uppermost hypocotyl section compared to plastids of cells in lower hypocoty] sections. The higher up on the seedling the more the ratio increased of protochlorophyll(ide) emitting at 657 nm to short-wavelength protochlorophyll(ide). After flash irradiation of the different sections, fluorescence emission spectra with maxima at 680 and 690 nm, respectively, were observed, indicating the formation of short- and long wavelength chlorophyll(ide) forms. The lower the ratio of protochlorophyll(ide) emitting at 657 nm to the short-wavelength protochlorophyll(ide), the less long-wavelength chlorophyll(ide) was formed after irradiation. However, after continuous irradiation long-wavelength chlorophyll(ide) was formed. In dark grown roots, where only short-wavelength protochlorophyll forms were present, it was not possible to transform protochlorophyll to chlorophyll by flash irradiation. Possible explanations for this phenomenon are discussed.     

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.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

Photochlorophyll Biosynthesis in Cucumber (Cucumis sativus, L.) Cotyledons

The formation of protochlorophyllide and protochlorophyllide phytyl ester was investigated during etioplast biogenesis in order to study the biosynthetic relation of these two compounds. Protochlorophyllide accumulates slowly during the first 2 days of germination, its rate of formation increases sharply during the 3rd day, and then it decreases. Protochlorophyllide phytyl ester starts accumulating a day later; its formation coincides with the initiation of xanthophyll biosynthesis. Kinetic analysis of specific radioactivities after ¹⁴C labeling of the protochlorophyll pools does not support the currently accepted conversion of protochlorophyllide into protochlorophyllide phytyl ester, but suggests that both compounds originate simultaneously from a common precursor pool.