The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655

Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A.     

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night.Abbreviations Chl chlorophyll - T _o time of transfer to darkness     

The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.     

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea ( Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m⁻² s⁻¹photon flux density (PFD) white light and subsequently incubated in total darkness for 4–24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form—dominating in epicotyls of dark-grown seedling—required 18–24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4–7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.     

Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro

It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber.     

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo)

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.     

Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.     

Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

Glucose and δ-Aminolevulinic Acid Stimulate the Dark Chlorophyll Synthesis of Rice Seedlings

This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ(Proto), Mg-protoporphyrin Ⅸ(Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark. Key words: angiosperm; rice; dark chlorophyll synthesis; glucose; δ-aminolevulinic acid; protoporphyrin Ⅸ; Mg-protoporphyrin Ⅸ; protochlorophyllide     

Chlorophyll synthesis in green leaves and isolated chloroplasts of barley (Hordeum vulgare)

The photoreduction of protochlorophyllide was studied in leaves and isolated chloroplasts of barley. Leaves of plants which had been preilluminated for varying lengths of time were incubated with [¹⁴C]-δ- aminolevulinic acid for 2 h in the dark. The subsequent photoreduction of [¹⁴C]-protochlorophyllide was analyzed by high performance liquid chromatography of pigments extracted from illuminated leaves and plastids. The plastids used in this study were isolated in the dark from leaves at the end of the 2 h labelling period. Three major results were obtained:

• 1

  The extent of protochlorophyllide reduction in vivo was rapidly reduced as a function of the preillumination period. In 24 h preilluminated plants only a small fraction of the radioactively labelled protochlorophyllide was reduced during the subsequent light period.
• 2

  The amount of NADPH-protochlorophyllide oxidoreductase (EC 1.6.99.-) present in plastids of fully-green plants was drastically reduced relative to levels in plastids of dark-grown plants as estimated by the methods of immunoblotting of plastid proteins and immunogold labelling of ultrathin sections of the leaf tissue.
• 3

  In etiolated plants light seemed to affect the reduction of protochlorophyllide directly through the excitation of protochlorophyllide. In fully green plants, however, light also affected chlorophyll formation indirectly by the supply of NADPH via photosynthetic electron transport.

     

Chlorophyll accumulation and breakdown in light-grown barley transferred to darkness: effect of seedling age

Diurnally grown barley (Hordeum vulgare L. cv. Clipper) seedlings of various ages (3–4, 5–6 and 10–11-days-old) were transferred to darkness for 17 h and changes in leaf fresh weight, chlorophyll a, chlorophyll b and protochlorophyllide measured. The results were consistent with previous evidence of a light-independent chlorophyll biosynthetic pathway in light-grown barley. There was a net gain in chlorophyll (μg leaf^-1) in 5–6- and 10–11-day-old plants after 17 h dark treatment. The amounts of chlorophyll that accumulated were similar (5.9 and 4.3 μg Chl leaf^-1), despite a twofold difference in leaf size at T₀. The rate of leaf expansion in 5–6-day-old plants greatly exceeded the rate of chlorophyll accumulation and leaves were noticeably paler after dark treatment i.e. there was a reduction in chlorophyll concentration (μg g fresh weight^-1) in spite of an increase in chlorophyll content (μg leaf^-1). The ability of light-grown barley to accumulate chlorophyll in darkness was a function of seedling age. Very young seedlings (3–4-day-old) generally lost chlorophyll in darkness. The decrease in chlorophyll per leaf resulted mainly from loss of chlorophyll b. Preferential loss of chlorophyll b resulted in dramatic increases in the chlorophyll a:b ratio. Since 3–4-day-old seedlings (1) accumulated 5-aminolevulinic acid in the presence of levulinic acid at a rate comparable to older seedlings, and (2) converted exogenous 5-aminolevulinic acid to chlorophyll in the absence of light, it is unlikely that failure of the youngest plants to accumulate chlorophyll in darkness was due to blocks at these steps in the pathway. Net loss of chlorophyll (μg leaf^-1) in 3–4-day-old seedlings in darkness was eliminated by the addition of chloramphenicol, which occasionally produced a small, but significant, gain in total chlorophyll. These results imply that chlorophyll degradation in young barley leaves is strongly influenced by the chloroplast genome, and is a major factor influencing changes in chlorophyll levels in darkness. The present findings are consistent with the suggestion that the failure of 3–4-day-old barley seedlings to accumulate chlorophyll in darkness may be due to chlorophyll turnover in which the rate of degradation exceeds the rate of synthesis.