The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein (LHCP) is investigated in wild-type barley (Hordeum vulgare L.) and in the chlorophyll b-less mutant chlorina f2. In dark-grown plants a short red light pulse triggers the appearance of mRNA activity for the LHCP. While the accumulation of this mRNA is controlled by phytochrome (Apel (1979) Eur. J. Biochem. 97, 183–188), the red light treatment is not sufficient to induce the appearance of the LHCP within the membrane. Thus, at least one of the subsequent steps in the biosynthetic pathway leading to the assembly of the LHCP is controlled by light. The red light-induced mRNA is taken up into the polysomes during the subsequent dark period and is translated in vitro in a cell-free protein synthesizing system. However, an accumulation of the freshly synthesized polypeptide within the plant is not observed. The apparent instability of the polypeptide might be explained by the deficiency of chlorophyll in the red light-treated plants. In the chlorophyll b-less barley mutant chlorina f2 an accumulation of the freshly synthesized apoprotein of the LHCP can be observed in the light. Thus, chlorophyll a formation seems to be a light-dependent step which is required for the stabilization of the LHCP.Abbreviations mRNA messenger RNA - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecylsulfate - LHCP light-harvesting chlorophyll a/b protein     

Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors

Light induction of light-harvesting chlorophyll a/b-binding protein (LHCP) mRNA accumulation was studied in light-dark synchronized cultures of Chlamydomonas reinhardi. LHCP mRNA accumulation was prevented by the chlorophyll-synthesis inhibitor alpha,alpha-dipyridyl which blocks late steps in the chlorophyll biosynthetic pathway and leads to the accumulation of the porphyrin intermediate magnesium protoporphyrin methyl ester. LHCP mRNA accumulated normally, however, when chlorophyll synthesis was blocked by inhibitors such as hemin and levulinic acid which interfere with early steps in the chlorophyll biosynthesis pathway prior to the formation of magnesium protoporphyrin methyl ester. Similar effects were observed in the light induction of LHCP mRNA levels in protoporphyrin IX-accumulating mutants, brc-1 and brs-1. These mutants have low levels of LHCP mRNA when grown under heterotrophic conditions in the dark where they accumulate protoporphyrin IX. However, LHCP mRNA is light-induced in brc-1 which synthesizes chlorophyll in the light and presumably consumes porphyrin intermediates in doing so. These results suggest that the chlorophyll-synthesis intermediates, magnesium protoporphyrin methyl ester and its immediate precursors, inhibit by a feedback mechanism the light induction of LHCP mRNA accumulation. Low magnesium protoporphyrin methyl ester levels permit the light-induced accumulation of LHCP mRNA, whereas high magnesium protoporphyrin methyl ester levels destabilize LHCP mRNA regardless of the illumination conditions. Preliminary experiments show that LHCP mRNA accumulation in C. reinhardi is stimulated by blue light, and not by red light which stimulates LHCP mRNA accumulation in higher plants.     

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.     

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH: protochlorophyllide oxidoreductase

During the illumination of dark-grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr 44000. This component was identified as a precursor of the NADPH:protochlorophyllide oxidoreductase. The precursor has an Mr larger than the authentic protein by approximately 8000. The light-induced change in the level of translatable mRNA can be induced by a 15-s red-light pulse followed by 5 h of darkness. The red-light effect is reversed by a subsequent far-red-light treatment. It is concluded that the light-induced decline of translatable mRNA for the NADPH:protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light-dependent control of chloroplast development and chlorophyll synthesis is discussed.     

Effect of 1,10-phenanthroline on ultrastructure of pea leaves

Summary We have examined ultrastructural changes of mesophyll cells in pea leaves induced by the photodynamic herbicide 1,10-phenanthroline (Phe). Dark incubation of pea plants did not cause any damage in plants or changes in the ultrastructure of mesophyll cells. Two hours of illumination after pretreatment with Phe caused photooxidative damage in plant but was not sufficient to markedly change the ultrastructure, although dilation of endoplasmic reticulum (ER) cisternae occurred. Illumination for 12 h caused inhibition of grana formation in pretreated plants. These ultrastructural changes and the inhibition of chlorophyll (Chl) accumulation may be due to the inhibition of transport of certain proteins to the plastids, diminished accumulation of chlorophyll proteins (e.g., LHCP) and a decrease in activity of the chlorophyll synthetase.Abbreviations ALA 5-aminolevulinate - 2,2 2,2-bipyridyl - Chl chlorophyll - ER endoplasmic reticulum - 8H 8-hydroxyquinoline - LHCP light-harvesting chlorophyll a/b-binding proteins - PBs prolamellar bodies - Mg-Proto Mg-protoporphyrin - Mg-Proto-Me Mg-protoporphyrin monomethyl ester - Pchlide protochlorophyllide - Phe 1,10-phenanthroline - Proto protoporphyrin IX     

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.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient

The possible regulatory role of NADPH-protochlorophyllide oxidoreductase for chlorophyll accumulation has been investigated in barley plants. Within the primary leaf of etiolated plants the different maturation stages of etioplasts are found in a linear series with the youngest in cells near the base and the oldest in cells near the tip. This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide-oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination. The amount of enzyme and the rate of chlorophyll accumulation are highest in the mature etioplast in the tip of the leaf and both decline rapidly with decreasing age of the leaf tissue, being almost undetectable in the leaf base. The translatable mRNA coding for the enzyme shows a different distribution pattern within the leaf. The highest concentration is found in the middle part of the leaf while in the top part only traces of this mRNA are detectable. It is concluded that during leaf development the enzyme is synthesized rapidly only during a limited time period and that it is stored subsequently in the mature etioplast as a stable protein. The close correlation between the distribution of the enzyme within the barley leaf and that of the potential to accumulate chlorophyll during illumination would favour a control of chlorophyll accumulation by the amount of NADPH-protochlorophyllide oxidoreductase. Dark-grown plants which were exposed to far-red light were used to test this possibility. The far-red-absorbing form of phytochrome (P_fr) has an inverse effect on the kinetics of chlorophyll accumulation and the enzyme concentration. Our results indicate that the rate of chlorophyll accumulation in barley is not determined by the level of NADPH-protochlorophyllide oxidoreductase present in the leaves.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

The Circadian Oscillator Coordinates the Synthesis of Apoproteins and Their Pigments during Chloroplast Development

Greening has been studied at circadian times of maximal and minimal levels of mRNA for the light-harvesting chlorophyll a/b binding protein in photosystem II (Cab mRNA) after circadian synchronization of etiolated barley plantlets (Hordeum vulgare cv Apex) by heat-shock treatments. It was found that greening occurs faster and without a lag period when illumination was started at the time of maximal Cab mRNA accumulation. This holds true for the rate of accumulation of Cab and early light-inducible protein mRNAs, the levels of their correspondent proteins, and the levels of chlorophyll a and b. When illumination was started at the time of Cab mRNA minimum, a lag in the appearance of all components mentioned above was observed. Under these conditions, the lag in chlorophyll b accumulation was by far more pronounced than that found for chlorophyll a. The circadian oscillation in the capacity of chlorophyll synthesis appears to be controlled via [delta]-aminolevulinic acid ([delta]-ALA) synthesis. [delta]-ALA accumulation after levulinic acid treatment is itself under circadian control; the maxima in stationary concentrations coincide with those of Cab mRNA levels. The amounts of protochlorophyllide and photoconvertible protochlorophyllide showed only minor differences between circadian minima and maxima, the levels being slightly lower during the time of minimum.     

Effect of Cycloheximide on the Inverse Control by Phytochrome of the Expression of Two Nuclear Genes in Barley (Hordeum vulgare L.)

The accumulation of mRNAs encoded by two phytochrome-regulatedgenes in barley (Hordeum vulgare L.) was examined after a singlered light pulse in presence of cycloheximide. The initial increasein mRNA encoding the major light harvesting chlorophyll a/bbinding protein (LHCP) could still be observed indicating thatno protein synthesis-requiring step is essential for the transductionchain. The phytochrome-induced decrease in mRNA encoding NADPH:protochlorophyllideoxidoreductase was considerably slowed. (Received February 22, 1988; Accepted June 8, 1988)     

Tissue-specific and light-dependent changes of chromatin organization in barley (Hordeum vulgare)

The DNase I sensitivity of the nuclear genes encoding the NADPH-protochlorophyllide oxidoreductase, the light-harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15-kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.). A tissue-specific differentiation of chromatin structure was found for the LHCP, hordein and 15-kDa protein genes. The genes for the LHCP and the 15-kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15-kDa protein is there a remarkable change from a DNAse-I-sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH-protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.