Effect of White and Red Light on the Pigment Content and Functional Activity in Pine Chloroplasts

The pigment content and rates of primary photosynthetic reactions were determined in chloroplasts of 14-day-old pine (Pinus silvestris L.) seedlings grown in light and darkness. In addition, the functional activities were investigated in chloroplasts from dark-grown seedlings exposed to white, red ( = 670 nm), and red + far-red ( = 748 nm) light. Dark-grown seedlings were capable of performing the Hill reaction, noncyclic photophosphorylation, and phenazine methosulfate–supported photophosphorylation, although the reaction rates in chloroplasts from dark-grown plants were considerably lower than in preparations from light-grown plants. Light treatment of dark-grown seedlings rapidly activated the photoreduction of ferricyanide and photophosphorylation, while the additional accumulation of green pigments started only after a lag period of two hours. Preirradiation of dark-grown seedlings with red light stimulated the formation of pigments, especially chlorophyll b, as well as the functional activity of chloroplasts. When far-red light was applied after red-light exposure, the processes examined were inhibited. It is concluded that accumulation of the light-harvesting complex and functional activities of chloroplasts at the photosystem II level in pine seedlings are controlled by the phytochrome.     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons

Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b_[ill]/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex.     

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.     

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.Abbreviations Chl chlorophyll - Chlide chlorophyllide - NADPH-Pchlide oxidoreductase NADPH protochlorophyllide oxidoreductase - NC nitrocellulose - PBS phosphate buffered saline - Pchlide protochlorophyllide - SDS sodum dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Chloroplast photooxidation affects the accumulation of cytosolic mRNAs encoding chloroplast proteins in maize

Maize (Zea mays L.) seedlings were grown in the presence or absence of an herbicide, norflurazon (4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-pyridazinone), which prevents the accumulation of colored carotenoids. In the absence of carotenoids, plants grown in high light incur extensive photooxidative damage to their plastids, but relatively little damage elsewhere. Growth in very low light minimizes chlorophyll photooxidation and allows chloroplast development to proceed. We have previously reported that mRNA encoding light-harvesting chlorophyll a/b protein (LHCP) fails to accumulate in high-light-grown carotenoid-deficient seedlings, but accumulates normally in carotenoid-deficient seedlings grown in low light. Here we extend these results by examining the levels of translatable mRNAs encoding seven additional nuclear-encoded chloroplast proteins. When norflurazon-treated seedlings were grown in low light for 8 d and then transferred to high light for 24 h, three cytosolic mRNAs (plastocyanin, Rieske Fe–S protein, and the 33-kdalton (kDa) subunit of the photosystem II O₂-evolving complex) decreased to less than 1% the amount found in untreated seedlings. Two other mRNAs (NADP malic enzyme, EC 1.1.1.40, and the 23-kDa subunit of the photosystem II O₂-evolving complex) decreased significantly but not to levels as low as the first three. Levels of translatable mRNA for two other chloroplast proteins (pyruvate orthophosphate dikinase, EC 2.7.9.1, and ferredoxin NADP oxidoreductase, EC 1.18.1.2) were not reduced in nonflurazon-treated seedlings after 24 h in high light, but did not show the normal light-induced increase found in untreated plants. Photooxidative damage in the chloroplast thus affects the accumulation of a number of cytosolic mRNAs encoding proteins destined for the chloroplast.Abbreviations Da dalton - FNR ferredoxin NADP oxidoreductase - LHCP light-harvesting chlorophyll a/b-binding protein - poly(A)RNA polyadenylated RNA - PPDK pyruvate orthophosphate dikinase - PSII photosystem II - SDSPAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSu small subunit (of ribulose-1,5-bisphosphate carboxylase)     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Consequences of LHC II deficiency for photosynthetic regulation in chlorina mutants of barley

The light-harvesting chlorophyll a/b proteins associated with PS II (LHC II) are often considered to have a regulatory role in photosynthesis. The photosynthetic responses of four chlorina mutants of barley, which are deficient in LHC II to varying degrees, are examined to evaluate whether LHC II plays a regulatory role in photosynthesis. The efficiencies of light use for PS I and PS II photochemistry and for CO₂ assimilation in leaves of the mutants were monitored simultaneously over a wide range of photon flux densities of white light in the presence and absence of supplementary red light. It is demonstrated that the depletions of LHC II in these mutants results in a severe imbalance in the relative rates of excitation of PS I and PS II in favour of PS I, which cannot be alleviated by preferential excitation of PS II. Analyses of xanthophyll cycle pigments and fluorescence quenching in leaves of the mutants indicated that the major LHC II components are not required to facilitate the light-induced quenching associated with zeaxanthin formation. It is concluded that LHC II is important to balance the distribution of excitation energy between PS I and PS II populations over a wide range of photon flux densities. It appears that LHC II may also be important in determining the quantum efficiency of PS II photochemistry by reducing the rate of quenching of excitation energy in the PS II primary antennae.Abbreviations Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_p photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - ø_PSI, ø_PSII relative quantum efficiencies of PS I and PS II photochemistry - øCO₂ quantum yield of CO₂ assimilation     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic -helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.This study is dedicated to Prof. Dr. W Rüdiger on the occasion of his 60th birthday     

Fate of the porphyrin cofactors during the light-dependent turnover of catalase and of the photosystem II reaction-center protein D1 in mature rye leaves

The apoprotein of the enzyme catalase (EC 1.11.1.6) was shown to exhibit a light-dependent turnover in leaves. Present results indicate that photoinactivation of the enzyme was not accompanied by a synchronous destruction and new synthesis of its heme moiety. In rye (Secale cereale L.) leaves the catalase content was not depleted in light when porphyrin synthesis was inhibited by gabaculine. Photoinactivation of purified bovine liver or rye leaf catalase in vitro was not accompanied by concomitant damage to the heme groups. Both the incorporation of -[³H]aminolevulinic acid ([³H]ALA) into catalase-heme and its apparent turnover increased with irradiance. However, the apparent half-life of the catalase-heme was much longer than that of its apoprotein. It is probable that not only degradation but also an exchange with the free heme pool contributed to the apparent turnover of radioactivity of the catalase-heme. Part of the chlorophyll (Chl) associated with photosystem II (PS II) had a preferential light-induced turnover, and repair of PS II appeared to require new Chl synthesis also in mature green rye leaves. The activity of PS II, indicated by the ratio of variable to maximal fluorescence (F_v/F_m), rapidly declined in the presence of gabaculine in light and the reaction-center proteins D1 and D2 were depleted. When segments of mature green rye leaves were labeled with [³H]ALA and incorporation into Chl-protein complexes analysed after electrophoretic separation in the presence of Deriphat, the highest radioactivity was observed in the core complex of PS II, while PS I and the light-harvesting complex of PS II (LHC II) were unlabeled. In greening etiolated leaves highest incorporation was observed in LHC II. Both the incorporation of [³H]ALA into the PS II core complex of green rye leaves and its turnover increased with irradiance. However, the apparent half-life of the PS II-bound labeled porphyrin compounds (mainly Chl) was considerably longer than that of the reaction-center protein D1 under identical conditions.Abbreviations ALA -aminolevulinic acid - CII Core complex of PS II - Chl chlorophyll - DMSO dimethyl sulfoxide - F_v/F_m ratio of variable to maximal chlorophyll fluorescence - LHC light-harvesting complex - PAR photosynthetically active radiation We thank the Deutsche Forschungsgemeinschaft for financial support. Technical assistence by B. Kramer and Ch. van Oijen is greatly appreciated. We are grateful to Dr. Johanningmeier and Dr. Godde (Lehrstuhl für Biochemie der Pflanzen, Universität Bochum, Germany) for providing antisera against the D1 and D2 proteins and Dr. M. Schmidt (Botanisches Institut, Universität Frankfurt am Main, Germany) for valuable advice. Deriphat 160 was kindly supplied by Henkel Corp., Hoboken, N.J., USA.     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Acclimation of Arabidopsis thaliana to the light environment: Changes in composition of the photosynthetic apparatus

Acclimation to changes in the light environment was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Plants grown under four light regimes showed differences in their development, morphology, photosynthetic performance and in the composition of the photosynthetic apparatus. Plants grown under high light showed higher maximum rates of oxygen evolution and lower levels of light-harvesting complexes than their low light-grown counterparts; plants transferred to low light showed rapid changes in maximum photosynthetic rate and chlorophyll-a/b ratio as they became acclimated to the new environment. In contrast, plants grown under lights of differing spectral quality showed significant differences in the ratio of photosystem II to photosystem I. These changes are consistent with a model in which photosynthetic metabolism provides signals which regulate the composition of the thylakoid membrane.Abbreviations Aac1 gene encoding actin - Chl chlorophyll - F far-red-enriched light (R:FR = 0.72) - FR far-red light - H high light (400 mol · m^–2 · s^–1) - L low light (100 ml · m^–2 · s^–1) - LHCII light-harvesting complex of PSII - Lhcb genes encoding the proteins of LHCII - R red light - Rbcs genes encoding the small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - W white light (R:FR = 1.40) This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (University of Sheffield) for helpful discussions.     

Action Spectra for the Inhibition of Hypocotyl Growth by Continuous Irradiation in Light and Dark-Grown Sinapis alba L. Seedlings

Action spectra for the inhibition by continuous (24-hour) irradiation of hypocotyl growth in 54-hour-old Sinapis alba L. seedlings were measured using seedlings which had had four different pretreatments. These seedlings were either (a) dark-grown with a high total phytochrome level, (b) dark-grown with a low total phytochrome level, (c) light-grown with chlorophyll, or (d) light-grown with no chlorophyll [treated with 4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H) -pyridazinone (San 9789)].