Photooxidative destruction of chloroplasts and its consequences for expression of nuclear genes

Expression of nuclear genes involved in plastidogenesis is known to be controlled by light via phytochrome. Examples are the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b binding protein of photosystem II (LHCP). In the present study we show that, beside phytochrome, the integrity of the plastid is essential for the expression of the pertinent nuclear genes as measured at the level of translatable mRNA. When the plastids are severely damaged by photooxidation in virtually carotenoid-free mustard (Sinapis alba L.) seedling cotyledons (made carotenoid-free by the application of Norflurazon, NF), almost no SSU, no SSU precursor, LHCP and LHCP precursor can be detected by immunological assays, and almost no translatable mRNA of SSU and LHCP can be found, although the levels and rates of phytochrome-mediated syntheses of representative cytoplasmic, mitochondrial and glyoxisomal enzymes are not adversely affected and morphogenesis of the mustard seedling proceeds normally (Reiß et al. 1983; Planta 159, 518–528). Norflurazon per se has no effect on the amount of translatable mRNA of SSU and LHCP as shown by irradiation of NF-treated seedlings with far-red light (FR) which strongly activates phytochrome but does not cause photooxidation in the plastids. It is concluded that a signal from the plastid is required to allow the phytochrome-mediated appearance of translatable mRNA for SSU and LHCP. Seedlings not treated with NF show a higher level of translatable mRNA_LHCP in red light (RL) compared to FR, whereas the mRNA_SSU levels are the same in RL and FR. These facts indicate that the level of translatable mRNA_LHCP is adversely affected if the apoprotein is not incorporated into the thylakoid membrane.Abbreviations FR far-red light (3.5 W m^-2) - LHCP light harvesting chlorophyll a/b binding protein of photosystem II - LSU large subunit of RuBPCase - NF Norflurazon - RL red light (6.8 W m^-2) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase - WL white light (28 W m^-2)     

Expression of nuclear genes as affected by treatments acting on the plastids

In a preceding paper (Oelmüller and Mohr 1986, Planta 167, 106–113) it was shown that in the cotyledons of the mustard (Sinapis alba L.) seedling the integrity of the plastid is a necessary prerequisite for phytochrome-controlled appearance of translatable mRNA for the nuclear-encoded small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP). It was concluded that a signal from the plastid is essential for the expression of nuclear genes involved in plastidogenesis. The present study was undertaken to characterize this postulated signal. Chloramphenicol, an inhibitor of intraplastidic protein synthesis and Norflurazon, an inhibitor of carotenoid synthesis (to bring about photooxidative sensitivity of the plastids) were applied. We obtained the following major results. (i) After a brief period of photooxidative damage a rapid decrease of the above translatable mRNAs was observed. Conclusion: the signal is short-lived and thus required continually. (ii) Once the plastids became damaged by photooxidation, no recovery with regard to nuclear gene expression was observed after a transfer to non-damaging light conditions. Conclusion: even a brief period of damage suffices to prevent production of the signal. (iii) Chloramphenicol inhibited nuclear gene expression (SSU, LHCP) and plastidic development when applied during the early stages of plastidogenesis. Once a certain stage had been reached (between 36–48 h after sowing at 25° C) nuclear gene expression became remarkably insensitive toward inhibition of intraplastidic translation. Conclusion: a certain developmental stage of the plastid must be reached before the signal is released by the plastid. (iv) Under the growth conditions we adopted in our experiments the plastids in the mesophyll cells of mustard cotyledons developed essentially between 36 and 120 (-144) h after sowing. Only during this period could translatable mRNAs for SSU and LHCP be detected. Conclusion: the signal is released by the plastids only during this time span.Abbreviations CAP Chloramphenicol (D-threo) - cFR continuous far-red light - FR far-red light (3.5 W·m^-2) - GPD glyceraldehyde-3-phosphate dehydrogenase - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - LSU large subunit of RuBPCase - MDH malate dehydrogenase - NF Norflurazon - NIR nitrite reductase - Pfr physiologically active form of phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light (10 W·m^-2) - RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU small subunit of RuBPCase - WL_s strong white light (28 W·m^-2) - photoequilibrium of phytochrome at wavelength      

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

Ultrastructural detection of ribulose-1,5-bisphosphate carboxylase protein and its subunit mRNAs in wild-type and holoenzyme-deficient Nicotiana using immuno-gold and in-situ-hybridization techniques

In-situ-localization techniques have been adapted to the ultrastructural detection of the holoenzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) and its composite large- and smallsubunit mRNAs in wild-type and mutant RuBPCase deficient plantlets of Nicotiana tabacum L. Immuno-gold techniques which show the distribution of target proteins have confirmed visually the presence of the holoenzyme in the wild-type plastids and its total absence in the enzyme-less mutant. Using in-situ hybridization coupled with electron microscopy and biotinylated probes for the two subunits, we have directly visualized specific small-subunit mRNAs located in the cytoplasm and large-subunit mRNAs confined to plastids in the enzyme-deficient mutant, and with apparent distributions comparable to those visualized in the wild-type counterpart. These results show that (i) gene products can be visualized in situ by electronmicroscopy techniques under conditions where the respective cellular compartments are readily recognizable and (ii) that an accumulation of mRNAs corresponding to the composite subunits can occur without translation and-or assembly of the protein.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU RuBPCase small subunit - LSU RubBPCase large subunit     

Rapid variations in the content of the RNA of the small subunit of ribulose-1,5-bisphosphate carboxylase of mature tobacco leaves in response to localized changes in light quantity. Relationships between the activity and quantity of the enzyme

Mature green leaves from tobacco (Nicotiana tabacum L.) plants were submitted to contrasting light conditions; half of each leaf was shaded (changed from 60 to 25 mol photons· m^-2 ·s^-1=LL) and the other half was exposed to higher light (changed from 60 to 360 mol·m^-2· s^-1=HL) for 24 h. The activity and quantity of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were measured during the first 24 h in each leaf region and the variation was compared with that of small subunit (SSU)-and large subunit (LSU)-mRNA contents determined by a hybridot technique. Each leaf half responded separately to the actual light received. The activity of RuBPCase increased progressively in the HL zones and decreased in the LL zones. The RuBPCase-protein content was not significantly modified during the first 24 h but SSU-mRNA content responded very rapidly to the treatment. Within 2 h a significant difference in SSU mRNA appeared between LL and HL zones: at the end of the photoperiod the content in LL zones was approx. 25% of the initial value. The increase in the exposed zone, however, was not significant, indicating that there was a dissymmetry of the response to variation in incident white light. The LSU-mRNA contents from the same leaf extracts were totally unaffected by the light treatment. No day-night variations were noted in either SSU or LSU mRNAs in control plants.Abbreviation HL high-light irradiance - LL lower-ligh irradiance - LSU large subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU small subunit of RuBPCase     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Inhibition and promotion by light of the accumulation of translatable mRNA of the light-harvesting chlorophyll a/b-binding protein of photosystem II

The amount of in-vitro translatable mRNA of the light-harvesting chlorophyll a/b-binding protein (LHCP) of photosystem II strongly increases in darkness (D) after a 5-min red-light pulse while continuous illumination of mustard seedlings with far-red (FR), red or white light leads only to a slight increase in the amount of translatable LHCP-mRNA. No increase can be observed after a long-wavelength FR (RG9-light) pulse. However, a FR pretreatment prior to the RG9-light pulse strongly increase LHCP-mRNA accumulation in subsequent D. This is not observed in the case of the mRNA for the small subunit of ribulose-1.5-bisphosphate carboxylase. The increase of LHCP-mRNA in D after a FR pretreatment can be inhibited by a reillumination of the seedlings with FR. The inhibition of LHCP-mRNA accumulation during continuous illumination with FR and the strong increase in D following a FR illumination was found to be independent of chlorophyll biosynthesis since no correlation between chlorophyll biosynthesis and translatable LHCP-mRNA levels could be detected. Even strong changes in the amount of intermediates of chlorophyll biosynthesis caused by application of levulinic acid or 5-aminolevulinic acid did not affect LHCP-mRNA levels. Therefore, we conclude that the appearance of LHCP-mRNA is inhibited during continuous illumination, even though illumination leads to a storage of a light singal which promotes accumulation of translatable LHCP-mRNA in D.Abbreviations c continuous - Chl chlorophyll - D darkness - FR far-red light (3.5 W·m^-2) - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - NF Norfluration - PChl protochlorophyll(ide) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength FR (10 W·m^-2) - SSU small subunit of ribulose-1.5-bisphosphate carboxylase - WL white light - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from Euglena

Summary The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10^-9 per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.Abbreviations DABITC Dimethyl amino azobenzene isothiocyanate - HPLC high pressure liquid chromatography - Kd Kilo daltons - LSU large subunit - PITC phenyl isothiocyanate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - SSU small subunit - TFA trifluoric acetic acid     

Heat modification of ribulose-1,5-bisphosphate carboxylase/oxygenase by temperature pretreatment of wheat (Triticum aestivum L.) seedlings

The effect of low-, ambient- and high-temperature pretreatments (48 h at 4° C, 20° C or 36° C) of wheat seedlings (spring wheat Triticum aestivum L., cv. Kolibri) on the solubility properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase; EC 4.1.1.39) was studied. The extractable protein moiety of heat-pretreated plants exhibited increased solubility in dilute buffer (50 mM k-phosphate, pH 6.8), compared with protein extracted from 4° C- or 20° C-plants. The salting-out characteristics for ammonium-sulfate precipitation confirmed this finding since a delayed precipitation of extractable protein from 36°C-plants was observed. Using polyacrylamide gel electrophoresis, the in-vivo temperature-induced differences in protein solubility could be traced back to a change in the solubility of RuBPCase. The RuBPCase was purified from wheat seedlings, and the purified enzyme also exhibited differential solubility. In order to evaluate this further, purified RuBPCase was subjected to probing for conformational properties. A decrease of fluorescence of the RuBPCase 1-anilino-8-naphtalene sulfonate complex revealed that the RuBPCase from 36° C-plants had a more hydrophilic protein surface. Titration of the sulfhydryl groups of native RuBP-Case with 5,5-dithiobis (2-nitrobenzoic acid) pointed to a reduced accessibility of the R-SH groups in the case of the 36° C-type of RuBPCase. The large subunit of RuBPCase from 4° C/20° C-plants tended to give rise to an artificial lower-molecular-weight polypeptide which could not be found in crude or purified RuBPCase from heat-pretreated wheat seedlings.Abbreviations ANS 1-anilino-8-naphtalene sulfonate - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bis-phosphate carboxylase/oxygenase - RuBP ribulose-1,5-bis-phosphate     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Pyrenoid protein from the brown alga Pilayella littoralis

Characterization by peptide mapping and amino acid analysis of the two major pyrenoid polypeptides from the brown alga Pilayella littoralis shows that they are very similar to the subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) from this alga. The observed similarities are discussed in relation to previous pyrenoid protein characterization from members of the Chlorophyceae.Abbreviations DTT dithiothreitol - EDTA Na₂ ethylenediamine tetraacetic acid (disodium salt) - PMFS phenylmethylsul-phonylfluoride - PVPP polyvinylpyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TRIS 2-amino-2-(hydroxymethyl) propane-1,3-diol - TPCK L-1-tosylamido-2-phenylethylchoromethyl ketone     

Light-mediated control of translational initiation of ribulose-1, 5-bisphosphate carboxylase in amaranth cotyledons.

In cotyledons of 6-day-old amaranth seedlings, the large subunit (LSU) and the small subunit (SSU) polypeptides of ribulose-1,5-bisphosphate carboxylase are not synthesized in the absence of light. When dark-grown seedlings were transferred into light, synthesis of both polypeptides was induced within the first 3 to 5 hr of illumination without any significant changes in levels of their mRNAs. In cotyledons of light-grown seedlings and of dark-grown seedlings transferred into light for 5 hr (where ribulose-1,5-bisphosphate carboxylase synthesis was readily detected in vivo), the LSU and SSU mRNAs were associated with polysomes. In cotyledons of dark-grown seedlings, these two mRNAs were not found on polysomes. In contrast to the SSU message, mRNAs encoding the nonlight-regulated, nuclear-encoded proteins actin and ubiquitin were associated with polysomes regardless of the light conditions. Similarly, mRNA from at least one chloroplast-encoded gene (rpl2) was found on polysomes in the dark as well as in the light. These results indicate an absence of translational initiation in cotyledons of dark-grown seedlings which is specific to a subset of nuclear- and chloroplast-encoded genes including the SSU and LSU, respectively. Upon illumination, synthesis of both polypeptides, and possibly other proteins involved in light-mediated chloroplast development, was induced at the level of translational initiation.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase expression in melon plants infected with Colletotrichum lagenarium

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.Abbreviations LS (SS) Large (small) subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid