Comparative analysis of the action of cytokinin and light on the formation of ribulosebisphosphate carboxylase and plastid biogenesis

The role of cytokinin in plastid biogenesis was investigated in etiolated rye leaves (Secale cereale L.) and compared with the effect of white light. Cytokinin deficiency of the leaves was induced by early excision of the seedling roots and reversed by the application of kinetin. The cytokinin supply had a much greater influence on plastid biogenesis than on leaf growth in general. The activities of several chloroplastic enzymes were increased 200%–400% after kinetin treatment of cytokinin-depleted leaves. The activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and the amount of fraction-I protein even showed a sevenfold increase. In cytokinin-depleted leaves the development of ribulose-1,5-bisphosphate carboxylase and NADP-glyceraldehydephosphate dehydrogenase was specifically, and markedly inhibited by actinomycin D. The inhibition was partially or even completely overcome after treatment with kinetin. However, under all conditions, RNA synthesis of the leaves, was only partially inhibited by actinomycin D. According to immunologic studies, all dark-grown leaves, in addition to the complete enzyme, contained an excess of free small subunit of ribulose-1,5-bisphosphate carboxylase that was absent in mature light-grown leaves. The most striking accumulation of free small subunit, protein occurred in cytokinin-depleted dark-grown leaves, indicating a deficiency of the plastidic synthesis of the large subunit. The capacity as well as the activity of plastidic protein synthesis was preferentially increased by cytokinin and light. Cytokinin increased, the amount of plastidic ribosomes per leaf and relative to the amount of cytoplasmic ribosomes. While the percentage of cytoplasmic ribosomes bound as polyribosomes was little affected by the cytokinin supply, the proportion of plastidic polyribosomes was increased from 11% to 18% after kinetin treatment of cytokinin-depleted leaves. In the light, the proportion of plastidic polyribosomes reached 39% of the total plastidic ribosomes.Abbreviations RuBP carboxylase ribulose-1,5-bisphosphate carboxylase - NADP-GAP dehydrogenase NADP-dependent glyceraldehyde-3-phosphate dehydrogenase     

Tagetitoxin affects plastid development in seedling leaves of wheat

Ultrastructural and biochemical approaches were used to investigate the mode of action of tagetitoxin, a nonhost-specific phytotoxin produced by Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye and Wilkie, which causes chlorosis in developing — but not mature — leaves. Tagetitoxin has no effect on the growth rate or morphology of developing leaves of wheat (Triticum aestivum L.) seedlings. Its cytological effects are limited to plastid aberrations; in both light-and dark-grown leaves treated with toxin, internal plastid membranes fail to develop normally and plastid ribosomes are absent, whereas mitochondrial and cytoplasmic ribosomes are unaffected. The activity of a plastid stromal enzyme, ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), which is co-coded by nuclear and chloroplast genes, is markedly lower in extracts of both light-and dark-grown toxin-treated leaves, whereas the activity of another stromal enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-G-3P-DH, EC 1.2.1.13), which is coded only by the nuclear genome, is significantly lower in extracts of light-grown, but not of dark-grown, treated leaves. The mitochondrial enzymes fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) are unaffected by toxin in dark-grown leaves, but fumarase activity is reduced in light-grown ones. Four peroxisomal enzyme activities are lowered by toxin treatment in both light- and dark-grown leaves. Light- and dark-grown, toxintreated leaves contain about 50% and 75%, respectively, of the total protein of untreated leaves. There are threefold and twofold increases in free amino acids in light-grown and dark-grown treated leaves, respectively. In general, the effects of tagetitoxin are more extensive and exaggerated in light-grown than in dark-grown leaves. We conclude that tagetitoxin interferes primarily with a light-independent aspect of chloroplast-specific metabolism which is important in plastid biogenesis.Abbreviations NADP-G-3-DH NADP-glyceraldehyde-3-phosphate dehydrogenase - PLB prolamellar body - RuBP-Case ribulose-1,5-bisphosphate carboxylase - SADH shikimic acid dehydrogenase     

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     

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.     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.     

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)     

Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

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     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Identification and Properties of the Messenger RNA Activity in Chlamydomonas reinhardi Coding for the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

Properties of the mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi were determined. Large subunit synthesis, directed by RNA from partially purified whole cell extracts, was detected by specific immunoprecipitation of polypeptide products synthesized in a heterologous translation system derived from Escherichia coli. Large subunit synthesis showed sharp RNA concentration dependence in an E. coli translation system, and at optimal RNA concentrations, immunoprecipitable large subunit synthesis accounted for 2% of the total incorporation. Large subunit messenger activity sedimented at 12 to 14S on nondenaturing sucrose gradients and did not bind to oligo(dT)-cellulose suggesting the mRNA is not polyadenylated. The immunoprecipitable products translated in vitro are not complete polypeptide chains, but are smaller peptides identifiable as large subunit fragments by tryptic fingerprint analysis. No immunoprecipitable product was obtained when similar RNA fractions were tested in a wheat germ translation system.     

Molecular cloning of pea mRNAs encoding a shoot-specific polypeptide and light-induced polypeptides

Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of M_r27 500 and isoelectric point of 4.7.     

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     

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     

Cyanelle DNA from Cyanophora paradoxa

Summary Cyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.Abbreviations Kbp 10³ base pairs - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme