Transcriptional activity of isolated maize chloroplasts (original) (raw)
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Phytochrome control of RNA levels in developing pea and mung-bean leaves
Planta, 1983
We have examined phytochrome effects on the abundance of transcripts from several nuclear and chloroplast genes in buds of dark-grown pea seedlings and primary leaves of dark-grown mung-bean seedlings. Probes for nuclear-coded RNAs were selected from a library of cDNA clones and included those corresponding to the small subunit (SS) of ribulosebisphosphate carboxylase and a chlorophyll a/b binding protein (AB). Transcripts from chloroplast genes for RuBP carboxylase large subunit (LS) and a 32,000-dalton photosystem II polypeptide (PII) were assayed with cloned fragments of the chloroplast genome. In addition, we present data on transcripts from a number of other nuclear genes of unknown function, several of which change in abundance during light-induced development. Transcript levels were measured as a proportion of total RNA by a dot blot assay in which RNA from different tissues or stages is fixed to nitrocellulose and hybridized with 32p-labeled probes prepared from cloned DNAs. Several patterns of induction can be seen. For example, although both SS and AB RNAs show positive, red/far-red reversible responses in both pea and mung bean, in pea buds the induction ratio for SS RNA is much higher than that for AB RNA, while just the reverse is true for mungbean leaves. In addition, treatment with lowfluence red light produces full induction of the pea Abbreviations: AB=chlorophyll a/b polypeptide of the lightharvesting complex; FR= far-red light; PII=32,000-dalton photosystem II polypeptide; R = red light; SS = small subunit and LS=large subunit of ribulosebisphospate carboxylase (RuP2; 3-phospho-D~glycerate carboxylase [dimerizing], EC 4.1.1.39) AB RNA, while SS RNA in the same tissue does not reach a maximum steady-state level until after about 24 h of supplementary high-intensity white light. In pea buds, chloroplast genes (LS, PII) also show clear responses to phytochrome, as measured by the steady-state levels of their RNA products. Chloroplast DNA levels (as a fraction of the total cellular DNA) show the same response pattern, which may indicate that in peas many of the light effects we see are related to a general stimulation of chloroplast development. In mung beans, the levels of plastid DNA and RNA are already quite high in the leaves of 7-d dark-grown seedlings, and light effects are much less pronounced. The results are consistent with the notion that chloroplast development is arrested at a later stage in dark-grown mung-bean leaves than in etiolated pea buds.
Nuclear-encoded factors associated with the chloroplast transcription machinery of higher plants
Frontiers in plant science, 2014
Plastid transcription is crucial for plant growth and development. There exist two types of RNA polymerases in plastids: a nuclear-encoded RNA polymerase (NEP) and plastid-encoded RNA polymerase (PEP). PEP is the major RNA polymerase activity in chloroplast. Its core subunits are encoded by the plastid genome, and these are embedded into a larger complex of nuclear-encoded subunits. Biochemical and genetics analysis identified at least 12 proteins are tightly associated with the core subunit, while about 34 further proteins are associated more loosely generating larger complexes such as the transcriptionally active chromosome (TAC) or a part of the nucleoid. Domain analyses and functional investigations suggested that these nuclear-encoded factors may form several functional modules that mediate regulation of plastid gene expression by light, redox, phosphorylation, and heat stress. Genetic analyses also identified that some nuclear-encoded proteins in the chloroplast that are important for plastid gene expression, although a physical association with the transcriptional machinery is not observed. This covers several PPR proteins including CLB19, PDM1/SEL1, OTP70, and YS1 which are involved in the processing of transcripts for PEP core subunit as well as AtECB2, Prin2, SVR4-Like, and NARA5 that are also important for plastid gene expression, although their functions are unclear.
Plant Molecular Biology, 1993
The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-dayold dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability ofpsaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNAs, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.
The EMBO journal, 1993
Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat-bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species. Cells of white leaves of the mutant and cells of heat-bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2. The following results were obtained. (i) Plastid genes are transcribed despite the lack of chloroplast gene-encoded RNA polymerase subunits. The plastid origin of these transcripts was proven. This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes. (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI-H), were abundantly accumula...
Molecular cloning of a pea mRNA encoding an early light induced, nuclear coded chloroplast protein
Plant Molecular Biology, 1985
cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2-4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)+-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein (ELIP)-mRNA established the basis of our screening procedure~ Colony hybridization experiments were performed with 32P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M r 24 000. After posttranslational import in vitro, the processed product of M r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2 4 hours after onset of illumination. Our results correspond precisely to the in vivo characteristics and indicate positive identification of the sought clones.
Plant Molecular Biology, 1984
We have investigated the molecular basis of differential localization of enzyme activities in mesophyll (M) and bundle-sheath (B) cells of maize leaves. M protoplasts and B strands were prepared by enzymatic digestions and mechanical treatment of secondary leaves. Soluble and thylakoid membrane proteins from the two cell types were compared by one-and two-dimensional gel electrophoresis and quantitative rocket immunoelectrophoresis. In addition, several thylakoid polypeptides were identified by crossed immunoelectrophoresis using monospecific antibodies. M and B thylakoids show quantitative and qualitative differences in their polypeptide compositions. While the M thylakoids contain the normal complement of polypeptides, the B thylakoids are deficient in ferredoxin-NADP + reductase, photosystem II reaction center polypeptides, and the light-harvesting chlorophyll a/b-protein complex. Comparison of the soluble proteins by two-dimensional gel electrophoresis revealed marked differences between M and B cells. The major proteins of one cell type are clearly absent from the other. These differences are paralleled by differences in the in vitro translation products of poly A + RNA isolated from the two cell types. Immunoprecipitation experiments showed that mRNA encoding the small subunit of ribulose-l,5-bisphosphate carboxylase (rbcS) is localized exclusively in B cells, whereas mRNA encoding phosphoenolpyruvate carboxylase is detected only in M cells. cDNA clones encoding the carboxylase rbcS and the chlorophyll a/b binding protein were used as probes in Northern blot analysis. M ceils contain no detectable RNA encoding rbcS but have a higher steady state level of RNA encoding the chlorophyll a/b-binding polypeptide compared to B cells. Taken together, our results demonstrate that differential gene expression in the two leaf cell types is regulated at the level of translatable mRNA, and, for at least two proteins, at the level of steady-state RNA.
Plant molecular biology, 1998
A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase ...
Plastid transcription and translation are light-activated in 8day-old dark-grown barley (Hordeum vulgare L.) seedlings. Pretreatment of dark-grown seedlings with cycloheximide (inhibitor of cytoplasmic protein synthesis) abolished the activation of rbcL, psbA, and psaA-B transcription by light. In contrast, inhibition of plastid protein synthesis by chloramphenicol stimulated lightactivated transcription of rbcL, psbA, and psaA-B. Light-induced transcription of the plastid genome occurred normally in the chlorophyll-deficient mutant xan-J". These results suggest that although the light-induced activation of plastid transcription is modulated by cytoplasmic and organellar protein synthesis, transcriptional activation is not dependent on the absorption of light by protochlorophyllide or the attainment of photosynthetic competence. In addition, plastid translation increased dramatically when 8-day-old dark-grown seedlings were illuminated and activation was dependent on cytoplasmic protein synthesis. Blockage of light-activated plastid transcription by Tagetin treatment (inhibitor of plastid RNA polymerase) did not attenuate the activation of plastid translation by light. These results suggest that while light simultaneously activates plastid transcription and translation, the rapid burst in plastid protein synthesis is due mainly to cytoplasmic-denved changes that regulate the rate of translation of pre-existing mRNAs.
Plant Physiology, 1990
Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem 11 and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A 'high irradiance response' also controls the induction of these genes. Gernes encoding photosystem 11-and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light. These distinct expression characteristics are shown to reflect differences at the level of transcription. Higher plants have several photoreceptors which detect light quality and intensity. The major photoreceptors are phytochrome (26), which control induction and 'high irradiance responses' (HIR2) (10), a blue/UV-A photoreceptor (27), and a UV-B photoreceptor (34). Studies of expression of nuclear genes encoding CAB, SSU, and CHS proteins revealed that photoregulation of gene expression in higher plants occurs at transcriptional and posttranscriptional levels (33). The dependence ofgene expression on light quality and intensity varies for different genes and different species. For instance, in parsley cell suspension cultures excitation of the UV-B photoreceptor is essential for maximal expression of CHS, while the excitation of the blue/