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The Cytokinin-hypersensitive genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development.

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin-hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.

Arabidopsis↗

Expression of the chloroplast ribosomal RNA genes of Euglena gracilis during chloroplast development.

The cellular content and transcription program of the chloroplast ribosomal RNA genes of Euglena gracilis Z have been determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ([3H]ctrDNA). Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E. coli DNA polymerase I with [3H]TTP as a substrate. The [3H] DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA. The rRNA-[3H]ct rCNA hybrid was isolated by chromatography on hydroxylapatite. The [3H]ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of [3H]DNA-DNA and [3H]DNA-RNA hybrids. [3H]ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0,4,8,12,24,48, and 72 h after the onset of chloroplast development. From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made. Chloroplast rRNA increases from 2 to 26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases two- to threefold. Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than twofold, while the number of copies of rRNA per cell increases sixfold. The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental.

Animals↗

Breakdown of thylakoid pigments by soluble proteins of developing chloroplasts.

In the presence of Triton X-100 (TX-100) or imazalil, plastidic pigments were degraded by a soluble enzyme extracted from developing chloroplasts. This bleaching was not photochemical and required oxygen; it was not inhibited by superoxide dismutase or catalase, but was strongly inhibited by benzoquinone, quinol, phenazine methosulphate and, more weakly, by other reagents. Synthetic intermediates of chlorophyll biosynthesis, e.g. Mg(II)-protoporphyrin IX monomethyl ester, was also degraded. This reaction was compared with the bleaching catalysed by soybean (Glycine max) lipoxygenase. The plastidic system required TX-100 and was inhibited by unsaturated fatty acids, whereas lipoxygenase required a polyunsaturated fatty acid and was inhibited by TX-100. The bleaching capability of the stromal extract decreased with age if the seedlings were placed in the greenhouse to allow further development of the chloroplasts. A direct relationship was observed between the promotion of pigment bleaching by TX-100 and the inhibition of the in vitro synthesis of divinylprotochlorophyllide. This bleaching reaction is discussed on the basis of interference by TX-100 with the normal O2-requiring anabolic processes of developing chloroplasts.

Chlorophyll↗

A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana.

Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23, encodes a putative Na(+)(K(+))/H(+) exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na(+)(K(+))/H(+) exchanger important for pH homeostasis and chloroplast development and function.

Antiporters↗

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts.

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.

Aerobiosis↗

Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase.

Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC). We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance. The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development. Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones. The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants. Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts. The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts. The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid.

Acclimatization↗

Chilling stress suppresses chloroplast development and nuclear gene expression in leaves of mung bean seedlings.

Etiolated leaves of 28 degrees C-dark-grown mung bean (Vigna radiata L. cv. 2937) seedlings fail to turn green after being shifted to a light and cold environment. At the visible phenotypic level, incapability of leaf greening is the only failure event for the de-etiolation of mung bean seedlings at low temperature. Ultrastructural studies revealed that chloroplast development was completely suppressed by chilling treatment. A cDNA library originating from 28 degrees C-light-grown seedling leaves was constructed for screening cold-suppressed (cos) genes. Thirteen full-length cDNA clones were obtained, with 12 clones encoding chloroplast proteins, which, according to their known physiological functions, were important for chloroplast development and photosynthesis. Another cos cDNA encodes CYP90A2, which is a cytochrome P450 protein involved in the biosynthesis of brassinosteroid hormones. All cos genes are light-regulated at normal temperature. The influence of chilling stress on cos expression was examined in 10 degrees C-light- and 10 degrees C-dark-grown etiolated seedlings, and in 10 degrees C-light-grown green plants. The data show that cos expression in these three treatments is severely suppressed. This suppression is controlled at the transcriptional level, as demonstrated by nuclear runoff experiments, and is reversible because cos mRNAs accumulate again after the cold-treated plants have been transferred to 28 degrees C.

Cell Nucleus↗

Evidence that Isolated Developing Chloroplasts Are Capable of Synthesizing Chlorophyll b from 5-Aminolevulinic Acid.

Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating [(14)C]5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.

Journal Article↗

The Arabidopsis ClpB/Hsp100 family of proteins: chaperones for stress and chloroplast development.

The Casein lytic proteinase/heat shock protein 100 (Clp/Hsp100) proteins are chaperones that act to remodel/disassemble protein complexes and/or aggregates using the energy of ATP. In plants, one of the best-studied proteins from this family is cytosolic ClpB1 (At1g74310), better known in Arabidopsis as AtHsp101, which is a heat shock protein required for acclimation to high temperatures. Three other ClpB homologues have been identified in the Arabidopsis genome (ClpB2, ClpB3 and ClpB4; At4g14670, At5g15450 and At2g25140). To define further the roles of these chaperones in plants we investigated their intracellular localization, evolutionary relationships, patterns of expression and the phenotypes of corresponding T-DNA insertion mutants. We first found that ClpB2 was misannotated; there is no functional ClpB/Hsp100 gene at this locus. By fusing the putative transit peptides of ClpB3 and ClpB4 with GFP, we showed that these proteins are targeted to the chloroplast and mitochondrion, respectively, and we therefore designated them as ClpB-p and ClpB-m. Phylogenetic analysis supports two major lineages of ClpB proteins in plants, an 'eukaryotic', cytosol/nuclear-localized group containing AtHsp101, and an organelle-localized lineage, containing both ClpB-p and ClpB-m. Although AtHsp101, ClpB-p and ClpB-m transcripts all accumulate dramatically at high temperatures, the T-DNA insertion mutants of ClpB-p and ClpB-m show no evidence of seedling heat stress phenotypes similar to those observed in AtHsp101 mutants. Strikingly, ClpB-p knockouts were seedling lethals, failing to accumulate chlorophyll or properly develop chloroplasts. Thus, in plants, the function of ClpB/Hsp100 proteins is not restricted to heat stress, but a specific member of the family provides housekeeping functions that are essential to chloroplast development.

Amino Acid Sequence↗

Chloroplast development in green barley leaves transferred to darkness.

Evidence is presented that immature barley chloroplasts developing normally from proplastids in light-grown plants continue to synthesise thylakoids when the plants are transferred to darkness. This observation is consistent with the finding that normal light-grown barley plants continue to synthesis chlorophyll when transferred to total darkness (Popov and Dilova 1969, Adamson 1982). A physiological role for the light-independent protochlorophyllide reductase in higher plants is suggested.

Chloroplasts↗

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Chlamydomonas↗

[Genetic mapping of rice gene OsALB23 regulating chloroplast development].

The biogenesis of chloroplast from proplastid is the prerequisite of photosynthesis. Using electron microscope, we found that rice albino mutant Osalb23 had no thylakoid inside the chloroplast, only some empty vesicles could be observed (Fig. 2). Genetics analysis showed that albino phenotype was controlled by a single recessive locus. Using map-based cloning technique, OsALB23 has been mapped to a region of 280 kb between molecular markers R2M501 and R2M502 on chromosome 2 (Fig. 4). Homologous analysis indicated that this region contained six chloroplast protein genes.

Chloroplasts↗

Significant Changes in Cell and Chloroplast Development in Young Wheat Leaves (Triticum aestivum cv Hereward) Grown in Elevated CO2.

Cell and chloroplast development were characterized in young Triticum aestivum cv Hereward leaves grown at ambient (350 [mu]L L-1) or at elevated (650 [mu]L L-1) CO2. In elevated CO2, cell and chloroplast expansion was accelerated by 10 and 25%, respectively, in the first leaf of 7-d-old wheat plants without disruption to the leaf developmental pattern. Elevated CO2 did not affect the number of chloroplasts in relation to mesophyll cell size or the linear relationship between chloroplast number or size and mesophyll cell size. No major changes in leaf anatomy or in chloroplast ultrastructure were detected as a result of growth in elevated CO2, but there was a marked reduction in starch accumulation. In leaf sections fluorescently tagged antisera were used to visualize and quantitate the amount of cytochrome f, the [alpha]- and [beta]-subunits of the coupling factor 1 in ATP synthase, D1 protein of the photosystem II reaction center, the 33-kD protein of the extrinsic oxygen-evolving complex, subunit II of photosystem I, and ribulose-1,5-bisphosphate carboxylase/oxygenase. A significant finding was that in 10 to 20% of the mesophyll cells grown in elevated CO2 the 33-kD protein of the extrinsic oxygen-evolving complex of photosystem II and cytochrome f were deficient by 75%, but the other proteins accumulated normally.

Journal Article↗

An essential role of a TatC homologue of a Delta pH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana.

At least three transport systems function in targeting nuclear-encoded chloroplast proteins to the chloroplast thylakoid membrane. One of these systems requires a thylakoid pH gradient and is named the DeltapH-dependent protein transport system. A similar DeltapH export system of Escherichia coli contains four components, twin arginine translocation A (TatA), TatB, TatC, and TatE. TatC is a major component of the DeltapH-dependent protein transporter in E. coli and functions in the translocation of tightly folded proteins across membranes. We have isolated four transposon-inserted albino mutants named albino and pale green 2 (apg2) from Arabidopsis thaliana and showed that the transposons were inserted into different sites of a single gene. The APG2 gene product (named cpTatC) has sequence similarity with bacterial TatC and contains six putative transmembrane domains, including bacterial TatC proteins and a transit peptide in its N terminus. apg2 mutants showed albino phenotypes and could not grow in soil. The apg2 plastids were highly vacuolated, lacked internal membrane structures and lamellae of the thylakoid membrane, and contained many densely stained globule structures, like undifferentiated proplastids. Immunoblot analysis detected no thylakoid membrane proteins such as D1, light-harvesting complex, and OE23 in apg2 plastids, whereas soluble proteins such as rubisco large and small subunits were not decreased. These results indicate an essential role of cpTatC in chloroplast development, especially in thylakoid membrane formation.

Amino Acid Sequence↗

Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts : IV. An Endogenous Inhibitor from the Thylakoid Membranes.

5-Aminolevulinic acid synthesis in isolated, intact, developing chloroplasts from greening cucumber (Cucumis sativus) cotyledons was inhibited by broken chloroplast fragments. It was shown that the inhibitory constituent was associated with the thylakoid membrane system. The inhibitor was resistant to boiling, was not a form of ribonuclease, and did not inhibit Mg-chelatase, indicating that massive organelle destruction was not involved. The inhibitor was also found in etioplast and mature chloroplasts; and it was found in barley as well as cucumber. 5-Aminolevulinate synthesis in the dark with exogenous ATP and NADPH, or in the light without added cofactors, were inhibited approximately equally. In the dark, 5-aminolevulinate synthesis and protochlorophyllide synthesis from glutamate were inhibited to about equal extent. The inhibition was decreased when the membranes were washed with aqueous acetone prior to incubation. The inhibition by the unknown factor was compared to the inhibition by gabaculine, 4-amino-5-hexynoic acid, protoheme, and glutathione. The unknown inhibitor appeared to have a number of similarities with protoheme.

Journal Article↗

Light-induced biogenesis of light-harvesting complex I (LHC I) during chloroplast development in barley (hordeum vulgare). Studies using cDNA clones of the 21- and 20-kilodalton LHC I apoproteins.

The light-harvesting complex (LHC) Ib pigment-proteins form the major component of the LHC I complex in higher plants. They comprise chlorophylls a and b, xanthophylls, and at least two polypeptide subunits of 21 and 20 kD in barley (Hordeum vulgare). We have identified two cDNA clones, LHC Ib-21 and LHC Ib-20, encoding the 21- and 20-kD LHC Ib apoproteins, respectively. N-terminal protein sequences of the purified LHC Ib polypeptides were used for the unequivocal correlation of these clones to their respective apoproteins. The cDNA clones encode two proteins that have strong sequence similarity to other LHC I and LHC II pigment-binding polypeptides of photosystems I and II. The 21-kD polypeptide contains 201 amino acid residues (22.14 kD), and the 20-kD polypeptide contains 200 amino acid residues (22.18 kD). The biogenesis of the LHC Ib apoproteins and the pigmented LHC I during the light-induced development of the chloroplast was studied. Accumulation of the two LHC Ib mRNAs is induced by light, and their amount is regulated by phytochrome. LHC Ib polypeptide accumulation in the thylakoid membrane temporally lags behind transcript accumulation. The rates of accumulation of LHC Ib transcripts and of their apoproteins lag behind those of the major LHC II component, LHC IIb. Complete assembly of the LHC Ib pigment-protein, as observed by low-temperature fluorescence spectroscopy, requires exposure of dark-grown seedlings to 72 h or more of light.

Amino Acid Sequence↗

Protoplasts as a means of studying chloroplast development in vitro.

Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.In parallel, electron microscopic investigations showed a pronounced decrease in prolamellar body area from 89.5 square micrometers at 0 hours to 40.6 square micrometers at 6 hours, whereas the length of the thylakoid membranes (prothylakoids, thylakoids) increased from 21 micrometers at 0 hour to about 293 micrometers at 6 hours. This was accompanied by the formation of grana (bi- and polythylakoids).A comparison of plastid structure and function, developed within isolated protoplasts and those of organelles greened in intact leaves up to 6 hours, showed that there was only a slight lag in development of plastids illuminated in vitro to those illuminated in vivo. However, times of in vitro illumination longer than 6 hours resulted in signs of deterioration and the lack of further development, except for photosystem I activities.

Journal Article↗