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PubMed2026

Characterization and comparative analysis of the complete chloroplast genomes of twelve Allium species from Kazakhstan.

The genus Allium L. represents one of the largest and taxonomically complex groups of monocots, with Central Asia recognized as a major center of its diversity. Despite the high species richness of Allium in Kazakhstan, genomic data for many native taxa remain limited. In this study, we sequenced, assembled, and analyzed the complete chloroplast genomes of 12 Allium species from Kazakhstan. All chloroplast genomes exhibited a conserved quadripartite structure, with genome sizes ranging from 152,029 to 153,521 bp and a uniform gene content of 137 genes, including 88 protein-coding genes, 38 tRNAs, 8 rRNAs, and 3 pseudogenes. Comparative analyses revealed high structural conservation, with most sequence divergence concentrated in intergenic regions. Several highly variable regions, including ycf1, matK, rpoC2, and ycf2, were identified as potential molecular markers. Phylogenetic analyses based on chloroplast genome sequences using Maximum Likelihood and Bayesian approaches recovered three major chloroplast genome-based lineages within Allium, largely consistent with previous phylogenomic studies. Divergence-time analyses suggested that major chloroplast lineage diversification events within the genus occurred during the early Eocene (ca. 47.97 Mya). Overall, this study expands the currently available chloroplast genomic resources for Allium from Kazakhstan, provides insights into chloroplast genome evolution and chloroplast genome-based relationships, and establishes a valuable foundation for future phylogenetic, taxonomic, and evolutionary studies of this diverse genus.

Genome, Chloroplast↗

PubMed1999

The endosymbiotic origin of the protein import machinery of chloroplastic envelope membranes.

Chloroplasts have evolved a complex proteinaceous machinery to import nuclear-encoded proteins. The origin of this machinery, following the endosymbiotic events leading to chloroplasts, is an intriguing, unresolved problem. Given that cyanobacteria are the probable ancestors of chloroplasts, the genome sequence of Synechocystis sp. PCC 6803 offers a valuable resource to identify putative homologs for components of this protein import machinery and to gain insights into its possibly endosymbiotic origin. Detailed computational sequence analysis reveals that Synechocystis sp. PCC 6803 has homologs of three of the known membrane proteins of the chloroplastic translocon, namely Toc75, Tic20 and Tic22, as well as a homolog of the putative component Tic55. Thus, the chloroplastic protein import machinery is mainly derived from the endosymbiotic cyanobacterium, but, interestingly, not from any of the four main protein secretion systems of prokaryotes. The relatively high sequence variability between chloroplastic and Synechocystis proteins suggests that the ancestral proteins had different physiological roles and were modified significantly to fulfill the new demand of importing proteins into the evolving chloroplast. The fact that some chloroplastic protein import components are not related to any Synechocystis proteins (Toc159, Tic110 and Toc34) indicates that the chloroplastic protein import apparatus is of a dual evolutionary origin.

Journal Article↗

PubMed2002

Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea).

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [alpha-32P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [alpha-32P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.

Journal Article↗

PubMed1979

Chloroplast membrane alterations in triazine-resistant Amaranthus retroflexus biotypes.

The effectiveness of diuron, atrazine, procyazine, and cyanazine were compared in controlling growth of redroot pigweed (Amaranthus retroflexus L.) in hydroponic culture. A very marked differential inhibition response was observed for atrazine between resistant and susceptible biotypes. Procyazine and cyanazine exhibited less dramatic differential responses, whereas diuron was equally effective in controlling growth in both biotypes. Photosystem II activity of chloroplasts from both triazine-resistant and triazine-susceptible biotypes was inhibited by diuron but only the chloroplasts from triazine-susceptible biotypes were inhibited significantly by atrazine. The photochemical activity of chloroplasts from triazine-resistant biotypes was partially resistant to procyazine or cyanazine inhibition. The parallel lack of diuron differential effects, partial procyazine and cyanazine differential response, and very marked atrazine differential response in both whole plant and chloroplast assays indicates that the chloroplast is the site of selective herbicide tolerance in these triazine-resistant redroot pigweed biotypes.Photosystem II photochemical properties were characterized by analysis of chlorophyll fluorescence transients in the presence or absence of herbicides. Data with susceptible chloroplasts indicated that both diuron and atrazine inhibit electron flow very near the primary electron acceptor of photosystem II. Only diuron altered the fluorescence transient in resistant chloroplasts. In untreated preparations there were marked differences in the fast phases of the fluorescence increase in resistant vs. susceptible chloroplasts; these data are interpreted as showing that the resistant plastids have an alteration in the rate of reoxidation of the primary photosystem II electron acceptor. Electrophoretic analysis of chloroplast membrane proteins of the two biotypes showed small changes in the electrophoretic mobilities of two polypeptide species. The data provide evidence for the following herbicide resistance mechanism: genetically controlled modification of the herbicide target site.

Journal Article↗

PubMed1993

Maintenance of Chloroplast Components during Chromoplast Differentiation in the Tomato Mutant Green Flesh.

During ripening of tomato (Lycopersicon esculentum) fruit, chloroplasts develop into chromoplasts. The chloroplast-chromoplast transition is marked by the accumulation of carotenoids and the disappearance of chlorophyll, the degradation of the highly structured thylakoid membrane system, and a reduction in the levels of proteins and mRNAs associated with photosynthesis. In the tomato mutant green flesh (gf), detectable amounts of chlorophyll remain in the ripe, mutant fruit, giving rise to a rusty red fruit color and suggesting that at least chlorophyll degradation is defective in the mutant. We show here that the ultrastructure of the plastids in the ripe gf fruit maintained significant amouonts of the chloroplast thylakoid grana along with structures characteristic of tomato chromoplasts. The maintenance of chloroplast structure in the gf ripe fruit was paralleled on the molecular level by the retention of plastid photosynthetic components that normally decline significantly in ripening tomato fruits. These included the light-harvesting chlorophyll a/b-binding proteins of photosystem II, the second electron accepting plastoquinone of photosystem II binding protein, the large and small subunits of ribulose bisphosphate carboxylase/oxygenase, the 33-kD oxygen evolution protein, and cytochrome b559. Similarly, photosynthetic transcripts, cab, psbA, rbcL, rbcS, and psbE mRNAs, also accumulated to higher levels in ripening gf fruit than wild type. It is interesting that the levels of some of these transcripts, especially cab mRNA, were noticeably higher in the mature gf green fruit than in the corresponding wild-type fruit. This suggests that the onset of the effect from the gf mutation might be earlier than fruit ripening. We also observed that when chloroplast formation was blocked during the development and ripening of gf fruit, these mutant fruits were bright red and their chromoplasts were indistinguishable from those found in wild-type ripe fruits grown and ripened either in the dark or in the light. These results suggest that the lesion in gf may alleviate conditions associated with chloroplast deterioration during the chloroplast-chromoplast transition in tomato ripening but has no direct effect on chromoplast differentiation per se. The ultrastructure of gf provides unequivocal evidence that, in ripening tomato, chromoplasts indeed differentiate from preexisting chloroplasts; on the other hand, chromoplast differentiation in the dark-matured and -ripened tomato fruits indicates that chromoplast development can be a process entirely independent of the chloroplasts.

Journal Article↗

PubMed1995

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast.

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.

Journal Article↗

PubMed1972

The Ultrastructure of Chloroplasts in Mineral-deficient Maize Leaves.

The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral-deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogen-deficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium- or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen- and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity.

Journal Article↗

PubMed1977

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum.

The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C(4) plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg(2+) chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.

Journal Article↗

PubMed1977

Oxygen Concentration in Isolated Chloroplasts during Photosynthesis.

The O(2) concentration in intact and osmotically disrupted isolated spinach (Spinacia oleracea, L.) chloroplasts during photosynthesis was estimated. The chloroplasts were allowed to reduce 3-phosphoglycerate, CO(2), or ferricyanide in light until the rate of O(2) production was linear. When the light was turned off O(2) evolution from the chloroplasts continued for a few seconds. This prolonged O(2) evolution is due to an O(2) surplus inside the chloroplasts which equilibrates with that in the medium. From this surplus the O(2) concentration inside the chloroplasts at the moment when the light had been switched off was calculated. In all experiments the O(2) concentration inside the photosynthesizing chloroplasts was higher than that outside, but was dependent upon the O(2) concentration of the chloroplast medium. At low external O(2) concentration (30 mum) the ratio of the internal to the external O(2) concentration was about 5, whereas at concentrations corresponding to those in airsaturated water this ratio was close to 1. With osmotically broken chloroplasts this ratio was 1.2 at 30 mum O(2) and almost 1 from 150 mum onward. When the O(2) surplus found in broken chloroplasts during photosynthesis was related to the volume of the thylakoids, a ratio of about 2.3 was observed.

Journal Article↗

PubMed1979

Comparison of the Molecular Weights of Proteins Synthesized by Isolated Chloroplasts with Those Which Appear during Greening in Zea mays.

The proteins of prolamellar bodies of etioplasts and of thylakoid membranes of greening and mature chloroplasts from Zea mays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three classes of proteins were distinguished: those present in etioplasts and disappearing during greening, those absent in etioplasts and appearing during greening, and those present in both etioplasts and chloroplasts. The largest number of proteins belonged to this last class.The molecular weights of chloroplast thylakoid proteins were compared to the molecular weights of the membrane-associated proteins synthesized by isolated, mature chloroplasts. Thirteen of the 15 to 20 membrane-bound proteins made by isolated chloroplasts corresponded in size to proteins present in chloroplasts. Most of the 13 are present in both etioplasts and chloroplasts although a few were the same size as proteins which increase during greening. Production of most of the membrane proteins made in the plastids is not stringently regulated by light in vivo. The polypeptide subunits of the light-harvesting pigment-protein complex, the most abundant proteins of the chloroplast thylakoids, were absent from etioplasts. They were not synthesized by isolated chloroplasts.

Journal Article↗

PubMed1981

Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate.

Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O(2) evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.Extraction of enzymes from wheat protoplasts and treatment with IAP showed that nanomolar levels of the compound completely inhibited NAD and NADP glyceraldehyde-3-P dehydrogenase and the half-time for inactivation at 5 nanomolar IAP was about 1 minute. The inhibitory effect of IAP was not reversed by passing the enzyme extract through a column of Sephadex G-25. The concentration of IAP required for inhibition of the chloroplastic triose phosphate isomerase is about three orders of magnitude higher than that with glyceraldehyde-3-P dehydrogenase. Micromolar levels of IAP had no effect on ribulose-1,5-bisphosphate carboxylase. Inhibition of chloroplast photosynthesis and of glyceraldehyde-3-P dehydrogenase in protoplast extracts with IAP follows pseudo first-order kinetics.Pretreatment of chloroplasts with IAP did not inactivate the phosphate translocator of the chloroplast envelope. Iodoacetol phosphate may enter the chloroplasts through the phosphate translocator since a high concentration of IAP (0.5 millimolar) competitively inhibits uptake of (32)Pi. Iodoacetol phosphate had no effect on ferricyanide-dependent O(2) evolution with isolated chloroplasts. Also, IAP had no effect on light-dependent fixation of CO(2) through the carboxylation phase of the C(4) pathway with protoplast extracts of crabgrass mesophyll cells. The site of IAP inhibition of photosynthesis with wheat chloroplasts is suggested to be through the inactivation of glyceraldehyde-3-P dehydrogenase.

Journal Article↗

PubMed1984

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop.

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O(2) evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C(4) mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O(2) evolution in the range of 90 to 135 micromoles O(2) per milligram chlorophyll per hour, and up to 180 micromoles O(2) per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O(2) evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K(m) (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O(2) evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O(2) evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O(2) evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C(4) mesophyll chloroplast. C(3) spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K(i) values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.

Journal Article↗

PubMed1988

Isolation of Intact Chloroplasts from Dunaliella tertiolecta.

Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO(2) was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O(2) evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O(2) and fixed 21 micromoles of CO(2) per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO(2) in air for 24 hours. On low CO(2) the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations.

Journal Article↗

PubMed1989

Evidence for a light dependent increase of phosphoglucomutase activity in isolated, intact spinach chloroplasts.

Phosphoglucomutase (PGM) activity was measured in spinach (Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glc 1,6-P(2)) in the assay. Initial PGM activity increased 2- to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Initial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, dl-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3-phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glc 1,6-P(2) was maximal between pH 7.5 and 8.5. Stromal concentrations of Glc 1,6-P(2) were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc 1,6-P(2) concentration.

Journal Article↗

PubMed1989

Relationship between Gene Dosage and Gene Expression in the Chloroplast of Chlamydomonas reinhardtii.

Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rpl2 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were largely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO(2) fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms.

Journal Article↗

PubMed1992

Differential Synthesis of Photosystem Cores and Light-Harvesting Antenna during Proplastid to Chloroplast Development in Spirodela oligorrhiza.

Proplastids and etioplasts are common starting points for monitoring chloroplast development in higher plants. Although proplastids are the primary precursor of chloroplasts, most proplastid to chloroplast systems are cumbersome to study temporally. Conversely, the etioplast to chloroplast transition is initiated by light and is readily examined as a function of time. Etioplasts, however, are found mostly in plants germinated in the dark and are not an obligatory step in chloroplast development. We have chosen to study chloroplast ontogeny in Spirodela oligorrhiza (Kurtz) Hegelm (a C(3)-monocot) because of its unique ability to grow indefinitely in the dark. Ultrastructural, physiological, and molecular evidence is presented in support of a temporal, light-triggered proplastid to chloroplast transition in Spirodela. The dark-grown plants are devoid of chlorophyll, and upon illumination synchronously green over a 3- to 5-day period. Synthesis of chloroplast proteins involved in photosynthesis is coincident with thylakoid assembly, chlorophyll accumulation, and appearance of CO(2) fixation activity. Interestingly, the developmental sequence in Spirodela was slow enough to reveal that biosynthesis of the D1 photosystem II reaction center protein precedes biosynthesis of the major light-harvesting antenna proteins. This, coupled with the high chlorophyll a/b ratio observed early in development, indicated that reaction center assembly occurred prior to accumulation of the light-harvesting complexes. Thus, with Spirodela one can study proplastid to chloroplast conversions temporally in higher plants and follow the process on a time scale that enables a detailed dissection of plastid maturation processes.

Journal Article↗

PubMed1994

Gene Amplification Can Correct a Photosynthetic Growth Defect Caused by mRNA Instability in Chlamydomonas Chloroplasts.

Chlamydomonas reinhardtii chloroplast transformants that lack an inverted repeat normally found at the 3[prime] end of the chloroplast atpB gene have a slow phototrophic growth phenotype due to reduced accumulation of atpB mRNA and the chloroplast ATPase [beta] subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderate frequency (~1% relative to slow-growing transformants). Quantitative DNA blot analysis indicated that in one class of these robust photosynthetic transformants, the introduced plasmid DNA is maintained at high copy number-~25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladder with at least 15 visible fragments, indicating that most of the transforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase [beta] subunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DNA was stably maintained at high copy number under mixotrophic growth conditions. It was inherited uniparentally from the mt+ parent, and its synthesis was sensitive to 5-fluoro-2[prime]-deoxyuridine, an inhibitor of chloroplast DNA synthesis. Therefore, we conclude that the tandem repeat is maintained in the chloroplast. Restriction enzymes that fail to digest the transforming plasmid but have recognition sites in chloroplast DNA did not alter the electrophoretic mobility of the tandem repeat, suggesting that it is not integrated in the chloroplast genome. We conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.

Journal Article↗

PubMed1987

Chloroplast genome characterization in the red alga Griffithsia pacifica.

It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes. In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoechst-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.

Chloroplasts↗