Isolation of chloroplasts and chloroplast DNA from pea leaves]

A method of isolating DNA from pea chloroplasts (ch-DNA) in CsCl density gradient is described. DNA preparations are free of 5-methylcytosine and have a melting temperature of 86.5 degrees. Denatured DNA molecules completely reassociate for 3 hours at 60 degrees C. It is concluded that the preparations obtained are pure ch-DNA.     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Insertion polymorphism in pea chloroplast DNA

Summary The chloroplast DNA of higher plants is suitable for restriction endonuclease analysis due to its size and homogeneity. We have analysed 48 different cultivars of pea (Pisum sativum) with EcoRI and HindIII. Of these, only 24 show the standard genotype, the remaining 24 comprise four different classes of short insertions, three of which are found at the same site. Even though this kind of insertion polymorphism has not been detected elsewhere in the plant kingdom, it is consistent with the discovery that the chloroplast DNA of pea is destabilised through the loss of an inverted repeat.     

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.     

Phytochrome-mediated plastid development in etiolated pea stem apices

The effects of red and far-red light on growth and plastid development in the stem apices of etiolated pea seedlings have been examined. Changes were determined in various growth parameters (DNA, soluble protein and fresh weight) and also in the activities of the plastid-localized enzymes ribulose-1,5-bisphosphate carboxylase, NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline-1,6-bisphosphatase and the non-photosynthetic (cytoplasmic) enzymes NADP-isocitrate dehydrogenase, enolase and NAD-malate dehydrogenase. Changes in the amounts of Fraction I protein were also measured. Brief daily irradiation with low intensity red light increased growth 5·1–7·6-fold which was correlated with increases of about 3·5-fold in activities of the non-photosynthetic enzymes. The chloroplast enzymes, however, showed much greater increases in activity ranging from 15- to 91-fold. Fraction I protein increased 11·7-fold. These increases approached the levels attained in fully green leaves. All these responses were largely prevented by far-red light indicating that they were mediated by phytochrome. In experiments with red light given at daily intervals there was a lag of 24 hr before the initially very low activity of ribulose-1,5-bisphosphate carboxylase increased. Fraction I protein which was initially present in significant amounts showed a similar lag in its synthesis. However, for 3 days after the initial irradiation, the rate of increase of the enzymic activity was much greater than the rate of net synthesis of Fraction I protein. A single initial red irradiation was as effective as 3 daily irradiations in increasing the activity of ribulose-1,5-bisphosphate carboxylase. A fourth irradiation, however, gave an additional response which exceeded that of the single initial irradiation. It was shown that there was a rapid activation of ribulose-1,5-bisphosphate carboxylase by either continuous white or 3 min of red light. The red light response was slowly reversed in the dark. These results are discussed with particular emphasis on the relation between growth and plastid development in a phytochrome-mediated system.     

Phytochrome-mediated phototropism inAdiantum cuneatum young leaves

Phototropism of youngAdiantum fern leaves is induced by red light as well as blue light. The red light response is mediated by phytochrome. This is the first evidence of phytochrome action in diploid fern tissue. The blue light response is mainly mediated not by phytochrome, but probably by a blue light-absorbing pigment as in the case of almost all plants and fungi. The red light-induced phototropism becomes detectable within 2 hr after the onset of unilateral light. The highest bending rate is about 10 degrees/hr, which occurs between 3–5 hr after the induction of the tropic response. The bending region is about 6–8 mm from the highest point of the coiled crozier where the growth rate becomes slow.     

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.     

Phytochrome-mediated regulation of a monooxygenase hydroxylating cinnamic acid in etiolated pea seedlings

Cinnamic acid is hydroxylated by the mixed-function oxidase trans-cinnamic acid 4-hydroxylase (CA4H). The hydroxylation reaction involves the transfer of electrons from reduced pyridine nucleotides via the enzyme NADPH cytochrome P-450 reductase to the terminal oxidase cytochrome P-450. This multi-enzyme complex is localized in the microsomal fraction. Isopycnic and velocity gradient centrifugation suggest that in the apical bud of etiolated pea seedlings this complex is restricted to the endoplasmic reticulum membranes. CA4H activity which develops in dark germinating pea seedlings was found to be stimulated by light, an effect mediated by phytochrome. CA4H and NADPH cytochrome c reductase activities, cytochromes P-450 and b 5 contents were measured in seedlings submitted to either short pulses of red and far-red light, or to continuous far-red or blue irradiation. The results are discussed in terms of a specific effect of phytochrome on the different parts of the multi-enzyme complex.     

Cloning chloroplast DNA in escherichia coli. I. Construction and selection of recombinant plasmids containing fragments of pea chloroplast DNA]

Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Benzyladenine-induced increase in DNA content per chloroplast in intact bean leaves

Benzyladenine (BA) treatment was found to induce chloroplast DNA (ctDNA) synthesis after it had stopped in primary leaves of light-grown intact bean plants (Phaseolus vulgaris L.). The leaves were treated with BA from 7 days after sowing. Chloroplasts were isolated and the ctDNA content per chloroplast was determined. Chloroplast division occurred until 13 days after sowing in untreated leaves. BA stimulated the division keeping the level of ctDNA content per chloroplast the same as that in the untreated controls. After the division period, the ctDNA content per chloroplast increased in BA-treated leaves, but not in controls. Consequently, ctDNA per leaf (or per cell) increased immediately after the beginning of BA treatment, but remained constant in the control leaves.     

Amylopectin degradation in pea chloroplast extracts

Phosphorolysis rather than phosphorylation of amylolysis products was found to be the major pathway of sugar phosphate formation from amylopectin by pea (Pisum sativum L.) chloroplast stromal proteins. The K_m for inorganic phosphate incorporation was 2.5 mm, and ATP did not stimulate amylopectin-dependent phosphate incorporation. Arsenate (10 mm) inhibited phosphate incorporation into glucose monophosphates up to 46% and phosphoglucomutase activity 96%, resulting in glucose 1-phosphate accumulation as a product of amylopectin degradation. The intracellular distribution of enzymes of starch utilization was determined. Phosphorylase, phosphoglucomutase, and hexokinase were found in the chloroplast and cytoplasm, while β-amylase was restricted to the cytoplasm. Maltase was not detectable; maltose phosphorylase was active in the chloroplast.     

Evidence that the amount of chloroplast DNA exceeds that of nuclear DNA in mature leaves

Cell-free homogenates containing intact chloroplasts and nuclei were allowed to settle for up to 1 h before the top 2 ml of the 5-ml homogenate was withdrawn. Whereas less than 18% of the chloroplasts moved from the top to the bottom portions, the ratio of nuclei to chloroplasts in the top portion changed from approximately 1/200 to 1/900. The total numbers of chloroplasts and nuclei were counted in the homogenate before settling and in the top 2 ml and bottom 3 m1 after settling. The total DNA content of the homogenate and the top and bottom portions after settling was determined by the diphenylamine colorimetric assay. By simultaneous equations, the absolute amount of DNA in chloroplasts and nuclei was determined. The results are consistent with previous observations of chloroplast DNA by fluorescence microscopy which indicated that the amount of chloroplast DNA per chloroplast is a function of chloroplast size. In addition, the results show that the amount of chloroplast DNA per average chloroplast in large leaves is 0.14 times 10(-12) g, a magnitude higher than previous reports in the literature, and that large leaves contain about twice as much chloroplast DNA as nuclear DNA.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

Phosphorylation of pea chloroplast acetyl-CoA carboxylase

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea ( Pisum sativum ) chloroplasts were incubated in the presence of [γ- ³³ P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The β-subunit of the carboxyltransferase was found to be labeled by ³³ P. Phosphoamino acid analysis of the immunoprecipitated β-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of ³³ P into carboxyltransferase β-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase β-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.     

Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.