Broad Bean Chloroplast Research Articles

3D Confocal Laser Scanning Microscopy for the Analysis of Chlorophyll Fluorescence Parameters of Chloroplasts in Intact Leaf Tissues

We analyzed the chlorophyll fluorescence parameters in a 3D cellular arrangement in vivo by using a modified Nipkow disk-type confocal laser scanning microscope (CLSM). We first defined the 3D values of Phi(PSII) (photochemical yield of PSII) and NPQ (non-photochemical quenching) in mesophyll, epidermal and guard cell chloroplasts from the leaf surface to several tens of microns in depth. We also used this CLSM method to analyze the relationships between actinic light intensity and the chlorophyll fluorescence parameters for Boston fern and broad bean leaf specimens. As the actinic light intensity increased, the mean Phi(PSII) values decreased and the NPQ values increased in all chloroplasts of Boston fern and broad bean leaf. These values differed with cell type and species. The Boston fern chloroplasts had lower Phi(PSII) values than the broad bean chloroplasts, and vice versa for the NPQ values. The Phi(PSII) values of Boston fern chloroplasts decreased in the order mesophyll, epidermal and guard cell chloroplasts. The NPQ values decreased in the order guard cell, mesophyll and epidermal chloroplasts, except at 12 micromol m(-2) s(-1) actinic light, when the mesophyll value was slightly lower than that of the epidermis. The trend in the Phi(PSII) and NPQ values of broad bean mesophyll and guard cell chloroplasts was opposite to that of Boston fern chloroplasts. As 3D CLSM can provide the Phi(PSII) and NPQ values of each chloroplast in a 3D cellular arrangement, this method has potential for investigating differences in the functions of chloroplasts in vivo.

Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine (Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non‐embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non‐embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean (Vicia faba) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS‐PAGE and non‐denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light‐yellow non‐embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.

Isolation by asymmetric polymerase chain reaction and partial sequencing of the common bean chloroplast trnL (UAA) gene and pseudogene

AbstractA polymerase chain reaction (PCR) strategy has been developed which utilizes the common bean (Phaseolus vulgaris) chloroplast DNA as a template and primers from (a) the 5′ exon of the broad bean chloroplast trnL (UAA) gene (EMBL ac no. X51471) and (b) the 3′ exon of the common bean chloroplast trnL (UAA) pseudogene (EMBL ac no. X58537). A 600 bp PCR Product was reamplified using the 5′ exon primer and an internal primer from the 5′ end of the intron of the common bean chloroplast trnL (UAA) pseudogene. A 130 bp PCR Product was separated into 130 bp and 106 bp DNA fragments on an 8% non‐denaturing polyacrylamide gel. Both fragments were purified by phenol extraction, reamplified by asymmetric PCR reactions and directly sequenced by the Sanger method. When compared to the broad bean trnL (UAA) gene, the 130 bp PCR product showed sequence homologies of 100% with its 5′ exon and 79% over the first 58 nucleotides of the 5′ end of the intron. The 106 bp PCR product contained deletions in its 5′ exon region. These results indicated that the PCR technique had isolated the trnL (UAA) gene and its pseudogene.

Sequence of thetrnH gene and the inverted repeat structure deletion site of the broad bean chloroplast genome

Sequence of the<i>trn</i>H gene and the inverted repeat structure deletion site of the broad bean chloroplast genome

Points of rearrangements between plastid chromosomes: location of protein coding regions on broad bean chloroplast DNA

By using genes from spinach and tobacco chloroplast DNA as probes we have localized 22 protein genes on a refined map of Vicia faba (broad bean) chloroplast DNA. The genes are rbcL, rpoA, psaA,B, psbA–E, atpA,B,E,FH,I, pet-A,B,D, and the ribosomal protein genes rp12, rps7, rps12, and rps19. In addition, we mapped sequences homologous to a conserved open reading frame previously found in an intron of a trnK gene. The results were used to map the relative positions of rearrangements that distinguish the broad bean and spinach plastid DNAs. By hybridization with small probes several regions on both DNAs with sequence homology to more than one chromosomal position were found. Intermediate segments are rearranged in broad bean relative to spinach. A spinach chloroplast DNA fragment that functions as an autonomously replicating sequence in yeast is shown to be missing in broad bean chloroplast DNA. This ars is located at a position in spinach DNA which has been a target for rearrangement in the broad bean chromosome. These results can be interpreted by assuming that the DNAs contain short repeat sequences at some conserved positions. We suggest a model for chloroplast DNA divergence assuming that short repeats exist which are the substrate for a recombination system. The few rearrangements between chloroplast chromosomes from a variety of plants may be explained as the result of selection against short repeats.

Ubiquity of the genes for components of a NADH dehydrogenase in higher plant chloroplast genomes

Sequences homologous to the six tobacco chloroplast genes, ndhA-F, which correspond to the genes for components of the human mitochondrial NADH dehydrogenase, have been identified in rice, sugar beet and broad bean chloroplast genomes. The locations of the six ndh sequences are essentially conserved among these chloroplast genomes. The results indicate that the ndh sequences are widespread in chloroplast genomes of higher plants.

Gene organization of chloroplast DNA from the broad bean Vicia faba

Six protein genes have been mapped on broad bean chloroplast DNA by Southern hybridization using the tobacco chloroplast genes as probes. In broad bean chloroplast DNA, the genes for the β and e subunits of proton-translocating ATPase and the 32,000 dalton thylakoid membrane protein are located near the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase. The gene for the α subunit of proton-translocating ATPase is distantly located from the β and e genes. The gene for the ribosomal protein CS19 was found close to the ribosomal RNA operon. The gene organization of broad bean chloroplast DNA is therefore quite different from that of tobacco chloroplast DNA. The nucleotide sequence of the spacer region between the large subunit and the β genes of the broad bean has been determined. Conserved sequences are found among the putative promoter regions of the chloroplast protein genes.

Nucleotide sequence of the split tRNA UAA Leu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

The gene encoding the tRNA UAA Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA UAA Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5′-and 3′-flanking regions of the broad bean and maize chloroplast tRNA UAA Leu genes.

Nucleotide sequence of a 1.1 kb fragment of the pea chloroplast genome containing three tRNA genes, one of which is located within an open reading frame of 91 codons.

The nucleotide sequence of a 1082 bp fragment from the pea (Pisum sativum) chloroplast genome is presented. This fragment contains genes for tRNAGlu, tRNATyr and tRNAAsp as well as an open reading frame (ORF) of 91 codons on one strand and two ORFs of 52 and 59 codons on the complementary strand. The tRNAAsp gene is located entirely within the ORF of 91 codons. The first 366 bp of the fragment correspond to 376 bp at one end of a recently published (1) sequence from the broad bean (Vicia faba) chloroplast genome. These regions contain the tRNAGlu and tRNATyr genes, which are identical and separated by 60 bp in both species. These two genes are probably cotranscribed. The intergenic regions in the corresponding segments from the two species are, except for a 10 bp deletion in the pea sequence, 94% homologous.

Nucleotide sequence of a 2 kbp BamH I fragment of Vicia faba chloroplast DNA containing the genes for threonine, glutamic acid and tyrosine transfer RNAs.

The entire nucleotide sequence of a 2014 bp BamH I fragment from broad bean (Vicia faba) chloroplast DNA containing the genes for tRNAThr (trnT), tRNAGlu (trnE) and tRNATyr (trnY) has been determined. The tRNAGlu and tRNATyr genes are separated by only 60 bp and are probably part of the same transcriptional unit. The tRNAThr gene is located on the complementary strand, 876 bp away from the tRNAGlu gene. This fragment also contains an open reading frame of 82 codons, as well as a series of AT-rich, direct and inverted repeats.

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean ( Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.

Molecular cloning of the genes for ribosomal DNAs from broad bean chloroplast DNA.

Four Eco RI fragments from broad bean chloroplast DNA which were hybridized with chloroplast rRNAs have been cloned into E. coli plasmid pBR 325. Physical maps of the recombinant plasmids have been constructed by cleavage with several restriction endonucleases and Southern blot hybridization with chloroplast rRNAs. Plasmids pBC9, pBC14 and pBC52 contain a 16S rRNA sequence on the 3.5kb Eco RI fragment, a 23S rRNA sequence on the 2.0kb+1.7kb Eco RI fragments and 4.5S-5S rRNA sequences on the 1.8 kb Eco RI fragment, respectively.

Existence of the gene for 4.55 RNA in broad bean chloroplast DNA

Tobacco chloroplast 4.5S RNA hybridized specifically to the 1.16 × 106 dalton Eco RI fragment of broad bean chloroplast DNA. This indicates the existence of the gene for 4.5S RNA in broad bean chloroplasts which have been reported to lack 4.5S-size RNA.

Loss of hill activity in isolated chloroplasts is not directly related to free fatty acid release during ageing

Losses of functional activity in chloroplasts during ageing have often been attributed to the accumulation of free fatty acids released from membrane lipids by lipase activity. The rate of loss of Hill activity and the rate of release of endogenous free linolenic acid (FLA) were compared in broad bean chloroplasts aged under different conditions. The results suggested that there was little or no correlation between the two parameters. Furthermore, measurements of the loss of Hill activity due to the addition of FLA to freshly isolated chloroplasts indicated that the levels of exogenous FLA required to decrease Hill activity to the same extent as that observed in ageing chloroplasts were at least 10-fold higher than the corresponding endogenous levels. It is, therefore, suggested that free fatty acid (FFA) release is likely to be a symptom, rather than one of the prime causes, of ageing in chloroplasts.

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 106 dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.

Requirement of alkaline hydrolysis for estimating chloroplast RNA using the orcinol reaction

Considerable amounts of orcinol-reacting material other than RNA-ribose were extracted with hot PCA from isolated spinach and broad bean chloroplasts, while cold PCA only incompletely extracted the chloroplast RNA. The procedure utilizing alkaline hydrolysis followed by an orcinol test was found to be appropriate for measuring the chloroplast RNA.

Studies with Chloroplast and Mitochondrial DNA: I. Evidence of Sequence Homology between Chloroplast and Nuclear DNA (Broad Bean) and between Mitochondrial and Nuclear DNA (Rat Liver)

A mixture of broad bean chloroplast and nuclear DNA or rat liver mitochondrial and nuclear DNA was taken through a heating and annealing cycle, and examined by CsCl density gradient centrifugation. The formation of intermediates between the two DNAs during joint annealing was used as a method of detecting sequence homology in different DNA samples. Homology was found between chloroplast and nuclear DNA from broad bean and between mitochondrial and nuclear DNA from rat liver. Since this method produces only qualitative data no implication for possible nucleocytoplasmic relationship can be assessed.

Base composition of DNA from chloroplasts and nuclei of Phaseolus vulgaris.

WHEN the first reports of the isolation and biochemical study of chloroplast DNA from higher plants appeared in 1963, there was a discrepancy between the two sets of findings. J. T. O. K.1,2, using chemical analysis, reported that broad bean chloroplast DNA had a guanine-cytosine content (37.4 per cent GC) close to, but slightly (and significantly) lower than, that of the nuclear DNA (39.4 per cent GC). Chun, Vaughan and Rich3 using spinach and beet, and later other workers using swiss chard4, tobacco5 and other plants6, reported, however, that DNA from chloroplast preparations analysed by CsCl equilibrium density gradient centrifugation contained—in addition to a component of similar density to nuclear DNA—one or two components of much higher density (and therefore of much higher GC content): these dense components they considered to be the true chloroplast DNA. Recent and more rigorous studies7–9 have now cast considerable doubt on the identification of these high density bands with chloroplast DNA.

Inhibition of Photosynthetic Electron Transport by Ioxynil

THE herbicide ioxynil (4-hydroxy-3,5-diiodo benzonitrile)1 has been found to inhibit the photoreduction of ferricyanide by chloroplasts from a range of plants2 and also NADP photoreduction, the accompanying photophosphorylation and carbon dioxide fixation by the broad bean (Vicia faba) chloroplasts3. Recently, Paton and Smith4 further reported that ioxynil inhibited the photoreduction of endogenous plastoquinone in broad bean chloroplasts, even in the presence of ascorbate and indophenol. It was previously found5 that although o-phenanthroline and the herbicide DCMU inhibit the photoreduction of endogenous plastoquinone they do not inhibit the enhanced photoreduction in the presence of ascorbate and indophenol. Furthermore, ascorbate and indophenol reduce plastoquinone in darkness6, in a reaction which has been shown to be non-enzymatic7. No other inhibitors previously investigated have inhibited the dark reduction and we therefore decided to investigate the effects of ioxynil on plastoquinone reduction in more detail.

Studies on RNA synthesis in chloroplast preparations

It has been found (Kirk, 1964) that highly purified preparations of broad bean chloroplasts incorporate radio-activity from 14C-ATP into a substance having the properties of RNA. The reaction is similar in certain respects (requirement for all four nucleoside triphosphates, sensitivity to deoxyribonuclease and actinomycin D) to the RNA polymerase enzymes that have been found in other organisms ( Weiss, 1960; Hurwitz et al. , 1960 ; Stevens, 1960; Huang et al. , 1960 ; Chamberlin and Berg, 1962). It was suggested that the activity is due to a chloroplast RNA polymerase, working with chloroplast DNA as a template. Evidence has now been obtained by other workers (Dr. S.G. Wildman, personal communication) for RNA synthesis in tobacco chloroplast preparations. In this paper, further studies on the enzyme are described, with particular reference to apparent differences between the reactions catalyzed by the chloroplast and nuclear fractions.