Biomedical subjects

R M Leech

Publications and source records attributed to R M Leech.

At least 19 recordsLinked to original sources

PubMed2000

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes.

The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L.

Acyl-CoA Oxidase↗

PubMed1999

The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis.

ARC (accumulation and replication of chloroplasts) genes control different aspects of the chloroplast division process in higher plants. In order to establish the hierarchy of the ARC genes in the chloroplast division process and to provide evidence for their specific roles, double mutants were constructed between arc11, arc6, arc5, arc3 and arc1 in all combinations and phenotypically analysed. arc11 is a new nuclear recessive mutant with 29 chloroplasts compared with 120 in wild type. All the phenotypes of the double mutants are unambiguous. ARC1 down-regulates proplastid division but is on a separate pathway from ARC3, ARC5, ARC6 and ARC11. ARC6 initiates both proplastid and chloroplast division. ARC3 controls the rate of chloroplast expansion and ARC11 the central positioning of the final division plane in chloroplast division. ARC5 facilitates separation of the two daughter chloroplasts. ARC5 maps to chromosome 3 and ARC11 and ARC6 map approximately 60 cM apart on chromosome 5.

Arabidopsis↗

PubMed1999

Chloroplast acclimation in leaves of Guzmania monostachia in response to high light.

Acclimation of leaves to high light (HL; 650 micromol m(-2) s(-1)) was investigated in the long-lived epiphytic bromeliad Guzmania monostachia and compared with plants maintained under low light (LL; 50 micromol m(-2) s(-1)). Despite a 60% decrease in total chlorophyll in HL-grown plants, the chlorophyll a/b ratio remained stable. Additionally, chloroplasts from HL-grown plants had a much lower thylakoid content and reduced granal stacking. Immunofluorescent labeling techniques were used to quantify the level of photosynthetic polypeptides. HL-grown plants had 30% to 40% of the content observed in LL-grown plants for the light-harvesting complex associated with photosystems I and II, the 33-kD photosystem II polypeptide, and Rubisco. These results were verified using conventional biochemical techniques, which revealed a comparable 60% decrease in Rubisco and total soluble protein. When expressed on a chlorophyll basis, the amount of protein and Rubisco was constant for HL- and LL-grown plants. Acclimation to HL involves a tightly coordinated adjustment of photosynthesis, indicating a highly regulated decrease in the number of photosynthetic units manifested at the level of the content of light-harvesting and electron transport components, the amount of Rubisco, and the induction of Crassulacean acid metabolism. This response occurs in mature leaves and may represent a strategy that is optimal for the resource-limited epiphytic niche.

Chlorophyll↗

PubMed1998

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Journal Article↗

PubMed1996

Immunofluorescent quantitation of chloroplast proteins.

Using scanning light microscopy software to detect and measure immunofluorescence in leaf sections Rubisco concentration in situ in chloroplasts has been accurately determined throughout development. The fluorescence measurements were calibrated by comparison with values for Rubisco accumulation obtained from rocket immuno-electrophoresis profiles of soluble protein from isolated cells and from chloroplasts using a purified sample of Rubisco as the standard. It has been shown that in situ immunofluorescence can be used for cytoquantitation of proteins within individual chloroplasts to a sensitivity of 1fg and also for the comparison of the protein levels in adjacent chloroplasts and cells. Several important applications of this new technique are discussed.

Cellular Senescence↗

PubMed1996

A putative Mg chelatase subunit from Arabidopsis thaliana cv C24. Sequence and transcript analysis of the gene, import of the protein into chloroplasts, and in situ localization of the transcript and protein.

We have isolated and sequenced a cDNA from Arabidopsis thaliana cv C24 that encodes a putative Mg chelatase subunit. The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrhinum majus (olive and also high similarity to bchH, a bacterial gene involved in the Mg chelatase reaction of bacteriochlorophyll biosynthesis. We suggest that this gene be called CHL H. Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, ch-42, and ferrochelatase. The CHL H transcript was observed to undergo a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily to a minimum by the end of the light period; in contrast, transcripts for ch-42 and ferrochelatase remained constant. A model is proposed in which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle. In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the ch-42 gene. The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay. Immunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg+. In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo.

Arabidopsis↗

PubMed1996

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.

Arabidopsis↗

PubMed1996

Castor bean isocitrate lyase lacking the putative peroxisomal targeting signal 1 ARM is imported into plant peroxisomes both in vitro and in vivo.

To understand and manipulate plant peroxisomal protein targeting, it is important to establish the universality or otherwise of targeting signals. Contradictory results have been published concerning the nature and location of the glyoxysomal/peroxisomal targeting signal of isocitrate lyase (ICL). L.J. Olsen, W.F. Ettinger, B. Damsz, K. Matsudaira, A. Webb, and J.J. Harada ([1993] Plant Cell 5: 941-952) concluded that the last 5 amino acids (AKSRM) of Brassica napus ICL were sufficient and the last 37 amino acids were necessary for targeting to Arabidopsis leaf peroxisomes. In contrast, R. Behari and A. Baker ([1993]) J Biol Chem 268: 7315-7322) could find no requirement for the almost identical carboxy-terminal sequence AKARM for import of Ricinus communis ICL into isolated sunflower cotyledon glyoxysomes. To resolve this discrepancy, the import characteristics of a mutant R. communis ICL lacking the last 19 amino acids of the carboxy terminus was studied. ICL delta 19 was able to be imported by isolated sunflower glyoxysomes and by tobacco leaf peroxisomes when expressed transgenically. These results demonstrate that the in vitro import system faithfully reflects targeting in vivo, and that the source of the organelles (Arabidopsis versus sunflower, leaf peroxisomes versus seed glyoxysomes) is not responsible for observed differences between B. napus and R. communis ICL. The R. communis enzyme would therefore appear to possess an additional glyoxysome/peroxisome targeting signal that is lacking in the B. napus protein.

Biological Transport↗

PubMed1995

arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices.

The arc6 (accumulation and replication of chloroplasts) mutant of Arabidopsis has only two greatly enlarged chloroplasts per mature leaf mesophyll cell compared with ninety chloroplasts per cell in the wild type. The mutation is a single nuclear gene and the plant phenotype is normal. Shoot and root apical meristems of arc6 plants have been examined to determine how early during plastid development the mutant arc6 phenotype can be recognised. In the cells of the arc6 apical meristem there are only two proplastids, which are larger than wild type with a highly variable morphology. In the cells of the leaf primordia where differentiation of proplastids to chloroplasts occurs arc6 plastids are larger and at a more advanced developmental stage than wild-type plastids. In arc6 root cells statoliths and other plastids also show grossly abnormal morphology and the statoliths are greatly increased in size. During arc6 stomatal guard cell development the perturbation in proplastid population dynamics affects plastid segregation and 30% of stomata lack plastids in one or both guard cells. Our evidence would suggest that ARC6 is expressed throughout the vegetative cells of the Arabidopsis seedling with major effects on both the proplastid phenotype and the proplastid population. ARC6 is the first gene to be identified in Arabidopsis which has a global effect on plastid development in cells arising from both the shoot and root meristems, and is of major importance in the nuclear control of plastid differentiation in higher plants.

Arabidopsis↗

PubMed1992

Chloroplast Division and Expansion Is Radically Altered by Nuclear Mutations in Arabidopsis thaliana.

We have isolated three mutants of Arabidopsis thaliana in which there is a sevenfold change in chloroplast number in fully expanded leaf mesophyll cells and increases and decreases in chloroplast number are compensated for by changes in chloroplast size. The changes are stably inherited in sexual crosses for three generations and mutant phenotypes are effected by changes at single recessive nuclear loci, termed arc loci. This is the first report of large, stably inherited changes in chloroplast number in higher plants, and represents a major advance toward the genetic dissection of the control of chloroplast division.

Journal Article↗

PubMed1991

Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh.

To analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erecta are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell.

Journal Article↗

PubMed1986

Stability and plasticity during chloroplast development.

Chloroplast development occurs during cellular development. In non-limiting conditions chloroplast development is a highly conserved process, it is also complex and involves the continuous interaction of both chloroplast and nuclear genomes. In the first part of the paper the sequential and structural changes characteristic of chloroplast division and development in angiosperms are described. The synthesis of the major chloroplast components including chlorophylls a and b, lipids, nucleic acids and the major soluble and membrane proteins are then described. Chloroplast development in biochemical terms is a quantitative accretion of additional functional units. In development from proplastid to fully mature chloroplast the molecular changes are almost exclusively quantitative and the youngest plastids that can be analysed are already photochemically fully competent. In the second part of the paper the dominant role of the nuclear genome in chloroplast development is discussed. Recent work in the author's laboratory on the synthesis and accumulation of ribulose bisphosphate carboxylase-oxygenase in the developing chloroplasts of young wheat leaves is cited to illustrate the stable genomic and genotypic differences that can be recognized. In comparisons of wheat species of differing ploidy, in hexaploid cultivars and in artificially processed genetic lines, several genomic and genotypic effects have been detected. The possibilities for future investigation are discussed.

Cell Differentiation↗

PubMed1984

Biochemistry of photosynthesis in species of triticum of differing ploidy.

Illuminated flag leaves of Triticum monococcum(2X), T. urartu(2X), T. dicoccum(4X), T. dicoccoides(4X), and T. aestivum(6X) were exposed to (14)CO(2) for 10 seconds and subsequently allowed to continue photosynthesis in the ambient air for periods of up to 2 minutes. The relative distribution of (14)C among water-soluble products in the leaves was similar for each species at each sampling time. After the 10-second pulse of (14)CO(2), radioactivity was mainly in phosphate esters with less than 5% in C(4) acids. Subsequently, radioactivity increased in sucrose, glycine, and serine at the expense of that in phosphate esters. By 2 minutes, between 18% and 29% of the (14)C was in glycine plus serine. The results suggest rapid photorespiration in all species and an absence of C(4) photosynthesis.d-Ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) was partly purified from seedling leaves of each of the five Triticum species. Each preparation was assayed for simultaneous carboxylase and oxygenase activities in 2.1 millimolar NaHCO(3) and 265 micromolar O(2) at pH 8.2 and 25 degrees C. The mean ratio of carboxylase to oxygenase activities was 6.11 +/- 0.16 (standard error); differences between values for different species were not statistically significant. The results do not explain the faster rates of photosynthesis per unit leaf area reported for diploid and tetraploid species of Triticum compared to the hexaploid.

Journal Article↗

PubMed1982

Genome Expression during Normal Leaf Development : I. CELLULAR AND CHLOROPLAST NUMBERS AND DNA, RNA, AND PROTEIN LEVELS IN TISSUES OF DIFFERENT AGES WITHIN A SEVEN-DAY-OLD WHEAT LEAF.

Changes in genome expression during normal cellular and plastid development in the first leaf of young (7-day-old) wheat (Triticum aestivum var. Maris Dove) were investigated by examining homogeneous populations of leaf cells and plastids of several developmental ages present in the same leaf. The cells were characterized over a period immediately following the last cell division. All of the leaf cells had cytoplasmic contents and nuclei, and between 44% (young tissue) and 54% (older tissue) of the leaf cells were mesophyll cells. Chloroplast development was complete 36 hours after the chloroplasts had ceased dividing. Extremely large changes occurred in cellular constituents over a very short period of leaf development. Maximum rates of accumulation of ribulose bisphosphate carboxylase per mesophyll cell (80 picograms/hour), chlorophyll per mesophyll cell (9 picograms/hour), and 70S ribosomes per mesophyll cell (19 x 10(5)/hour) were recorded.Total cellular DNA varied from 40 to 60 picograms/cell, reflecting the changes in nuclear and chloroplast DNA synthesis during different phases of cellular and chloroplast division. The period of maximum accumulation of protein, total RNA, and both 80S and 70S ribosomes occurred between 36 and 48 hours after the last cell division. Between 48 and 60 hours, 70S rRNA per cell and protein content per cell continued to increase as 80S rRNA per cell declined. Ribulose bisphosphate carboxylase per cell increased 20-fold between 15 and 60 hours.

Journal Article↗

PubMed1982

Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves.

Plastids at different stages of development were isolated from light-grown wheat (Triticum aestivum, var. Maris Dove) seedling leaves, and the average chloroplast DNA (cpDNA) per plastid at each developmental stage was measured directly. In the earliest stages of development, the number of plastids per cell and the amount of cpDNA per cell increased with cell age, but cpDNA per plastid remained constant at between 800 and 1,000 genome copies per plastid. After this phase, plastids per cell continued to increase, but cpDNA per plastid decreased. Subsequently, both plastids per cell and cpDNA per plastid remained constant as cell age increased, the final DNA content being approximately 300 genome copies per plastid. These results are related to previous reports of cpDNA changes during the development of dicotyledonous plants, and to theories about the regulation of chloroplast numbers per cell.

Journal Article↗

PubMed1982

Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat.

The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2x), Triticum dicoccum (4x), and Triticum aestivum (6x). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2x, 4x, and 6x ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content.

Journal Article↗

PubMed1981

Localization of DNA in Mature and Young Wheat Chloroplasts Using the Fluorescent Probe 4'-6-Diamidino-2-phenylindole.

The spatial organization of chloroplast DNA in developing and dividing wheat chloroplasts was studied in the light microscope using the fluorescent probe 4'-6-diamidino-2-phenylindole, which binds specifically to DNA.The DNA of wheat chloroplasts was localized at the periphery of the plastid, frequently in a discrete band. No relocalization of the DNA was observed during plastid replication. This peripheral location of the DNA was shown to differ from the central random location of DNA in tobacco and spinach chloroplasts.

Journal Article↗

PubMed1981

Photosynthesis of Lipids from CO(2) in Spinacia oleracea.

Young expanding spinach leaves exposed to (14)CO(2) under physiological conditions for up to 20 minutes assimilated CO(2) into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the (14)C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO(2)in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H(14)CO(3) into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.

Journal Article↗