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J Browse

Publications and source records attributed to J Browse.

At least 55 records · Page 3Linked to original sources
PubMed1990

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism.

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degrees C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury-leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of (14)CO(2) into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.

Journal Article↗
PubMed1989

The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester.

A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P. D. Beremand et al. (1987) Arch. Biochem. Biophys. 256, 90-100). After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D. J. Guerra et al. (1988) J. Biol. Chem. 263, 4386-4391). We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation. We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis. This result reflects previous work (D. S. Guerra et al. (1986) Plant Physiol. 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants. The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E. coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.

Acyl Carrier Protein↗
PubMed1989

A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase.

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated fadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [(14)C]acetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid omega-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated-presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes.

Journal Article↗
PubMed1989

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation.

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C(16:1) and C(18:1) lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.

Journal Article↗
PubMed1989

Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity.

Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28 degrees C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

Journal Article↗
PubMed1989

A mutant of Arabidopsis deficient in desaturation of palmitic Acid in leaf lipids.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast omega9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast omega9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0.

Journal Article↗
PubMed1989

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation.

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast omega9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO(2) fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28 degrees C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.

Journal Article↗
PubMed1988

Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity.

The leaf membrane lipids of many plant species, including Arabidopsis thaliana (L.) Heynh., are synthesized by two complementary pathways that are associated with the chloroplast and the endoplasmic reticulum. By screening directly for alterations in lipid acyl-group composition, we have identified several mutants of Arabidopsis that lack the plastid pathway because of a deficiency in activity of the first enzyme in the plastid pathway of glycerolipid synthesis, acyl-ACP:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (where ACP is acyl carrier protein). The lesion results in an increased synthesis of lipids by the cytoplasmic pathway that largely compensates for the loss of the plastid pathway and provides nearly normal amounts of all the lipids required for chloroplast biogenesis. However, the fatty acid composition of the leaf membrane lipids of the mutants is altered because the acyltransferases associated with the two pathways normally exhibit different substrate specificities. The remarkable flexibility of the system provides an insight into the nature of the regulatory mechanisms that allocate lipids for membrane biogenesis.

Journal Article↗
PubMed1988

Characterization of an HSP70 Cognate Gene Family in Arabidopsis.

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5' regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.

Journal Article↗
PubMed1987

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis.

A mutant of Arabidopsis thaliana with reduced content of C(18:3) and C(16:3) fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO(2)-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.

Journal Article↗
PubMed1986

Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the '16:3' plant Arabidopsis thaliana.

The kinetics of [1-14C]acetate incorporation in Arabidopsis thaliana L. (Heyn) showed almost equal labelling of phosphatidylcholine (PC) and diacylgalactosylglycerol (DGG) at early times and the transfer of radioactivity from PC to DGG and diacyldigalactosylglycerol (DDG) at longer times. These kinetics demonstrated the parallel operation of the prokaryotic and eukaryotic pathways of lipid synthesis [Roughan & Slack (1982) Annu. Rev. Plant Physiol. 33, 97-132] in this tissue. At 2 h after the application of [1-14C]acetate, more than 85% of the radioactivity at the sn-2 position of each chloroplast lipid was in 16-carbon fatty acids. However, after 60 h, molecular species containing labelled C18 fatty acids at position sn-2 and presumably derived from microsomal PC made a large contribution (20-70%) to each chloroplast lipid except phosphatidylglycerol. These findings are consistent with the contention that the chain length of the fatty acid at the sn-2 position of glycerol is an accurate predictor of whether a particular lipid molecule has been synthesized by the prokaryotic or eukaryotic pathway. At 30 min after the start of [1-14C]acetate labelling, only 12.3% of the radioactivity in PC was in saturated fatty acids, but the proportion increased steadily to 24.3% after 142 h. It is suggested that steps involved in the conversion of PC to chloroplast lipids on the eukaryotic pathway discriminate against palmitate-containing species. The step involved does not appear to be transfer of PC to the chloroplast because extrachloroplastic and chloroplast membranes purified from Arabidopsis mesophyll protoplasts each contained PC with a fatty acid composition similar to that of the same lipid from leaves. Positional analysis of unlabelled lipids, together with the information summarized above, is used to construct a quantitative scheme of the fluxes through the prokaryotic and eukaryotic pathways during lipid synthesis in Arabidopsis. This scheme shows that 38% of the fatty acids synthesized de novo in the chloroplast enter the prokaryotic pathway in the chloroplast envelope. Of the 62% which are exported as acyl-CoA species to enter the eukaryotic pathway, 56% (34% of the total) are returned to complete synthesis of the chloroplast's complement of glycerolipids.

Acetates↗
PubMed1986

Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue.

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.

Chromatography, Gas↗
PubMed1986

A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids.

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [(14)C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26 degrees C but are relatively minor below 18 degrees C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites.

Journal Article↗
PubMed1985

Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid.

Several lines of evidence support the proposal that the unusual chloroplast-specific lipid acyl group Delta3,trans-hexadecenoic acid (trans-C(16:1)) stimulates the formation or maintenance of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C(16:1) by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C(16:1) with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C(16:1) is very subtle or is only conditionally expressed.

Journal Article↗
PubMed1984

Glycerolipid labelling kinetics in isolated intact chloroplasts.

Glycerolipid synthesis was studied in intact chloroplasts isolated from three different plant species. The sequential acylation of sn-glycerol 3-phosphate and lysophosphatidate (1-acyl-sn-glycerol 3-phosphate) was confirmed by monitoring the incorporation of oleate synthesized in situ into lysophosphatidate, phosphatidate and diacylglycerol. Lysophosphatidate was not only readily detected in these experiments, but was also present in the chloroplasts at the beginning of the time courses. The rate of glycerolipid synthesis depended primarily on sn-glycerol 3-phosphate supply, and given adequate sn-glycerol 3-phosphate, the proportion of newly synthesized fatty acids diverted into glycerolipids appeared to be determined by differing acyltransferase activities in the chloroplasts isolated from different plant species.

Acetates↗
PubMed1982

Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat.

A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-(14)C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid (14)C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of (14)C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast.

Journal Article↗
PubMed1981

Light control of fatty acid synthesis and diurnal fluctuations of fatty acid composition in leaves.

1. Although isolated spinach chloroplasts were almost entirely (greater than 99%) dependent on light for fatty acid synthesis, leaf discs were capable of fatty acid synthesis in the dark (up to 500nmol of 3H/h per mg of chlorophyll equivalent to approx. 400nmol of carbon/h per mg of chlorophyll), which represented 12-20% of the corresponding 'light rates'. 2. Net fatty acid accumulation by greening maize leaves occurred largely or entirely during the light period. 3. There was a diurnal fluctuation in the proportions of C18 unsaturated fatty acids in the lipids of developing spinach leaves, where an increase in the concentration of oleate during the day and a subsequent decline at night was observed; a complementary change occurred in the concentration of alpha-linolenate. The rhythm is interpreted as reflecting the continuation of oleate and linoleate desaturation at high rates when oleate synthesis is markedly decreased at night. 4. Changes in the fatty acid composition of 3-sn-phosphatidylcholine accounted for at least 60% of the total decrease in oleate over the dark period. This result is consistent with suggestions that this lipid is the substrate for the leaf microsomal oleate desaturase and an intermediate in leaf glycerolipid biosynthesis.

Chloroplasts↗