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°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.     

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.     

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.     

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 CO₂-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.     

Changes in cell permeability following a marked reduction of saturated fatty acid content of Escherichia coli K-12

The present study examines the extent to which the fatty acid composition of the membrane lipid can be altered by nutritional means in mutants of Escherichia coli defective in total fatty acid synthesis. These changes are compared to those observed in wild type cells subjected to the same conditions of fatty acid supplementation. Abnormalities in physiological behavior of whole cells and membranes are related to extremes in fatty acid composition that can be produced in the mutant but not the wild type cells. In particular, when the saturated fatty acid of the membrane lipid is reduced below approx. 15% the barrier properties of the membrane toward small molecules such as K⁺ and a lactose analog decreases abruptly. This change is also reflected in the diminished temperature dependence of passive permeability and of NADH oxidase activity associated with the cytoplasmic membrane. Detailed studies on the properties of specific membrane function in relation to the physical behavior of membrane lipids should be possible with this biological system possessing a relatively simple membrane lipid structure in which the mole percentage of specific lipid components can be systematically varied.     

Cadmium-Induced Changes in Chloroplast Lipids and Photosystem Activities in Barley Plants

Fatty acid content and composition of chloroplast membranes, ethylene production associated with thylakoid lipids degradation as well as photosynthetic electron transport involving photosystems 1 and 2 were used to determine the effects of increasing Cd concentrations in the growth medium [0, 14, 28, and 42 mg (Cd) kg^–1(sand)] on the photosynthetic performance of barley plants (H. vulgare L., cv. CE9704). High concentrations of Cd triggered serious disturbances of the chloroplast membranes. Ethylene production increased whereas a drop of 18:3 fatty acid content occurred, indicating that Cd mediates lipid peroxidation in the thylakoids. The enhanced ethylene production could be used as an early indicator of Cd-induced membrane degradation, yet at very high concentration (42 mg kg^–1) Cd decreased ethylene production.     

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 ω9 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 ω9 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.     

Chloroplast Ultrastructure in Mutant Strains of Chlamydomonas reinhardi Lacking Components of the Photosynthetic Apparatus

The fine structure of the chloroplast of wild-type and 9 photosynthetic mutant strains of Chlamydomonas reinhardi is described. The chloroplast phenotypes of the mutant strains are clearly distinct from the wild type in all but 2 cases. Moreover, strains with similar photosynthetic disabilities have structurally similar chloroplasts. These differences are apparently not the result of altered chlorophyll content, nor of photosynthetic inactivity. It is therefore proposed that the structural alterations are in some way related to the mutant strains' inability to synthesize active components of the photosynthetic electron transport chain.     

Relationship between Chloroplast Membrane Fatty Acid Composition and Photosynthetic Response to a Chilling Temperature in Four Alfalfa Cultivars

The photosynthetic responses of four alfalfa (Medicago sativa L.) cultivars to 10 and 22 C air temperatures were examined and the relationship between the photosynthetic response at 10 C and the fatty acid composition of the chloroplast membranes was determined. Chilling-resistant cultivars exhibited moderate reductions in photosynthesis at 10 C, compared to 22 C, and contained a significantly greater percentage of polyunsaturated fatty acids in the chloroplast membrane and a greater double bond index than the chilling-sensitive cultivars. The chilling-sensitive cultivars exhibited severe reductions in photosynthesis at 10 C, compared to 22 C. The reduction in photosynthesis at 10 C is shown to be negatively correlated (r = −0.94) with the double bond index of the chloroplast membranes of the cultivars observed.

The results support the hypothesis that reduced photosynthesis due to chilling temperatures is influenced by the unsaturated fatty acid composition of the chloroplast membrane which affect temperature-induced phase changes in chloroplast membrane lipids.

     

Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis

A mutant strain, Y₉, of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y₉ cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y₉ cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y₉. The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed.     

Trienoic fatty acids are required to maintain chloroplast function at low temperatures

The chloroplast membranes of all higher plants contain very high proportions of trienoic fatty acids. To investigate how these lipid structures are important in photosynthesis, we have generated a triple mutant line of Arabidopsis that contains negligible levels of trienoic fatty acids. For mutant plants grown at 22 degrees C, photosynthetic fluorescence parameters were indistinguishable from wild type at 25 degrees C. Lowering the measurement temperature led to a small decrease in photosynthetic quantum yield, Phi(II), in the mutant relative to wild-type controls. These and other results indicate that low temperature has only a small effect on photosynthesis in the short term. However, long-term growth of plants at 4 degrees C resulted in decreases in fluorescence parameters, chlorophyll content, and thylakoid membrane content in triple-mutant plants relative to wild type. Comparisons among different mutant lines indicated that these detrimental effects of growth at 4 degrees C are strongly correlated with trienoic fatty acid content with levels of 16:3 + 18:3, approximately one-third of wild type being sufficient to sustain normal photosynthetic function. In total, our results indicate that trienoic fatty acids are important to ensure the correct biogenesis and maintenance of chloroplasts during growth of plants at low temperatures.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.     

Photosynthetic Properties of ac-31, a Mutant Strain of Chlamydomonas reinhardi Devoid of Chloroplast Membrane Stacking

A pale-green mutant strain of Chlamydomonas reinhardi, ac-31, is characterized by the absence of any stacking of its chloroplast membranes. The capacity for photosynthetic electron transport, phosphorylation, and CO₂ fixation in ac-31 is substantial, and it is concluded that these photosynthetic activities occur within the single membrane. The photosynthetic capacities of wild type and ac-31 as a function of increasing light intensity are compared. Saturation is attained at higher light intensities in ac-31, and the kinetics of the 2 sets of curves are distinctly different. The possibility that energy transfer is enhanced by membrane stacking is suggested by these results. The repeatedly-observed correlation between reduced stacking and disfunctional Photosystem II activities is discussed in view of the observation that ac-31 has no stacking but retains a functional Photosystem II.     

Structural Characteristics of a Photosynthetic Mutant of Euglena gracilis Blocked in Photosystem II

The techniques of thin sectioning and freeze etching were employed in comparing the chloroplast structure of the wild type and photosynthetic mutant P₄ of Euglena gracilis, Z strain. The mutant chloroplasts were characterized by a lack of thylakoid pairing even under high salt conditions. In addition the mutant thylakoids were more varied in size and fewer in number than those of the wild type. No differences between the mutant and wild type were observed in the size and distribution of the particles within the chloroplast membranes seen by the freeze-etching technique.     

Membrane Polypeptides associated with Photochemical Systems

WE wish to report a specific relationship between certain chloroplast membrane polypeptides and functional properties of the chloroplast usually associated with one or the other of the two photochemical systems (designated PSI and PSII). We have known this by the analysis of the chloroplast membrane polypeptides of the wild type strain of the unicellular green alga Chlamydomonas reinhardi and of mutant strains derived from it which have lost the capacity to carry out normal photosynthesis. These mutants have been characterized by the loss of particular membrane-bound components of the photosynthetic electron transport chain^1,2. This relationship is supported by analysis of the membrane polypeptides obtained from chloroplast fractions of wild type C. reinhardi and spinach enriched for reactions characteristic of either PSI or PSII by fractionation of the membranes either with digitonin^3,4 or ‘Triton X-100’⁵.     

Expression of the Yeast Delta-9 Fatty Acid Desaturase in Nicotiana tabacum

To examine the processes of plant cytoplasmic fatty acid desaturation and glycerolipid biosynthesis, the protein coding sequence of the endoplasmic reticulum cytochrome b₅-dependent, Δ-9 fatty acid desaturase gene from Saccharomyces cerevisiae was introduced into Nicotiana tabacum via Agrobacterium transformation. All transformed plants expressing the yeast gene at the mRNA level exhibited an approximately 10-fold increase in the levels of palmitoleic acid (16:1) in leaf tissue. This fatty acid species is found in very low levels (less than 2%) in wild-type plants. These results indicate that the yeast desaturase can function in plants, presumably by using a leaf microsomal cytochrome b₅-mediated electron transport system. Lipid analysis demonstrated that the overproduced 16:1 is incorporated into most of the major polar lipid classes, including the cytoplasmically produced “eukaryotic” fraction of the chloroplast galactolipids. 16:1 was not found, however, in phosphatidyl glycerol, which is considered to be produced almost exclusively in the chloroplast. Despite these changes in membrane lipid composition, no obvious phenotypic differences were apparent in the transformed plants. Positional analysis shows that the cytoplasmically produced 16:1 is found primarily in the sn-2 position of phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol. The positional data suggest that the sn-2 acyltransferases responsible for the “eukaryotic” arrangement of 16- and 18- carbon fatty acids in glycerolipids are selective for unsaturated fatty acids rather than chain length.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.     

Homogeneous catalytic hydrogenation of lipids in the photosynthetic membrane: Effects on membrane structure and photosynthetic activity

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the homogeneous catalytic hydrogenation of the unsaturated fatty acid residues of membrane lipids. The modified membrane was investigated by electron microscopy, electron-spin resonance and fluorescence polarization methods. Alteration in the functional characteristics of the hydrogenated membrane was monitored by the measurement of photophosphorylation and electron-transport activities. The following results were found. (a) Saturation of 10% of the fatty acyl double bonds induced a definite decrease in the dimension of both thylakoids and loculi. Microdensitometry showed that these structural changes arose from a thickening of the single membranes with a simultaneous decrease in the spacing between membranes. These changes might be accounted for by the alignment of the hydrocarbon chains of saturated lipids and the increased hydrophobicity of the membranes. (b) The orientational pattern of chlorophyll-a molecules was not altered by saturating up to 50% of fatty acyl double bonds in membrane lipids, indicating that the energy-transfer processes amongst the chlorophyll molecules remained functional after hydrogenation. (c) Saturation of double bonds of lipids inhibited whole electron transport prior to the inhibition of Photosystem II and Photosystem I activity, which may suggest that the unsaturation level of fatty acids plays a crucial role by ensuring the lateral mobility of plastoquinone between Photosystem II and Photosystem I.     

Growth temperature effects on thylakoid membrane lipid and protein content of pea chloroplasts

The lipid composition and level of unsaturation of fatty acids has been determined for chloroplast thylakoid membranes isolated from Pisum sativum grown under cold (4°/7°C) or warm (14°/17°C) conditions. Both the relative amounts of lipid classes and degree of saturation were not greatly changed for the two growth conditions. In cold-grown plants, there was a slightly higher linolenic and lower linoleic acid content for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol. In contrast to thylakoid membranes, a non-thylakoid leaf membrane fraction including the chloroplast envelope, had a higher overall level of fatty acid unsaturation in cold-grown plants due mainly to an increase in the linolenic acid content of MGDG, DGDG, phosphatidylglycerol, and phosphatidylcholine. The most clear cut change in the thylakoid membrane composition was the lipid to protein ratio which was higher in the cold-grown plants.