E. Maissan

Chloroplast biogenesis at cold-hardening temperatures. Kinetics of trans-Δ3-hexadecenoic acid accumulation and the assembly of LHCII.

Etiolated seedlings developed at cold-hardening temperatures (5°C) exhibited etioplasts with considerable vesiculation of internal membranes compared to etioplasts developed at 20°C regardless of the osmotic concentration employed during sample preparation. This vesiculation disappeared during exposure to continuous light at 5°C. This transformation of 5°C and 20°C etioplasts to chloroplasts under continuous light at 5° and 20°C respectively proceeded normally with the initial development of non-appressed lamellae and the subsequent appearance of granal stacks. However, chloroplasts developed at 5°C exhibited fewer lamellae per granum than chloroplasts developed at 20°C.Although the polypeptide complements of etioplasts and chloroplasts developed at 5° or 20°C were not significantly different, monomeric light harvesting complex (LHCII3) was assembled into oligomeric light harvesting complex (LHCII1) during chloroplast biogenesis at 20°C (oligomer:monomer =1.8) whereas monomeric LHCII predominated at 5°C (oligomer:monomer =0.3). Low temperature fluorescence emission spectra of isolated thylakoids indicated that both the F685/F735 and F695/F735 were significantly higher after greening at 5°C than at 20°C. In addition, chloroplast biogenesis at 5°C was associated with a low ratio of trans-Δ3-hexadecenoic acid (0.5) in phosphatidylglycerol whereas at 20°C biogenesis was associated with a high ratio (1.6). Comparative kinetics indicated that the maximization of the trans-Δ3-hexadecenoic acid level precedes the assembly of monomeric LHCII into oligomeric LHCII during biogenesis at 20°C. It is suggested that low developmental temperatures modulate the assembly of LHCII by reducing the trans-Δ3-hexadecenoic acid content of phosphatidylglycerol such that monomeric or some intermediate form of LHCII predominates.

Membrane Assembly during Acclimation to Low Temperature: Lipid-protein Interaction

It is the prolonged exposure of plants to low temperature under natural conditions which is an absolute requirement for the initiation of a cold acclimation process and the subsequent establishment of a cold-hardy state which imparts the property of freezing resistance to the plant. Huner (1985b) has suggested that cold-acclimation in winter cereals under natural conditions, is a direct consequence of plant growth and development at low, cold-hardening temperatures. Furthermore, Huner (1985b) has shown that leaves of winter rye (Secale cereale L. cv Puma and cv Muskateer) undergo distinct morphological, anatomical and biochemical changes during growth and development at low temperature. The thylakoid membranes of chloroplasts have been used as a model system for identifying the primary lesions induced by freezing (Garber and Steponkus, 1976a; 1976b; Klosson and Krause, 1981). However, we have utilized the thylakoid membrane to examine membrane assembly during growth and development at low temperature in order to understand the role(s) that this process may play in plant acclimation to low temperature. We have reported that the gross polypeptide, pigment and lipid compositions are not significantly altered during development at cold-hardening temperatures.

Phosphatidylglycerol Content and Composition Influence in Vitro Oligomerization of Purified Lhcii From Winter Rye

Williams and co-workers (1) reported that growth and development of winter rye at low, cold-hardening temperatures (5°C) resulted in a specific 72% decrease in the transhexadecenoic acid (trans-16;l) level of PG although the general thylakoid lipid content was not significantly different. The microenvironment of the thylakoid buyer was not significantly altered as indicated by the rotational correlation times of the spin probe, 16-doxyl stearic acid but differential scanning calorimetry (DSC) indicated that both the number of endotherms resolved and their respective transition temperatures were generally reduced in thylakoid membranes developed at 5°C compared to the temperature transitions in thylakoids developed at 20°C. Specifically, the transitions at 60°C and 73°C were not resolved in 5°C thylakoids and have been associated with PSII and LHCII organization respectively (Low, personnal communication). In addition, in situ results based on freeze fracture (2) and Chl fluorescence (3) are consistent with in vitro results (1) which indicated that the oligomeric form of LHCII predominates during development at 20°C whereas the monomeric or an intermediate form predominates upon development of rye at 5°C. Williams et al (1) concluded that low developmental temperature modulates the organization of LHCII by specifically affecting the fatty acid composition of PG.

3-Transhexadecenoic Acid Content and Lhcii Organization During Chloroplast Biogenesis at Low Temperature

Recently we have reported low temperature (5°C) development of winter rye (Secale cereale L. cv Puma) results in a specific 72% decrease in the level of 3-transhexadecnoic acid leves (3-trans-16:l) associated with phosphatidlyglycerol (PG). This was correlated with a 2-fold decrease in the ratio of oligomeric (LHCII1): monomeric (LHCII3) in 5°C thylakoids (1). Purified LHCII from both 5° and 20°C thylakoids exhibited a 5-fold enrichment in PG. However, purified LHCII from 5°C thylakoids exhibited a 50% decrease in the level of 3-trans-16:l associated with its PG compared to purified LHCII from 20°C thylakoids. This was also correlated with a 60% lower LHCII1: LHCII3 for purified 5°C LHCII than purified 20°C LHCII (2). It was concluded that oligomeric LHCII predominates in 20°C thylakoids whereas a monomeric or an intermediate form of LHCII predominates upon development at 5°C. Low developmental temperature modulates LHCII organization by specifically lowering the 3-trans-16:l content of rye thylakoid PG. In this report we examine the biogenesis of thylakoid membranes in the primary leaves of winter rye during the transition from etioplast to mature chloroplast under conditions of continuous light at either 5° or 20°C.

The Dynamic Properties of Anacystis nidulans Thylakoids Containing Increased Levels of Oleic Acid in the Membrane Glycerolipids

Summary The aim of this study was to investigate the extent to which changes in fatty acid composition of the glycerolipids of Anacystis nidulans elicit changes in fluidity of the thylakoid membranes measured by two different spin probes. After the addition of oleic acid (18:1) to the culture medium of A. nidulans , the level of 18:1 in monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG) and sulphoquinovosyldiacylglycerol (SL) in the cells increased continuously for a period up to 96 hours. Levels of 18:1 up to 70 % were found in all glycerolipids while concomitant decreases occurred in the levels of the natural palmitic (16:0) and palmitoleic (16:1) acids. Following this modulation of the membrane fatty acid composition, the fluidity of the thylakoid membranes was determined by measuring the mobility of nitroxide spin probes 7- and 16-SASL. The fluidity of A. nidulans thylakoid membranes increases with the increase of the 18:1 incorporation to the membrane lipids. For example, an decrease in the rotational correlation time ( c ) of 16-SASL to 2.5 ns at 10 °C was found in cells grown for 72 hours in a medium enriched with 18: 1 compared to 4.0 ns of the same spin probe embedded in normal membranes of A. nidulans . Characteristic break points were determined at 10 °C and 25 °C in Arrhenius plots of c of 16-SASL, outer hyperfine splitting (2T ∥ ) and order parameter S of 7-SASL in normal control cells of A. nidulans . After 24 hours culture in 18:1-enriched media the increase in the content of 18:1 resulted in an increase in membrane fluidity and continued to display a clear break point at 25 °C. However, the samples after 72 or 96 hours growth in 18:1-enriched media show no break points and a monotonous increase in fluidity with temperature. On the basis of this experiment it was concluded that the induced change in lipids causes all together an increase in the membrane fluidity of the cells.

In Vivo Low Temperature-Induced Decrease in 3-Transhexadecenoic Acid Influences Oligomerization of LHCII

Winter rye (Secale cereale L cv Puma) is dependent on low growth temperature in order to develop a photosynthetic apparatus capable of efficient processing of light energy and reduction of CO2 to carbohydrates (1). Functionally, Puma rye leaves developed at 5°C exhibit a 70% greater capacity to utilize CO2 at low temperature than rye leaves developed at 20°C (2). This was correlated with light-saturated rates of whole chain electron transport (H2O ----> MV) which were 40% higher in thylakoids isolated from 5°C leaves than those isolated from 20°C leaves (3). In addition, 77°K fluorescence emission spectra and room temperature fluorescence induction measurements in the presence of DCMU indicated that the energy distribution between LHCII and PSII reaction centres and between PSI and PSII reaction centres had been altered upon growth and developement at low temperature (4). Griffith et al (5) concluded that development of rye leaves at low temperature results in an alteration in protein-protein interactions associated with LHCII. This is consistent with an earlier report which indicated that 5°C thlakoids exhibited a decrease in particle size on the EF fracture face (6). However, Huner and co-workers (6, 7) have reported that no significant differences between 5°C and 20°C thylakoids exist with respect to pigment or polypeptide composition. In this report we present preliminary evidence which indicates that low temperature developemnt results in a specific alteration in the fatty composition of thylakoid phosphatidylgycerol which, in turn, affects the structural organization of LHCII.