Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Low Temperature Development Induces a Specific Decrease in trans-Delta-Hexadecenoic Acid Content which Influences LHCII Organization

Lipid and fatty acid analyses were performed on whole leaf extracts and isolated thylakoids from winter rye (Secale cereale L. cv Puma) grown at 5°C cold-hardened rye (RH) and 20°C nonhardened rye (RNH). Although no significant change in total lipid content was observed, growth at low, cold-hardening temperature resulted in a specific 67% (thylakoids) to 74% (whole leaves) decrease in the trans-Δ³-hexadecenoic acid (trans-16:1) level associated with phosphatidyldiacylglycerol (PG). Electron spin resonance and differential scanning calorimetry (DSC) indicated no significant difference in the fluidity of RH and RNH thylakoids. Separation of chlorophyll-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the ratio of oligomeric light harvesting complex:monomeric light harvesting complex (LHCII₁:LHCII₃) was 2-fold higher in RNH than RH thylakoids. The ratio of CP1a:CP1 was also 1.5-fold higher in RNH than RH thylakoids. Analyses of winter rye grown at 20, 15, 10, and 5°C indicated that both, the trans-16:1 acid levels in PG and the LHCII₁:LHCII₃ decreased concomitantly with a decrease in growth temperature. Above 40°C, differential scanning calorimetry of RNH thylakoids indicated the presence of five major endotherms (47, 60, 67, 73, and 86°C). Although the general features of the temperature transitions observed above 40°C in RH thylakoids were similar to those observed for RNH thylakoids, the transitions at 60 and 73°C were resolved as inflections only and RH thylakoids exhibited transitions at 45 and 84°C which were 2°C lower than those observed in RNH thylakoids. Since polypeptide and lipid compositions of RH and RNH thylakoids were very similar, we suggest that these differences reflect alterations in thylakoid membrane organization. Specifically, it is suggested that low developmental temperature modulates LHCII organization such that oligomeric LHCII predominates in RNH thylakoids whereas a monomeric or an intermediate form of LHCII predominates in RH thylakoids. Furthermore, we conclude that low developmental temperature modulates LHCII organization by specifically altering the fatty composition of thylakoid PG.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma)

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding β-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of β-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.     

Low Temperature-Induced Decrease in trans-Delta-Hexadecenoic Acid Content Is Correlated with Freezing Tolerance in Cereals

The effect of growth at 5°C on the trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol was examined in a total of eight cultivars of rye (Secale cereale L.) and what (Triticum aestivum L.) of varying freezing tolerance. In these monocots, low temperature growth caused decreases in the trans-Δ³-hexadecenoic acid content of between 0 and 74% with concomitant increases in the palmitic acid content of phosphatidyl(d)glycerol. These trends were observed for whole leaf extracts as well as isolated thylakoids. The low growth temperature-induced decrease in the trans-Δ³-hexadecenoic acid content was shown to be a linear function (r² = 0.954) of freezing tolerance in these cultivars. Of the six cold tolerant dicotyledonous species examined, only Brassica and Arabidopsis thaliana L. cv Columbia exhibited a 42% and 65% decrease, respectively, in trans-Δ³-hexadecenoic acid content. Thus, the relationship between the change in trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol and freezing tolerance cannot be considered a general one for all cold tolerant plant species. However, species which exhibited a low growth temperature-induced decrease in trans-Δ³-hexadecenoic acid also exhibited a concomitant shift in the in vitro organization of the light harvesting complex II from a predominantly oligomeric form to the monomeric form. We conclude that the proposed role of phosphatidyl(d)glycerol in modulating the organization of light harvesting complex II as a function of growth temperature manifests itself to varying degrees in different plant species. A possible physiological role for this phenomenon with respect to low temperature acclimation and freezing tolerance in cereals is discussed.     

Differential Detergent Stability of the Major Light-Harvesting Complex II in Thylakoids Isolated from Monocotyledonous and Dicotyledonous Plants

A survey of isolated thylakoids from 11 different higher plant species (Spinacia oleracea L., Pisum sativum L., Vicia faba L., Brassica napus L., Vigna sinensis L., Vinca minor L., Secale cereale L., Triticum aestivum L., Triticosecale Wittn., Hordeum vulgare L., Zea mays L.) indicated that the ratio of the oligomeric:monomeric form of the light-harvesting complex II was twofold higher for the dicots (3.16 ± 0.35) than the monocots (1.64 ± 0.25) examined under identical separation procedures. Under conditions specifically designed to stabilize the oligomeric form in vitro, we show that the oligomeric form of dicot light-harvesting complex II is twice as stable to solubilization in the presence of sodium dodecyl sulfate (SDS) than that observed for monocots. This decreased stability of monocot light-harvesting complex II is associated with a twofold increase in the trienoic fatty acid level of thylakoid phosphatidylglycerol but with no significant changes in the trienoic fatty acid levels in the major galactolipids. In addition, SDS polyacrylamide gel electrophoresis and western blot analyses with monoclonal antibodies indicated that monocots exhibited greater heterogeneity in the polypeptide complements associated with subfractions of light-harvesting complex II than the dicots examined. The data indicate that the oligomeric form of the light-harvesting complex II is not the result of a simple oligomerization of a common monomeric unit. We suggest that the difference in stability of the oligomeric form of light-harvesting complex II in isolated thylakoids of monocots and dicots is probably due to a differential accessibility to SDS. The differential SDS accessibility may be due to differences in thylakoid protein-protein and/or protein-lipid interactions.     

Development at Cold-Hardening Temperatures : The Structure and Composition of Purified Rye Light Harvesting Complex II

Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mm KCl and 10 mm MgCl₂ resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-Δ³-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII₁:LHCII₃) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII₁:LHCII₃ of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LCHII observed in situ and in vitro.     

Effect of cold hardening on sensitivity of winter and spring wheat leaves to short-term photoinhibition and recovery of photosynthesis

Photoinhibition of photosynthesis and its recovery were studied in wheat (Triticum aestivum L.) leaves grown at nonhardening (20°C) and cold-hardening (5°C) temperatures. Cold-hardened wheat leaves were less susceptible to photoinhibition at 5°C than nonhardened leaves, and the winter cultivars, Kharkov and Monopol, were less susceptible than the spring cultivar, Glenlea. The presence of chloramphenicol, a chloroplastic protein synthesis inhibitor, increased the susceptibility to photoinhibition, but cold-hardened leaves still remained less susceptible to photoinhibition than nonhardened leaves. Recovery at 50 μmol m⁻² s⁻¹ photosynthetic photon flux density and 20°C was at least biphasic, with a fast and a slow phase in all cultivars. Cold-hardened leaves recovered maximum fluorescence and maximum variable fluorescence in the dark-adapted state during the fast phase at a rate of 42% h⁻¹ compared with 22% h⁻¹ for nonhardened leaves. The slow phase occurred at similar rates (2% h⁻¹) in cold-hardened and nonhardened leaves. Full recovery required up to 30 h. Fast-recovery phase was not reduced by either lowering the recovery temperature to 5°C or by the presence of chloramphenicol. Slow-recovery phase was inhibited by both treatments. Hence, the fast phase of recovery does not require de novo chloroplast protein synthesis. In addition, only approximately 60% of the photochemical efficiency lost through photoinhibition at 5°C was associated with lost [¹⁴C]atrazine binding and, hence, with damage to the secondary quinone electron acceptor for photosystem II-binding site. We conclude that the decrease in susceptibility to photoinhibition exhibited following cold hardening of winter and spring cultivars is not due to an increased capacity for repair of photoinhibitory damage at 5°C but reflects intrinsic properties of the cold-hardened photosynthetic apparatus. A model to account for the fast component of recovery is discussed.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Leucine transport in cells isolated from cold-hardened and nonhardened winter rye

The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25°C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5°C efflux from the cells was inhibited.     

Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications

The effects of chilling in the light (4 days at 5°C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40°C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze-fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light-harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light-harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Δ³-hexadecenoic acid in the phosphatidyldiacylglycerol fraction.     

Cold-hardening results in increased activity of enzymes involved in carbon metabolism in leaves of winter rye (Secale cereale L.)

Light- and CO₂-saturated photosynthesis of nonhardened rye (Secale cereale L. cv. Musketeer) was reduced from 18.10 to 7.17 mol O₂·m^–2·s^–1 when leaves were transferred from 20 to 5°C for 30 min. Following cold-hardening at 5°C for ten weeks, photosynthesis recovered to 15.05 mol O₂·m^–2·s^–1,comparable to the nonhardened rate at 20°C. Recovery of photosynthesis was associated with increases in the total activity and activation of enzymes of the photosynthetic carbon-reduction cycle and of sucrose synthesis. The total hexose-phosphate pool increase by 30% and 120% for nonhardened and cold-hardened leaves respectively when measured at 5°C. The large increase in esterified phosphate in coldhardened leaves occurred without a limitation in inorganic phosphate supply. In contrast, the much smaller increase in esterified phosphate in nonhardened leaves was associated with an inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose-phosphate synthase activation. It is suggested that the large increases in hexose phosphates in cold-hardened leaves compensates for the higher substrate threshold concentrations needed for enzyme activation at low temperatures. High substrate concentrations could also compensate for the kinetic limitations imposed by product inhibition from the accumulation of sucrose at 5°C. Nonhardened leaves appear to be unable to compensate in this fashion due to an inadequate supply of inorganic phosphate.Abbreviations DHAP dihydroxyacetone phosphate - Fru6P fructose-6-phosphate - Fru 1,6BP fructose-1,6-bisphosphate - Fru1,6BPase fructose-1,6-bisphosphatase - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - CH cold-hardened rye grown at 5°C - NH nonhardened rye grown at 24°C - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - UDPGlc uridine 5-diphosphoglucose This work was supported by operating grants from the Swedish Natural Sciences Research Council to G.Ö. and P.G.     

Characterization of Chloroplasts Isolated from Triazine-Susceptible and Triazine-Resistant Biotypes of Brassica campestris L

Chloroplasts isolated from triazine-susceptible and triazine-resistant biotypes of Brassica campestris L. were analyzed for lipid composition, ultrastructure, and relative quantum requirements of photosynthesis. In general, phospholipids, but not glycolipids in chloroplasts from the triazine-resistant biotype had a higher linolenic acid concentration and lower levels of oleic and linoleic fatty acids, than chloroplasts from triazine-susceptible plants. Chloroplasts from the triazine-resistant biotype had a 1.6-fold higher concentration of t-Δ3-hexadecenoic acid with a concomitantly lower palmitic acid concentration in phosphatidylglycerol. Phosphatidylglycerol previously has been hypothesized to be a boundary lipid for photosystem II. Chloroplasts from the triazine-resistant biotype had a lower chlorophyll a/b ratio and exhibited increased grana stacking. Light-saturation curves revealed that the relative quantum requirement for whole chain electron transport at limiting light intensities was lower for the susceptible biotype than for the triazine-resistant biotype. Although the level of the chlorophyll a/b light-harvesting complex associated with photosystem II was greater in resistant biotypes, the increased levels of the light-harvesting complex did not increase the photosynthetic efficiency enough to overcome the rate limitation that is inherited concomitantly with the modification of the Striazine binding site.     

Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves

Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Δ^3t-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue.     

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 Δ3,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.     

Overwintering Periwinkle (Vinca minor L.) Exhibits Increased Photosystem I Activity

The effects of natural, overwintering conditions on photosystem I and photosystem II activity were examined in isolated thylakoids of periwinkle (Vinca minor L.), an endemic, cold-tolerant, herbaceous evergreen. DCMU-Insensitive photosystem I activity (ascorbate/dichlorophenolindophenol → methylviologen) exhibited a twofold increase in light-saturated rates upon exposure to low temperature and freezing stress with no effect on the apparent quantum yield of this reaction. DCMU-Sensitive photosystem II activity (H₂O → dichlorlophenolindophenol) exhibited only minor fluctuations in light-saturated rates but a 50% decrease in the apparent quantum yield of this reaction upon exposure to overwintering conditions. This was correlated with a decrease in the 77°K fluorescence emission at 694 nanometers. These functional changes occurred with no detectable changes in the relative chlorophyll contents of the chlorophyll-protein complexes or the chlorophyll-thylakoid protein. The chlorophyll a/b varied less than 10% during any single growth year. Analyses of total leaf extracts indicated that all lipid classes exhibited increased levels of linoleic and linolenic acid. Neither the trans-Δ³-hexadecenoic acid level nor the ratio of oligomeric:monomeric light harvesting of photosystem II was affected by exposure to winter stress. The content of the major chloroplast lipids monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidyl-diacyl-glycerol, and sulfoquinovosyldiacylglycerol exhibited minor fluctuations, whereas phosphatidylcholine and phosphatidylethanolamine content doubled on a mole percent or chlorophyll basis. We conclude that the previously reported increase in photosystem I activity during controlled, low temperature growth is observed during exposure to natural overwintering conditions. This appears to occur with minimal changes in the structure and composition of the photosynthetic apparatus of periwinkle.     

Importance of trans-Δ3-hexadecenoic acid containing phosphatidylglycerol in the formation of the trimeric light-harvesting complex in Chlamydomonas

Possible roles of trans-Δ³-hexadecenoic acid containing phosphatidylglycerol (PG) in the organisation of photosynthetic complexes were studied using two mutants of Chlamydomonas reinhardtii, mf1 and mf2, that totally lack this lipid and in which the level of the others remaining PG was consequently reduced to about 30% of the wild-type. Both the mutants have lost the capacity to stabilise the light-harvesting chlorophyll a/b–protein complex LHC II in a trimeric state and display an increased instability of the PS I light-harvesting-core complex after detergent mediated solubilisation. In this paper, we show that a very reduced growth rate of the mutant cells largely reduces the extent of these defects, allowing a significant formation of trimeric LHC II and a stabilisation of the PS I complex, in the absence of synthesis of trans-Δ³-hexadecenoic acid or of increased level of PG. These results seem to be at variance with the generally accepted role of trans-Δ³-hexadecenoic fatty acid (16:1(3t)) in the formation of the PS II light-harvesting antenna. On the other hand, they appear to be consistent with the observation that trimeric LHC II can be formed in the presence of 16:1(3t)-lacking PG in a mutant of Arabidopsis thaliana and in chloroplasts from cotyledons of some Orchideae. We conclude that 16:1(3t)-PG is indeed required for the stabilisation of the trimeric LHC II and of the PS I complex under conditions of high biosynthesis rate, and that it is not essential when these components of the photosynthetic membrane are synthesised at low rates.     

Cadmium-induced changes in the composition and structure of the light-harvesting chlorophyll a/b protein complex II in radish cotyledons

Five-day-old etiolated radish ( Raphanux salivux L. cv. Saxa) seedlings exposed to white continuous light in the presence of Cd²⁺ (0.2 mM) showed characteristic changes in their light-harvesting chlorophyll a/b protein complex II after 48 h of greening. The content of its oligomeric supramolecular form was greatly diminished with a concomitant increase in the level of the monomer. The isolation of highly purified light-harvesting chlorophyll a/b protein complex II from control and Cd²⁺ treated radish cotyledons and a detailed analysis of its structure and composition revealed that first of all, Cd²⁺ altered the content of the specific phosphatidylglycerol fatty acid - trans -Δ³-hexadecenoic acid, widely accepted as a component responsible for the oligomerization of this chlorophyll-protein complex. This fatty acid in the thylakoid membrane phosphatidylglycerol pool seems to be very sensitive to different environmental stresses lowering its content, which indicates the vital significance of this component for the supramolecular organization and proper functioning of the light-harvesting chlorophyll a/b protein complex II.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ∼4 × 10⁷ CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ∼3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10⁷ CFU ml⁻¹ of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ∼3 × 10⁹ CFU ml⁻¹ of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.