Cleavage of Chlorophyll-Porphyrin (Requirement for Reduced Ferredoxin and Oxygen)

The chemical structures of some colorless catabolites that accumulate in senescent leaves have been established recently (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346; W. Muhlecker, B. Krautler, S. Ginsburg, P. Matile [1993] Helv Chim Acta 76: 2976-2980). Such studies suggest that oxygenolytic cleavage of chlorophyll-porphyrin may occur by the action of a dioxygenase. We have attempted to demonstrate such an enzyme activity and to explore the requirements of the cleavage reaction in a reconstituted system of chloroplast (Chlpl) components prepared from senescent rape (Brassica napus L.) cotyledons. Intact senescent Chpls (also referred to as gerontoplasts) contain small amounts of two fluorescent chlorophyll catabolites, Bn-FCC-1 and Bn-FCC-2, probably representing primary cleavage products. Upon the incubation of Gpls in the presence of glucose-6-phosphate (Glc6P) or ATP, these catabolites (predominantly FCC-1) were produced in organello. In a reconstituted system of thylakoids and stroma fraction the FCCs (predominantly FCC-2) were produced in the presence of ferredoxin (Fd) and cofactors (NADPH, Glc6P) helping to keep Fd in the reduced state. Reduced Fd could not be replaced by other electron donors, suggesting that the putative dioxygenase requires Fd for the operation of its redox cycle. Production of FCC-2 did not occur in the absence of oxygen and it was inhibited by chelators of Fe2+. The contributions to the production of FCCs from both parts of the reconstituted system, thylakoids and stroma, are heat labile. The enzymic process in the thylakoids yields pheophorbide a, the presumptive precursor of FCCs. However, native senescent thylakoids could not be replaced as a "substrate" by free pheophorbide a. The stromal enzyme appears to have an affinity for senescent thylakoids; thus, "loaded" thylakoids capable of FCC production in the presence of Fd and cofactors were obtained upon homogenization of senescent cotyledons in a medium containing sorbitol and ascorbate. Such thylakoids were inactive if prepared from mature green cotyledons. As senescence was induced, the capacity to generate FCCs appeared and peaked when about half of the chlorophyll had disappeared from the cotyledons. The effectiveness of a relevant inhibitor showed that cytoplasmic protein synthesis was required for inducing the catabolic machinery in the loaded thylakoids. Thylakoids from mature Chlpls were ineffective as substrate of the stromal enzyme prepared from Gpls. However, senescent thylakoids yielded FCCs if challenged with stroma from either Chlpls or Gpls. Therefore, the stromal part of the system is likely to be a constitutive enzyme, and the pace-setting step of the pathway of chlorophyll breakdown seems to be located in the thylakoids.     

Production of a presumptive chlorophyll catabolite in vitro: requirement for reduced ferredoxin

Senescent chloroplasts (gerontoplasts) isolated from primary leaves of barley (Hordeum vulgare L.) contained a group of fluorescent chlorophyll (Chl) compounds designated as FCC-2, FCC-3 and FCC-4. Compound FCC-2 represents an established catabolite of Chl-porphyrin and was the most abundant constituent of this group. One of the minor constituents, FCC-4, was produced in a reconstituted system composed of thylakoids and stroma. The generation of FCC-4 depended on oxygen and required reduced ferredoxin (Fd) which probably acts as a reductant of the putative oxygenating enzyme responsible for the cleavage of Chl-porphyrin. A typical assay mixture consisted of thylakoids and stroma (equalling 10⁸ gerontoplasts), Fd, NADPH and glucose-6-phosphate for NADPH-regeneration. The oxygenating enzyme appeared to be a stromal protein. However, enzyme activities associated with the thylakoids were also required for the production of FCC-4. The senescence-specific part of the reconstituted system resided in the thylakoids. Thus, FCC-4 was produced in assay mixtures of senescent thylakoids and stroma from presenescent chloroplasts, whereas combinations with presenescent thylakoids failed to yield appreciable amounts of this putative primary Chl-catabolite.     

Light-dependent cleavage of the gamma subunit of coupling factor 1 by trypsin causes activation of Mg2+-ATPase activity and uncoupling of photophosphorylation in spinach chloroplasts

Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.     

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Isolation and characterization of an alkaline phosphatase from pea thylakoids

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.     

Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids

Ascorbate peroxidase, a key enzyme for the scavenging of hydrogenperoxide in chloroplasts, was found in a thylakoid-bound formin spinach chloroplasts at comparable activity to that in thestroma. The activity of peroxidase was detectable in the thylakoidsonly when prepared by an ascorbate-containing medium, and enrichedin the stroma thylakoids. The thylakoid enzyme was not releasedfrom the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or2 M NaSCN, but was solubilized by detergents. Enzymatic propertiesof the thylakoid-bound ascorbate peroxidase were very similarto those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogenperoxide either added or photoproduced by the thylakoids. Nophotoreduction of hydrogen peroxide was observed, however, inthe thylakoids whose ascorbate peroxidase was inhibited by KCNand thiol reagents or inactivated by the treatment with ascorbate-depletion.The primary oxidation product of ascorbate in a reaction ofascorbate peroxidase, monodehydroascorbate (MDA) radical, wasphotoreduced in the thylakoids, as detected by the quenchingof chlorophyll fluorescence, disappearance of EPR signals ofthe MDA radicals and the MDA radical-induced oxygen evolution.Thus, ascorbate is photoregenerated in the thylakoids from theMDA radicals produced in a reaction of ascorbate peroxidasefor the scavenging of hydrogen peroxide. (Received March 26, 1992; Accepted April 22, 1992)     

The reconstituted NADP photoreducing system by rebinding of the large form of ferredoxin-NADP reductase to depleted thylakoid membranes

The large form of ferredoxin-NADP reductase (FNR-L) was prepared by reassociating the small form of the enzyme (FNR-S) and connectein isolated from spinach leaves. The re-formed FNR-L could be rebound to depleted thylakoids from which most of the "built-in" FNR-L had been extracted. This rebinding of FNR-L brought about good restoration of the diminished NADP photoreducing activity of depleted thylakoids. Although rebinding of FNR-S to the depleted thylakoids took place with or without connectein, restoration of the NADP photoreducing activity required involvement of connectein. It becomes clear that involvement of connectein in the binding of FNR to thylakoids is indispensable for giving the physiological function of NADP photoreducing activity to the flavin enzyme on the surface of thylakoid membranes. It is most likely that FNR-L is the functional entity at the final step of the photosynthetic electron transport system in chloroplasts.     

Analyzing the Light Energy Distribution in the Photosynthetic Apparatus of C4 Plants Using Highly Purified Mesophyll and Bundle-Sheath Thylakoids

The chlorophyll fluorescence characteristics of mesophyll and bundle-sheath thylakoids from plant species with the C4 dicarboxylic acid pathway of photosynthesis were investigated using flow cytometry. Ten species with the NADP-malic enzyme (NADP-ME) biochemical type of C4 photosynthesis were tested: Digitaria sanguinalis (L.) Scop., Euphorbia maculata L., Portulaca grandiflora Hooker, Saccharum officinarum L., Setaria viridis (L.) Beauv., Zea mays L., and four species of the genus Flaveria. This study also included three species with NAD-ME biochemistry (Atriplex rosea L., Atriplex spongiosa F. Muell., and Portulaca oleracea L.). Two C4 species of unknown biochemical type were investigated: Cyperus papyrus L. and Atriplex tatarica L. Pure mesophyll and bundle-sheath thylakoids were prepared by flow cytometry and characterized by low-temperature fluorescence spectroscopy. In pure bundle-sheath thylakoids from many species with C4 photosynthesis of the NADP-ME type, significant amounts of photosystem II (PSII) emission can be detected by fluorescence spectroscopy. Simulation of fluorescence excitation spectra of these thylakoids showed that PSII light absorption contributes significantly to the apparent excitation spectrum of photosystem I. Model calculations indicated that the excitation energy of PSII is efficiently transferred to photosystem I in bundle-sheath thylakoids of many NADP-ME species.     

Modifications of the gamma subunit of chloroplast coupling factor 1 alter interactions with the inhibitory epsilon subunit.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.     

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.     

Comparison of violaxanthin de-epoxidation from the stroma and lumen sides of isolated thylakoid membranes from Arabidopsis: implications for the mechanism of de-epoxidation

The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.     

Further enzymatic characteristics of a thylakoid protein kinase

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.     

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.     

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.     

ATP and light regulate D1 protein modification and degradation. Role of D1* in photoinhibition.

We have recently shown that during in vivo photoinhibition the D1 protein is degraded via a modified form, designated D1*. Depending on light conditions, the amount of D1* varies in leaves between 0 and 50% of total D1 content. By isolating thylakoids from leaves acclimated to different light levels, and performing photoinhibition experiments on these thylakoids, the following results on D1 protein degradation were obtained: (i) the protease involved in D1 degradation requires activation by light; (ii) neither acceptor nor donor side photoinhibition of PSII induces formation of D1* in vitro; (iii) in isolated thylakoids, the transformation of D1 to D1* can be induced in low light in the presence of ATP, which suggests that D1* is a phosphorylated form of the D1 protein; (iv) D1*, induced either in vivo or in vitro, is much less susceptible to degradation during illumination of isolated thylakoids than the original D1 protein. We suggest that the modification to D1* is a means to prevent disassembly of photodamaged photosystem II complex in appressed membranes.     

Carboxyl-terminal processing of the D1 protein and photoactivation of water-splitting in photosystem II. Partial purification and characterization of the processing enzyme from Scenedesmus obliquus and Pisum sativum.

The D1 polypeptide of photosystem II (PSII) is synthesized as a precursor that is processed by cleavage at the carboxyl terminus during assembly of the active PSII complex. A mutant of the green alga Scenedesmus obliquus, LF-1, inactive in water-splitting, lacks the D1 processing activity but assembles otherwise normal PSII complexes containing the precursor D1 molecule. We have isolated and partially purified a soluble protease from sonicated thylakoids of both wild-type S. obliquus and Pisum sativum which will process the precursor D1 molecule in PSII-enriched membranes from the LF-1 mutant to the mature size. After processing (but not before), photoactivation of these PSII membranes in the presence of manganese restores water-splitting to levels seen after photoactivation of PSII membranes from dark-grown, wild-type, cells. The protease is unable to process D1 in intact thylakoids from the LF-1 mutant but processes D1 if present during sonication of the thylakoids, indicating that processing of the carboxyl-terminal extension of D1 occurs in the lumen of the thylakoid. The processing protease from both S. obliquus and P. sativum is a single subunit enzyme of native molecular mass 33-35 kDa. Processing rate is optimal at pH 6.5. Processing in vitro is evident within 5 min and is markedly inhibited by millimolar concentrations of divalent cations (Cu, Zn greater than Mn greater than Ca, Mg) but not by any known inhibitors of the major classes of proteases. The protease is inactive against the precursors of other thylakoidal proteins and is thus distinct from the thylakoidal amino-terminal processing enzyme involved in the removal of transit peptides from cytoplasmically-synthesised proteins imported into the thylakoid lumen.     

Quality control of Photosystem II: cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 degrees C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Effects of frost hardening on the composition of galactolipids and phospholipids occurring during isolation of chloroplast thylakoids from needles of scots pine

Frost hardening of seedlings of Scots pine (Pinus sylvestris) at a non-freezing temperature of 4°C resulted in a 2-fold increase of the acyl lipids of the needles. This was because of increases in phospholipids and triglycerides. The galactolipid content of the needles was almost the same in unhardened and frost-hardened seedlings. In unhardened seedlings the mol ratio of monogalactosyl diacylglycerol (MGDG) to digalactosyl diacylglycerol (DGDG) was 1.7 ± 0.3 and 0.9 ± 0.2 in needles and isolated thylakoids, respectively. Corresponding ratios for frost-hardened seedlings were 1.5 ± 0.2 and 0.3 ± 0.03. The lower ratios found in isolated thylakoids, particularly in thylakoids from frost-hardened seedlings, are suggested to depend on the enzyme galactolipid: galactolipid galactosyltransferase being active during the isolation procedure. This is deduced from the result that the content of MGDG decreased and that of DGDG and 1.2 diglycerides increased. Needles of Scots pine also contain phospholipidase D. This enzyme was active during thylakoid preparation, particularly after frost hardening, as judged from the large amount of phosphatidic acid found the in thylakoid fraction isolated from frost-hardening needles. The fatty acid composition of the acyl lipids showed no major changes due to hardening at non-freezing temperature.     

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

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.