Multiple deletions of small Cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: consequences for pigment biosynthesis and accumulation

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.     

Small CAB-like proteins prevent formation of singlet oxygen in the damaged photosystem II complex of the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial small CAB-like proteins (SCPs) are single-helix membrane proteins mostly associated with the photosystem II (PSII) complex that accumulate under stress conditions. Their function is still ambiguous although they are assumed to regulate chlorophyll (Chl) biosynthesis and/or to protect PSII against oxidative damage. In this study, the effect of SCPs on the PSII-specific light-induced damage and generation of singlet oxygen ((1)O(2)) was assessed in the strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking PSI (PSI-less strain) or lacking PSI together with all SCPs (PSI-less/scpABCDE(-) strain). The light-induced oxidative modifications of the PSII D1 protein reflected by a mobility shift of the D1 protein and by generation of a D1-cytochrome b-559 adduct were more pronounced in the PSI-less/scpABCDE(-) strain. This increased protein oxidation correlated with a faster formation of (1)O(2) as detected by the green fluorescence of Singlet Oxygen Sensor Green assessed by a laser confocal scanning microscopy and by electron paramagnetic resonance spin-trapping technique using 2, 2, 6, 6-tetramethyl-4-piperidone (TEMPD) as a spin trap. In contrast, the formation of hydroxyl radicals was similar in both strains. Our results show that SCPs prevent (1)O(2) formation during PSII damage, most probably by the binding of free Chl released from the damaged PSII complexes.     

Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C

Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated beta-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: their involvement in chlorophyll biogenesis for Photosystem II

The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the family of stress-induced light-harvesting-like proteins, but are constitutively expressed in a mutant deficient of Photosystem I (PSI). Using absorption, fluorescence and thermoluminescence measurements this PSI-less strain was compared with a mutant, in which all SCPs were additionally deleted. Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q(B) site was modified. Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. Although the excess PBS were functionally disconnected, their fluorescence was quenched under high irradiance by the activated Orange Carotenoid Protein (OCP). Additionally the amount of OCP, but not of the iron-stress induced protein (isiA), was higher in this SCP-depleted mutant compared with the control. As previously described, the lack of SCPs affects the chlorophyll biosynthesis (Vavilin, D., Brune, D. C., Vermaas, W. (2005) Biochim Biophys Acta 1708, 91-101). We demonstrate that chlorophyll synthesis is required for efficient PSII repair and that it is partly impaired in the absence of SCPs. At the same time, the amount of chlorophyll also seems to influence the expression of ScpC and ScpD.     

Cyanobacterial small chlorophyll-binding protein ScpD (HliB) is located on the periphery of photosystem II in the vicinity of PsbH and CP47 subunits

Cyanobacteria contain several genes coding for small one-helix proteins called SCPs or HLIPs with significant sequence similarity to chlorophyll a/b-binding proteins. To localize one of these proteins, ScpD, in the cells of the cyanobacterium Synechocystis sp. PCC 6803, we constructed several mutants in which ScpD was expressed as a His-tagged protein (ScpDHis). Using two-dimensional native-SDS electrophoresis of thylakoid membranes or isolated Photosystem II (PSII), we determined that after high-light treatment most of the ScpDHis protein in a cell is associated with PSII. The ScpDHis protein was present in both monomeric and dimeric PSII core complexes and also in the core subcomplex lacking CP43. However, the association with PSII was abolished in the mutant lacking the PSII subunit PsbH. In a PSII mutant lacking cytochrome b(559), which does not accumulate PSII, ScpDHis is associated with CP47. The interaction of ScpDHis with PsbH and CP47 was further confirmed by electron microscopy of PSII labeled with Ni-NTA Nanogold. Single particle image analysis identified the location of the labeled ScpDHis at the periphery of the PSII core complex in the vicinity of the PsbH and CP47. Because of the fact that ScpDHis did not form any large structures bound to PSII and because of its accumulation in PSII subcomplexes containing CP47 and PsbH we suggest that ScpD is involved in a process of PSII assembly/repair during the turnover of pigment-binding proteins, particularly CP47.     

Localization of the small CAB-like proteins in photosystem II

The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.     

Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis

delta-Aminolevulinic acid (ALA), the first committed precursor to the tetrapyrrole components of hemes and chlorophylls, is synthesized by two different routes in the photosynthetic phytoflagellate Euglena gracilis: directly from glutamate, mediated by a 5-carbon pathway, and via condensation of glycine and succinyl-CoA, catalyzed by the enzyme ALA synthase. The physiological roles of the two pathways were determined by administration of specifically 14C-labeled ALA precursors to cultures growing under different physiological conditions. Relative activities of the ALA synthase and 5-carbon pathways were monitored by incorporation of radioactivity from [2-14C] glycine and [1-14C]glutamate into highly purified protoheme, heme a and chlorophyll a derivatives. Wild type cells grown photoautotrophically or photoheterotrophically synthesized chlorophyll and incorporated radioactivity from [1-14C]glutamate into the tetrapyrrole nucleus of the pigment. [2-14C]Glycine was incorporated primarily into the nontetrapyrrole-derived portions of chlorophyll. In the same cultures both [2-14C]glycine and [1-14C]glutamate were efficiently incorporated into protoheme, while only [2-14C] glycine was incorporated into heme a. In dark-grown wild type or light-grown aplastidic cells, no chlorophyll was formed, and both protoheme and heme a were labeled exclusively from [2-14C]glycine. These results indicate: (a) ALA synthase and the 5-carbon pathway operate simultaneously in growing green cells; (b) the 5-carbon pathway provides ALA for chloroplast protoheme and chlorophyll, and is associated with chloroplast development; (c) ALA synthase provides ALA only for nonplastid heme biosynthesis; and (d) the two ALA pathways are separately compartmentalized along with complete sets of enzymes for subsequent tetrapyrrole synthesis from each ALA pool. The protoheme that was synthesized from [1-14C] glutamate had a higher specific radioactivity than chlorophyll synthesized from the same precursor. This result together with calculated specific radioactivities of the products synthesized during the incubation period, suggest that both protoheme and heme a undergo metabolic turnover.     

Identification of an intermediate of delta-aminolevulinate biosynthesis in Chlamydomonas by high-performance liquid chromatography

The first committed intermediate of the chlorophyll biosynthetic pathway is delta-aminolevulinic acid (ALA). In plant cells, ALA is formed from glutamate by a pathway not yet clearly defined. One of the proposed pathways involves the reduction of glutamate to glutamate-1-semialdehyde (GSA) via a glutamyl-tRNA intermediate. GSA is then converted to ALA by an aminotransferase. We are studying this pathway using partially purified components from Chlamydomonas reinhardtii in in vitro reactions with [3H]L-glutamate as the substrate and analysis of the radioactive reaction products via HPLC. In reactions either lacking GSA-aminotransferase or containing gabaculine (an inhibitor of aminotransferase), a radioactive intermediate is formed which cochromatographs with synthetic GSA. As observed previously for ALA synthesis, the synthesis of this intermediate has an absolute requirement for RNA, ATP, and active enzymes, while the requirement for NADPH is less stringent. Both the accumulated intermediate and the synthetic GSA can be converted to ALA by the aminotransferase without any additional substrates or cofactors. These results support previous observations that GSA or a very similar compound is an intermediate of ALA synthesis.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

不同氮浓度和形态比例对水曲柳幼苗叶绿素合成、光合作用以及生物量分配的影响 (英文)

 水曲柳（Fraxinus mandshurica）是我国东北林区重要的工业用材树种,在东北林区广泛种植,因而其培育近来日益得到高度重视。在水曲柳的种植区域内,尽管林地内凋落物丰富,但该地区气温低,冬季长,氮素矿化速度低,氮素供给显得不足。本研究采用沙培的方式,在为幼苗提供完     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes

We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

Ubiquinone biosynthesis by mitochondria, sonicated mitochondria, and mitoplasts of rat liver.

Ubiquinone was biosynthesized when rat liver mitochondria were incubated with S-adenosyl-L-methionine, solanesyl diphosphate, and [U-14C]p-hydroxybenzoate. The intermediates of ubiquinone biosynthesis but not ubiquinone were accumulated in mitochondria incubated without S-adenosyl-L-methionine and the accumulated intermediates were converted to ubiquinone by the addition of the methyl group donor and an excess of cold p-hydroxybenzoate. No solaneylated compounds except nonaprenyl p-hydroxybenzoate were found in sonicated mitochondria, while the biosynthesis of ubiquinone was observed in the sonicated preparation of mitochondria in which the intermediates accumulated. The results indicate that the initial decarboxylation reaction is completely abolished and the subsequent reactions of hydroxylation and methylation are not completely inhibited by the sonication treatment and therefore the decarboxylation reaction is the next step after nonaprenylation of p-hydroxybenzoate. Mitoplasts could biosynthesize ubiquinone with activity comparable to that of intact mitochondria, suggesting that components of the outer membrane and the intermembranous space of mitochondria are not involved in ubiquinone biosynthesis.     

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris)

The basis of the increased resistance to photoinhibition upon growth at low temperature was investigated. Photosystem II (PSII) excitation pressure was estimated in vivo as 1 - qp (photochemical quenching). We established that Chlorella vulgaris exposed to either 5[deg]C/150 [mu]mol m-2 s-1 or 27[deg]C/2200 [mu]mol m-2 s-1 experienced a high PSII excitation pressure of 0.70 to 0.75. In contrast, Chlorella exposed to either 27[deg]C/150 [mu]mol m-2 s-1 or 5[deg]C/20 [mu]mol m-2 s-1 experienced a low PSII excitation pressure of 0.10 to 0.20. Chlorella grown under either regime at high PSII excitation pressure exhibited: (a) 3-fold higher light-saturated rates of O2 evolution; (b) the complete conversion of PSII[alpha] centers to PSII[beta] centers; (c) a 3-fold lower epoxidation state of the xanthophyll cycle intermediates; (d) a 2.4-fold higher ratio of chlorophyll a/b; and (e) a lower abundance of light-harvesting polypeptides than Chlorella grown at either regime at low PSII excitation pressure. In addition, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 exhibited resistance to photoinhibition comparable to that of cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 and were 3- to 4-fold more resistant to photoinhibition than cells grown at either regime at low excitation pressure. We conclude that increased resistance to photoinhibition upon growth at low temperature reflects photosynthetic adjustment to high excitation pressure, which results in an increased capacity for nonradiative dissipation of excess light through zeaxanthin coupled with a lower probability of light absorption due to reduced chlorophyll per cell and decreased abundance of light-harvesting polypeptides.     

Evaluation of two biosynthetic pathways to delta-aminolevulinic acid in Euglena gracilis.

delta-Aminolevulinic acid (ALA), which is an intermediate in the biosynthesis of chlorophyll a, can be biosynthesized via the C5 pathway and the Shemin pathway in Euglena gracilis. Analysis of the (13)C-NMR spectrum of (13)C-labeled methyl pheophorbide a, derived from 13C-labeled chlorophyll a biosynthesized from d-[1-(13)C]glucose by E. gracilis, provided evidence suggesting that ALA incorporated in the (13)C-labeled chlorophyll a was synthesized via both the C5 pathway and the Shemin pathway in a ratio of between 1.5 and 1.7 to one. The methoxyl carbon of the methoxycarbonyl group at C-132 of chlorophyll a was labeled with (13)C. The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3(1), C-P4, C-P6, C-P7(1), C-P8, C-P10, C-P11(1), C-P12, C-P14, C-P15(1) and C-P16.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Dynamics of Photosystem Stoichiometry Adjustment by Light Quality in Chloroplasts

Long-term imbalance in light absorption and electron transport by photosystem I (PSI) and photosystem II (PSII) in chloroplasts brings about changes in the composition, structure, and function of thylakoid membranes. The response entails adjustment in the photosystem ratio, which is optimized to help the plant retain a high quantum efficiency of photosynthesis (W.S. Chow, A. Melis, J.M. Anderson [1990] Proc Nat Acad Sci USA 87: 7502-7506). The dynamics of photosystem ratio adjustment were investigated upon the transfer of pea {Pisum sativum} plants from a predominantly PSI-light to a predominantly PSII-light environment and vice versa. The concentration of functional components (primary electron accepting plastoquinone of PSII [QA], P700) and that of constituent proteins were monitored during acclimation by A difference spectrophotometry and immunoblot analysis, respectively. Fully reversible changes in photosystem ratio occurred with a half-time of about 20 h. They involved closely coordinated changes in the concentration of the QA, reaction center protein D1, D2, and the 9-kD apoprotein of the cytochrome b559 for PSII. Similarly, closely coordinated changes in the relative concentration of P700 and reaction center proteins of PSI were observed. The level of chlorophyll b and that of the light-harvesting complex II changed in accordance with the concentration of PSII in the acclimating thylakoids. Overall, adjustments in the photosystem ratio in response to PSI- or PSII-light conditions appeared to be a well-coordinated reaction in the chloroplast. The response was absent in the chlorophyll b-less chlorina f2 mutant of barley (Hordeum vulgare) and in a phycobilisomeless mutant of Agmenellum quadruplicatum, suggesting that photosystem accessory pigments act as the light-quality perception molecules and that PSI and PSII themselves play a role in the signal transduction pathway.     

Cellular levels of glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase do not control chlorophyll synthesis in Chlamydomonas reinhardtii

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNA(Glu). Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 micromol photons s(-1) m(-2)), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.