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1.
PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP+ photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (KD) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced QA. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex. 相似文献
2.
Poul Erik Jensen Lisa Rosgaard Jurgen Knoetzel Henrik Vibe Scheller 《The Journal of biological chemistry》2002,277(4):2798-2803
PSI-G is a subunit of photosystem I in eukaryotes. The function of PSI-G was characterized in Arabidopsis plants transformed with a psaG cDNA in antisense orientation. Several plants with significantly decreased PSI-G protein content were identified. Plants with reduced PSI-G content were indistinguishable from wild type when grown under optimal conditions, despite a 40% reduction of photosystem I. This decrease of photosystem I was correlated with a similar reduction in state transitions. Surprisingly, the reduced photosystem I content was compensated for by a more effective photosystem I because the light-dependent reduction of NADP(+) in vitro was 48% higher. Photosystem I antenna size determined from flash-induced P700 absorption changes did not reveal any significant effect on the size of the photosystem I antenna in the absence of PSI-G, whereas a 17% reduction was seen in the absence of PSI-K. However, nondenaturing green gels revealed that the interaction between photosystem I and the light-harvesting complex I was less stable in the absence of PSI-G. Thus, PSI-G plays a role in stabilizing the binding of the peripheral antenna. The increased activity in the absence of PSI-G suggests that PSI-G could have an important role in regulation of photosystem I. 相似文献
3.
The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex. 相似文献
4.
Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes. 相似文献
5.
Zygadlo A Robinson C Scheller HV Mant A Jensen PE 《The Journal of biological chemistry》2006,281(15):10548-10554
The PSI-G subunit of photosystem I (PSI) is an 11-kDa membrane protein that plays an important role in electron transport between plastocyanin and PSI and is involved in the stability of the PSI complex. Within the complex, the PSI-G subunit is bound to PSI-B and is in contact with Lhca1. PSI-G has two transmembrane spans connected by a positively charged stromal loop. The loop is inaccessible to proteases, indicating a tightly bound location within the PSI complex. Here, we have studied the insertion mechanism and assembly of PSI-G. We show that the protein inserts into thylakoids by a direct or "spontaneous" pathway that does not involve the activities of any known chloroplast protein-targeting machinery. Surprisingly, the positively charged stromal loop region plays a major role in this process. Mutagenesis or deletions within this region almost invariably lead to a marked lowering of insertion efficiency, strongly indicating a critical role for the loop in the organization of the transmembrane regions prior to or during membrane insertion. Finally, we have examined the assembly of newly inserted PSI-G into the PSI complex, since very little is known of the assembly pathway for this large multimeric complex. Interestingly, we find that inserted PSI-G can be found within the full PSI complex within the import assay time frame after insertion into thylakoids, strongly suggesting that PSI-G normally associates at the end of the assembly process. This is consistent with its location on the periphery of the complex. 相似文献
6.
Single and double knockouts of the genes for photosystem I subunits G,K, and H of Arabidopsis. Effects on photosystem I composition,photosynthetic electron flow,and state transitions
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Varotto C Pesaresi P Jahns P Lessnick A Tizzano M Schiavon F Salamini F Leister D 《Plant physiology》2002,129(2):616-624
Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions. 相似文献
7.
Electron transport between the two photosynthetic reaction centres of high plants is mediated by plastoquinone, a rieske iron-sulfur centre, cytochrome f and plastocyanin. Measurements of redox equilibration amongst these have produced confusing results, with apparent equilibrium constants being estimated that are inconsistent with in vitro measurements of redox midpoint potentials of the components concerned. We have critically reexamined methods for deconvoluting cytochrome f absorbance signals in intact leaves. We have determined the decay of cytochrome f+ following light to dark transitions from steady state and compared this with the decay of the oxidised photosystem I primary donor, P700+. Measurements across a wide range of different irradiances and CO2 concentrations were all consistent with cyt f and P700 existing in redox equilibrium, with a potential difference of around 117 mV. These results are discussed in relation to our understanding of the organisation of the photosynthetic electron transport. They also have implications for measurements of PSI electron flux--provided more than about 20% of P700+ is oxidised in the light, then the initial decay in the concentration of P700+ following a light to dark transition provides a good estimate of electron flux through PSI. Where P700 is largely reduced in the light, net reduction of cyt f+ might need to be corrected for. 相似文献
8.
Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport. 相似文献
9.
Interaction of plastocyanin with photosystem I: a chemical cross-linking study of the polypeptide that binds plastocyanin 总被引:10,自引:0,他引:10
Plastocyanin has been covalently cross-linked to photosystem I (PSI) by using a water-soluble cross-linker, N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linking reaction is light stimulated and results in the disappearance of a single 19-kDa subunit of PSI with the formation of a new protein-staining component of 31 kDa. The new product at 31 kDa reacts with both plastocyanin and 19-kDa subunit antibodies. Carboxyl group modified plastocyanin does not form a cross-linked product with PSI, implying that the negatively charged surface-exposed groups on plastocyanin are necessary to stabilize binding. These results demonstrate a specific interaction of plastocyanin with PSI and further implicate a specific protein to which plastocyanin binds to facilitate electron transfer to the P700 reaction center. 相似文献
10.
11.
PSI-H is an intrinsic membrane protein of 10 kDa that is a subunit of photosystem I (PSI). PSI-H is one of the three PSI subunits found only in eukaryotes. The function of PSI-H was characterized in Arabidopsis plants transformed with a psaH cDNA in sense orientation. Cosuppressed plants containing less than 3% PSI-H are smaller than wild type when grown on sterile media but are similar to wild type under optimal conditions. PSI complexes lacking PSI-H contain 50% PSI-L, whereas other PSI subunits accumulate in wild type amounts. PSI devoid of PSI-H has only 61% NADP+ photoreduction activity compared with wild type and is highly unstable in the presence of urea as determined from flash-induced absorbance changes at 834 nm. Our data show that PSI-H is required for stable accumulation of PSI and efficient electron transfer in the complex. The plants lacking PSI-H compensate for the less efficient PSI with a 15% increase in the P700/chlorophyll ratio, and this compensation is sufficient to prevent overreduction of the plastoquinone pool as evidenced by normal photochemical quenching of fluorescence. Nonphotochemical quenching is approximately 60% of the wild type value, suggesting that the proton gradient across the thylakoid membrane is decreased in the absence of PSI-H. 相似文献
12.
Haldrup A Naver H Scheller HV 《The Plant journal : for cell and molecular biology》1999,17(6):689-698
The PSI-N subunit of photosystem I (PSI) is restricted to higher plants and is the only subunit located entirely in the thylakoid lumen. The role of the PSI-N subunit in the PSI complex was investigated in transgenic Arabidopsis plants which were generated using antisense and co-suppression strategies. Several lines without detectable levels of PSI-N were identified. The plants lacking PSI-N assembled a functional PSI complex and were capable of photoautotrophic growth. When grown on agar media for several weeks the plants became chlorotic and developed significantly more slowly. However, under optimal growth conditions, the plants without PSI-N were visually indistinguishable from the wild-type although several photosynthetic parameters were affected. In the transformants, the second-order rate constant for electron transfer from plastocyanin to P700+, the oxidized reaction centre of PSI, was only 55% of the wild-type value, and steady-state NADP+ reduction was decreased to a similar extent. Quantum yield of oxygen evolution and PSII photochemistry were about 10% lower than in the wild-type at leaf level. Photochemical fluorescence quenching was lowered to a similar extent. Thus, the 40-50% lower activity of PSI at the molecular level was much less significant at the whole-plant level. This was partly explained by a 17% increase in PSI content in the plants lacking PSI-N. 相似文献
13.
14.
The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed. 相似文献
15.
Shinya Wada Yuji Suzuki Daisuke Takagi Chikahiro Miyake Amane Makino 《Photosynthesis research》2018,137(3):431-441
Under CO2-limited conditions such as during stomatal closure, photorespiration is suggested to act as a sink for excess light energy and protect photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. In this study, this issue was directly examined with rice (Oryza sativa L.) plants via genetic manipulation of the amount of Rubisco, which can be a limiting factor for photorespiration. At low [CO2] of 5 Pa that mimicked stomatal closure condition, the activity of photorespiration in transgenic plants with decreased Rubisco content (RBCS-antisense plants) markedly decreased, whereas the activity in transgenic plants with overproduction of Rubisco (RBCS-sense plants) was similar to that in wild-type plants. Oxidation of P700 was enhanced at [CO2] of 5 Pa in wild-type and RBCS-sense plants. PSI was not damaged by excess light stress induced by repetitive saturated pulse-light (rSP) in the presence of strong steady-state light. On the other hand, P700 was strongly reduced in RBCS-antisense plants at [CO2] of 5 Pa. PSI was also damaged by rSP illumination. These results indicate that oxidation of P700 and the robustness of PSI against excess light stress are hampered by the decreased activity of photorespiration as a result of genetic manipulation of Rubisco content. It is also suggested that overproduction of Rubisco does not enhance photorespiration as well as CO2 assimilation probably due to partial deactivation of Rubisco. 相似文献
16.
Photosynthetic regulation under fluctuating light in field-grown Cerasus cerasoides: A comparison of young and mature leaves 总被引:1,自引:0,他引:1
Photosystem I (PSI) is a potential target of photoinhibition under fluctuating light. However, photosynthetic regulation under fluctuating light in field-grown plants is little known. Furthermore, it is unclear how young leaves protect PSI against fluctuating light under natural field conditions. In the present study, we examined chlorophyll fluorescence, P700 redox state and the electrochromic shift signal in the young and mature leaves of field-grown Cerasus cerasoides (Rosaceae). Within the first seconds after any increase in light intensity, young leaves showed higher proton gradient (ΔpH) across the thylakoid membranes than the mature leaves, preventing over-reduction of PSI in the young leaves. As a result, PSI was more tolerant to fluctuating light in the young leaves than in the mature leaves. Interestingly, after transition from low to high light, the activity of cyclic electron flow (CEF) in young leaves increased first to a high level and then decreased to a stable value, while this rapid stimulation of CEF was not observed in the mature leaves. Furthermore, the over-reduction of PSI significantly stimulated CEF in the young leaves but not in the mature leaves. Taken together, within the first seconds after any increase in illumination, the stimulation of CEF favors the rapid lumen acidification and optimizes the PSI redox state in the young leaves, protecting PSI against photoinhibition under fluctuating light in field-grown plants. 相似文献
17.
Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress 总被引:1,自引:0,他引:1
Marek Zivcak Marian Brestic Zuzana Balatova Petra Drevenakova Katarina Olsovska Hazem M. Kalaji Xinghong Yang Suleyman I. Allakhverdiev 《Photosynthesis research》2013,117(1-3):529-546
The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO2 exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700 absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks. 相似文献
18.
Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. 相似文献
19.
Previous work in our laboratory (Krueger, Miles 1981 Plant Physiol 68: 1110-1114) indicated that a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) which exhibits unusually high net photosynthesis rates also had high potential rates of photosynthetic electron transport through photosystem I (PSI) compared to the typical hexaploid genotype (V6-802). Analysis of electron transport activity revealed that the oxidizing side of PSI as the major site of difference. Examination of the whole thylakoids and subchloroplast particle protein components of the common hexaploid and the decaploid genotypes had major polypeptide differences at 30, 21, and 12.5 kilodaltons. These differences could not be assigned to a specific physiological function in PSI. The decaploid had increased P700 and plastocyanin content on a chlorophyll basis. Antibodies raised against fescue plastocyanin were used to quantitate plastocyanin in crude (Triton X-100) solubilized extracts of plant material. Results showed that the decaploid had 16% and 40% more plastocyanin on a weight and area basis, respectively. The antibodies did not inhibit electron transport (diaminodiurene to methyl viologen) in isolated thylakoids strengthening the hypothesis of plastocyanin as an internal mobile electron shuttle. The trend of inhibition of plastocyanin by KCN was similar in the two genotypes but the decaploid had 15 to 20% higher rates of electron flow under nearly all inhibiting conditions. 相似文献
20.
Bateman RL Ashworth J Witte JF Baker LJ Bhanumoorthy P Timm DE Hurley TD Grompe M McClard RW 《The Biochemical journal》2007,403(2):251-260
The functions of several small subunits of the large photosynthetic multiprotein complex PSI (Photosystem I) are not yet understood. To elucidate the function of the small plastome-encoded PsaJ subunit, we have produced knockout mutants by chloroplast transformation in tobacco (Nicotiana tabacum). PsaJ binds two chlorophyll-a molecules and is localized at the periphery of PSI, close to both the Lhca2- and Lhca3-docking sites and the plastocyanin-binding site. Tobacco psaJ-knockout lines do not display a visible phenotype. Despite a 25% reduction in the content of redox-active PSI, neither growth rate nor assimilation capacity are altered in the mutants. In vivo, redox equilibration of plastocyanin and PSI is as efficient as in the wild-type, indicating that PsaJ is not required for fast plastocyanin oxidation. However, PsaJ is involved in PSI excitation: altered 77 K chlorophyll-a fluorescence emission spectra and reduced accumulation of Lhca3 indicate that antenna binding and exciton transfer to the PSI reaction centre are impaired in DeltapsaJ mutants. Under limiting light intensities, growth of DeltapsaJ plants is retarded and the electron-transport chain is far more reduced than in the wild-type, indicating that PSI excitation might limit electron flux at sub-saturating light intensities. In addition to defining in vivo functions of PsaJ, our data may also have implications for the interpretation of the crystal structure of PSI. 相似文献