Thylakoid Membrane Development and Differentiation: Assembly of the Chlorophyll a-b Light-Harvesting Complex and Evidence for the Origin of Mr=19, 17.5 and 13.4 kDa Proteins

Pea plants were grown under intermittent illumination (ImL)conditions. The low dosage of light given to ImL plastids limitedthe rate of chlorophyll (Chl) a and Chl b biosynthesis and,therefore, it retarded the rate of photosynthetic unit formationand thylakoid membrane development. Depending on the developmentalstage of the photosynthetic unit, ImL plastids had variableChl a/Chl b ratios (2.7 <Chl a/Chlb<20) and showed distinctintermediates in the assembly of the chlorophyll a–b light-harvestingcomplex (LHC) of photosystem-II (PSII). The results are consistentwith a step-wise increment in the PSII antenna size involvingthree distinct forms of the PSII unit: (i) a PSII-core formwith about 37 Chl a molecules; (ii) a PSIL_ß form containingthe PSII-core and the LHC-II-inner antenna with a total of about130 Chl (a + b) molecules, and (iii) the mature PSII_a form containingPSII_ß and the LHC-II-peripheral antenna with a totalof 210–300 Chl (a + b) molecules. The thylakoid membranecontained polypeptide subunits b, c and d (the Lhcb1, 2 and3 gene products, respectively) when only the LHC-II-inner waspresent. Polypeptide subunit a, (the apoprotein of the chlorophyll-proteinknown as CP29), along with increased amounts of b and c appearedlater in the development of thylakoids, concomitant with theassembly of the LHC-II-peripheral. The results suggest thatpolypeptide subunit d has priority of assembly over subunita. It is implied that, of all LHC-II constituent proteins, subunitd is most proximal to the PSII-core complex and that it servesas a linker in the transfer of excitation energy from the bulkLHC-II (subunits b and c) to the PSII-core. The work also addressesthe origin of low-molecular-weight proteins (M_r = 19, 17.5 and13.4 kDa) which co-isolate with intact developing plastids andwhose abundance decreases during plastid development. Aminoacid compositional and immunoblot analyses show a nuclear histoneorigin for these low-molecular-weight proteins and suggest co-isolationof histone-containing nuclear vesicles along with intact developingplastids. ¹Present address: Plant Physiology Research Group, The Universityof Calgary, Department of Biological Sciences, 2500 UniversityDrive N.W., Calgary, Alberta CANADA T2N 1N4.     

Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat

Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [³⁵S]methionine, mild solubilization of the thylakoid membrane with Triton X-100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed.     

Photo-inhibition and the Effects of Protein Phosphorylation on Electron Transport in Wheat Thylakoids

The kinetics of changes in photosystem I (PSI), photosystemII (PSII), and whole chain (PSII and PSI) electron transport,chlorophyll fluorescence parameters, the capacity to bind atrazineand the polypeptide profiles of thylakoids isolated from wheatleaves on exposure to a photon flux density of 2000 µmolm^–2 s^–1 were determined. Severe and similar levelsof photo-inhibitory damage to both PSII and whole chain electrontransport occurred and were correlated with decreases in theratio of variable to maximal fluorescence, the proportionalcontribution of the rapid a phase of the fluorescence kineticsand the capacity to bind atrazine. Severe photo-inhibition ofelectron transport was not associated with a major loss of chlorophyllor total thylakoid protein. However, a small decrease in a 70kDa polypeptide together with increases in a number of low molecularmass polypeptides (8–24 kDa) occurred. Phosphorylation of thylakoid polypeptides alleviated photo-inhibitionof PSII electron transport but stimulated photoinhibitory damageto whole chain electron transport. The consequences of suchphosphorylation-induced effects on photoinhibition in vivo areconsidered. Key words: Chlorophyll fluorescence, electron transport, photo-inhibition, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Chromatic Regulation in Chlamydomonas reinhardtii: Time Course of Photosystem Stoichiometry Adjustment Following a Shift in Growth Light Quality

Photosystem stoichiometry adjustments in Chlamydomonas reinhardtiiwere induced upon a sudden shift in the light quality duringcell growth. Reversible changes in the PSI/PSII ratio were acompensation response to changes in the balance of light absorptionby the two photosystems. Quantitations of PSII, Cyt b₆-f complexand PSI revealed a constancy in the cellular content of PSIIand the Cyt b₆-f complex, and variable amounts of PSI in C.reinhardtii. These results strengthen the notion that PSI isthe thyla-koid component subject to chromatic regulation andresponsible for the adjustment and optimization of the PSI/PSII ratio in the thylakoid of oxygenic photosynthesis. Additionalresults, obtained upon the use of protein biosynthesis translationinhibitors (chloramphenicol and cyclohex-imide), suggested thata chromatically-induced lowering of the PSI/PSII ratio in C.reinhardtii occurs by suppression of de novo biosynthesis ofPSI components and, therefore, by dilution of the PSI complexin the thylakoid membrane, rather than by active degradationof assembled PSI in chlo-roplasts. (Received November 8, 1996; Accepted December 6, 1996)     

Changes in Stoichiometry among PSI, PSII and Cyt b6-f Complexes in Response to Chromatic Light for Cell Growth Observed with the Red Alga Porphyra yezoensis

Stoichiometry among 3 thylakoid components, PSI and PSII andCyt b6-f complexes, was determined with the red alga Porphyrayezoensis with special reference to the regulation of PSI/PSIIstoichiometry in response to light regime. The ratio of PSIto PSII abundance was four times greater in thalli grown underorange light which excites mainly phycobilisome, thus PSII,than that under red light which excites preferentially Chl a,thus PSI. Cyt b₆-f abundance remained almost constant. The PSIand PSII content was regulated separately under the two growthlight conditions as was also observed with the red alga Porphyridiumcruentum by Cunningham et al. [(1990) Plant Physiol. 93: 888].This differs from the cyanophyte Synechocystis PCC 6714 whereadjustment occurs only in the PSI content [(1987) Plant CellPhysiol. 28: 1547]. However, results on the marine cyanophyteSynechococcus NIBB 1071 indicate that changes in the PSI/PSIIsoichiometry is similar to red algae. In this species, as inthe red algae, more than one PSII is associated with each phycobilisome.The light regime also induced changes in the phycobiliproteincomposition in Porphyra yezoensis. Under PSII light, phycoerythrinincreased, and phycocyanin decreased, while under PSI lightthe response was reversed. The change suggests an occurrenceof complementary chromatic adaptation. (Received April 8, 1994; Accepted June 1, 1994)     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

Effect of Supra-High Irradiation on the Photosynthetic System of the Cyanophyte Synechocystis PCC 6714

Stability of thylakoid components under supra-high irradiancewas studied with the cyanophyte Synechocystis PCC 6714. Theactivity of overall photosynthesis was quickly inactivated (T_1/2=20min) under supra-high irradiance (300 W m^–2, white light).In parallel with the inactivation of photosynthesis, QA in PSII was also inactivated. Both inactivations were acceleratedby chloramphenicol (CAP) addition. The reactivation of PS IIrequired weak irradiation and was suppressed by CAP. However,PS I measured as P700 was very stable. The level of PS I measuredas P700 was not significantly reduced by the irradiation for12 h even in the presence of CAP while the level of Cyt b₅₅₉,component of PS II, was decreased markedly. The function ofPS I before and after supra-high irradiation with CAP was examinedby comparing sizes of P700 oxidation induced by a short flash,by a continuous light, and by determination of O₂-and ferredoxin-reduction.No difference was observed in PS I actions before and afterthe irradiation treatment. These results indicate that the PSI complex is very tolerant of supra-high irradiation. However,the cells grown under supra-high irradiance contained much fewerPS I and PS II complexes than Cyt b₆–f complexes. Theformer levels were reduced to a half to one fourth of thosebefore growth while the level of Cyt b₆–f complex wasnot reduced so much. A possible mechanism for changes in thylakoidcomposition under supra-high irradiation was discussed. (Received February 16, 1991; Accepted June 12, 1991)     

Changes in Photosystem Stoichiometry in Response to Environmental Conditions for Cell Growth Observed with the Cyanophyte Synechocystis PCC 6714

Changes in photosystem stoichiometry in response to shift ofenvironments for cell growth other than light regime were studiedwith the cyanophyte Synechocystis PCC 6714 in relation to thechange induced by light-quality shift. Following two environment-shiftswere examined: the shift of molecular form of inorganic carbonsource for photosynthesis from CO₂ to HCO₃^– (CO₂ stress)and the increase in salinity of the medium with NaCl (0.5 M)(Na⁺ stress). Both CO₂ and Na⁺ stresses induced the increasein PSI abundance resulting in a higher PSI/PSII stoichiometry.CO₂ stress was found to elevate simultaneously Cyt c oxidaseactivity (V_max). The feature was the same as that caused bylight-quality shift from preferential excitation of PSI to PSII(light stress) though the enhancement by either stress was smallerthan that by light stress. Under our experimental conditions,PSI/PSII stoichiometry appeared to increase at a fairly constantrate to the basal level even when the basal level had been differentlydetermined by the light-quality. Enhancing rates for PSI/PSIIstoichiometry and for Cyt c oxidase activity were also similarto each other. Since the two stresses affect the thylakoid electrontransport similarly to the shift of light-quality, we interpretedour results as follows: three environmental stresses, CO₂, Na⁺,and light stresses, cause changes in electron turnover capacityof PSI and Cyt c oxidase under a similar, probably a common,mechanism for monitoring redox state of thylakoid electron transportsystem. ¹On leave from Department of Biology, College of Natural Science,Kyngpook National University, Taegu 702-701, Korea. ₂Present address: Department of Marine Bioscience, Fukui Pre-fecturalUniversity, Obama, Fukui, 917 Japan.     

Photosynthetic Adaptation to Salt Stress in Three-Color Leaves of a C4 Plant Amaranthus tricolor

We examined the photosynthetic adaptation mechanisms for saltstress in Amaranthus tricolor, which has leaves with green,yellow and red regions, in relation to the accumulation of glycinebetaineas osmoprotectants. The content of Chl, especially of Chl bin the red and yellow regions was 3{small tilde}4% of that inthe green region. The levels of Chl proteins such as LHCII,PSI and PSII were significantly lower than those in the greenregion. However, the contents of other photosynthetic proteinsin these regions seem to be relatively high. We observed thenet photosynthetic CO₂ fixation activity in the red and yellowregions which was about 40% of that in the green region. Uponsalt stress (0.3 M NaCl) for 5 d the levels of Chl, PSI, PSII,ribulose 1,5-bis phosphate carboxygenase and oxygenase, andthe CO₂ fixation rate in the green region decreased by about20{small tilde}35% whereas those in the non-green regions remainedalmost at the same levels. A. tricolor was found to accumulatesglycinebetaine, betainealdehyde dehydrogenase and choline monooxygenaseat similar levels in all three color regions and their contentsincreased upon salt stress. These results suggest that the lowcapacity of light harvesting in non-green regions would be favorof salt stress since the photosynthetic components in theseregions were retained at relatively high levels under high salinity. (Received February 9, 1999; Accepted April 16, 1999)     

Regulation of Synthesis of PSI in the Cyanophytes Synechocystis PCC6714 and Plectonema boryanum during the Acclimation of the Photosystem Stoichiometry to the Light Quality

The effects of light quality on the formation of the PSI complexwere examined in Synechocystis PCC6714 and in Plectonema boryanum.The rate of increase in levels of core polypeptides of PSI,PsaA/B, doubled upon shift from Chl a-absorbed light (PSI light)to phycobilisome-ab-sorbed light (PSII light). The elevatedrate was decreased upon the reverse shift. Half time of theacceleration was approximately 10 min, and that of the decreasewas approximately 4 min. The rate of degradation of the polypeptideswas far lower than the rate of the increase under either lightregime. Neither synthesis nor degradation of the PsbA and PsbCpolypeptides of PSII was significantly altered by the lightquality. We conclude that synthesis of the PSI complex is chromaticallyregulated to allow adjustments in photosystem stoichiometry.The level of mRNA for PsaA/B was not altered by the light regime.Anomalous inhibition by chloramphenicol suggested that the regulationoccurs at a step(s) other than the peptide elongation step,perhaps at the initiation of the ribosome cycle or at the insertionof Chl a for the stabilization of the polypeptides. The pho-toreductionof protochlorophyllide (Pchlide) was compared with the synthesisof the polypeptides in a mutant of Plectonema boryanum thatlacked Pchlide dark reductase (YFC1004). The results indicatedthat the synthesis of stable PsaA/B polypeptides was not limitedby the reduction of Pchlide, although the synthesis did dependon a supply of Chl a. ¹Present address: Department of Plant Biology, University ofMaryland at College Park, MD 20742, U.S.A. ²Present address: Department of Marine Bioscience, Fukui Pre-fecturalUniversity, Obama, Fukui, 917 Japan     

The Accumulation of Protochlorophyllide in Cells of Synechocystis PCC 6714 with a Low PSI/PSII Stoichiometry

Changes in intracellular levels of Chl a precursors were examinedin relation to changes in the PSI/PSII stoichiometry in thecyanophyte Synechocystis PCC 6714. Protochlorophyllide (Pchlide)accumulated markedly in cells with a low PSI/PSII stoichiometrygrown under light that is absorbed by Chl a (PSI light) whereasno accumulation occurred in cells with a high PSI/PSII stoichiometrygrown under light absorbed by phycobilisomes (PSII light). Levelsof Pchlide in cells grown under PSI light decreased rapidlyupon a shift to PSII light. The rapid decrease in Pchlide accompanieda transient increase in chlorophyllide a, indicating that reductionof Pchlide was enhanced by shift to PSII light. The action spectrumindicated that the Pchlide decrease upon the shift to PSII lightdepended on excitation of Pchlide, suggesting that the accumulationof Pchllide was due to limited excitation of Pchlide, so thatPchlide photoreduction, under PSI light. However, comparisonof levels of Pchlide and the photosystem complexes in wild-typePlectonema boryanum with those in a mutant that lacked the darkPchlide reductase (YFC 1004) indicated that dark reduction compensatedfor the limited photoreduction under PSI light. Similar compensationby dark reduction was confirmed with Synechocystis PCC 6714.In cultures of Synechocystis under conditions where Pchlidecould not be photoreduced, accumulation of Pchlide and low PSI/PSIIstoichiometry occurred only when cells were illuminated withlight that preferentially excited PSI. The results indicatethat the low PSI/PSII stoichiometry in cells grown under PSIlight is not a result of inefficient synthesis of Chl a witha reduced rate of Pchlide photoreduction. They suggest furtherthat accumulation of Pchlide under PSI light results from retardationof the Chl a synthesis due to suppression of PSI synthesis. ¹Present address: Tsurukawa 5-15-11, Machida, Tokyo, 195 Japan.     

Changes in the photosynthetic characteristics and photosystem stoichiometries in wild-type and Chl <Emphasis Type="Italic">b</Emphasis>-deficient mutant rice seedlings under various irradiances

By using a wild-type rice (Oryza sativa L. cv. Norin No. 8) and the chlorophyll (Chl) b-deficient mutant derived from Norin No. 8 (chlorina 11), the present study monitored the oxygen evolution, contents of Chl a and b, β-carotene, and lutein in leaf and the contents of cytochrome f, and the reaction centres of photosystem I (PSI) and photosystem II (PSII) in thylakoids. The oxygen evolution, maximal quantum yield of PSII (F_v/F_m) and Chl concentration remained constant in both Norin No. 8 and chlorina 11 under 5 and 2% of full sunlight for six days. On the other hand, on the thylakoid level, the PSII reaction centre of chlorina 11 was more stable even under high irradiance, while approximately 40% decrease in levels of the PSII reaction centre occurred under 2% of full sunlight for six days. However, under such conditions, by regulating the stoichiometry of active PSII and PSI centres, the light absorption balance in both rice types was adjusted between the two photosystems. The present study attempted to examine whether the light absorption balance between PSII and PSI is altered to effectively conduct photosynthesis in the wild-type and Chl b-deficient mutant rice seedlings.     

Regulation of Stoichiometry between PSI and PSII in Response to Light Regime for Photosynthesis Observed with Synechocystis PCC 6714: Relationship between Redox State of Cyt b6-f Complex and Regulation of PSI Formation

The effect of the Cyt b₆-f redox state on the PSI formationwas examined with the cyanophyte Synechocystis PCC 6714 by usinga Q-cycle inhibitor, HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide).HQNO inhibited the rapid reduction of flash-oxidized Cyt f,the reaction correlating with the stimulation of PSI formation,on one hand, and accumulated reduced Cyt b₆, on the other, indicatingthat the electron flow in the Q-cycle correlates with regulationof PSI synthesis. HQNO also inhibited the stimulation of PSIformation under PSII light, resulting in a low PSI/PSII ratioeven under PSII light, while the PSI formation under PSI lightwas not suppressed by HQNO. Simultaneous inhibition of Cyt b₆oxidation through the Q-cycle and the stimulated PSI formationby HQNO suggests that an HQNO-sensitive Cyt b₆ oxidation isinvolved in the mechanism of monitoring the state of electrontransport system for regulation of PSI formation. (Received March 3, 1993; Accepted August 9, 1993)     

Formation of Chlorophyll-Protein Complexes during Greening. 2. Redistribution of Chlorophyll among Apoproteins

The formation of Chl-protein complexes (CPs) in cucumber cotyledonsduring a dark period after a brief illumination was studied.SDS-PAGE analysis showed that the P700-Chl a-protein complex(CP1) and Chl a-protein complex of the PS II core (CPa) increased,with a concomitant decrease in the light-harvesting Chl a/6-proteincomplex of PS II (LHCII), during 24-h dark incubation of cotyledonsafter 6h of continuous illumination. In agreement with theseresults, curve analysis revealed that spectral components characteristicof CP1 and CPa increased while those of Chi b decreased duringthe dark incubation. Since Chl is not synthesized in the dark,Chl must be released from LHCII and re-incorporated into CP1and CPa. The amounts of apoproteins of CP1 and 43 kDa protein(one of the apoproteins of CPa) increased during the dark incubation,and the increase could be inhibited by chloramphenicol (CAP).CP1 did not increase in the dark when tissues were incubatedwith CAP which inhibited the synthesis of apoproteins of CP1,indicating that CP formation by Chl redistribution needs newlysynthesized apoproteins. The decrease in LHCII apoproteins duringdark incubation was inhibited by CAP probably because Chl wasnot removed from LHCII by apoproteins of CP1 and CPa, whosesynthesis was blocked by the presence of CAP. When intermittently-illuminatedcotyledons containing a little LHCII were incubated with CaCl₂in the dark, Chl b and LHCII apoproteins accumulated with thedisappearance of 43 kDa protein; Chl of 43 kDa protein may beutilized for LHCII formation. We concluded that Chl moleculesonce bound with their apoproteins are redistributed among theapoproteins. (Received October 17, 1990; Accepted December 6, 1990)     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Thylakoid Membrane-Bound, NADPH-Specific Pyridine Nucleotide Dehydrogenase Complex Mediates Cyclic Electron Transport in the Cyanobacterium Synechocystis sp. PCC 6803

The donation of electrons from NADPH to the intersystem chain,as monitored by an increase in Chl fluorescence, occurred inthe isolated thylakoid membranes of Synechocystis PCC 6803.The stimulation by NADPH of the methyl viologen-dependent photoreductionof dioxygen and of the reduction of P700⁺ after photooxidationin the presence of DCMU also confirmed the donation of electronsfrom NADPH to the electron carriers in the intersystem. Thesereactions were sensitive to rotenone, capsaicin, l-(2-thenoyl)-3,3,3-trifluoroacetoneand HgCl₂ but not to antimycin A or flavone. In contrast tothe thylakoid membranes from the wild type, those from a mutant,designated M55, in which a gene of a subunit of the pyridinenucleotide dehydrogenase complex (NDH) had been inactivated,did not show evidence of such reactions. These results supportour previous hypothesis that the transport of electrons fromNADPH to the intersystem chain is mediated by NDH [Mi et al.(1994) Plant Cell Physiol. 35: 163] and indicate the bindingof an NADPH-specific NDH to the thylakoid membranes. The Chlfluorescence was quenched transiently by addition of ferredoxinand NADP₊ to the thylakoid membranes but showed a subsequentincrease. This result suggests the reduction of plastoquinoneby the photoreduced NADP₊ and initiation of the NADPH-mediatedcyclic flow of electrons around PSI. Furthermore, a similarresponse of Chl fluorescence was observed upon the additionof ferredoxin only, demonstrating the ferredoxin-dependent cyclicflow of electrons. Both pathways of cyclic electron transportwere inhibited by rotenone, and were not detected in the NDH-defectedthylakoid membranes from M55, indicating the participation ofthe NDH complex. These results confirm that, in Synechocystis,the thylakoid-bound NDH complex mediates the ferredoxin-dependentcyclic electron flow, as well as the NADPH-dependent cyclicelectron flow. (Received November 24, 1994; Accepted March 16, 1995)     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.