Senescence-induced alterations in the photosystem II functions of Cucumis sativus cotyledons: probing of senescence driven alterations of photosystem II by chlorophyll a fluorescence induction O-J-I-P transients

Senescence-induced alterations in photosystem II (PS II) structure and photofunctions were probed in cucumber (Cucumis sativus) cotyledons, using fast O-J-I-P Chlorophyll a (Chl a) fluorescence transients. Analysis of measured and derived parameters of the fast fluorescence O-J-I-P transient revealed senescence-induced alterations in (i), PS II acceptor side electron transfer equilibrium between QA and QB, the primary stable and secondary acceptors of PS II; (ii), intersystem PQ pool size and (iii), affected electron transfer from PS II to PS I. Also, senescence of cotyledons triggered conversion of QA-reducing (fully active) to non- QA-reducing PS II (heat sink) centres. Further, some of the remaining active PS II centres showed a high apparent trapping efficiency due to clustering and energetic connectivity (grouping) between the antennae of active and inactive centers. The overall density of active PS II reaction centers showed a temporal decrease due to the onset of foliar senescence. Thus, the fast Chl a fluorescence transients, with a time resolution of at least 50 mircosec and use of the equations of JIP-test, provide a valuable, non-invasive rapid biophysical probe to study the ageing in plants in terms of detecting photosynthetic activities and the heterogeneity of different types of photosynthetic units. Further, these results were found to be in agreement with the earlier in vitro studies using thylakoids isolated from senescing cotyledons where it was shown that senescence induced heterogeneity in PS II centers affected acceptor side QA<-->QB equilibrium.     

Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons

Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b_[ill]/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Photoactivation of Oxygen-evolving System in Dark-grown Spruce Seedlings

Plastids prepared from dark-grown spruce seedlings showed a negligible activity of photosystem II, and no fluorescence variation was observed during actinic illumination. The photosystem II reaction centre, however, was present in primary thylakoids. Exposure of such seedlings to continuous light induced the development of photosystem II activity via three stages (rapid, lagged and gradual), and the variable fluorescence appeared. The rapid development of photosystem II may be attributed to the activation of the oxygen-evolving system, possibly the manganese-catalyzing site, and the lagged and gradual developments may be closely related to the formation of thylakoid membranes and their assembly to grana.     

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.     

Age dependent alterations in photosystem II acceptor side in Cucumis sativus cotyledonary leaf thylakoids: analysis of binding characteristics of herbicide [14C]-atrazine.

Senescence induced temporal changes in photosystems can be conveniently studied in cotyledonary leaves. We monitored the protein, chlorophyll and electron transport activities in Cucumis sativus cv Poinsette cotyledonary leaves and observed that by 20th day, there was a 50%, 41% and 30-33% decline in the chlorophyll, protein and photosystem II activity respectively when compared to 6th day cotyledonary leaves taken as control. We investigated the changes in photosystem II activity (O2 evolution) as a function of light intensity. The photosystem II functional antenna decreased by 27% and the functional photosystem II units decreased by 30% in 20-day old cotyledonary leaf thylakoids. The herbicide [14C]-atrazine binding assay to monitor specific binding of the herbicide to the acceptor side of photosystem II reaction centre protein, D1, showed an increase in the affinity for atrazine towards D1 protein and decrease in the QB binding sites in 20th day leaf thylakoids when compared to 6th day leaf thylakoids. The western blot analysis also suggested a decrease in steady state levels of D1 protein in 20th day cotyledonary leaf thylakoids as compared to 6th day sample which is in agreement with [14C]-atrazine binding assay and light saturation kinetics.     

Expression of 5-amino levulinic acid induced photodynamic damage to the thylakoid membranes in dark sensitized by brief pre-illumination

The 5-amino levulinic acid treated cucumber (Cucumis sativus L., CV. Pointsette) plants upon exposure to light (≃30,000 lux) wilted within 6 h and died after 36 h due to photodynamic reactions. Thylakoid membranes, the site of accumulation of porphyrins, were damaged due to photodynamic reactions leading to the inhibition of membrane linked functions of photosystem II, photosystem I and the whole chain electron transport. Photosystem II was more susceptible to photodynamic damage than photosystem I. The exogenous electron donors Mn²⁺, diphenyl carbazide and NH₂OH failed to donate electrons to photosystem II suggesting that the damage has taken place close to P680. The 5-amino levulinic acid treated plants exposed to 30 min of light did not show any damage to the thylakoid membranes. However, when the above plants were transferred to dark for 12 h there was substantial damage to the thylakoid membrane system.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.Abbreviations CHLOX linolenic acid-dependent oxidative chlorophyll bleaching activity - CHLPX chlorophyll peroxidase - CPI chlorophyll-protein complex I - LHC light-harvesting complex - LNA linolenic acid - PSI photosystem I - PSII photosystem II - S relative senescence rate - t _1/2 lialf time for degradation     

Light‐harvesting superstructures of green plant chloroplasts lacking photosystems

The light‐harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures – stacks of dozens of thylakoids or supergrana. Freeze‐fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light‐harvesting membranes, organized into three‐dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self‐assembly of the three‐dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.     

Stabilization of Thylakoid Membranes by Spermine during Stress-induced Senescence of Barley Leaf Discs

The effect of spermine on photochemical activity and polypeptide composition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show photosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water. Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential loss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence.     

Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5–5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii

To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments.     

Mobilization of photosystem II induced by intense red light in the Cyanobacterium Synechococcus sp PCC7942

We use confocal fluorescence microscopy and fluorescence recovery after photobleaching to show that a specific light signal controls the diffusion of a protein complex in thylakoid membranes of the cyanobacterium Synechococcus sp PCC7942 in vivo. In low light, photosystem II appears completely immobile in the membrane. However, exposure to intense red light triggers rapid diffusion of up to approximately 50% of photosystem II reaction centers. Particularly intense or prolonged red light exposure also leads to the redistribution of photosystem II to specific zones within the thylakoid membranes. The mobilization does not result from photodamage but is triggered by a specific red light signal. We show that mobilization of photosystem II is required for the rapid initiation of recovery from photoinhibition. Thus, intense red light triggers a switch from a static to a dynamic configuration of thylakoid membrane protein complexes, and this facilitates the rapid turnover and repair of the complexes. The localized concentrations of photosystem II seen after red light treatment may correspond to specific zones where the repair cycle is active.     

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the 'H' subunit of bacterial reaction centres?

Although the amino acid sequence of the 9 kDa (phospho)protein of chloroplasts has been determined, the function of this thylakoid membrane protein in photosynthetic electron transport and the reason for its physiological control remains unclear. In this paper, I briefly review the evidence which indicates that the phosphorylation of the 9 kDa protein results in a partial inhibition of photosynthetic oxygen evolution by increasing the stability of the semiquinone bound to QA the primary, plastoquinone-binding site of photosystem II (PS II). I propose that in its dephosphorylated state, the 9 kDa thylakoid membrane protein may serve PS II to ensure efficient photochemical charge separation by aiding the transfer of reducing equivalents out of the reaction centre to the attendant plastoquinone pool. This function is analogous to that proposed for the H-subunit of the reaction centre of photosynthetic eubacteria. Whether these two proteins have evolved from a common ancestral reaction centre protein is discussed in the light of a comparison of their amino acid sequences and predicted secondary structures.     

Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.     

Changes of the photosystem 2 activity and thylakoid proteins in spruce seedlings during water stress

We subjected spruce [Picea glauca (Moench) Voss × P. engelmanni Parry hybrid complex] seedlings to a severe water stress (shoot water potential ≤-3.5 MPa) to permit assessment of stress effects on photosystem 2 (PS2) in isolated photosynthetic membranes. The thylakoids and Triton-treated membranes isolated from stressed seedlings showed declines in O2-evolving capacity (H2O → 2,6-dichloro-p-benzoquinone, DCBQ) and electron transport activity (H2O → 2,6-dichlorophenol indophenol, DCIP). A partial restoration of O2-evolution by adding CaCl2 suggested an effect of water stress on the oxygen-enhancing extrinsic (OEE) polypeptides. Water stress had an additional impact on the reaction centre, shown by the inability of 1,5-diphenylcarbazide (DPC) to restore the electron transport (DPC → DCIP) to the levels seen in control membranes. Quantification of specific photosynthetic membrane proteins by immunoblots strengthened the above suggestions: after drought stress, concentrations of OEE1 and OEE2 declined by 40 %, and amount of the reaction centre protein D1, ATP synthetase, and cytochrome f also declined. The specific effect of stress on these proteins was confirmed by the fact that the amount of chlorophyll-protein complex CP2 was unchanged in membranes isolated from drought-stressed seedlings.     

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.     

Plant plasma membrane. Correlation between glucan synthase II activity and a mannosyl transferase activity.

In cotyledons of germinating cucumber seeds (Cucumis sativus), plasma membranes were investigated biochemically and partially characterized. Glucan synthease II was utilized as a marker to locate plasma membrane vesicles within fractions obtained by differential centrifugation or within sucrose gradients used either in zonal centrifugations or in sedimentations to equilibrium density. Thorough homogenization led to a homogeneous population of plasma membrane vesicles which could be clearly separated from mitochondria by centrifugation at 150000 x g for 4 h in a zonal rotor. The profiles of glucan synthase II activity and naphthylphthalamic acid binding coincided with that of a mannosyl transferase activity, monitored by direct transfer of mannose from GDPmannose to endogeneous acceptors.