Native Green Gel Research Articles

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

Mechanism of interaction of Al3+ with the proteins composition of photosystem II.

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al 3+, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn4O5Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (Fv/Fm) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F0 and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al 3+ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al 3+-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.

Isolation and Characterization of Homodimeric Type-I Reaction Center Complex from Candidatus Chloracidobacterium thermophilum, an Aerobic Chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ∼483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.

Suppression of the phytoene desaturase gene influence on the organization and function of photosystem II (PSII) and antioxidant enzyme activities in tobacco

Carotenoid compounds play a key role in plant photosynthesis. Some are integrated within the photosynthetic system, where they serve as regulators of light harvesting. In this study, we found pigment alterations in transgenic tobacco leaves following RNAi mediated suppression of pds (phytoene desaturase). Native green gel analysis and pigment determinations showed that the ratios of Chl a/b in PSI and PSII cores and in LHCII were unaffected by gene silencing. SDS-PAGE indicated that the Chl-binding complexes of PSI and PSII cores were unchanged in the transgenic plants, but the LHCII levels were changed. A new band around 50 kDa was found, with a protein density around 18 kDa increased. Chl fluorescence kinetics showed that energy transfer and photosynthesis in the pds-silenced plants were inhibited under both high and moderate light, and that both Fv′/Fm′ and qP were decreased when qN was increased. The number of Chl molecules in every LHCII protein complex was decreased, resulting in less efficient energy transfer and a severe decline in PSII activity. Antioxidant enzymes no longer worked in concert, which could explain the appearance of the leaf albinism.

Loss of peripheral polypeptides in the stromal side of photosystem I by light-chilling in cucumber leaves.

Photosystem I (PSI) is severely damaged by chilling at 4 degrees C in low light, especially in the chilling sensitive plant cucumber. To investigate the early events in PSI photoinhibition, we examined structural changes in the level of pigment-protein complexes in cucumber leaves in comparison with pea leaves. The complexes were separated on a native green gel and an increase in the intensity of a band was observed only in light-chilled cucumber leaves. The 77 K fluorescence emission spectrum of this green band indicated that the band was mainly composed of PSI with light-harvesting complex I. Each lane was cut from the green gel and separated on a fully denaturing SDS-PAGE in the second dimension. The new green gel band observed after light-chilling in cucumber leaves lacked 19, 18, and 16.5 kDa polypeptides. These results suggest that light-chilling facilitates the release of three peripheral polypeptides as an early event of chilling stress in vivo, which results in the inactivation of PSI in intact cucumber leaves.

Changes in Thermostability of Photosystem II and Leaf Lipid Composition of Rice Mutant with Deficiency of Light‐harvesting Chlorophyll a/b Protein Complexes

AbstractWe studied the difference in thermostability of photosystem II (PSII) and leaf lipid composition between a T‐DNA insertion mutant rice (Oryza sativa L.) VG28 and its wild type Zhonghua 11. Native green gel and SDS‐PAGE electro‐phoreses revealed that the mutant VG28 lacked all light‐harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSII photochemistry (Fv/ Fm) and oxygen‐evolving activity of PSII in leaves significantly decreased with increasing temperature. However, the PSII activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of 16:1t and a marked increase in the content of 18:3 compared with the wild type. The effects of lipid composition and unsaturation of membrane lipids on the thermostability of PSII are discussed.

Heat Stress Induces an Aggregation of the Light-Harvesting Complex of Photosystem II in Spinach Plants

Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25 degrees C-50 degrees C) in the dark for 30 min. At temperatures higher than 35 degrees C, CO2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45 degrees C and decreased only slightly at 50 degrees C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25 degrees C to 50 degrees C in the dark for 10 min. At temperatures over 35 degrees C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25 degrees C to 50 degrees C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35 degrees C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

Differential changes in degradation of chlorophyll–protein complexes of photosystem I and photosystem II during flag leaf senescence of rice

The stability of chlorophyll–protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO 2 assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/ b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll–protein complexes of PSI was greater than in chlorophyll–protein complexes of PSII. The fluorescence yields for all chlorophyll–protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll–protein complex was different and the order for the stability of chlorophyll–protein complexes during leaf senescence was: LHCII > CP43 > CP47 > LHCI > PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll–protein complexes.

Increased stability of LHCII by aggregate formation during dark-induced leaf senescence in the Arabidopsis mutant, ore10.

Leaf senescence in a stay-green mutant of Arabidopsis thaliana, ore10, was investigated during dark-incubation of its detached leaves. During this dark-induced senescence (DIS), Chl loss was delayed in ore10 mutants, as compared with wild type, but the rate of decline in the photochemical efficiency of PSII was not delayed in mutant leaves. After 2 d of DIS, native green gel electrophoresis of ore 10 leaf proteins resulted in a significant amount of pigment remaining as aggregates on top of the stacking gel. In addition, the accumulation of aggregates coincided with the emergence of a new band near 700 nm (F(699)) in the 77 K fluorescence emission spectrum of the aggregates. At 4 d, F(699) became a major band, both in the isolated aggregates and in intact leaves. Prolonged treatment with detergents revealed that light-harvesting complex II (LHCII) remaining after 2 d was highly stable, and the accumulation of aggregates coincided with the appearance of truncated LHCII in senescing ore10 leaves. These results suggest that increased LHCII stability is due to the formation of aggregates of trimmed LHCII. Thus, the LHCII protein degradation step that follows proteolysis of its terminal peptides is a possible lesion site of the ore10 mutant.

Photoinhibition of photosystem I is accelerated by dimethyldithiocarbamate, an inhibitor of superoxide dismutase, during light-chilling of spinach leaves

In vivo photoinhibition of photosystem I (PS I) was investigated at chilling temperature using the leaves of the chilling-resistant spinach plant treated with an inhibitor of superoxide dismutase, diethyldithiocarbamate (DDC). When spinach leaves were treated with DDC during chilling at 4 °C for 12 h with a light intensity of 120 μmol m −2 s −1, the activity of PS I and the content of iron–sulfur centers declined to about 50% and 25% of the non-DDC-treated controls, respectively. A native green gel analysis of thylakoid membranes isolated from the DDC-treated leaves resolved a novel chlorophyll–protein complex, which was identified as the light-harvesting complex I (LHC I)-deficient PS I complex when examined by 77 K fluorescence spectroscopy and two-dimensional sodium dodecyl sulfate gel electrophoresis. The possible dissociation of LHC I as an early structural change in the PS I complex after DDC-induced photoinhibition of PS I is discussed.

ANALYSIS OF THE CHLOROPLAST PROTEIN COMPLEXES BY BLUE-NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b6f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.

Appearance of type 1, 2, and 3 light-harvesting complex II and light-harvesting complex I proteins during light-induced greening of barley (Hordeum vulgare) etioplasts.

Monospecific antibodies directed against typical domains of type 1, 2, and 3 light-harvesting complex (LHC) II apoproteins have been used (a) to identify these apoproteins on denaturing sodium dodecyl sulfate gels of barley (Hordeum vulgare) thylakoids, (b) to determine their distribution between grana and stroma membranes, and (c) to follow their accumulation during light-induced greening of etioplasts. In addition, we have studied the light-induced assembly of chlorophyll-protein complexes with a native green gel system (K.D. Allen, L.A. Staehelin [1991] Anal Biochem 194: 214-222). Western blot analysis of the three major LHCII apoprotein bands has identified the highest molecular mass band at 27.5 kD as containing the type 2 LHCII apoproteins, the middle band at 26.9 kD as containing the type 1 LHCII apoproteins, and the lowest band at 26.0 kD as containing the type 3 LHCII apoproteins. During light-induced greening of 6-d-old etiolated barley seedlings, the type 1, 2, and 3 LHCII apoproteins accumulate simultaneously and at similar rates but appear somewhat sooner (< 4 h) in thylakoids from apical than from basal (4-8 h) leaf segments. LHCI polypeptides accrue with similar kinetics, whereas the 33-kD oxygen-evolving complex polypeptides can be detected already in the 0-h light samples. During the most rapid phase of thylakoid development (8-24 h), two slightly larger (28.3 and 28.7 kD) type 2 LHCII apoproteins (precursor intermediates?) also accumulate in the thylakoids. No corresponding higher molecular mass forms of type 1 and 3 LHCII apoproteins could be detected. It is interesting that differences are still apparent in the composition of chlorophyll-protein complexes of light-control plants and those of etiolated plants greened for 8 d.

Resolution of 16 to 20 chlorophyII-protein complexes using a low lonic strength native green gel system

Conventional native “green gel” systems resolve at most 10 chlorophyII-protein complexes from thylakoid membranes of higher plants and green algae. Such analyses suggest a simplicity of the thylakoid membrane that is not supported by a growing body of evidence on the heterogeneity of photosystems I and II (PSI and PSII) and their associated antennae (LHCI and LHCII). We report here the development and characterization of a low ionic strength native “green gel” system that resolves from 16 to 20, mostly large chlorophyII-protein complexes from a variety of higher plant and green algal species with very little release of free pigment. In Chlamydomonas, this system resolves multiple PSI-LHCI complexes, multiple PSII-LHCII complexes, four oligomeric LHCII complexes, as well as several low electrophoretic mobility reaction center complexes, and a number of small complexes. We have obtained similar resolution with a large number of higher plant and green algal species. We also demonstrate how this system can be used as a sort of “fingerprinting” technique to distinguish thylakoids of different species, and for the analysis of photosynthetic mutants, using the chlorophyII b-less chlorina f2 mutant of barley as an example.