High-light induced superoxide radical formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy.

The generation of superoxide radical (O2·â») in Cyt b6f of Bryopsis corticulans under high light illumination was studied using electron paramagnetic resonance (EPR) spectroscopy. This could be evidenced by the addition of SOD which specifically reacted with O2·â». The generation of O2·â» was lost in the absence of oxygen and was found to be suppressed in the presence of NaN3 and be scavenged by extraneous antioxidants such as ascorbate, ß-carotene and glutathione which could also scavenged ¹O2*. These results indicated that O2·â» which produced under high light illumination in Cyt b6f of B. corticulans might rise from a reaction which ¹O2* could participated in. Also the photo-protection mechanism to Cyt b6f complex by antioxidants which might contain in thylakoid was speculated.

High-light induced singlet oxygen formation in cytochrome b(6)f complex from Bryopsis corticulans as detected by EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen ((1)O(2)*) in the intact and the Rieske-depleted cytochrome b(6)f complexes (Cyt b(6)f) from Bryopsis corticulans, as well as in the isolated Rieske Fe-S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b(6)f can be bleached by light-induced (1)O(2)*, and that the (1)O(2)* production can be promoted by D(2)O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, beta-carotene and glutathione. Under similar experimental conditions, (1)O(2)* was also detected in the Rieske-depleted Cyt b(6)f complex, but not in the isolated Rieske Fe-S protein. The results prove that Chl a cofactor, rather than Rieske Fe-S protein, is the specific site of (1)O(2)* formation, a conclusion which draws further support from the generation of (1)O(2)* with selective excitation of Chl a using monocolor red light.

Singlet oxygen formation and chlorophyll a triplet excited state deactivation in the cytochrome b6f complex from Bryopsis corticulans.

We have attempted to investigate the correlation between the detergent-perturbed structural integrity of the Cyt b (6) f complex from the marine green alga Bryopsis corticulans and its photo-protective properties, for which the nonionic detergents n-octyl-beta-D-glucopyranoside (beta-OG) and n-dodecyl-beta-D-maltoside (beta-DM), respectively, were used for the preparation of Cyt b (6) f, and the singlet oxygen ((1)O(2)*) production as well as the triplet excited-state chlorophyll a ((3)Chl a*) formation and deactivation were examined by spectroscopic means. Near-infrared luminescence of (1)O(2)* (approximately 1,270 nm) on photo-irradiation was detected for the beta-OG preparation where the complex is mainly in oligomeric state, but not for the beta-DM one in which the complex exists in dimeric form. Under anaerobic condition, photo-excitation of Chl a in the beta-DM preparation generated (3)Chl a* with a lower quantum yield of Phi(T) approximately 0.02 and a longer lifetime of approximately 600 micros with respect to those as in the case of beta-OG preparation, Phi(T) approximately 0.12 and 200-300 micros. These results prove that the enzymatically active and intact Cyt b (6) f complex on photo-excitation tends to produce little (3)Chl a* or (1)O(2)*, which implies that the pigment-protein assembly of Cyt b (6) f complex per se is crucial for photo-protection.

Characterization of chlorophyll-protein complexes isolated from a Siphonous green alga, Bryopsis corticulans.

Six chlorophyll-protein complexes are isolated from thylakoid membranes of Bryopsis corticulans by dodecyl-beta-D: -maltoside polyacrylamide gel electrophoresis. Unlike that of higher plants, the 77 K fluorescence emission spectrum of the CP1 band, the PSI core complexes of B. corticulans, presents two peaks, one at 675 nm and the other at 715-717 nm. The emission peak at 715-717 nm is slightly higher than that at 675 nm in the CP1 band when excited at 438 or 540 nm. However, the peak at 715 nm is obviously lower than that at 675 nm when excited at 480 nm. The excitation spectra of CP1 demonstrate that the peak at 675 nm is mainly attributed to energy from Chl b while it is the energy from Chl a that plays an important role in exciting the peak at 715-717 nm. Siphonaxanthin is found to contribute to both the 675 nm and 715-717 nm peaks. We propose from the above results that chlorophyll a and siphonaxanthin are mainly responsible for the transfer of energy to the far-red region of PSI while it is Chl b that contributes most of the transfer of energy to the red region of PSI. The analysis of chlorophyll composition and spectral characteristics of LHCP(1 )and LHCP(3) also indicate that higher content of Chl b and siphonaxanthin, mainly presented in LHCP(1), the trimeric form of LHCII, are evolved by B. corticulans to absorb an appropriate amount of light energy so as to adapt to their natural habitats.

Thermal denaturation of CP43 studied by Fourier transform-infrared spectroscopy and terahertz time-domain spectroscopy.

Thermal denaturation of CP43 was studied by Fourier transform-infrared (FT-IR) spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and terahertz time-domain spectroscopy (THz-TDS). Under heat treatment, the secondary structure of CP43 changed, and the main thermal transition occurred at 59 degrees C. During the process, CP43 aggregated at first, and then with increasing temperature degraded. The low-frequency collective vibrational modes of CP43 changed with increasing temperature and decreasing mass. THz-TDS is a new technique used to study the conformational state of a molecule, and it is the first use of this technique to study the photosynthesis membrane proteins in this paper. The results presented here demonstrate that THz-TDS has both advantages and disadvantages in monitoring the thermal denaturation of membrane proteins, which is important in applying THz-TDS technique to study of biomolecules.

The effects of detergents DDM and beta-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts.

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b(6)f (Cyt b(6)f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b(6)f complexes from different sources ( approximately 200 and approximately 600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-beta-D-maltoside (DDM) and n-octyl-beta-D-glucopyranoside (beta-OG), but has nothing to do with the different sources of Cyt b(6)f complexes. Compared with the Cyt b(6)f dissolved in beta-OG, the Cyt b(6)f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, approximately 200 ps, of the Chl a in Cyt b(6)f complex in DDM is closer to the true in vivo.

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy.

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.

Study on energy transfer between carotenoid and chlorophyll a in cytochrome b6f complex from Bryopsis corticulans.

The excitation energy transfer between carotenoid and chlorophyll (Chl) in the cytochrome b ( 6 ) f complex from Bryopsis corticulans (B. corticulans), in which the carotenoid is 9-cis-alpha-carotene, was investigated by means of fluorescence excitation and sub-microsecond time-resolved absorption spectroscopies. The presence of efficient singlet excitation transfer from alpha-carotene to Chl a was found with an overall efficiency as high as approximately approximately 24%, meanwhile the Chl a-to-alpha-carotene triplet excitation transfer was also evidenced. Circular dichroism spectroscopy showed that alpha-carotene molecule existed in an asymmetric environment and Chl a molecule had a certain orientation in this complex.

Ultrafast carotenoid-to-chlorophyll singlet energy transfer in the cytochrome b6f complex from Bryopsis corticulans.

Ultrafast carotenoid-to-chlorophyll (Car-to-Chl) singlet excitation energy transfer in the cytochrome b(6)f (Cyt b(6)f) complex from Bryopsis corticulans is investigated by the use of femtosecond time-resolved absorption spectroscopy. For all-trans-alpha-carotene free in n-hexane, the lifetimes of the two low-lying singlet excited states, S(1)(2A(g)(-)) and S(2)(1B(u)(+)), are determined to be 14.3 +/- 0.4 ps and 230 +/- 10 fs, respectively. For the Cyt b(6)f complex, to which 9-cis-alpha-carotene is bound, the lifetime of the S(1)(2A(g)(-)) state remains unchanged, whereas that of the S(2)(1B(u)(+)) state is significantly reduced. In addition, a decay-to-rise correlation between the excited-state dynamics of alpha-carotene and Chl a is clearly observed. This spectroscopic evidence proves that the S(2)(1B(u)(+)) state is able to transfer electronic excitations to the Q(x) state of Chl a, whereas the S(1)(2A(g)(-)) state remains inactive. The time constant and the partial efficiency of the energy transfer are determined to be 240 +/- 40 fs and (49 +/- 4)%, respectively, which supports the overall efficiency of 24% determined with steady-state fluorescence spectroscopy. A scheme of the alpha-carotene-to-Chl a singlet energy transfer is proposed based on the excited-state dynamics of the pigments.

Hydrogen peroxide-induced chlorophyll a bleaching in the cytochrome b6f complex: a simple and effective assay for stability of the complex in detergent solutions.

The instability of cytochrome b ( 6 ) f complex in detergent solutions is a well-known problem that has been studied extensively, but without finding a satisfactory solution. One of the important reasons can be short of the useful method to verify whether the complex suspended in different detergent is in an intact state or not. In this article, a simple and effective assay for stability of the complex was proposed based on the investigation on the different effects of the two detergents, n-octyl-beta-D: -glucopyranoside (OG) and dodecyl-beta-D: -maltoside (DDM), on the properties of the complex. DDM stabilizes the complex preparation more effectively whereas OG denatures the interactions of the heme groups and pigment molecules with the protein environment, leading to the bleaching of chlorophyll a induced by addition of hydrogen peroxide. The assay of the use of hydrogen peroxide to characterize the complex by studying the bleaching of chlorophyll induced by hydrogen peroxide and the peroxidase activity of the complex was discussed. This simple method will probably be useful to study the stability of the complex.

Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor.

Photosystem I (PSI), which consists of a core complex and light-harvesting complex I (LHCI), is an important multisubunit pigment-protein complex located in the photosynthetic membranes of cyanobacteria, algae and plants. In the present study, we described a rapid method for isolation and purification of PSI and its subfractions. For purification of PSI, crude PSI was first prepared by differential centrifugation, which was applicable on a large scale at low cost. Then PSI was purified by sucrose gradient ultracentrifugation in a vertical rotor to reduce the centrifugation time from more than 20 h when using a swinging bucket rotor to only 3 h. Similarly, for subfractionation of PSI into the core complex and light-harvesting complex I, sucrose gradient ultracentrifugation in a vertical rotor was also used and it took only 4 h to obtain the PSI core, LHCI-680, and LHCI-730 at the same time. The resulting preparations were characterized by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectroscopy, and 77 K fluorescence spectroscopy. In addition, their pigment composition was analyzed by high-performance liquid chromatography and the results showed that each Lhca could bind 1.5-1.6 luteins, 1.0 Violaxanthins, and 0.8-1.1 beta-carotenes on average, demonstrating that fewer carotenoids were released than with the slower traditional centrifugation. These results showed that the rapid isolation procedure, based on differential centrifugation and sucrose gradient ultracentrifugation in a vertical rotor, was efficient, and it should significantly facilitate preparation and studies of plant PSI. Moreover, the vertical rotor, rather than the swinging bucket rotor, may be a good choice for isolation of some other proteins.

Characterization of the cytochrome b(6)f complex from marine green alga, Bryopsis corticulans.

A pure, active cytochrome b(6)f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b(6)f complex, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt (6) to Cyt f was 2.01 : 1. The cytochrome b(6) f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin-ferricyanide per Cyt f per second. alpha-Carotene, one kind of carotenoid that has not been found to present in cytochrome b(6)f complex, was discovered in this preparation by reversed phase HPLC. It was different from beta-carotene usually found in cytochrome b(6)f complex. The configuration of the major alpha-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this alpha-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophyll a, cis and all-trans-alpha-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.

[Effects of Triton X-100 on the oxygen uptake rate of photosystem I particles treated at 70 degrees C].

The characteristics including oxygen uptake rates, fluorescence spectra and absorption spectra of photosystem I particles with or without Triton-X 100 treatment before or after the incubation at 70 degrees C for 10 min were compared. The oxygen uptake rates of photosystem I particles decreased after being incubated at 70 degrees C for 10 min, which could be recovered by the addition of Triton-X 100. Singlet oxygen was formed when the light-harvesting complex I was separated from the core complex of photosystem I, which resulted in high oxygen uptake rate. There was much difference in the fluorescence spectra of photosystem I particles between photosystem I particles treated with Triton-X 100 after the incubation at 70 degrees C for 10 min or not, which implies the ability of Triton-X 100 to promote the recovery of photosystem I particles after the incubation at 70 degrees C for 10 min.

Effects of acid and alkali on the light absorption, energy transfer and protein secondary structures of core antenna subunits CP43 and CP47 of photosystem II.

The effects of acid and alkali treatment on the light absorption, energy transfer and protein secondary structure of the photosystem II core antenna CP43 and CP47 of spinach were investigated by the absorption spectra, fluorescence emission spectra and circular dichroism spectra. It has been found that acid treatment caused the appearance of absorption characteristic of pheophytin a (Pheo a), whereas alkali treatment induced a new absorption peak at 642 nm. The energy transfer between beta-carotene and chlorophyll a (Chl a) in CP43 was easily disturbed by alkali, whereas in CP47 was readily affected by acid. As to the effects on the secondary structure of proteins in CP43 and CP47, effects of acid were far less than those of alkali. Both acid and alkali disturbed the microenvironment of Chl a and interfered exciton interaction between Chl a molecules. It was suggested that acid and alkali affect the light absorption, energy transfer and protein secondary structure of CP43 and CP47 in a different way. H+ can permeate into the internal space of alpha-helix, change Chl a into Pheo a and disturb the microenvironment of pigments without damaging the secondary structure of protein, whereas OH- can induce the protein unfolding at first, then saponify Chl a to chlorophyllide and disturb the microenvironment of pigments.

Effect of various temperatures on phosphatidylglycerol biosynthesis in thylakoid membranes.

Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of phosphatidylglycerol (PG) in thylakoid membranes during spinach (Spinacia oleracea) and squash (Cucurbita pepo) cotyledon growth to investigate how the growth temperature affects the PG biosynthesis. Of the 10 molecular species detected, temperature affected mainly the relative content of molecular species containing linolenic acid (18:3) and those containing palmitic acid (16:0) at the sn-1 position of glycerol backbone. Lowering the temperature induced an increase in the former and a decrease in the latter. The relative content of molecular species containing 18:3 or 16:0 at the sn-1 position of the glycerol backbone were correlated with temperature. Our results indicate that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or oleic acid (18:1) and the activity of fatty acid desaturases are greatly affected by temperature. In addition, changes in the relative content of PG molecular species induced by variations in growth temperature depended mainly on the substrate selectivity of GPAT.

The composition and spectral properties of three different forms of light-harvesting complex II.

Different aggregates of LHC II play a very important role in regulating the light absorption and excitation energy transfer of plant. Trimeric LHC II was purified from spinach thylakoid membrane. In order to obtain the dimeric and monomeric LHC II, the trimer was treated with the mixture of 2% OGP and 10 microg/mL PLA(2), then loaded onto the sucrose density gradient in the presence of 0.06% triton X-100. The LHC II trimer, dimer and monomer isolated by sucrose density gradient all contained three polypeptides with molecular weight of 29, 28 and 26 kd respectively. The pigment composition showed much difference in the content of Chl b and xanthophyll among three forms of LHC II. To study the light capture and excitation energy transfer in different forms of LHC II, the absorption and fluorescence spectra were analyzed. The results clearly showed that the efficiency of energy absorption and transfer was different in the three kinds of LHC II, the highest for trimeric LHC II, intermediate for dimeric LHC II, and the lowest for monomeric LHC II. It was suggested that there might be a physiological homeostasis of different aggregates of LHC II in plants, which is significant for the plant self-regulating upon exposure to variable light environment.

Photosynthesis research in the People's Republic of China.

The first research paper on photosynthesis in China was published by T.T. Li(2) in 1929. Two photosynthesis laboratories were established in Shanghai and Beijing in the 1950s and the 1960s, respectively. A photophosphorylation 'intermediate' was discovered after the energy conversion process was separated into light and dark phases in the 1960s. Since the 1980s, research has accelerated at several different levels through efforts of a large number of scientists in China.

Relationship between leaf photosynthetic function at grain filling stage and yield in super high-yielding hybrid rice (Oryza sativa. L).

The characteristics of dry matter production before and after heading and the relationships between photosynthesis of flag leaves and dry matter accumulation in panicles were investigated on super high-yielding rice cv. Xieyou 9308 (the yield of up to 12 t/ha) with rice cv. Xieyou 63 as a control. The results showed that (i) the capacity of dry matter production before and after heading in Xieyou 9308, i.e. biomass and daily dry matter production, was remarkably higher than that in Xieyou 63, especially after heading; (ii) CO(2) assimilation capacity in flag leaves in Xieyou 9308, namely Leaf Source Capacity (LSC), was also significantly higher than that in Xieyou 63, and the supply of photosynthate in leaves and the demand of grain filling were completely synchronous in Xieyou 9308, but photosynthetic function in flag leaves in Xieyou 63 declined sharply 20 days after heading and it was not enough to meet the demand of grain filling. These results confirmed that high efficient photosynthetic function in leaves after heading and its complete synchronization with grain filling are the key approaches to super high yield of rice.