The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b_6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f com-plexes from different sources (~200 and ~600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-β-D- maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f 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, ~200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

Cloning and characterization of the gene cluster required for beauvericin biosynthesis in Fusarium proliferatum

Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.     

The effects of detergents DDM and β-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 (Chi a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f complexes from different sources (～200 and ～600 ps are the two measured results). The present study demonstrated that the singiet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f 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, ～200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

Transient Decrease of Light-harvesting Complex Ⅱ Phosphorylation Level by Hypoosmotic Shock in Dark-adapted Dunaliella salina

This study investigated the regulation of major light harvesting chlorophyll a/b protein （LHCⅡ） phosphorylation by hypoosmotic shock in dark-adapted Dunaliella salina cells. When the external NaCI concentration decreased in darkness, D. salina LHCⅡ phosphorylation levels transiently dropped within 20 min and then restored gradually to basal levels. The transient decrease in LHCII phosphorylation levels was insensitive to NaF, a phosphatase inhibitor. Inhibition of intracellular ATP production by addition of an uncoupler or an ATP synthase inhibitor increased LHCⅡ phosphorylation levels in D. salina cells exposed to hypoosmotic shock. Taken together, these results indicate that hypoosmotic shock inhibits the LHCⅡ phosphorylation process. The related mechanism and physiological significance are discussed.     

Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Reconstitution of Photosystem Ⅱ Reaction Center with Cu-ChlorophyⅡ a

An Isolated photosystem （PS） II reaction center （RC） with altered pigment content was obtained by chemical exchange of native chlorophyll a （Chl） with externally added Cu-Chl a （Cu-Chl）. Pigment composition and spectroscopic properties of the RC exchanged with Cu-Chl were compared with native RC and RC treated with Chl In the same way. High-performance liquid chromatography analysis showed approximately 0.5 Cu-Chl per two pheophytln in the Cu-Chl-reconstltuted RC preparation. Insertion of Cu-Chl resulted in a decrease In absorption at 670 nm and an Increase at 660 nm, suggesting that the peripheral Chl may have been displaced. Fluorescence emission spectra of the Cu-Chl-reconstituted RC displayed a marked decrease In fluorescence yield and a blue shift of the band maximum, accompanied by the appearance of a broad peak at a shorter wavelength, Indicating that energy transfer In the modified RC was disturbed by Cu-Chl, a quencher of the excited state. However, there were few differences in the circular dichrolsm （CD） spectra, suggesting that the arrangement of pigments and proteins responsible for the CD signal was not significantly affected. In addition, no obvious change In peptlde components was found after the exchange procedure.     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum （Bacillariophyta）

Responses of the photosynthetic activity of Phaeodactylum tricornutum （Bacillariophyta） to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem （PS） Ⅰ and PS Ⅱ and the values of F685/F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

Overexpression of sweet pepper glycerol-3-phosphate acyltransferase gene enhanced thermotolerance of photosynthetic apparatus in transgenic tobacco

In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of pho-tosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase (GPA T) gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol (MGDG), suifoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T1 lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.0% in T1-2 and T1-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem Ⅱ the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSII in transgenic tobacco compared with wild-type plants, the entire capability of PSII recovered faster in transgenic tobacco, which was shown in Increase in saturated extent of thylakoid membrane Iipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.     

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

We 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 Zhonghuau. Native green gel and SDS-PAGE electrophoreses 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 （FJ 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.     

Center of Forensics,Beijing Genomics Institute

Center of Forensics of Beijing Genomics Institute is a neutral professional forensic identification organization that has been authorized by the Ministry of Justice of China .She is supported by the Genomics & Bioinformatics Center of Chinese Academy of Sciences, Beijing Genomics Institute, and Hangzhou Genomics Institute. She possesses the outstanding predominance in DNA analysis, apparatus and equipments, high performance computation and huge information processing. She     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

西方蜜蜂四个亚种酯酶同工酶型和苹果酸脱氢酶Ⅱ同工酶基因型的遗传差异

The esterase (EST) and the malate dehydrogenase Ⅱ (MDHⅡ) isozymes in A. mellifera carpatica (C), A. mellifera ssp.(D), “Zhejiang Agricultural University No.1” A. mellifera ligustica (Ea) and A. mellifera carnica (K), and the genotypes of MDHⅡ isozymes in four subspecies of A. mellifera were analysed by IEF PAGE. The results indicate that the four subspecies of A. mellifera showed the same EST zymogram, while A. cerana showed a different EST zymogram, which suggests that the genotypes of EST isozymes in A. mellifera are different from those in A. cerana. The genotypes of the MDHⅡ isozymes in four subspecies of A. mellifera were aa, ab, ac, bb, bc and cc. The C and Ea subspecies exhibited high homozyosity, while D and K displayed high heterozyosity. The allele b appeared to be the highest frequency in C, while the allele c had the highest frequency in Ea. The frequencies of the alleles a, b and c in sub species D were very similar. The alleles a and c were common, while allele b was rare in sub species K. There were highly significant differences in genotype frequencies, allele frequencies, homozyosity and heterozyosity among these four subspecies.     

Clonal differences in the response of dark and light reactions of photosynthesis to elevated atmospheric CO2 in poplar

Two hybrid poplar (Populus) clones (i.e., fast growing clone Beauprè and slow growing clone Robusta) were grown for two years from cuttings at close spacings in open top chambers (OTCs) under ambient (AC) and elevated [EC = AC + 350 μmol(CO2) mol-1] CO2 treatments. For clone Beauprè no down-regulation of photosynthesis was observed. Two years of growing under EC resulted in an increase in quantum yield of photosystem 2 (PS2), steady state irradiance saturated rate of net photosynthesis (P _Nmax), chlorophyll (Chl) content, and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC) activity for this clone. We suppose that under non-limiting conditions of nitrogen and phosphorus content the response to EC was by building up light-harvesting complexes of PS2 and increasing photochemical efficiency of PS2. Due to a high rate of the primary reactions of photosynthesis and a high RuBPCO activity the end product of the response to EC was an increase in PNmax and a larger saccharides content. The Robusta clone showed a depression in the primary reactions of photosynthesis under EC. We found a decrease in quantum yield of PS2, Chl and phosphorus contents, and in RuBPCO activity. However, an increase in PNmax, saccharides content and Chl a/b ratio was observed. We speculate (1) that the phosphorus deficiency in combination with an increase in CO2 concentrations may lead to a potential damage of the assimilation apparatus of the primary reactions of photosynthesis and to a decrease in photochemical efficiency of PS2; (2) that the primary target of "down-regulation" takes place at PS2 for irradiances above 150 μmol m-2 s-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

Prasinoxanthin-constaining Prasinophyceae Discovered in Jiaozhou Bay, China

The class Prasinophyceae （Chlorophyta） contains some photosynthetic eukaryotic ultraplankton species characterized by containing prasinoxanthin. The existence and abundance of these organisms can be estimated by the diagnostic pigment. We detected the unique pigments of prasinoxanthin-containing Prasinophyceae in Jiaozhou Bay, China using high performance liquid chromatography （HPLC）. This was the first finding of this kind in Chinese seas. Using the ratio of prasinoxanthin to chlorophyll a, the abundance of prasinoxanthin-containing Prasinophyceae has been calculated. The average contribution of prasinoxanthin-containing Prasinophyceae to the chlorophyll a pool was 8.5% and 17.0% in May and August 2004 in Jiaozhou Bay, and the maximums were 25.9% and 36.3%. Size fractionated pigment analysis suggested that more than 80% of prasinoxanthin were in the fraction of 2-20 μm. According to the results of pigment and morphological analysis, the possible genera of prasinoxanthin-containing Prasinophyceae and the reasons for causing this high abundant phytoplankton in Jiaozhou Bay were discussed. This kind of phytoplankton can not be discovered in traditional biological investigation, but its contribution to the coastal ecosystem is significant enough to be studied further.     

Characteristics of photosensitization of Pheophorbide a in liposomal media

Pheophorbide a (PPa), a decomposition product of chlorophyll a, is a photosensitizer. The photosensitization mechanisms (Type Ⅰ and Type Ⅱ) of PPa in simple buffer solutions and in buffer solutions containing double-layered DPPC liposomes have been studied using techniques of ESR, spin-trapping, spin-counteraction and laser flash photolysis. The results showed that adding DPPC liposomes to the buffer solution caused an increase of efficiency of generating ~1O_2 and PPa~- by photoactivating PPa. The increase could be ascribed to the disaggregation of hydrophobic PPa caused by the addition of liposomes and the protective effect of liposomal media on the triplet state of PPa. It is concluded that the photosensitization of PPa in liposomal systems is different from that in simple aqueous solutions, and shows higher efficacy. The results will be useful to elucidating the mechanisms of photodynamic therapy of cancer.     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Purification of full-length human Pregnane and Xenobiotic Receptor： polyclonal antibody preparation for immunological characterization

Pregnane and Xenobiotic Receptor （PXR; or Steroid and Xenobiotic Receptor, SXR）, a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body＇s homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21（DE3） cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.