A Comparative Study on PSⅡ Light Harvesting Chlorophyll a/b Protein Complexes Between Spinach and Cucumber

PS Ⅱ light harvesting chlorophyll a/b protein complexes (LHC Ⅱ ) were isolated from chloroplast of spinach (Spinacia oleracea Mill. ) and cucumber (Cucumis sativus L. ). Comparative studies were made on the polymerized forms. Chl a/b ratio, spectral characteristics and polypeptide components of these two kinds of LHC Ⅱ. Experimental results showed that the LHC Ⅱ from spinach had a Chl a/b ratio of 1.33 and the LHC Ⅱ from cucumber had a Chl a/b ratio of 1.77. The spectral characteristics of the LHC Ⅱ from cucumber also indicated the enrichment of Chl b in this LHC Ⅱ . There was also obvious differences in the polypeptide components between these two kinds of LHC Ⅱ, the LHC Ⅱ of spinach contained a 27 kD and a 25 kD polypeptides, while the LHC Ⅱ of cucumber contained only a 27 kD polypeptide. This showed that the 25 kD polypeptide contained less Chl b. The analysis of the chlorophyll protein complexes showed that the monomer, dimer and trimer of the LHC Ⅱ of spinach were composed of two polypeptides, while all the polymerized forms of cucumber’s LHC Ⅱ were composed of one polypeptide.     

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.     

The Effects of Water Stress on Photosystem Ⅱ in Wheat

The fluorescence yield at room temperature, the capacity of excitation energy distribution between photosystem Ⅰ and Ⅱ by Mg2+, variable fluorescence yield, variable fluorescence quenching rate and fluorescence complementary area were decreased under water stress. These indicated that photosystem Il was impaired. The inhibited variable fluorescence yield could be partly recovered by the addition of artificial electron donor DPC. Therefore, water stress inhibited not only the oxidizing site of photosystem Ⅱ but also impaired partly the reaction center of photosystem Ⅱ.     

Photoreduction of Cytochrome b559 in the Photosystem Ⅱ Reaction Center Complex

The isolated and purified photosystem Ⅱ (PS Ⅱ ) reaction center D1/D2/Cyt b559 complex was taken as the experimental system. It was observed that under anaerobic conditions, cytochrome b559 (Cyt b559) could be reduced by exposure to strong illumination, suggesting Cyt b559 could accept electrons directly from reduced pheophytin (Pheo-). And the photoreduction of Cyt b559 was irreversible. When the isolated D1/D2/Cyt b559 complex reconstituted with exogenous secondary electron acceptor 2,6-dimethyl-benzoquinone (DMBQ), the photoreduction of Cyt b559 was delayed in the function of illumination time. Meanwhile, the electrons transferred mainly through DMBQ and photoreduced Cyt b559 could be partially reoxidized in the dark incubation following illumination. It was concluded that the quinone-independent electron transfer via Cyt b559 was a new, secondary electron pathway, which represented one of the protective pathes for PS Ⅱ reaction center to dissipate excess excitation energy.     

Study on the Binding State of Calcium in Photosystem ⅡOxygen-Evolution Complex

Washing spinach PSII oxygen-evolution complex (OEC) with 2 mmol/L EGTA or extraction medium caused a 28.4% and 25.0% loss of oxygen evolution activities respectively, but the loss of polypeptide components of OEC did not take place, whereas washing with 1 mol/L NaCI caused both a 90.0% loss of oxygen evolution activity and loss of 17, 23kD polypeptides. Adding 5–10 mmol/L CaC12 could restore oxygen evolution activities of OEC by various washing to a great extent, but had no effect on control OEC, whereas adding 5–10 mmol/L EGTA had no effect on the OEC by various' washing, but caused the loss of oxygen evolution mixtures, which could induce the release of of 17, 23kD polypeptides from OEC, caused 54.3% loss of oxygen evolution activity, under this circumstance, adding 2 mmol/L of EGTA could only maintain a weak oxygen evolution activity of OEC, but adding 10 mmol/L of CaCl2 could restore oxygen evolution activity of OEC to the control level. These findings' suggest a two way loose binding of Ga2+ to PSⅡ OEC in one way Ca2+ is loose bound to the surface of PSⅡOEC and in other, the Ca2+-binding site is wrapped by 17, 23kD polypeptides. Both of them have effect on oxygen evolution activity of PSⅡ OEC. By way, Mn2+ can antagonize the restoration of oxygen evolution activity by Ca2+ to the NaCl-washing PSⅡ OEC.     

Novel Evidence for a Reversible Dissociation of Light-harvesting Complex Ⅱ from Photosystem Ⅱ Reaction Center Complex Induced by Saturating Light Illumination in Soybean Leaves

After saturating light illumination for 3 h the potential photochemical efficiency of photosystem Ⅱ (PSⅡ) (Fv/Fm, the ratio of variable to maximal fluorescence) decreased markedly and recovered basically to the level before saturating light illumination after dark recovery for 3 h in both soybean and wheat leaves, indicating that the decline in Fv/Fm is a reversible down-regulation. Also, the saturating light illumination led to significant decreases in the low temperature (77 K) chlorophyll fluorescence parameters F685 (chlorophyll a fluorescence peaked at 685 nm ) and F685/F735 (F735, chlorophyll a fluorescence peaked at 735 nm) in soybean leaves but not in wheat leaves. Moreover,trypsin (a protease) treatment resulted in a remarkable decrease in the amounts of PsbS protein (a nuclear gene psbS-encoded 22 kDa protein) in the thylakoids from saturating light-illuminated (SI), but not in those from darkadapted (DT) and dark-recovered (DRT) soybean leaves. However, the treatment did not cause such a decrease in amounts of the PsbS protein in the thylakoids from saturating light-illuminated wheat leaves. These results support the conclusion that saturating light illumination induces a reversible dissociation of some light-harvesting complex Ⅱ (LHCⅡ) from PSⅡ reaction center complex in soybean leaf but not in wheat leaf.     

Effects of Cadmium on Polar Lipid Composition and Unsaturation in Maize （Zea mays） in Hydroponic Culture

This study aimed to evaluate the effect of Cd exposure （100 μmol/L） on polar lipid composition, and to examine the level of fatty acid unsaturation in maize （Zea mays L.）. In roots, the level of 16：0 and monounsaturated fatty acids （16：1 ＋ 18：1） decreased in phosphatidylcholine （PC） and phosphatidylethanolamine （PE）. In contrast, the proportion of unsaturated 18-C fatty acid species showed an opposite response to Cd. The content, on the other hand, of PC, PE, digalactosyldiacylglycerol （DGDG）, and steryl lipids increased in roots （2.9-, 1.6-, 5.3-, and 1.7-fold increase, respectively）. These results suggest that a more unsaturated fatty acid composition than found in control plants with a concomitant increase in polar lipids may favor seedling growth during Cd exposure. However, the observed increase in the steryl lipid （SL） ： phospholipid （PL） ratio （twofold）, the decrease in monogalactosyldiacylglycerol （MGDG） ： DGDG ratio, as well as the induction of lipid peroxidation in roots may represent symptoms of membrane injury. In shoots, the unsaturation level was markedly decreased in PC and phosphatidylglycerol （PG） after Cd exposure, but showed a significant increase in sulfoquinovosyldiacylglycerol （SQDG）, MGDG and DGDG. The content of PG and MGDG was decreased by about 65%, while PC accumulated to higher levels （4.4-fold increase）. Taken together, these changes in the polar lipid unsaturation and composition are likely to be due to alterations in the glycerolipid pathway. These results also support the idea that the increase in overall unsaturation plays some role in enabling the plant to withstand the metal exposure.     

Light-Induced Damage of Pheophytin a Molecule in the Isolated Photosystem Ⅱ Reaction Center D1/D2/Cyt b559 Complex

Photodamage of some pigments in the isolated photosystem Ⅱ (PS Ⅱ ) reaction center D1/D2/Cyt b559 complex from spinach has been investigated by means of high performance liquid chromatography. The light-induced damage of pheophytin a (pheo a) in the complex was observed for the first time. The content of pheo a decreased about 47 % by illumination, suggesting only one of the two pheo a molecules in the PS Ⅱ reaction center complex was damaged. No damage of β-carotene was found.     

(—) S-adenosyl-L-methionine-magnesium Protoporphyrin Methyltransferase, an Enzyme in the Biosynthetic Pathway of Chlorophyll in Zea mays

The enzyme (—) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (—) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays.

Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin.

The following scheme of chlorophyll synthesis in higher plants is proposed: δ-aminolevulinic acid → → → protoporphyrin → magnesium protoporphyrin → magnesium protoporphyrin monomethyl ester → → → chlorophyll a.

     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

Mutation of Residue Arginine18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the ?subunit of cytochrome b559 (Cyt b559) except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine18 (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high light intensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSII (Fv/Fm) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the ?subunit of Cyt b559 and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt b559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.     

Are Bryophytes Shade Plants? Photosynthetic Light Responses and Proportions of Chlorophyll a,Chlorophyll b and Total Carotenoids

BACKGROUND AND AIMS: Data are presented from 39 species of mosses and 16 liverworts for ratios of chlorophylls and total carotenoids, and light saturation of photosynthetic electron flow or photosynthetic CO2 uptake, in relation to the postulate that bryophyte cells in general show shade-plant characteristics. METHODS: Pigment concentrations were measured by spectrophotometer in 80 % acetone extracts. Light-saturation curves were constructed by (modulated) chlorophyll florescence and for some species by infra-red gas analysis. KEY RESULTS: The pigment measurements were widely variable but broadly in line with the findings of previous authors. Median values (mosses/liverworts) were: total chlorophyll, 1.64/3.76 mg g(-1); chlorophyll a : b, 2.29/1.99; chlorophylls : carotenoids, 4.74/6.75). The PPFD values at 95 % saturation (estimated from fitted curves) also ranged widely, but were almost all <1000 micromol m(-2) s(-1); the median for mosses was 583 and for liverworts 214 micromol m(-2) s(-1). The two highest PPFD95% values were from Polytrichum species with lamella systems forming a ventilated photosynthetic tissue. Total chlorophyll, chlorophyll a : b and chlorophylls : carotenoids all correlated significantly with PPFD95%. CONCLUSIONS: Bryophytes include but are not inherently shade plants. Light-saturation levels for species of open sun-exposed habitats are lower than for vascular sun plants and are probably limited by CO2 diffusion into unistratose leaves; this limit can only be exceeded by bryophytes with ventilated photosynthetic tissues which provide increased area for CO2 uptake.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light （800μmol photons/m^2 per s）-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center （PSII RC） under aerobic conditions （in the absence of electron donors or acceptors） was studied using high-pressure liquid chromatography （HPLC）, absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSII RC as determined by HPLC after light treatment were as follows： with Increasing illumination time chlorophyll （Chl） a and β-carotene （β-car） content decreased. However, decreases in pheophytin （Pheo） could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illuminatlon, the initial bleaching occurred maximally at 680 nm but that with Increasing Illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed Initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 mln light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination. After Illumination, the fluorescence emission Intensity changed and the fluorescence maximum blue shifted, showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm^-1 （υ101）, 1 159 cm^-1 （υ2）, 1 006 cm^-1 （υ3）, 966 cm^-1 （υ4） for 488.0 nm excitation and 1 525 cm^-1 （υ1）, 1 159 cm^-1 （υ2）, 1 007 cm^-1 （υ3）, 968 cm^-1 （υ4） for 514.5 nm excitation. It was confirmed that two spectroscopically different β-car molecules exist In the PSII RC. After light treatment for 20 mln, band positions and bandwidths were unchanged. This indicates that carotenoid configuration Is not the parameter that regulates photoprotectlon in the PSII RC.     

Studies on Femtosecond Fluorescence Properties of Light-harvesting Complex in Photosystem ￠ò

Fluorescence properties of light-harvesting complex LHCⅡ） in photosystem Ⅱ isolated from spinach (Spinacia oleracea L.) was investigated with the time-resolved fluorescence spectroscopic technique. The sample was excited by double frequency Ti:Al203 laser with 400 nm wavelength and 120 fs width. Fluorescence signal was detected by Boxcar. Three life-time components （320 fs,4.0 ps and 20.0 ps） were obtained by multi-exponential curve model and nonlinear least-square fitting method. Three fluorescence peaks （652 nm, 672 nm, 691 nm） were obtained by Global analysis and Gauss curve fitting. These components were analyzed according to the structural and fluorescence spectroscopic property of LHCⅡ. The fluorescence ratio of each component to that of all the component was 3.4%, 50% and 46.6% respectively. The possible model of energy transfer in LHCⅡ was discussed.     

Embryo Rescue and Induction of Somatic Embryogenesis as a Method to Overcome Seed Inviability in Zea mays ssp. mays × ; Zea mays ssp. parviglumis Crosses

Zea mays ssp. mays (2n=40) and Z. mays ssp. parviglumis (2n=20) were crossed to obtain hybrid plants by embryo rescue. Hybrid embryos were isolated and cultured on García et al. (1992) basic medium supplemented with 2,4-dichlorophenoxyacetic acid and/or kinetin in different concentrations. Caryopses harvested 23 d after pollination (DAP) were turgid, with 0.3 to 0.5 mm long embryos, while those harvested 30 DAP were shrunken, with 1 to 1.5 mm long embryos. Twenty days after plating, 100 % of the younger embryos gave rise to white, compact embryogenic calli. Subsequently, coleoptiles, leaf-like structures, shoots and roots originated from them and 35 hybrid plants were regenerated from 60 embryos. Embryogenic or organogenic calli frequencies did not differ among hormonal treatments, but they decreased, on average, from 90.5 to 44.3 %, comparing 50 and 120-d-old cultures. The older embryos regenerated plants only by germination, although they gave rise to organogenic callus with low frequencies. Regenerated plants showed a somatic chromosome number of 2n=30, pollen fertility of 40 to 80 % and 15 % viable naked caryopses.     

Effects of Exogenous Nitric Oxide on Photochemical Activity of Photosystem Ⅱ in Potato Leaf Tissue Under Non-stress Condition

Under non-stress condition, effects of exogenous nitric oxide (NO) on chlorophyll fluorescence parameters in detached leaves and leaf discs of potato (Solanum tuberosum L.) were surveyed. Results showed that the maximal quantum efficiency (Fv/Fm) and the effective quantum efficiency (F P_SⅡ) of photosystem Ⅱ (PSⅡ) were reduced by exogenous NO under illumination (150 mmol.m^-2.s^-1, 25 ℃). This influence was related not only to the concentration of sodium nitroprusside (SNP, a NO donor) solution, but also to the active duration of NO on leaf tissue. Results with leaf discs showed that the effects of SNP on F P_SⅡ could be prevented by bovine hemoglobin (a powerful NO scavenger), while a mixture of NO^2- and NO^3- (the decomposition product of NO or its donor SNP) had much less influence on F P_SⅡ than SNP, indicating that effects of exogenous SNP on PSⅡ photochemical activity was mainly due to NO generation. Under light (150 mmol.m^-2.s^-1, 25 ℃) for 4 h or longer period, the non-photochemical quenching (NPQ) in SNP-soaked leaves was statistically similar to that in H₂O-soaked control, but F P_SⅡ and the proportion of open reaction centers (measured as qP) were lower than control, respectively. After 25 min dark-adaptation, the maximal fluorescence (Fm) in SNP treatment (8 and 12 h illumination duration) was significantly lower than the control, while the initial fluorescence (F_o) in SNP and H₂O-treated leaves had no significant difference. Therefore this indicated that under the present experimental condition, the NO-affected site might not be the PSⅡ reaction centers. On the donor side of PSⅡ, NO putatively influenced the light-harvesting capacity of leaves under light; on the acceptor side, NO-affected sites were some components of electron transport chain after Q_A, i.e. NO enhanced the reductive degree of reaction centers through blocking the electron transport after Q_A, thus reducing the photochemical activity of PSⅡ.     

Effects of γ-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles

The effects of γ-irradiation on elongation and the level of indole-3-acetic acid (IAA) of maize (Zea mays) coleoptiles were investigated. When 3-day-old seedlings of maize were exposed to γ-radiation lower than 1 kGy, a temporal retardation of coleoptile elongation was induced. This retardation was at least partly ascribed to a temporal decrease in the amount of free IAA in coleoptile tips on the basis of the following facts: (1) the reactivity to IAA of the elongating coleoptile cells was not altered by irradiation; (2) endogenous IAA level in the tip of irradiated coleoptiles was at first unchanged, but then declined before returning to nearly the same level as that of the non-irradiated control; and (3) the amount of IAA that diffused from coleoptile tip sections showed a similar pattern to that of endogenous IAA. The rate of conversion between free and conjugated IAA was not significantly affected by irradiation. These results suggest that a temporal inhibition of maize coleoptile elongation induced by γ-irradiation can be ascribed to the reduction of endogenous IAA level in the coleoptile tip, and this may originate from the modulation in the rate of IAA biosynthesis or catabolism.