Changes of Photosystem Ⅱ Electron Transport in the Chlorophyll-deficient Oilseed Rape Mutant Studied by Chlorophyll 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 de creased the amount of light-harvesting complexes with an increased amount of some core polypeptides of PSII,including CP43 and CP47. By means of chlorophyll fluorescence and thermoluminescence, 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 b-deficient mutant, the decay of the primary electron acceptor quinones (QA-) reoxidation was decreased, measured by the fluorescence. Furthermore, the thermolumlnescence studies in the chlorophyll b deficient mutant showed that the B band (S2/S3QB-) decreased slightly and shifted up towards higher temperatures.In the presence of dichlorophenyl-dimethylurea, which is inhibited in the electron flow to the second electron acceptor quinines (QB) at the PSII 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 chlorophyll 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.     

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.     

Fluorescence Transients as Indicator for the Existence of Regulatory Mechanisms in Photosynthetic Electron Transport

In green algae several characteristic differences in the slope of the fast 685 nm fluorescence transient indicate the existence of different mechanisms for the regulation of the photosynthetic electron transport in vivo with respect to the requirements for ATP and NADPH. Autotrophically cultivated Chlamydobotrys stellata exhibits a normal time curve of the fluorescence yield. Anaerobiosis and C0₂-deficiency raise the O-, I- and S-level, whereas the P- level is lowered and the I-D-decay disappears. The readdition of oxygen increases the fluorescence significantly. Supplementation of aerobic cells with CO₂ restores the normal fluorescence transients. The replacement of carbon dioxide by acetate as a carbon source in the light lowers the overall fluorescence emission and abolishes the D-P-increase and the P-S-decline. The presence of DCMU increases fluorescence only at high intensities of incedent light. Anaerobiosis in these photoheterotrophic algae lowers the fluorescence emission. In this case DCMU increases fluorescence even at low light intensities. In Gonium multicoccum, which shows a normal fluorescence transient when cultivated autotrophically, CO₂-deficiency abolishes the O-level and increases the I- and S-niveau. Additional anaerobiosis in CO₂-deficient cells raises the steady state emission. Readdition of oxygen to these cells raises the I- and S-level even more and prevents the build up of the P-level. In Gonium     

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S₂₍₃₎Q_B ^– (B band), S₂Q_A ^– (Q band), Tyr-D⁺Q_A ^– (C band) and Tyr-Z⁺(His⁺)Q_A ^– (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S₂₍₃₎Q_B ^– recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DT desiccation tolerant - PS II Photosystem II - TL thermoluminescence - P₆₈₀ reaction center Chl of PS II - Q_A and Q_B puinone electron acceptors of PS II - S₀,...,S₄ the redox states of the water-oxidizing complex - Tyr-Z and Tyr-D redox-active tyrosine electron donors of PS II     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

Decrease of Fluorescence Intensity After the K Step in Chlorophyll a Fluorescence Induction is Suppressed by Electron Acceptors and Donors to Photosystem 2

Chlorophyll a fluorescence induction measured by a fluorometer with a high temperature stressed plant material shows a new K step which is a clear peak due to fast fluorescence rise and subsequent decrease of fluorescence intensity. We focused on an explanation of the decrease of fluorescence after the K step using artificial electron acceptors and donors to photosystem 2 (PS2). Addition of the artificial electron acceptors or donors suppressed the decrease of fluorescence after the K step. We suggest that the decrease mainly reflects (by more than 81 %) an energy loss process in the reaction centre of PS2 which is most probably a nonradiative charge recombination between P680⁺ (oxidised primary electron donor in PS2) and a negative charge stored on either Pheo⁻ or Q_A ⁻ (reduced primary electron acceptor of PS2 and reduced primary quinone electron acceptor of PS2, respectively). We suggest that the energy loss process is only possible when the inhibition of both the donor and the acceptor sides of PS2 occurs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Three-Dimensional Electron Microscopy of the Photosystem II Core Complex

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochromeb-559, andpsbI gene product was reconstructed at 20-Å resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had ap22₁2₁symmetry. One PSII core complex was measured to be 80 × 80 Å in the membrane plane and 88 Å normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.     

空间飞行微重力降低裸藻光合电子传递并改变光合能量分配

利用神舟8号飞船的SIMBOX发射机会,对真实微重力影响裸藻光合作用活性进行了研究．我们发现,微重力降低了光合活性(Fv/Fm),提高了细胞内叶绿素a和胡萝卜素含量．快速叶绿素荧光动力学研究显示微重力降低了叶绿素荧光强度,但快速叶绿素荧光动力学曲线的形状(O-J-I-P)没有改变．在微重力处理下裸藻的最大光化学效率(φPo)、用于电子传递的量子产额(φEo)和光合作用性能指数(PIABS and PICS)都明显降低,但单位反应中心吸收的光能(ABS/RC)和单位反应中心耗散的能量(DIo/RC)都明显升高．77K低温荧光光谱实现微重力改变了能量在PSⅠ和PSⅡ之间的分配并出现了红移现象．这些结果表明真实微重力降低光合作用的活性有可能通过两个途径,即抑制裸藻抑制光合电子传递中PSⅡ的受体端和改变PSⅠ的结构从而引起流向PSⅠ的能量传递减少．