P515的测量原理、方法和应用

光谱技术已广泛应用于光合研究领域,如光吸收信号P515和P700氧化还原动力学以及叶绿素荧光等,可快速、准确地检测植物的光合活性。P515信号广泛存在于高等植物和藻类中,是类囊体膜上的色素分子吸收光能后,其吸收光谱发生位移造成。利用光诱导的P515快速和慢速动力学,可检测PSI和PSII反应中心的比值、ATP合酶的质子...     

两种常绿阔叶植物越冬光系统功能转变的特异性

通过叶绿素荧光和P700氧化还原动力学同步测定,研究大叶黄杨(Euonymus japonicus)和锦熟黄杨(Buxus spervirens L.)的阳生叶和阴生叶在北京地区越冬进程,光系统Ⅱ(PSⅡ)和光系统Ⅰ(PSⅠ)功能转变机制的特异性.结果表明,入冬前0℃以上低温条件下,各叶片PSⅡ有效光量子效率Y(Ⅱ)(Effective quantum yield of PSⅡ)处于同一水平,但阳生叶Y(Ⅰ)(Effective quantum yield of PSⅠ)均高于阴生叶,同时各叶片Fo(Minimal fluorescence)和OJIP水平不完全相同:大叶黄杨两种叶片差异不显著,锦熟黄杨阳生叶显著低于其阴生叶;同步测定P700氧化还原变化表明,两种阴生叶在0-20 ms的P700氧化过程分两个阶段,尤其是锦熟黄杨2 ms后的氧化速率显著降低,而两种阳生叶0-20 ms基本保持同一氧化速率;两种阴生叶的两个光系统量子效率比Y(Ⅰ)/Y(Ⅱ)＜1,两种阳生叶Y(Ⅰ)/Y(Ⅱ)=1.入冬后,各类叶片PSⅡ受到不同程度光抑制,而PS Ⅰ光量子效率Y(Ⅰ)均先增加后减小,Y(Ⅰ)/Y(Ⅱ)发生不同程度增加,Y(Ⅱ)和两个光系统的平衡能力均依次为锦熟黄杨阴生叶＞锦熟黄杨阳生叶＞大叶黄杨阴生叶＞大叶黄杨阳生叶;冬季大叶黄杨阴生叶J相的相对强度高于锦熟黄杨阴生叶,而两种阳生叶OJIP动力学变化几乎消失;同步测定P700氧化还原变化表明,锦熟黄杨阴生叶2 ms即达到Pm(Maximal P700 change),其他叶片0-20 ms保持同一氧化速率,阳生叶Fo、P700氧化速率和Pm均低于阴生叶.返青后,各叶片两个光系统功能逐渐恢复.可见,冬季低温或低温强光逆境会导致阳生叶和阴生叶的两个光系统功能和互动机制发生不同转变.     

高温胁迫对烟草叶绿体NADPH脱氢酶复合体活性的促进

为探讨叶绿体NAD(P)H脱氢酶复合体(NDH)在植物抵御热胁迫中的生理意义,比较了烟草ndhJK基因缺失突变体(ΔndhJK)和野生型对50℃高温胁迫的响应.高温下,野生型中一条NBT-NADPH氧化还原酶活性带有所增加,免疫印迹分析确定了此活性染色带是NDH亚复合体,该活性带中的NDH-K表达量也在热胁迫条件下明显地增加.与ΔndhJK相比,在高温胁迫下,野生型中远红光诱导的P700 氧化速率明显地变慢,而远红光关闭后的P700暗还原速率则显著地变快,表明高温促进NDH介导的围绕光系统I的循环电子传递.根据这些结果推测,在热胁迫条件下野生型中对NADPH底物专一的NDH活性的增加可能有利于减少NADPH的积累,减轻叶绿体间质的过度还原.     

氧化还原伴侣工程：P450低效问题的解决方案之一

细胞色素P450酶(CYPs或P450s)可将O₂的一个原子插入有机底物同时将另一个原子还原为水，广泛参与各种合成代谢和分解代谢过程，所以一直以来都是生物技术领域关注的焦点。在催化循环底物的氧化依赖于氧化还原伴侣向血红素铁传递电子，因此电子转移是P450s催化过程中的限速步骤。利用不同方法优化蛋白质-蛋白质相互作用以提高P450系统的电子转移效率，被称为“氧化还原伴侣工程”，是目前工程化P450s的重要手段之一，并取得了卓有成效的进展。本文将着重介绍关于氧化还原伴侣组分替换组装、P450酶与氧化还原伴侣融合及P450酶与氧化还原伴侣作用界面修饰等方面的进展，期望为未来该方面的工作提供一定的指导作用。     

不同叶色矢竹叶绿体结构和光系统特性差异

以矢竹(Pseudosasa japonica)、花叶矢竹(P. japonica f. akebonosuji)和曙筋矢竹(P. japonica f. akebono)为研究对象, 借助叶绿体超微结构和荧光动力学曲线的变化揭示不同叶色矢竹的光系统活性及光合特性差异。结果表明: 3个竹种的光合色素含量差异明显, 除花叶矢竹条纹叶白色部分叶绿体内无完整类囊体片层结构外, 花叶矢竹绿条纹和曙筋矢竹的基粒数明显少于矢竹, 叶绿体发育成熟度不一致; OJIP曲线及参数表明, 花叶矢竹条纹绿叶和曙筋矢竹光系统II (PSII)反应中心开放降低程度低于矢竹, 捕获能量用于电子传递的份额变小, PSII活性变弱; 而曙筋矢竹叶片P700至初级电子受体(Q_A)的电子传递链氧化还原平衡偏向于还原侧, 推测其光系统I (PSI)反应中心P700至PSII Q_A电子传递链受损。因此, PSII活性变化导致叶绿体发育不成熟, 可能是引起矢竹类叶色差异的直接原因。     

琥珀酸细胞色素c还原酶系中细胞色素b的多相氧化还原反应

呼吸链中从泛醌到细胞色素c一段一向受到人们的重视。尤其是细胞色素b的动力学行为,虽然文献上报道很多但结果却不一致。早在工929年Keilin就已经观察到细胞色素b的氧化还原速度比细胞色素系统其它组份都慢一些。1952年Chance也观察到这一现象。但他发现在不具氧化磷酸化能力的制剂中细胞色素b的还原速度才比c_1或c慢,而在具有氧化磷酸化能力的制剂中细胞色素b的还原速度并不比c_1慢。于是他认为也许是磷酸化系统受到损害而影响到细胞色素b的还原速度。Chance还发现b的还原在动力学上是快慢两相。后来有一些作者也都报道了b的两相还原现象。     

光诱导叶片P-700氧化还原的测量

依据光合作用反应原理 ,介绍了光诱导的叶片P 70 0氧化还原的测量方法。并分别以烟草和菠菜为例 ,给出了典型的远红光诱导的P 70 0氧化还原曲线和不同波长的作用光诱导下P 70 0的氧化还原动力情况     

半胱氨酸氧化还原修饰组分析:进展与展望

半胱氨酸巯基上的氧化还原修饰能够可逆、可控地调节蛋白质活性、互作与定位,进而实现对诸多生物学进程与信号通路的精细调控.在蛋白质组层面上分析此类修饰位点及其动态转换有助于系统描绘并解析氧化还原调控网络.近年来,随着化学选择性标记试剂的不断涌现,质谱技术的升级换代,氧化还原修饰组分析的覆盖度与通量均得以极大提升,为氧化还原...     

硫氧还蛋白的结构及在生物抗氧化中的功能

硫氧还蛋白(thioredoxin,Trx)是广泛存在于原核与真核生物体内的氧化还原调节蛋白。Trx通过对目标蛋白质进行还原,从而调节机体的氧化还原平衡。Trx与硫氧还蛋白还原酶(thioredoxin reductase,TrxR)及NADPH共同组成硫氧还蛋白系统参与众多生理过程。细胞中的活性氧是导致生物氧化胁迫的一个主要方面。Trx可以通过对细胞内被氧化的二硫键的还原来修复机体的氧化损伤,并通过这种方式防止机体衰老。同时,Trx系统可以与其它氧化还原系统如谷胱甘肽(GSH)系统协调配合,并消除体内过多的活性氧。     

检测细胞即时影像共振传感器

日前,美国伊利诺大学基因组生物学研究所开发成功用于来检测细胞内的氧化还原动力学的即时影像的共振传感器。     

苜蓿国际发展态势分析

苜蓿是草食动物的优质饲草, 被誉为“牧草之王”。发展苜蓿产业对提升我国草食畜牧业具有重要意义。该研究采用定性调研与定量分析相结合的方法, 从创新链角度, 研究了全球苜蓿科技产出、代表性国家苜蓿产业格局和全球苜蓿市场贸易等状况及我国苜蓿产业存在的问题, 旨在为我国苜蓿产业发展提供参考。分析发现, 美国是全球最重要的苜蓿生产国, 在苜蓿基础研究、技术开发、品种培育和商业化种植等方面均具有很强的优势, 引领了全球苜蓿产业的发展。欧美等跨国企业掌控着全球苜蓿产业链的各个关键环节, 是苜蓿产品的主要出口市场, 而亚洲苜蓿产品消费缺口最大。近10年来, 我国在苜蓿科技领域表现活跃, 科技成果产出呈快速增长趋势, 但在成果数量和影响力方面与欧美国家差距明显, 且苜蓿育种进程缓慢, 优质苜蓿产品对外依存度仍然较高。综合来看, 我国应持续加大苜蓿的研发力度和科技投入, 推进苜蓿产业化发展, 提升苜蓿产品的自给率, 保障草食畜牧业健康、稳定发展。     

Species dependence of the redox potential of the primary electron donor p700 in photosystem I of oxygenic photosynthetic organisms revealed by spectroelectrochemistry

The redox potential of the primary electron donor P700, E(m)(P700/P700(+)), of Photosystem I (PSI) has been determined for 10 oxygenic photosynthesis organisms, ranging from cyanobacteria, red algae, green algae to higher plants, by spectroelectrochemistry with an optically transparent thin-layer electrode (OTTLE) cell to elucidate the scattering by as much as 150 mV in reported values of E(m)(P700/P700(+)). The E(m)(P700/P700(+)) values determined within error ranges of ± 1-4 mV exhibited a significant species dependence, with a span >70 mV, from +398 to +470 mV vs. the standard hydrogen electrode (SHE). The E(m)(P700/P700(+)) value appears to change systematically in going from cyanobacteria and primitive eukaryotic red algae, then to green algae and higher plants. From an evolutionary point of view, this result suggests that the species believed to appear later in evolution of photosynthetic organisms exhibit higher values of E(m)(P700/P700(+)). Further, the species dependence of E(m)(P700/P700(+)) seems to originate in the species-dependent redox potentials of soluble metalloproteins, Cyt c(6) and plastocyanin, which re-reduce the oxidized P700 in the electron transfer chain.     

Equilibrium or disequilibrium? A dual-wavelength investigation of photosystem I donors

Oxidation of photosystem I (PSI) donors under far-red light (FRL), slow re-reduction by stromal reductants and fast re-reduction in the dark subsequent to illumination by white light (WL) were recorded in leaves of several C₃ plants at 810 and 950 nm. During the re-reduction from stromal reductants the mutual interdependence of the two signals followed the theoretical relationship calculated assuming redox equilibrium between plastocyanin (PC) and P700, with the equilibrium constant of 40 ± 10 (ΔE _m = 86–99 mV) in most of the measured 24 leaves of nine plant species. The presence of non-oxidizable PC of up to 13% of the whole pool, indicating partial control of electron transport by PC diffusion, was transiently detected during a saturation pulse of white light superimposed on FRL or on low WL. Nevertheless, non-oxidizable PC was absent in the steady state during fast light-saturated photosynthesis. It is concluded that in leaves during steady state photosynthesis the electron transport rate is not critically limited by PC diffusion, but the high-potential electron carriers PC and P700 remain close to the redox equilibrium.     

The kinetic behavior of P-700 during the induction of photosynthesis in algae

The kinetics of P-700 were examined spectrophotometrically during the induction of photosynthesis in algae. A pronounced oscillation was observed in the redox level of P-700 upon illumination of dark-adapted cells. The dark adaptation required approximately 1 min. The oscillation may be described as an initial rapid oxidation reaching a peak at approx. 50 ms followed by complete reduction of the pool of P-700. A subsequent slower oxidation resulted in attainment of the final state around 1 s. The main features of the oscillation were qualitatively the same in a wide variety of algae.The modulation in redox level of P-700 required high intensity activation of both photosystems and was eliminated by pre-illumination of the cells with weak short wavelength light but not by longer wavelengths absorbed primarily by Photosystem I. We propose that the P-700 modulation is directly related to the fast redox changes in Photosystem II which occur during the induction of photosynthesis.Cells incubated with methyl viologen did not show the P-700 oscillation confirming the suggestion previously advanced that exhaustion of Photosystem I acceptor and kinetic limitations in the carbon reduction cycle partially control the fast phase of photosynthetic induction.     

Effects of oxygen and photosynthesis carbon cycle reactions on kinetics of P700 redox transients in cyanobacterium <Emphasis Type="Italic">Arthrospira platensis</Emphasis> cells

Effects of oxygen and photosynthesis and respiration inhibitors on the electron transport in photosystem I (PSI) of the cyanobacterium Arthrospira platensis cells were studied. Redox transients of P700 were induced by illumination at 730 nm and monitored as kinetics of the absorption changes at 810 nm; to block electron influx from PSII, the measurements were performed in the presence of 30 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Inhibitors of terminal oxidases (potassium cyanide and pentachlorophenol) insignificantly influenced the fast oxidation of P700 under aerobic conditions, whereas removal of oxygen significantly decelerated the accumulation of P700(+). In the absence of oxygen the slow oxidation of P700 observed on the first illumination was accelerated on each subsequent illumination, suggesting an activation of the carbon cycle enzymes. Under the same conditions, pentachlorophenol (an uncoupler) markedly accelerated the P700 photooxidation. Under anaerobic conditions, potassium cyanide (an inhibitor of carbon dioxide assimilation) failed to influence the kinetics of redox transients of P700, whereas iodoacetamide (an inhibitor of NADP(H)-glyceraldehyde-3-phosphate dehydrogenase) completely prevented the photooxidation of P700. Thus, the fast photooxidation of P700 in the A. platensis cells under aerobic conditions in the presence of DCMU was caused by electron transport from PSI onto oxygen, and complicated transient changes in the P700 photooxidation kinetics under anaerobic conditions (in the presence of DCMU) were due to involvement of NADP+ generated during the reducing phase of the carbon cycle.     

Significant species-dependence of P700 redox potential as verified by spectroelectrochemistry: comparison of spinach and Theromosynechococcus elongatus

The redox potentials of P700, the primary electron donor of photosystem (PS) I, of spinach and Thermosynechococcus elongatus were determined by means of spectroelectrochemistry with an error range of +/-2-3 mV, to find that the redox potential of P700 in T. elongatus is lower by ca. 50 mV as compared with spinach. The shift in the P700 redox potential of PS I core particles prepared by harsh detergent treatments remained to within 10 mV for both organisms. These results show that the 50 mV difference in the P700 redox potential between the two organisms is not a detergent-induced artifact but reflects an intrinsic property of each PS I.     

Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Reduction of the primary donor P700 of photosystem I during steady-state photosynthesis under low light in <Emphasis Type="Italic">Arabidopsis</Emphasis>

During steady-state photosynthesis in low-light, 830-nm absorption (A₈₃₀) by leaves was close to that in darkness in Arabidopsis, indicating that the primary donor P700 in the reaction center of photosystem I (PSI) was in reduced form. However, P700 was not fully oxidized by a saturating light pulse, suggesting the presence of a population of PSI centers with reduced P700 that remains thermodynamically stable during the application of the saturating light pulse (i.e., reduced-inactive P700). To substantiate this, the effects of methyl viologen (MV) and far-red light on P700 oxidation by the saturating light pulse were analyzed, and the cumulative effects of repetitive application of the saturating light pulse on photosynthesis were analyzed using a mutant crr2-2 with impaired PSI cyclic electron flow. We concluded that the reduced-inactive P700 in low-light as revealed by saturating light pulse indicates limitations of electron flow at the PSI acceptor side.