光合磷酸化的研究 Ⅷ.黄化小麦幼苗变綠时光合磷酸化活力的发生

(1)黄化小麦幼苗初变绿时,光合磷酸化活力之发生远较叶绿素的生成为迟。在实验条件下,照光变绿3小时后,才可测得光合磷酸化活力,且其按叶绿素为基础计算的活力随照光变绿时间的增加而增加,至照光变绿7—8小时后,叶绿体上叶绿素含量尚在继续增加,但光合磷酸化活力则趋向恒定。(2)在黄化幼苗变绿初期,测得的循环光合磷酸化ATP形成能力较非循环光合磷酸化ATP形成能力高得多,以后较接近;但将循环光合磷酸化之ATP形成能力与非循环光合磷酸化之放氧能力相比较,则其比例在不同时期相差不大。这说明,在变绿初期非循环光合磷酸化之ATP形成能力特别小的原因,主要是由于当时它的偶联程度特别低,并不是因为它较循环光合磷酸化多牵涉到放氧等步骤,而这些步骤可能发生得较晚所致。以DCPIPH_2作氢供体的氧化光合磷酸化活力的最初增长情况与以Fe(CN)_6~≡作氢受体的非循环光合磷酸化ATP形成能力的增长情况一样,均比以PMS促进的循环光合磷酸化活力增长时间为晚,这结果也有助于证明非循环光合磷酸化ATP形成能力增长较晚的原因与它牵涉到放氧步骤无关。(3)使黄化变绿幼苗光合磷酸化、希尔反应活力达到饱和所需的光强度与绿苗所需的相仿。变绿初期的叶绿体,其光合磷酸化作用有很强的“光强效应”,卽弱光下电子传递速度慢、PSP活力低时,与磷酸化的偶联程度会急剧下降。这现象可能是造成变绿初期测得的非循环光合磷酸化ATP形成能力特别低的原因。(4)黄化幼苗变绿时,同化CO_2能力之发生时间与光合磷酸化活力之发生时间差别不大,但以叶绿素为基础计算,前者的活力较早达到恒定。     

循环光合磷酸化

文章在回顾循环光合磷酸化和循环电子传递链发现的基础上,分析了循环光合磷酸化在光合作用中的地位,并对影响循环光合磷酸化的内外因素及其调控作了述评,为进一步开展相关研究提供参考.     

光合磷酸化的量子需要量

用菠菜叶绿体悬浮液,在红光下(620—660mμ,6—8×10~3尔格/厘米~2-秒)测定同位素P~(32)标记的无机磷酸进入ATP的强度,并根据吸收的光能量换算为形成一个分子ATP所需要的红光量子数。结果指出: (1)循环光合磷酸化作用,不论用何种辅助因素(PMS,维生素K_3,FMN),形成一个分子ATP的量子需要量均在4—5之间(最低一次获得2.9)。叶提取液代替辅助因素,结果亦同。(2)与希尔反应偶联的光合磷酸化作用(希尔氧化剂为K_3Fe(CN)_6或TPN)的量子需要量亦是4—6。同时测定的还原作用指出希尔反应中每放出一个分子O_2,需要8—12个红光量子,表示在试验条件下,二者是完全偶联的(P/2e?1)。没有磷酸化(不加ADP及P_i)时,希尔反应的量子需要量不变,表示偶联的ATP形成不需额外的光量子。(3)光强度减低,则循环与非循环光合磷酸化作用的效率随之降低,量子需要量增加,而希尔反应的效率则不变。从上述结果推论,两种光合磷酸化作用均是通过同一的电子传递系统,在此系统中仅有一个磷酸化部位,除非另有一个部位是极易破坏的。试验结果也对光合作用的量子需要量问题,供给可能的解释。在弱光下光合作用效率高,可能是由于部份ATP来自呼吸;而在强光下效率减低,则是呼吸所供给的ATP不足而必需依靠循环光合磷酸化所致。     

低浓度硫酸甲酯吩嗪（PMS）对叶片光合放氧的促进作用

外加低浓度循环光合磷酸化电子递体硫酸甲酯吩嗪(PMS)对菠菜、大豆、水稻和小麦叶片光合放氧有促进作用,与此同时叶片ATP含量也得到增加。PMS对经8 mmol L~(-1)NH_4Cl处理过的菠菜叶片的光合放氧也有促进,最适促进浓度比未经NH_4Gl处理的叶片高,促进的幅度也大。幼龄叶与成长叶相比,幼龄叶的光合磷酸化活性和P/O比值低于成长叶片,其光合放氧速率受PMS促进的幅度大于成长叶片。因此光合磷酸化也可以成为光合作用的一个重要限制因素。     

低浓度NaHSO3促进田间水稻的光合磷酸化和光合作用

用1～2 mmol/L NaHSO3喷施于水稻(Oryza sativa L.)叶面可以提高叶片的光合速率,并能持续3 d以上.在此条件下,光下叶片中的ATP含量明显增高,叶片的叶绿素毫秒延迟荧光加强,反映与光合磷酸化活力有关的跨类囊体膜质子梯度增加.乳熟期喷施2次1 mmol/L NaHSO3后,水稻产量提高约10%.研究表明NaHSO3的主要作用和PMS(phenazine methosulfate)促进光合速率的原因可能类似,都是增加了ATP的供应.与此同时,观察到低浓度NaHSO3可促进水稻中反映循环电子传递的叶绿素荧光在作用光关闭后的短时上升.以上现象表明低浓度NaHSO3的促进作用很可能是通过促进围绕PSⅠ的循环电子传递及其耦联的光合磷酸化而促进水稻光合作用的.     

抑制AX-3表达改变本氏烟氧化和光合磷酸化ATP积累

基因组注释预测了AX-3普遍存在于不同植物种类,但其功能鲜有报道。本研究通过StAX-3基因反义cDNA在本氏烟(Nicotiana benthamiana)体内表达,实现了其AX-3基因表达下调;蛋白质组表达谱测定分析显示,伴随AX-3积累减少,168个DAPs (表达倍绝对值大于1.5)的积累随之改变;其中15个密切关联DAPs网络分析显示,AX-3下调表达,主要降低了9种线粒体内代谢酶类的积累,包括氧化磷酸化ATP合成酶和TCA循环核心脱氢酶,同时,增加了光合作用代谢酶类合成,包括光合磷酸化ATP合成酶和叶绿素合成底物萜类酶等。叶绿素和ATP含量等表型测定结果,证实了萜类底物和叶绿素合成积累增加,促进光合磷酸化ATP合成积累。结果表明,StAX-3调控氧化和光合磷酸化ATP合成积累模式。该结果不仅明确了St AX-3具有调控线粒体和叶绿体能量代谢的功能,而且为进一步研究植物ataxin-3功能提供理论指导。     

叶绿体结构和功能的研究——Ⅳ、红霉素对叶绿体能量转换的效应

研究了红霉素对叶绿体能量转换的效应,获知:10(-4)M红霉素抑制循环和非循环光合磷酸化,这种抑制是与反应底物ADP非竞争性的。在抑制ATP合成的浓度范围内,红霉素对基础电子传递的速率并无影响,但它抑制因偶联磷酸化而促进的那部分电子传递。红霉素还抑制膜上Mg~(2 )-ATP酶的活性。以上结果表明,红霉素似有光合磷酸化能量传递抑制剂的特点,它的作用部位可能接近膜上CF_2,或ATP酶的催化部位。     

亚硫酸氢盐怎样促进光合作用

低浓度亚硫酸氢盐可以促进光合作用的现象和有关的机理早已引起人们的关注。通过归纳和分析相关文献,认为低浓度亚硫酸氢盐对光合作用的促进效应可能是由于它通过促进围绕PS I的循环电子传递及其耦联的光合磷酸化,增加ATP的供应（因为ATP的供应常常是光合机构运转的限制因素）;而不是由于它抑制了光呼吸,或促进气孔开度,或作为光合器官的生长发育以及运转所必需的硫肥而起作用的。     

完整叶绿体中的NADP及NADPH测定

NADP是植物体内重要的氢递体,叶绿体通过光合电子传递和偶联光合磷酸化反应形成NADPH和ATP,再利用它们去同化CO_2。因此对光合器官内NADP及NADPH的含量分析,在光合作用研究中显得十分重要。一般测定NADPH形成的非循环光合电子传递活性时,是在无被膜的离体叶绿体反应系统中加入外源NADP及铁氧还蛋白,光还原形成的NADPH的量直接由波长340     

光合磷酸化的研究 Ⅵ.2,6-二氯酚靛酚光还原过程中的偶联磷酸化

1958年,Arnon首先发现在辅酶Ⅱ的光还原过程中,同时有ATP的形成,即所谓偶联光合磷酸化或非循环光合磷酸化。随后又肯定了K_3Fe(CN)_6及苯醌作为希尔氧化剂时的偶联磷酸化。在常用的希尔氧化剂中,DCPIP却是一个例外。大家知道,     

Effects of Light and 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea on Levels of ATP in Lemna paucicostata 6746 and a Photosynthetic Mutant with Abnormal Flowering Responses

The effects of light, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and ammonium ion on pool sizes of ATP were studied in Lemna paucicostata 6746 (wild type) and a photosynthetic mutant (strain 1073) with abnormal flowering responses. Wild type fronds were capable of endogenous and phenazine methosulfate-catalyzed cyclic photophosphorylation. The endogenous cyclic photophosphorylation was inhibited by DCMU. The mutant fronds showed little endogenous but appreciable rates of phenazine methosulfate-catalyzed cyclic photophosphorylation. Treatment with DCMU during prolonged exposure to light did not result in elevated levels of ATP. Ammonium ion in the medium did not inhibit light-induced increases in pool sizes of ATP. It is concluded that the previously reported effects on flowering of DCMU, the photosynthetic mutation or ammonium ion, were not due to altered pool sizes of ATP.     

The ATP Levels Obtained in Photophosphorylation, Glycolysis and Oxidative Phosphorylation in Scenedesmus Titrated with Desaspidin

The ATP levels in photophosphorylation, glycolysis and oxidative phosphorylation, in the unicellular green alga Scenedesmus obtusiusculus, were titrated with narrow concentration intervals of desaspidin in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which allows the differentiation between non-cyclic, pseudocyclic and true cyclic photophosphorylation. The data on photophosphorylative ATP levels were compared with earlier data on total binding of phosphate. In the true cyclic process, both parameters are equally sensitive towards desaspidin. Under pseudocyclic conditions and in non-cyclic photophosphorylation, the level of ATP is more sensitive towards desaspidin than is total binding of phosphate. This suggests a structural difference between the cyclic and the two non-cyclic (one of which is also pseudocyclic) sites. The non-cyclic ATP level is more sensitive towards desaspidin than is pseudocyclic. This may be connected with the higher ATP level under pseudocyclic as compared to non-cyclic conditions.     

The Effect of Light Intensity on Total Photophosphorylation and the ATP Level in Scenedesmus

The dependence of in vivo photophosphorylation on light intensity was studied in the unicellular green alga Scenedesmus obtusiusculus. By selective use of the inhibitor DCMU, phosphorylation in (I) the complete system, (II) the pseudocyclic system alone, and (III) the true cyclic system alone, were followed. When the total binding of phosphate was studied, all reaction types became light saturated in about the same manner. The effect of DCMU on the level of ATP varied according to light intensity. As for the specific systems of photophosphorylation, the following ATP data were found: (I) In the complete system the level of ATP decreases with light intensity. (II) Under pseudo-cyclic conditions light first increases and then decreases the ATP level. Under the atmospheric conditions used (i.e. CO₂-free nitrogen) this indicates a regulation between photophosphorylation and glycolysis, for which possible explanations are discussed. (III) In the true cyclic conditions light has little effect on the ATP level. The possibility is indicated that there is a structural difference between the non-cyclic (site 1) and the pseudocyclic (site 2) sites of photophosphorylation on the one hand and the true cyclic site (3) on the other.     

The role of cyclic photophosphorylation in vivo

When cyclic photophosphorylation is inhibited in Chlorella vulgaris cells by carbonylcyanide-trifluoromethoxy phenylhy-drazone, photosynthetic CO₂-fixation under anaerobic conditions exhibits a distinct lag. Under the same conditions, the light-dependent formation of ribulose diphosphate shows also this lag. It is concluded that cyclic photophosphorylation is required to fill up the pools of phosphorylated intermediates of the Calvin cycle at a time when noncyclic photophosphorylation cannot yet efficiently operate. Under aerobic conditions, the initial energy demand can be accommodated by respiratory ATP or cyclic photophosphorylation or both. Evidence for stoichiometric participation of cyclic photophosphorylation in photosynthesis is still lacking.     

Cyclic and Non-cyclic Photophosphorylation as Energy Sources for Active K Influx in Hydrodictyon africanum

Under anaerobic conditions in the light, active K influx inHydrodictyon africanum is supported by cyclic photophosphorylation.The use of selective inhibitors shows that, in the presenceof CO₂, a considerable portion of the ATP used by the K pumpis supplied by noncyclic photophosphorylation. The rest of theATP in these conditions comes from cyclic photophosphorylation.This is true under light-limiting as well as light-saturatedconditions. If non-cyclic photophosphorylation is inhibited (by removalof carbon dioxide, by the addition of cyanide which interfereswith the carboxylation reaction, or by inhibition of photosystemtwo with DCMU or supplying only far-red light), the K influxat low light intensities is stimulated, and its characteristicsbecome those of a process powered by cyclic photophosphorylationalone. These results are interpreted in terms of a competitionfor ATP between K influx and CO₂ fixation. Implicit in thisexplanation is a requirement for a switch of excitation energyabsorbed by photosystem one from cyclic photophosphorylationto non-cyclic photophosphorylation whenever conditions (presenceof CO₂and photosystem two activity) allow CO₂ fixation to occur. Further evidence for such a switch of excitation energy absorbedby photosystem one was obtained in experiments in which redand far-red light were applied separately and together. It wasfound that CO₂ fixation showed the Emerson enhancement effect,while K influx (in the presence of CO₂) shows a ‘de-enhancement’.This suggests that far-red light alone powers cyclic photophosphorylation;if red light is also present, some of the far-red quanta arediverted to non-cyclic photophosphorylation. The nature of the interaction between cyclic and non-cyclicphotophosphorylation is discussed in relation to these and otherpublished results.     

ATP regeneration by cyclic photophosphorylation using spinach thylakoid

Summary ATP photophosphorylation by spinach thylakoid was examined to evaluate its use as an ATP regeneration reaction in biosynthetic reactors that consume ATP. Initial rate of cyclic photophosphorylation mediated by phenazine methosulfate was found to be 218 mole ATP/h.mg Chlorophyll. This activity was stable for over 3 months at –85°C. When phosphoryl transfer reactions were coupled to cyclic photophosphorylation, ATP was continuously regenerated by thylakoid between 14–24 times in batch reactors.     

The Role of Cyclic and Pseudocyclic Photophosphorylation in Photosynthetic 14CO2 Fixation in Hydrodictyon africanum

Experiments are reported in which the effects on photosynthesisof various inhibitors of cyclic photophosphorylation were investigated.These inhibitors, generally had only a small inhibitory effecton photosynthesis, and the inhibition was not increased by conditionswhich inhibit pseudocyclic photophosphorylation. These inhibitorsdo not inhibit the Emerson enhancement effect. From these resultsit was concluded that photosynthesis does not need any ATP otherthan that produced in non-cyclic photophosphorylation. The effectsof these inhibitors on active K influx in light-anaerobic conditionsin the presence or absence of CO₂ suggest that some of the ATPproduced by non-cyclic photophosphorylation can be used to supportactive K influx. The results are discussed in relation to themechanism of the Emerson effect, the stoichiometry of non-cyclicphotophosphorylation, and the ATP requirements for autotrophicgrowth.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Photophosphorylation Associated with Photosystem II: I. Photosystem II Cyclic Photophosphorylation Catalyzed by p-Phenylenediamine

Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p-phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p-phenylenediamine is completely inactive. When photosystem II is not inhibited, p-phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations.