LOCALIZATION OF LIGHT-INDUCED STRONTIUM ACCUMULATION IN SPINACH CHLOROPLASTS

Electron-microscopic evidence showing the location of strontiumand calcium deposits on the lamellae of spinach chloroplastsis presented. The chloroplast limiting membrane did not actas a barrier for this ion accumulation, since the chloroplastswere ruptured during the isolation and incubation, especiallyin the light. The grana stacks also were extensively swollen.The number of electrondense deposits attached on the lamellaewas greatly increased by light under conditions favoring thehydrolysis of ATP. The average diameter of the deposits aftera 6 minute illumination with 1 mM SrCl₂ was 14 mµ, after15 minutes with 1 mM SrCl₂ was 21 mµ, and after 15 minuteswith 20 µM SrCl₂ was 13 mµ. The findings are inharmony with the results of ion-uptake experiments using radioactivestrontium and calcium. ¹ United States National Science Foundation Postdoctoral Fellow.     

LIGHT-INDUCED HIGH-AMPLITUDE SWELLING AND SHRINKING IN ISOLATED SPINACH CHLOROPLASTS

It was found that light accelerates the swelling of spinachchloroplasts suspended in a solution containing NaCl and Tris-HCl,and that, in about 60 min, the swollen chloroplasts begin toshrink rapidly. The volume of chloroplasts reached, on swelling,270 per cent of the original volume on the average and thenshrank to 17 per cent. The rate of swelling and the maximumvolume were affected markedly by the tonicity of NaCl. NH₄Cldid not affect the volume changes, while phenazine methosulfateaccelerated the swelling remarkably. The relationship betweenphotoshrinkage and this light-induced high-amplitude swellingwas discussed. (Received April 2, 1966; )     

COMPARISON OF THE OSMOTIC VOLUME CHANGES IN CHLOROPLASTS ISOLATED FROM DIFFERENT PLANTS

Chloroplasts were isolated from leaves of four plant species(spinach, New. Zealand spinach, Swiss Chard and tomato) andtheir osmotic behaviour was compared. Maximal contraction ofchloroplasts from all plant sources occurred at 0.3–0.4M sucrose. The volume of the particles at this sucrose concentrationwas also similar in all cases; 300 mm³ per 10¹⁰ chloroplasts.At higher and lower sucrose concentrations, the chloroplastsswell. The degree of swelling, especially in higher sucroseconcentrations, differed in chloroplasts from different sources.Tomato chloroplasts are most sensitive, and the spinach chloroplastsare the most resistant to all types of treatment. Tomato chloroplastsrequire rather high phosphate concentrations for good preservation. Changes in optical density at 520 mµ can be used to geta rough idea about the condition of the particles. (Received February 7, 1966; )     

LIGHT-INDUCED FORMATION OF ASCORBIC ACID IN ISOLATED CHLOROPLASTS

Investigations were made to determine the nature of a reducingsubstance which was formed on illuminating a reaction systemconsisting of chloroplast and cytoplasmic fraction of spinachleaves. From the results of spectrophotometric examinationsit was concluded that the photoproduct in question, or at leastits major portion, was ascorbic acid. The precursor of ascorbicacid, which was found to be heat unstable, was contained inthe cytoplasmic fraction. (Received October 12, 1960; )     

THE VISUALIZATION OF THE PHOTOSYNTHETIC COUPLING FACTOR IN EMBEDDED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were stained with aqueous uranyl acetate immediately after glutaraldehyde-osmium fixation but before dehydration and embedding. Under these conditions, the lamellae are shown in thin sections to have 95-Å x 115-Å coupling factor particles on their surfaces. The particles can be seen only on the matrix side of nonopposed thylakoids, and are shown to occur on both stromal and granal lamellae, regardless of the organization of the lamellae into stacks. It is estimated that, in native, fully coupled chloroplast lamellae, there is on the average one coupling factor for every 500 chlorophyll molecules. The morphological appearance of the particles is not affected by a variety of buffers, by changes in illumination or temperature, or by alterations in the energy state of the membranes during preparation. The particles can be removed from the membranes with low concentrations of Na₂EDTA, and the photophosphorylating activity of the membranes is concomitantly lost. Both the activity and the appearance of the particles can be restored to the membranes by rebinding EDTA-extracted coupling factors to the uncoupled membranes.     

FRACTIONATION OF SPINACH CHLOROPLASTS BY FLOW SEDIMENTATION-ELECTROPHORESIS

A separation of spinach chloroplasts in vitro into fractions according to size (volume) and activity (light-dependent shrinkage and NADP reduction) has been achieved by stable-flow free boundary sedimentation-electrophoresis. The salient features of this chloroplast study are: (a) separation is achieved within 30 min; (b) only small density gradients are required, thus minimizing osmotic effects; (c) the fractions are collected continuously, with size fractionation being evidenced; and (d) particles are separated into fractions of higher and lower activities as compared with the control population.     

LIPIDS AND LIGHT-DEPENDENT SWELLING OF ISOLATED SPINACH CHLOROPLASTS

The light-dependent high-amplitude swelling of spinach chloroplastssuspended in 175 mM NaCl buffered with 50 mM Tris-HCl (pH 7.9)was investigated by a packed weight technique and by electronmicroscopy. Endogenous neutral and charged lipids were releasedby the thylakoid membrane to about the same extent during chloroplastincubation in both the light and the dark. The addition of laurate,oleate, stearate, lecithin and lysolecithin increased chloroplastswelling in the dark, but inhibited swelling in the light. Acetoneincreased chloroplast swelling at low concentrations and inhibitedat high concentrations in both the light and the dark. For 20%acetone, the grana stacks disappeared and a newly formed membranesystem appeared which was similar to a myelin structure. Therole of lipid components in the conformation of the membranestructure is discussed and a mechanism for lightinduced chloroplastswelling based on a hydrogen-sodium exchange is proposed. ¹ Present address: Department of Botanical Sciences, Universityof California, Los Angeles, California 90024.     

镉离子对菠菜叶绿体色素蛋白质复合物及激发能分配的影响

Cd~(2+)使叶绿体低温(77K)荧光发射光谱中 F686/F736及 F696/F736和激发光谱中F480/F436比值降低,说明 Cd~(2+)不利于激发能向 PSⅡ传递。SDS-PAGE 分析表明,Cd(2+)处理后叶绿体类囊体膜中光系统Ⅱ捕光叶绿素蛋白质复合物 LHCⅡ的部分寡聚体解聚成单体,且 LHCⅡ的总量也减少了。分析表明 Cd~(2+)使属于 LHCⅡ的多肽减少。已知LHCⅡ在光能吸收、传递以及激发能在两个光系统间的分配和调节方面起着重要作用,Cd~(2+)引起部分 LHCⅡ解聚和总量减少,必然导致由 LHCⅡ捕获和向光系统Ⅱ中心传递的能量减少。     

菠菜叶绿体类囊体膜磷酸酯酶的分离和性质

通过正丁醇抽提、离心和柱层析等方法,从菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）叶绿体类囊体膜片分离出磷酸酯酶,此酶水解磷酸单酯类物质。酶活性的最适ｐＨ 在７．０ 以下,属酸性磷酸酯酶。反应温度增至６０℃时反应速度达最高,具有高温酶的特性。ＡＴＰ和无机磷盐均抑制此酶活性。经ＳＤＳ－聚丙烯酰胺凝胶电泳表明,此酶制剂出现两条主要的蛋白带     

EFFECT OF {alpha}-HYDROXYSULFONATES ON PHOTOCHEMICAL REACTIONS OF SPINACH CHLOROPLASTS AND PARTICIPATION OF GLYOXYLATE IN PHOTOPHOSPHORYLATION

1. The effect of -hydroxy sulfonates and sulfite, inhibitors ofglycolate oxidase, on the photochemical reactions of spinachchloroplasts was studied. The photo reduction of ferricyanideand NADP was not affected by the poisons, whereas the photophosphorylationand ¹⁴CO₂ fixation were inhibited.
2. Glyoxylate was photoreducedby the chloroplasts in the presenceof PPNR and glyoxylate reductase,and this reduction was acceleratedby the addition of NADP.ATP formation accompanied with thereduction of glyoxylate bythe illuminated chloroplasts wasobserved. It is supposed thatglyoxylate oxidizes the photoreducedNADPH₂ or PPNR and thusthe photophosphorylation is stimulated.

¹A part of this paper was presented at the annual meeting ofAgricultural Society of Japan, in August, 1964. ²Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka.     

ENDOGENOUS CHANGES OF PHOTOCHEMICAL ACTIVITIES OF SPINACH LEAVES

The activities of green cell-free extracts of spinach leavesin performing photochemical transphosphorylation, photosyntheticphosphorylation, the HILL reaction and the light-induced formationof the endogenous reducing substance (ascorbic acid) were followedin parallel during the growth process of the plant. There wasa certain parallelism between the development of the activitiesof photochemical transphosphorylation, of photo-synthetic phosphorylationand of the HILL reaction, activities being low in the earlierstage of growth, reaching a maximum just before efflorescence,and showing thereafter a more or less sharp decline. The activityin the light-induced formation of endogenous reducing substancewas undetectable for the first 35 day-period of growth, reacheda maximum about one week earlier than the other three activities,and again disappeared after 60 days of growth. (Received September 9, 1960; )     

ULTRASTRUCTURAL CHANGES IN SUNFLOWER CHLOROPLASTS FOLLOWING INOCULATION WITH PSEUDOMONAS SYRINGAE PV. TAGETIS

Severe chlorosis and ultrastructural modifications of chloroplasts occur in sunflower in response to infection by Pseudomonas syringae pv. tagetis. Chlorosis became apparent within 2 days after the cotyledons of 10-day-old sunflower seedlings were inoculated with the bacteria. The first symptoms generally appeared in the center of leaves at the second node above the cotyledons. Leaves above the second node lost essentially all of their pigmentation but remained turgid and continued to expand. Grana thylakoids became dilated and separated from the granal stacks. These thylakoid membranes did not chemically breakdown as in the case in chromoplast formation or normal chloroplast senescence. Both grana and stroma thylakoid membranes coalesced to form a large membrane sheet within the plastid. The ultrastructural changes are unlike those reported to be caused by other chlorosis-inducing bacteria or chlorosis associated with normal senescence.     

ULTRASTRUCTURAL CHANGES OF CHLOROPLASTS IN ATTACHED AND DETACHED,AGING PRIMARY WHEAT LEAVES

Degradation of chloroplasts is shown in mesophyll cells of primary leaves of wheat. The sequence of ultrastructural changes in chloroplasts of naturally senescing leaves is compared with that of detached, aging leaves. In chloroplasts of naturally senescing leaves, the first indications of aging are the appearance of osmiophilic globuli and reorientation of the thylakoidal system. The membranes of the grana and intergrana lamellae then become distended and later dissociate into distinct vesicles. Concurrent with these membrane changes, osmiophilic globuli increase in size and number, and the stroma breaks down. Finally, the chloroplast envelope ruptures and plastid contents disperse throughout the cell's interior. In chloroplasts of mesophyll cells in detached, aging leaves, initial changes also include appearance of osmiophilic globuli, but later stages of chloroplast degradation are different. The chloroplast envelope ruptures before the lamellae break down. Swelling of grana and intergrana lamellae is not pronounced and, additionally, the thylakoidal system degenerates without forming vesicles or numerous osmiophilic globuli. These differences in the sequence of chloroplast degradation indicate that naturally senescing leaves rather than detached, aging leaves should be used in studies of chloroplast senescence.     

叶绿体中电色效应慢上升相的起源,质子释放过程及细胞色素b_6、f的氧化还原反应

P515吸收变化中的慢上升相(P515s),可用Tris,NH_4Cl或尼日利亚菌素,加在低盐浓度的叶绿体中形成。在叶绿体和叶片样品中,不管有无P515s,都有明显的细胞色素b_6和f的氧化还原变化。在有解联剂存在下,中性红染料可测到膜内质醌醇氧化释放质子的过程,释放速度与P515s形成的速度接近。高盐浓度中暗适应的叶绿体,释放H~+的速度比P515s形成的速度快,其中质醌醇释放H~+的速度只有1～2 ms。在接近生理条件下(无外加供受体、抑制剂),P515s的起源与细胞色素b_6、f的氧化还原反应造成的电子跨膜流动无直接联系,而质醌醇释放H~+的过程及随后的侧向扩散,可能是P515s形成的直接起因。     

ULTRASTRUCTURE OF CHLOROPLASTS AND CHROMOPLASTS IN CAPSICUM ANNUUM I. THYLAKOID MEMBRANE CHANGES DURING FRUIT RIPENING

Developing chromoplasts in the fruit of Capsicum annuum were examined by electron microscopy. Special attention was given to changes in the thylakoid system. All grana and some intergranal thylakoids in the mature chromoplasts of the seven cultivars studied underwent lysis. The particulate nature of the granal membranes disappeared during lysis before the relationship between the partitions and locules was obscured. The changes during lysis support the globular concept of membrane structure. The selective lysis of the synaptic membranes of the granal partitions may be attributed to their distinctive composition and structure. Lipid globules (osmio-philic) did not accumulate in the immediate region of granal lysis, indicating that they are not directly derived from membranes undergoing degradation. During and following granal lysis a profuse development of intergranal thylakoid membranes occurred in several cultivars. In some instances a thylakoid plexus (prolamellar body) was formed. This specialized structure of the thylakoid system occurs in the chromoplasts of other species as well as in other types of plastids. Extensive, concentrically arranged thylakoid sheets with specific interspaced membrane relationships were frequently associated with the plexus. Several types of membrane associations and interrelationships in the plastid are described. An analysis of one type of membrane configuration, the thylakoid sheets, indicated that one method of growth may be through intussusception into the original membrane. The development of thylakoid plexes and of extensive thylakoid sheets during or after granal lysis indicates that dynamic synthetic activities occur in the chromoplasts of some cultivars of pepper during fruit ripening.