EFFECT OF LIPASE-DIGESTION ON PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

In order to elucidate the role of lipids in photosynthesis,chloroplasts were digested with lipase, and the effect of lipase-digestionon some photochemical activities was studied. The HILL reactionwas sensitive to the digestion, but chloroplasts having intactmembrane were somewhat resistant to the action of lipase. Theinactivation by lipase digestion seems to be due to the destructionof a component necessary for the Hill reaction to proceed. Thechloroplasts treated with lipase showed the following activities. (1) Active photooxidation of reduced cytochrome c and menadione. (2) Photooxidation of ascorbate, which was enhanced in the presenceof DPIP, and retarded in the absence of the dye. (3) NADP-photoreduction in the presence of the DPIP-ascorbatecouple, as the electron donor. These facts suggested that the site attacked with lipase wasresponsible for the photochemical oxygen evolution. The decrease in the fluorescence intensity of chlorophyll awas also observed during the digestion. ¹Present address : Biological Laboratory, General EducationDeparment, Kyushu University, Otsubo-machi, Fukuoka.     

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.     

SYNTHESIS OF CHLOROPLAST DNA IN ISOLATED CHLOROPLASTS

Chloroplasts isolated from Euglena gracilis incorporated both tritiated thymidine 5'-triphosphate and tritiated deoxyadenosine 5'-triphosphate into an acid-stable fraction. The incorporation was dependent on the presence of all four deoxynucleoside triphosphates and was sensitive to treatment with deoxyribonuclease and actinomycin D. It was demonstrated that bacterial contamination could not account for the incorporation of label. Extraction of DNA from the chloroplasts and subsequent density gradient centrifugation of the DNA in CsCl₂ showed that the incorporation was into chloroplast DNA (ρ = 1.686) of high molecular weight.     

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; )     

OSMOTIC EFFECT ON THE PHOTOSHRINKAGE OF CHLOROPLASTS

Shrinkage of spinach chloroplasts by illumination and that byexposure to a high tonicity raised by addition of sucrose wereinvestigated by means of the rapid measurement of chloroplastvolume with a COULTER counter. The osmotic shock in the darkinduced two steps of volume change; almost instantaneous shrinkagefor the osmotic shock and follow-up gradual swelling. The finalvolume attained after equilibration was smaller than the originalvolume below 0.6 M, and greater above this concentration. Whenchloroplasts under osmotic shock were illuminated, the photoshrinkagecompeted with the swelling induced by the osmotic shock, andthese reverse effects were balanced at a certain volume. Photoshrinkageactivity measured after equilibration decreased with increasingconcentrations, and the activity curve plotted against sucroseconcentration showed a stationary level of 50% of the originalactivity between 0.2 and 0.6 M, indicating the resistance ofa structure in chloroplasts to the denaturation by the osmoticeffect of sucrose. The osmotic effect in the dark as well asin the light was completely reversible below 0.2 M and was partiallyreversible or irreversible above this concentration. Glucoseshowed qualitatively the same osmotic effect as sucrose. ¹ Present address: Laboratory of Chemistry of Natural Products,Tokyo Institute of Technology, Meguroku, Tokyo.     

OXIDATION OF PHLORIDZIN BY ISOLATED CHLOROPLASTS

Phloridzin was shown to be oxidized by chloroplast fragmentsfrom swiss-chard. From inhibitor studies, kinetics and affinitytoward oxygen, it was inferred that the oxidation was mediatedby a phenolase in a "cresolase" type reaction. Atebrin was foundto inhibit the enzymatic oxidation of phloridzin and of 4-methylcatechol. (Received November 2, 1966; )     

草甘膦对叶绿体能量转换的影响

除草剂草甘膦抑制植物叶绿体光合磷酸化活力,促进希尔反应活力,表现出明显的解偶联效应。它对叶绿体膜上腺三磷酶(ATPase)活力也起抑制效应,说明ATP合成被抑制不是由ATP酶活力变化所引起。这种解偶联现象主要是因光下质子转移受到抑制,在较低浓度的草甘膦影响下,先抑制质醌转移的质子进入膜内腔,浓度增加到20 mM,对水释放质子也有抑制。所以草甘膦对叶绿体能量转换的影响主要反映在质子转移被抑制,引起磷酸化活力受抑制。     

类根瘤对烟草叶片中叶绿体及其淀粉粒含量的影响

目的：探讨类根瘤对烟草叶片中的叶绿体及其淀粉粒含量的影响。方法：无菌培养烟草再生植株,其中一部分经过一定浓度的2,4－D与豇豆根瘤菌512快生型突变株菌液诱导处理,另外一部分为对照。运用常规电镜技术制作叶片厚切片,通过显微观察,对两组烟草叶片细胞中的叶绿体及其淀粉粒含量进行对比分析。结果：结瘤植株叶片细胞内的叶绿体含量虽然与对照之间无明显差异,但叶片栅栏组织和海绵组织细胞内淀粉粒的含量却均较对照减少,其中前者减少更为明显（P＜0.05)。结论：结瘤烟草叶片内光合产物的大量同化可能与类根瘤的形成有关。     

THE EFFECT OF LOW TEMPERATURE ON THE DEVELOPMENT OF THE LAMELLAR SYSTEM IN CHLOROPLASTS

The influence of low temperature (3°C.) on development of submicroscopic structure in plastids of Zea m. leaves was studied. Leaves from 8-day old etiolated plants, with plastids showing the prolamellar body and few lamellae, were floated for 1 day on tap water both in the dark and in the light, at 26°C and at 3°C. The structures remain unchanged in the dark, independent of temperature. Whereas in the light at 26°C., normal development of parallel compound lamellae and formation of grana occurs, in light at 3°C. ring structures are formed. Under the latter conditions protochlorophyll is converted to chlorophyll, although the in situ absorption maximum is different from the one for chlorophyll in plants grown in light at 26°C. When leaves were transferred from light at 3°C. to light at 26°C., ring structures in the plastids disappeared and normal development occurred. The possibility is discussed that development of parallel-arranged compound lamellae is due both to photochemical and synthetic processes, involving not only accumulation of chlorophyll, but also synthesis of other compounds.     

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; )     

锌对番茄叶绿体亚显微结构的影响与光强度的关系(简报)

番茄植物缺锌时,茎尖停止生长,叶部和茎上出现褐色斑点,有明显的小叶病症状。此外,叶绿素含量减少,光合强度降低,叶绿体结构遭到破坏。陈保生和崔澂发现叶绿体含锌量显著降低,并提出缺锌阻碍了叶绿体的发育。Hoagland发现锌在植物中的生理作用与光有关,此后,不少人对植物需锌与光的关系进行了研究,证明植物在光下较暗中需锌较多。通过进一步研究,李佳格和崔澂证明锌对自养生长的绿色植物的作用与光有关,而对异养的非绿色植物的作用与光无关。Spencer还报道锌和光影响叶绿体希尔反应活性。由此看来,锌与叶绿体的结构和功能是密切相关的。但锌在     

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; )     

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.     

STUDIES ON PHOTOPHOSPHORYLATION: I. TWO-STEP EXCITATION KINETICS OF PHOTOPHOSPHORYLATION

Based on the observations on the time lag and the low efficiencyof photophosphorylation at low light intensities, a ‘two-stepexcitation kinetics’ of photophosphorylation of chloroplastswas developed, which could explain some of the hitherto unexplainedfacts concerning photophosphorylation. By analyses of the experimental results along the line of thetheory, it was inferred that the assumed substance (X) responsiblefor ATP formation occurs in a relatively large quantity of about1 mole/5–20 moles of chlorophyll. It was concluded thatthe high energy intermediate (X^**) of ATP formation which wasassumed to accumulate on pre-illuminating the chloroplasts (inthe absence of ADP and Pi) does not represent a side pool ofextra energy, but a substantial intermediate in the normal courseof ATP formation. Alternative possibilities for the explanation of the experimentalfacts were also discussed. ¹This paper is submitted to the University of Tokyo to fulfilla part of the requirements for the doctorate of H. SAKURAI.