Localization of the NaCl-Sensitive Membrane Fraction in Cucumber Roots by Centrifugation on Sucrose Density Gradients

Cucumber plants (Cucumis sativus L. cv. Shogoin) were treatedwith 200 HIM NaCl for one day and the root microsomal membraneswere fractionated by centrifugation on a continuous densitygradient of 10% to 45% sucrose, and the activity of K⁺-Mg²⁺-ATPasewas studied. The most significant difference in the ATPase activitybetween control and NaCl-stressed roots was detected in themembranes sedimented at sucrose concentrations of 40% to 45%.The ATPase activity associated with those fractions was vanadate-sensitive,and the membranes contained much lower levels of calcium andphospholipids after the treatment with NaCl. The putative plasmamembrane fraction collected after centrifugation on a discontinuousgradient of 34% and 45% sucrose appeared to originate from theplasma membrane, as judged by sensitivity to inhibitors andpH optimum of the ATPase activity. NaCl-stress caused a significantreduction in K⁺-Mg²⁺- ATPase activity despite the similarityin polypeptide components detected by SDS-polyacrylamide gelelectrophoresis on disc gels. The effects of NaCl-stress onthe levels of calcium and phospholipids in membranes are discussed,with reference to the disintegration of the membranes. (Received April 7, 1989; Accepted September 19, 1989)     

Evidence for Decrease in Vanadate-Sensitive Mg2+-ATPase Activity of Higher Plant Membrane Preparations in Sucrose Solution

The effects of inhibitors (vanadate, nitrate and azide) of threetypes of higher plant H⁺-ATPase on membrane-associated Mg²⁺-ATPaseactivity are often examined in the presence of sucrose. We reporthere that sucrose itself decreases in vanadate-sensitive ATPaseactivity of higher plant membranes. (Received October 20, 1993; Accepted February 21, 1994)     

Effect of abscisic acid on differentiation and ribulose diphosphate carboxylase of chloroplasts

Daucus carota tissues were grown on Murashige-Skoog medium (MS)at different concentrations with abscisic acid (ABA). Sevenbands of chloroplast fractions were obtained on a sucrose gradient.At 10^–5M, ABA highly increased chlorophyll and proteinnitrogen content of medium density chloroplasts. With increasingage of the tissues, the most active chloroplasts according totheir ¹⁴CO₂ fixation were found in smaller numbers. When treatedwith 10^–5 M ABA, 34 day-old tissues cultivated in vitroshowed the chloroplast pattern of 110 day-old tissues. The effectof ABA—given to the tissues during a short pretreatmentor continuously present in the culture medium—on the ribulosediphosphate carboxylase activity was analysed. It was foundthat ABA at 10^–5 M strongly inhibited ¹⁴CO₂ fixation. (Received December 20, 1977; )     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Photosynthetic carbon metabolism in wild, primitive and cultivated forms of wheat at three levels of ploidy: Role of the glycolate pathway

¹⁴CO₂ assimilation was studied with diploid, tetraploid, hexaploidspecies of the genera Triticum and their wild relatives Aegilops.Attached mature leaves of 3–4 weekold plants were allowedto undergo photosynthesis under air at ambient temperature.The pattern of distribution of ¹⁴C was notably similar in Triticumand Aegilops species whatever the level of ploidy. Sucrose wasthe sink for photosynthetic carbon. ¹⁴C for sucrose synthesis was supplied either through the glycolatepathway by glycolate, the product of the photorespiration orby the Calvin cycle intermediates exported into the cytoplasm.Depending on the species, the glycolate pathway provided 40to 75%of the sucrose ¹⁴C. The higher labeling of sucrose was associated with the greaterparticipation of the glycolate pathway in the wild diploid (DD)A. squarrosa and in the cultivated hexaploid (AABBDD) T. aestivum.The results suggest that the expression of the male D genomeis dominant over the female AB genome in T. aestivum. In T. aestivum under ambient conditions lowering (low temperature)or hindering (1% O₂ ) photorespiration, sucrose labeling decreased,but serine and glycine labeling was favoured. We propose thatin wheat leaves, the role of photorespiration is to drain artof the carbon exported from the chloroplast as glycolate, towardssucrose synthesis. (Received March 16, 1979; )     

Conditions de formation d'agr?gats de chlorophylle {alpha} dans des solvants polaires aqueux: polarit? du milieu

The absorbance and emission spectroscopy of chlorophyll a (10^–5M) in water-miscible alcohols, acetone and dioxane were studiedto determine the properties of the chloroplast membranes whichsupport the aggregation of chlorophyll in vivo. It appears thatthe formation of the chlorophyll aggregates is closely dependenton the polarity of their molecular environment. (Received April 16, 1974; )     

Studies on nucleic acids in chloroplasts isolated from Chlorella protothecoides

1. 1. Subcellular fractions of Chlorella protothecoides were separatedby fractional centrifugation of the algal cell homogenate inmixtures of cyclohexane and CCl₄. The base composition, meltingprofiles and IRC-50 column chromatographic patterns of DNA preparationsfrom the chloroplast and non-chloroplast fractions were examined.It was shown that the algal chloroplast contains at least oneDNA species which is different from the nuclear DNA.
2. 2. RNApreparations from the subcellular fractions were subjectedtoMAK column chromatography, sucrose density gradient centrifugationand analysis for base composition. It was demonstrated thatthe chloroplast contains ribosomal RNA and soluble RNA. Twocomponents of the chloroplast ribosomal RNA were found to havethe same patterns as those of the E. coli ribosomal RNA in MAKcolumn chromatography and zone centrifugation. The major componentof the chloroplast ribosomal RNA was distinctly different fromthat of the non-chloroplastic (cytoplasmic) ribosomal RNA inall properties examined.

¹This work was partly reported at the Symposium on Mitochondriaand Chloroplasts as Self-duplicating Units sponsored by theBotanical Society of Japan in August, 1966, and at the Symposiumon Biogenesis of Subcellular Particles, the 7th Internatl. Congressof Biochemistry, Tokyo, 1967.     

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplastby a discontinuous sucrose density gradient and high speed centrifugation.These membranes catalyzed the transfer of [¹⁴C]glucose fromUDP-[¹⁴C]glucose to endogenous sterol acceptors and, in turn,catalyzed the esterification of steryl glucosides with fattyacids from an endogenous acyl donor. The synthesis of sterylglucosides was stimulated in the presence of Triton. X-100,while formation of acyl steryl glucosides was inhibited by thedetergent. However, in the presence of an added sterol acceptorand Triton X-100, the inhibition of acyl steryl glucoside synthesiswas overcome by the addition of phosphatidylethanolamine. Theenzyme involved in steryl glucoside formation was solubilizedby treatment of the envelope membranes with 0.3% Triton X-100.The solubilized enzyme had an almost absolute requirement forsterol acceptors. Key words: Solanum tuberosum, Sterol glucosylation, Steryl glucoside acylation, Amyloplast membrane     

Production and Secretion of Sucrose by Sugar-cane Leaf Tissue

Leaf discs, cut from mature plants of Saccharum officinarumwere found to accumulate large quantities of starch and sugar,amounting to some 25 per cent of their initial dry weight, during3 days of constant illumination. Secretion into the externalmedium of about one-third of the accumulated sucrose was effectedby 10^–4 M sodium iodoacetate. Concomitantly the inhibitorincreased apparent photosynthesis. Various factors, includingother respiratory inhibitors, failed to induce sucrose secretion,indicating a selective action of iodoacetate on the cell membranes.     

Changes in ribosomal patterns and a related membrane fraction during induction of cold hardiness in mimosa epicotyl tissues

Sucrose gradient profiles of polyribosomes from epicotyl tissuesof mimosa seedlings (Albizzia julibrissin Durazzini) were followedthroughout induction of cold hardiness. An unusual fractionappeared in the polyribosome region of sucrose gradients withadvancing stages of cold hardiness. Treatment of this unusualfraction and polyribosome fractions with RNase, Triton X-100and deoxycholate suggests that the unusual fraction consistedof chloroplast or mitochondrial membrane fragments. Free polyribosome patterns were retained throughout inductionof cold hardiness. However, the presence of polyribosomes duringadvancing stages of cold hardiness does not necessarily insureactive protein synthesis. ¹ This investigation was supported in part by National ScienceFoundation Grant GB 8692. ² Contribution from the Missouri Agricultural Experiment Station.Journal Series Number 6253. (Received November 30, 1971; )     

Isolation of Native Pyrenoid Core Matrix Having Ribulose 1,5-bisphosphate Caxrboxylase Activity from the Green Alga Bryopsis maxima

The native pyrenoid core matrix of the green alga Bryopsis maximawas isolated by diethyl ether treatment and sucrose densitygradient centrifugation using 1.8 M phosphate buffer. The purityof the pyrenoids was examined by microscopy, polyacrylamidegel electrophoresis and marker materials. The purified pyrenoidscontained the large subunit and the small subunit of ribulose1,5-bisphosphate carboxylase (RuBPCase) and more than 10 minorpolypeptides. They also showed RuBPCase activity when solubilizedon being transferred to a low-concentration buffer. The specificactivity was 0.62 µmol CO₂ fixed (mg protein)^–1min^–1. This isolation method is suitable for obtainingintact pyrenoids not covered by starch sheaths or membraneswithout the need for chloroplast fixation. (Received July 27, 1987; Accepted October 20, 1987)     

He-Ne激光对增强UV-B辐射小麦幼苗叶绿体的影响

对“晋麦8号”小麦幼苗分别采用5 mW·mm^-2 He-Ne激光辐照、10.08 kJ·m^-2·d^-1增强UV-B辐射及二者组合进行处理,研究各处理组小麦幼苗叶绿体膜透性、叶绿体蛋白质含量以及叶绿体偶联因子CF-1的ATP酶活性、希尔反应的活性变化。结果表明：UV-B辐射后小麦幼苗叶绿体膜透性增加,叶绿体蛋白质含量有一定的下降,而ATP酶活性、希尔反应的活性均受到抑制。经过He-Ne激光辐照可使叶绿体膜透性降低、叶绿体蛋白质含量有一定的升高,同时ATP酶活性、希尔反应的活性也受到部分激活。这些变化说明增强UV-B辐射引起小麦幼苗叶绿体损伤,而一定剂量的He-Ne激光辐照可部分修复增强UV-B对小麦幼苗光合系统的损伤。     

Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

Oligomycin-insensitive incorporation of 32P into chloroplast protein by illumination

³²P incorporation into the protein fraction of chloroplast fragmentsby short illumination was investigated under various phosphorylatingconditions. ³²P incorporation was generally accompanied by cyclic and non-cyclicphotophosphorylations and also by formation of a high energyintermediate "X_E". However, the addition of a DPIP-ascorbatecouple caused inhibition of ³²P incorporation, while ATP formationproceeded. Effects of inhibitors and uncouplers of photophosphorylationon the formation of protein-bound ³²P were generally similarto those on ATP formation. AT³²P was not utilized for protein-bound ³²P formation in thedark by chloroplast fragments, but its radioactivity was transferredinto the chloroplast protein fraction in the light. Oligomycininhibited ATP formation but did not inhibit protein-bound ³²Pformation. m-Cl-CCP blocked both reactions. This suggests thatprotein-bound ³²P is not an actual intermediate in the phosphorylativeprocess leading to formation of ATP. It is probably formed ona side pathway from an intermediate of ATP formation. Analyses of protein-bound ³²P after digestion with proteaseand lipase showed that the ³²P incorporated was bound to peptidesin chloroplast lamellae. The possible form of this bound ³²Pis discussed. (Received November 22, 1971; )     

Separation of K+-and Cl --selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K⁺channels, and a 148-pS Cl^– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K ⁺-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K ⁺ channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K⁺ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl^–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K⁺ channel, an 8-pS K⁺channel and a 4-pS K⁺ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl^–) channel, potassium (K⁺) channel, planar lipid bilayer, root, rye, Secale cerealeL.     

Polypeptide Compositions of Stroma, Internal Membranes, and Inner and Outer Envelope Membranes of Amyloplast from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Intact amyloplasts isolated from liquid-cultured white-wildcells of sycamore (Acer pseudoplatanus L.) were further subfractionatedinto internal membranes (d=1.05g/ml), envelope membranes (d=1.12g/ml)and stromal fraction, which contained each characteristic polypeptidecomposition as revealed by the Na-dodecyl sulfate polyacrylamidegel electrophoresis. Absorption spectra of internal and envelopemembranes were distinctly different. By the immunoblotting analysis,it was shown that the amyloplast envelope membranes contain31 kDa P_i-translocator, although it is not the predominant polypeptidecomponent in contrast to the case of chloroplast envelope. Onehundred kDa -l,4-glucan phosphorylase (plastid type) was detectedin the stromal fraction of amyloplasts using the specific antibodyraised against the major form of -l,4-glucan phosphorylase frompotato tuber. Amyloplast envelopes were further separated into inner and outermembrane fractions by the freezing-thawing method originallydeveloped for the separation of chloroplast envelope membranesby Cline and associates (1981) (Proc. Natl. Acad. Sci. USA 78:3595–3599). Nadodecylsulfate gel electrophoretic analysisrevealed that the inner and outer envelope membranes containthe distinctly different polypeptide compositions. ¹Supported by grants from the Ministry of Education, Scienceand Culture (Mombusho) of Japan. This is paper No. 77 in theSeries "Structure and Function of Chloroplast Proteins". ²Recipient of a predoctoral student fellowship from the Japanesegovernment (Mombusho). Permanent address: Department of Biochemistry,Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ³Permanent address: Department of Biology, Southwest AgriculturalUniversity, BeiBei Chongqing, People's Republic of China. Holderof the Chinese Government Scholarship (1987) (Received May 27, 1988; Accepted August 30, 1988)     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

Starch Synthesis from Exogenous Sugars in Tobacco Leaf Discs

Sets of discs were taken from leaves of destarched tobacco plants(Nicotiana tabacum L. cv. xanthii) and floated on solutionsof sucrose or glucose in the dark. Abundant starch was formedin the youngest leaves but there was a marked decline with leafage.By contrast, when replicate sets of discs were floated on waterand illuminated, photosynthetic starch formation was similarin the differently aged leaves. Uptake of sugar, measured bydry weight increases and incorporation of [¹⁴C]sucrose, wasnot dependent on leaf age. The possibility that physiologicalchanges, relating to ageing and import/export status of theleaf, regulate the metabolism of sugar to starch was examined.Increasing retention of sugar in the minor veins is likely tobe a major factor. Invertase activities were measured and foundto be similar in the differently aged leaves. Respiration ratesdeclined with increasing leaf age. Speculations concerning changesin selective permeability of the chloroplast membrane are alsodiscussed.     

The Effect of Sucrose Supply on the Energization of Amino Acid Uptake by Cotyledons of Euphorbia lathyris L

The cotyledons of Euphorbia lathyris L. take up sucrose andamino acids from the endosperm. The interaction between theuptake of sucrose and that of amino acids by cotyledons of intactseedlings was investigated. Sucrose (100 mol m^–3) reducedvaline uptake to 75% of the control rate; the active uptakecomponent of valine uptake was reduced from 45 to 25 % of thetotal uptake rate. In a reverse experiment, 100 mol m^–3valine inhibited sucrose uptake by 25%. At 500 mol m^–3sucrose, valine uptake was completely restored to the controlrate, whereas high valine concentrations failed to restore sucroseuptake. The stimulation of valine uptake by sucrose is linkedto the role of sucrose as a primary respiratory substrate. Whenthe cotyledons were bathed in sucrose concentrations rangingfrom 0 to 100 mol m^–3 (these concentrations are non-saturatingwith respect to sucrose uptake), a constant 1.8% of the sucrosetaken up was respired. The K_m of the concentration-dependentsucrose oxidation (44±6 mol m^–3) agreed reasonablywell with that for sucrose uptake (29±6 mol m^–3).When the external sucrose concentration was increased from 100to 600 mol m^–3, the sucrose uptake increased by 30% again,while sucrose oxidation was increased by 300%. This increasewas not due to an increased engagement of the alternative (cyanide-resistant)pathway for respiration. Alternative pathway, Euphorbia lathyris L., fermentation, seedling, sucrose uptake, valine uptake     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)