Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Involvement of Cell Wall Characteristics in Growth Processes along the Mung Bean Hypocotyl

To detect the cell wall characteristics involved in the regulationof growth responses, the composition of wall polysaccharidesand the kinetic behaviour of wall-bound glucanases on mung beanhypocotyls were investigated. In the parts of the hypocotyllocated below the "elongation zone", relationships have beenshown between the characteristics of pectins and the first phaseof auxin- or acid-induced growth responses. Only small pecticmolecules with a low calcium content, are compatible with acid-inducedwall loosening. The breakage of acid-labile bonds between uronide chains andhemicelluloses is believed to be responsible for the early burstof growth. In young tissues, acid-induced modifications in thecell wall structure might then produce changes in the kineticbehaviour of cell wall polysaccharidases that depend on thisstructure for support. In old tissues, the number of glycosyllinkages is not sufficient to permit glucanase activities. Enzymaticrupture of covalent bonds may indeed regulate the supply ofwall material requisite for sustained growth. (Received March 13, 1982; Accepted June 30, 1982)     

Functional Reconstitution of Plasma Membrane H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids

The activity of solubilized plasma membrane ATPase is affectedby the nature of exogenously added molecular species of phospholipids.To examine the role of the polar head group and of the molecularspecies of phospholipids in H⁺-pumping, the ATPase solubilizedfrom plasma membranes of mung bean (Vigna radiata L.) hypocotylswas reconstituted in liposomes prepared with a variety of phospholipids. The extent of activation of solubilized plasma membrane ATPasedue to the addition of 1-palmitoyl 2-oleoyl-phospholipids (PO-phospholipids)and asolectin decreased in the following order: POPS POPC asolectin POPG > POPE > POPA (see List of Abbreviations). H⁺-pumpinginto proteoliposomes reconstituted with asolectin and plasmamembrane ATPase was demonstrated by quinacrine fluorescencequenching in the presence of ATP-MgSO₄. H⁺-pumping was inhibitedby VO₄ and gramicidin D. When plasma membrane ATPase was reconstitutedin liposomes prepared with various PO-phospholipids, the abilityof PO-phospholipids to support H⁺-pumping into the proteoliposomesdecreased in the following order: POPG POPS > asolectin POPC. POPE and POPA failed to support any H⁺-pumping. A remarkablyhigh rate of H⁺-pumping was observed in proteoliposomes preparedwith 1-saturated 2-unsaturated fatty acids, such as POPC, butH⁺-pumping could hardly be detected in proteoliposomes preparedwith 1-, 2-unsaturated or 1-, 2-saturated fatty acids, suchas PSPC or DLPC. ATPase activity in proteoliposomes was dependenton the species of PO-phospholipids used for reconstitution anddecreased in the following order: POPS > POPG > POPC asolectin > POPA > POPE. DLPC (see List of Abbreviations)which includes a 1-, 2-unsaturated fatty acid supported onlymarkedly depressed activity. Both H⁺-pumping and the hydrolysis of ATP by the plasma membraneATPase are strongly affected by the polar head group and compositionof the fatty acyl chain of phospholipids used to prepare liposomesfor reconstitution of the ATPase. (Received May 31, 1991; Accepted September 18, 1991)     

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.     

High Temperature Sensitivity of Auxin-induced Ethylene Production in Mung Bean Hypocotyl Sections

High temperature sensitivities of IAA-induced and 1-aminocyclopropane-1-carboxylicacid (ACC)-dependent ethylene production in etiolated mung bean(Vigna radiata [L] Wilczek) hypocotyl sections were comparedat 30,40, 42.5°C. When ethylene production at 30°C wastaken as control, IAA-induced production at 40°C was firstenhanced and then suppressed after 3 h, whereas ACC-dependentproduction was enhanced two-fold throughout the 8 h experimentalperiod. However, when hypocotyl sections treated with 1 mM ACCat 30°C for several hours were transferred to 40°C,the ACC-dependent production rate fell below that at 30°C.An initial transient enhancement of IAA-induced ethylene productionat 40°C was supported by increased ACC synthase activityand thus by ACC content. At 42.5°C, both IAA-induced andACC-dependent production were almost completely suppressed.The results indicate that auxin-induced ethylene productionis affected by high temperatures in two different steps: a)at 40°C, the auxin action gradually deteriorates althoughconversion of ACC to ethylene is not affected at all, and at42.5°C, the conversion is nearly completely suppressed. (Received July 8, 1985; Accepted January 24, 1986)     

脱落酸对绿豆下胚轴不定根发生的影响

以10^-4 mol/L脱落酸(ABA)处理绿豆种子24 h,在幼苗下胚轴长6 cm时,切除根部作为插条,研究ABA对插条不定根发生及插条基部细胞周期时相的影响。结果表明,ABA可促进下胚轴插条不定根发生,增加生根数和生根范围;ABA提高插条基部细胞色氨酸转氨酶、吲哚丙酮酸脱羧酶和吲哚乙醛脱氢酶的比活性,增加吲哚乙酸含量,同时进入细胞周期S期的基部细胞数目增加,促进DNA合成,有利于不定根的发生。     

A Possible Regulation of Ethylene Production by the Epidermis in Mung Bean Hypocotyl Sections

IAA-induced and l-aminocyclopropane-l-carboxylic acid (ACC)-dependentethylene production in etiolated mung bean (Vigna radiata [L]Wilczek) hypocotyl sections does not occur in epidermal cells(Todaka and Imaseki 1985). Mung bean hypocotyls contain a proteinwhich inhibits auxin-induced ethylene biosynthesis in hypocotylsections (Sakai and Imaseki 1975a, b). This inhibitory proteinwas also found to inhibit ACC-dependent ethylene productionin hypocotyl sections, but not in hypocotyl sections from whichthe epidermis had been removed. Uptake of ACC by both unpeeledand peeled sections was not inhibited by the protein. Similarly,IAA-induced ethylene production was inhibited by the proteinin unpeeled hypocotyl sections, but not in peeled sections.The protein was not inactivated in peeled sections, as proteinsynthesis by peeled sections was inhibited to the same extentas in unpeeled sections. The protein inhibited incorporationof 3,4-[¹⁴C]-methionine into ACC and ethylene in unpeeled sections,but not in peeled sections, whereas oxidation of the labeledmethionine into CO₂ was inhibited by the protein to a similarextent in both types of hypocotyl sections. KCN, a potent inhibitorof ethylene production, inhibited both IAA-induced and ACC-dependentethylene production in both peeled and unpeeled hypocotyl sections.It is likely that the epidermis plays some role in controllingethylene production which occurs in stem cells other than epidermalcells. (Received July 16, 1985; Accepted October 21, 1985)     

Effects of the Inhibitory Protein of Ethylene Synthesis on ATP Levels in Mung Bean Hypocotyl Segments

The inhibitory protein of ethylene synthesis purified from mungbean seeds reduced ATP levels in mung bean hypocotyl segments.When the segments were incubated with 0.5mM IAA for 6 hr toinduce ethylene-producing activity, the presence of the inhibitoryprotein suppressed the ethylene production and ATP content inthe tissue about 82 and 60%, respectively. Similar suppressiveeffects were also observed for endogenous ethylene productionand ATP contents in tissue not treated with IAA. (Received June 20, 1981; Accepted October 24, 1981)     

纳米化的二氧化钛促进绿豆下胚轴不定根形成

5～200mg·L-1纳米化的TiO2能明显增加绿豆下胚轴不定根的数目、根干重和生根范围;光照条件下显著促进绿豆下胚轴不定根的形成;不同时间促生根效果不同,以6～18 h的效果最好.     

红光对绿豆下胚轴线粒体Ca~(2+)积累、ATPase及NAD激酶活性的影响

绿豆下胚轴切段经红光处理后10min,其线粒体的Ca~(2+)积累下降15％,Ca~(2+)-ATPase 及 Mg~(2+)-ATPase活性也分别下降29％和10％,切段CaM含量增加近1倍。Ca~(2+)存在时,红光能促进线粒体NAD 激酶活性。说明Ca~(2+)-ATPase及一部分Mg~(2+)-ATPase可作为钙泵控制Ca~(2+)进入线粒体。     

Regulation of Phytochrome on Swelling of Protoplasts Isolated from Hypocotyl of Etiolated Mung Bean Seedlings

Protoplasts from hypocotyls of etiolated mung bean (Phaseolus raditus L. ) seedlings were maintained at a constant osmotic potential at 20±2℃, and they were found to swell gradually after being pulsed with red light (R) (10.5 W · m-2, 3 min) when CaCl2 was present in the medium. The volume reached maximum during 30--60 min after R-irradiation and decreased swelling afterwards. Farred light (FR) irradiation in presence or absence of Ca2+ did not influence the protoplast volume. The R-effect was photoreversible by subse- quent FR (2.5 W · m-2, 5 min) irradiation, usually seen over two R-FR cycles. Furthermore, swelling response was in positivecorrelation with red light intensity and duration of R pulse, indicating the involvement of phytoehrome. FR became less effective in reversing the effect of R after 10 min in dark between R and FR. Protoplast swelling occurred only when Ca2+ ions (1 mmol/L) then Ca2+ ions (1 mmol/L) is added to the medium 5 rain after R. The effect of Ca2+ could not be replaced by Mg2+, Ba2+, Zn2+, or K+. The time course of water (3H20) uptake into protoplasts after R-irradiation was consistent with the trend of protoplast swelling, indicating the existence of certain relationship between the swelling and water uptake of the protoplasts.     

Ferredoxins in Developing Spinach Cotyledons: The Presence of Two Molecular Species

An antibody for ferredoxin was used to investigate the developmentof ferredoxin during the greening of spinach cotyledons. Ferredoxinwas present in 8-day-old etiolated cotyledons and increasedwith illumination, which means that the synthesis of ferredoxinwas both light dependent and independent. The ferredoxin purified from etiolated cotyledons, greeningcotyledons, and mature leaves was a mixture of two chemicallydistinct molecular species; ferredoxin I and II. The relativecontents of these two species varied with the stage of developmentand the conditions used. Ferredoxin I was identical with that isolated previously asvalidated by its amino acid sequence [Matsubara and Sasaki (1968)J. Biol. Chem. 243: 1732]. The complete amino acid sequenceof the second component, ferredoxin II, was determined as well.It was composed of 97 amino acid residues and differed fromferredoxin I by 25 residues. (Received October 16, 1982; Accepted December 14, 1982)     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

蓝光对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响

蓝光、白光和黑暗对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响不同。培养后３～１８ ｄ ,蓝光处理材料的可溶性蛋白质含量明显高于白光处理,更高于黑暗培养的材料。蓝光和白光明显促进３Ｈ亮氨酸掺入蛋白质,而蓝光和白光处理后游离氨基酸含量与黑暗对照相比,下降时间早,幅度大。在培养过程中,蛋白酶活性的变化与游离氨基酸相似。蛋白质合成抑制剂环己酰亚胺（ＣＨＭ） 抑制愈伤组织生长,其中以蓝光最大,白光次之,黑暗最小。在培养基中加入ＣＨＭ 愈早,抑制程度愈大。实验表明,ＣＨＭ 抑制愈伤组织蛋白质合成,也是以蓝光最甚。由此可见,蓝光促进绿豆下胚轴愈伤组织的形成、生长和蛋白质合成。     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

绿豆下胚轴质膜微囊的提取和H+-ATPase活性研究

以两相法提取纯化绿豆下胚轴质膜微囊,材料与两相体系重量之比为32:8时,一次洗膜就可以得到纯度较高的质膜微囊.提取缓冲液中牛血清白蛋白的浓度对质膜H -ATPase的潜在活性有影响.质膜H -ATPase水解活性依赖于Mg2 ,Ca2 对酶活性有明显的促进作用.壳梭孢素(fusicoccin,FC)对酶有明显的刺激作用,活体条件最大刺激达到72%,而离体条件下刺激为30%.     

绿豆下胚轴质膜微囊的提取和H+-ATPase 活性研究

以两相法提取纯化绿豆下胚轴质膜微囊,材料与两相体系重量之比为32∶8时,一次洗膜就可以得到纯度较高的质膜微囊。提取缓冲液中牛血清白蛋白的浓度对质膜H+-ATPase的潜在活性有影响。质膜H+-ATPase水解活性依赖于Mg2+,Ca2+对酶活性有明显的促进作用。壳梭孢素（fusicoccin, FC）对酶有明显的刺激作用,活体条件最大刺激达到72％,而离体条件下刺激为30％。     

Presence of a Na+-activated ATPase in the Plasma Membrane of the Marine Raphidophycean Heterosigma akashiwo

Highly purified plasma membranes were isolated from Heterosigmaakashiwo cells, a marine raphidophycean unicellular biflagellate,by the silica microbead method, and the ATPase activity of themembranes was characterized. The ionic requirements and spectrumof effective inhibitors enable us to identify a novel Na⁺-activatedATPase in the plasma membrane of this organism. Furthermore,we detected two phosphorylated intermediate forms of ATPases,with molecular weights of 150 kDa and 95 kDa as judged by acidSDS-polyacrylamide gel electrophoresis of extracts of isolatedplasma membrane. The 150 kDa intermediate was phosphorylated in the presenceof both Mg²⁺ and Na⁺, while the 95 kDa intermediate was phosphorylatedin the presence of Mg²⁺ alone. Both were dephosphorylated inthe presence of monovalent cations. These results indicate thatthe former intermediate was a Na⁺-activated ATPase, similarto Na⁺,K⁺-ATPases from animals, and the latter was similar toH⁺,K⁺-ATPases from higher plants. The physiological significanceof the two kinds of ATPase in the plasma membrane of marinealgae. (Received March 15, 1989; Accepted June 23, 1989)