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.     

Immobilized and Free Apoplastic Pectinmethylesterases in Mung Bean Hypocotyl

The nature and the action pattern of apoplastic pectinmethylesterase (PME) isoforms were investigated in mung bean [Vigna radiata (L.) Wilzeck] hypocotyls. Successive extractions of neutral and alkaline PME isoforms present in hypocotyl native cell walls (referred to as PE1, PE2, PE3, PE4, with increasingly basic isoelectric points) revealed that solubilization of PE1, PE2, and PE4 did not induce any significant decrease in the cell-wall-bound PME activity. The in vitro de-esterification occurring when isolated cell walls were incubated with pectin resulted, then, from the activity of PE3. In addition, pH control of PME activity was shown to be much stronger for enzymes bound to cell walls, in their native state or reintroduced after solubilization, than for enzymes in solution. Mature cell walls showed much more activity than young cell walls, and were relatively enriched in two acidic PME isoforms missing in young cell walls. One acidic PME was also detected in the extracellular fluid. The acidic and neutral isoforms that could be easily transferred from their binding sites to their substrate might be those involved in the demethylation process developing along the mung bean hypocotyl.     

Boron-induced Bioelectric Field Change in Mung Bean Hypocotyl

Low concentrations of boron were found to affect the bioelectric field potentials of hypocotyls excised from 4-day-old mung bean (Phaseolus aureus cv. Oklahoma 612) seedlings grown in darkness and irradiated with red light. The significance of this effect is discussed in relation to the possible role of boron in some membrane function.     

UVB诱导的绿豆下胚轴原生质体收缩效应

ＵＶ－Ｂ处理外起绿豆幼苗下胚轴原生质体的收缩;绿豆幼苗的下胚轴的伸长亦受ＵＶ－Ｂ处理的显著抑制。统计分析证实两者呈显著正相关（ｒ＾２＝０．８０６６）。这一结果表明,ＵＶ－Ｂ对绿豆下胚轴生长的抑制作用与不胚轴细胞伸长受到抑制相关。     

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)     

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)     

Composition, Properties and Localisation of Pectins in Young and Mature Cells of the Mung Bean Hypocotyl

Two pectic fractions were extracted along the growth gradientof the mung bean hypocotyl. The first one (PF₁) contained pectinssoluble in boiling water and characterized by low uronic acids/cationsratio, high esterification degree and high neutral sugars/acidicsugars ratio. The second one (PF₂) contained pectins insolublein boiling water characterized by high uronic acids/cationsratio, low esterification degree, very low neutral sugar contentand a high selectivity for calcium. Water soluble pectins werepresent in all the wall area whereas the other ones were detectedmainly in the middle lamella. The PF₁/PF₂ ratio was high infast growing tissues but low in mature, slowly growing tissues.The development of the cell wall exchange properties along thegrowth gradient was related to the changes observed in the PF₁/PF₂balance. (Received July 31, 1985; Accepted January 20, 1986)     

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)     

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.

     

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。     

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.     

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.     

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。     

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)     

Inactivation of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Etiolated Mung Bean Hypocotyl Segments by its Substrate, S-Adenosyl-L-methionine

ACC synthase, isolated from mung bean hypocotyl segments treatedwith IAA and BA, was inactivated by its substrate, SAM, duringits catalytic action. The reaction products, ACC and MTA, hadno effect on ACC synthase activity. The half-life of the enzymewas 12 min with an initial concentration of 150µM SAM,but this was extended to 23.5 min when the SAM concentrationwas reduced to 40 µM, near to the endogenous concentrationof SAM in mung bean hypocotyl tissue. Addition of AVG, a competitiveinhibitor of ACC synthase, to the reaction mixture containing40 µM SAM, prevented ACC synthase inactivation and increasedthe half-life about 2-fold. We suggest that ACC synthase inactivationis caused by SAM acting as an enzyme-activated irreversibleinactivator (k_cat-type inactivator), besides being the substratefor the enzyme. This SAM-dependent inactivation of ACC synthasemay explain the rapid inactivation of the enzyme in intact mungbean hypocotyl segments previously found by Yoshii and Imaseki(1982). (Received October 15, 1985; Accepted December 6, 1985)     

绿豆上胚轴细胞中BR的胶体金免疫电镜定位

利用葡萄球菌A蛋白与胶体金连接的复合物为探针的免疫电镜定位技术对绿豆上胚轴细胞中BR定位的结果表明,在用抗BR抗体处理的超薄切片中,叶绿体、核仁和液泡内有大量的金颗粒标记,细胞膜和淀粉粒也有金颗粒标记,但细胞壁中没有观察到金颗粒。在不用EDC固定的切片中,金颗粒标记密度非常低,而在用正常兔血清处理的切片中,所有细胞器内几乎没有金颗粒.该实验为绿豆上胚轴细胞中BR的分布提供了直接的证据。     

乙烯利、ACC、AOA和AgNO3对绿豆下胚轴插条不定根形成的作用

以绿豆下胚轴插条为实验材料,研究了乙烯利、ACC、AOA和AgNO,对其不定根形成的影响。结果表明：乙烯利和ACC能促进绿豆下胚轴插条的生根,最适浓度分别为50μmol／L和10μmol／L;AOA和AgNO3明显抑制不定根形成,随浓度增加,抑制作用增强。插条离体后24h内对ACC的促进作用和AOA的抑制作用敏感。插条在0-6h和18-24h用ACC处理,在0-2h和22-24h用50μmol／L乙烯利处理的生根效果好。乙烯在不定根形成的诱导期和起始晚期起促进作用。     

Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments: II. Fluxes of Chloride, Protons, and Orthophosphate in Apical and Subhook Segments

The active form of phytochrome (Pfr) decreased CI⁻ uptake by subhypocotyl hook segments of Phaseolus aureus Roxb. and increased uptake by apical segments. Pfr had similar effects on Pi [³²Pi] uptake. Modulations of Pi [³²Pi] uptake were detectable 10 minutes following photoconversion. Pfr may modulate Pi influx across the plasmalemma. Pfr inhibited H⁺ extrusion by subhook segments and enhanced extrusion by apical hook segments. No rapid effects on H⁺ extrusion were found. Phytochrome may regulate a K⁺ -H⁺ exchange process. The differential responses of the two regions of the hypocotyl are discussed with respect to Pfr-mediated changes in growth and development.