Effect of copper on peroxidase activity and lignin content in Raphanus sativus

Copper (Cu²⁺) significantly inhibits the growth of radish (Raphanus sativus) seedlings at the concentration of 1 μM. As far as the relationship between the growth of radish roots and peroxidase (POD) activity is concerned, the reduction of radish roots is correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise in lignin content in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (K_m of 57.9 μM) for syringaldazine and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 and 0.12 μM^–1 s^–1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment can be a good candidate for lignification in radish roots.     

Peroxidase genes differentially respond to auxin during the formation of adventitious roots in soybean hypocotyl

In our investigation, auxins (IAA, IBA and NAA) effectively promoted rooting in soybean hypocotyls. The activity of anionic peroxidase (POX) (pI 3.7) and cationic POX (pI 8.5) was significantly suppressed by exogenous auxins on day 2 (the inductive phase). Some particular anionic POXs (pI 4.0 and pI 5.3) significantly increased in IBA-treated tissues as compared with the control when the incubation time was prolonged to day 3 and day 4 (the initiation phase). We sequenced 5′-flanking region of pI 8.5 and pI 5.3 POX genes using the PLACE and PlantCARE databases to identify several potential cis-regulatory elements. The pI 8.5 POX gene promoter contained two sites that were homologous to sequences commonly found in auxin response elements; motifs ARF/AuxRE and CATATGGMSAUR. During the inductive phase, the activity of pI 8.5 POX was significantly suppressed by the exogenously applied auxins. The pI 8.5 POX gene promoter contained both ARF/AuxRE and CATATGGMSAUR motifs that responded to auxins earlier than the pI 5.3 POX gene. Hence, the pI 8.5 POX gene might belong to primary auxin response genes. The pI 5.3 POX gene, which responded to auxins a day or two later, contained only ARF/AuxRE motif. Moreover, unlike pI 8.5 and pI 3.7 POXs that were suppressed by auxins, the pI 5.3 POX was induced or enhanced by the applied auxins, especially IBA. The pI 5.3 POX might generate H₂O₂ which caused the auxin-induced growth at the initiation phase during the formation of adventitious root in soybean hypocotyls.     

Peroxidases, lignin and anatomy during in vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots

In vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots with or without indolic-3-butyric acid (IBA) was studied in order to improve acclimatization of microplants after root formation and transplantation. Peroxidase (POD) activity and isoforms, lignin content and anatomical observations were evaluated in the course of the three interdependent phases (induction, initiation and expression) of microshoot rooting. Microshoots treated or not treated with IBA achieved high rooting percentages both in vitro and ex vitro. At the end of the 2-week acclimatization period, the percentage of surviving microplants ranged from 80% to 100%, for in vitro and ex vitro rooted microshoots, respectively. Microshoots rooted in vitro and ex vitro showed a relationship between rooting and POD activity but in a different time course. It appeared that root formation occurred after the microshoots had reached and passed a peak of maximum enzyme activity. In all treatments, electrophoretic analysis (native PAGE) of PODs revealed the appearance of one anionic and three cationic POD isoforms (C(1), C(3) and C(4)). An additional cationic POD isoform (C(2)) appeared only in the ex vitro rooting. The lignin content was similar in microshoots rooted both in vitro and ex vitro. The sequential anatomical changes during the rooting process were similar in both in vitro and ex vitro rooting treatments. In the case of in vitro rooting, pith cells had vacuoles entirely filled with a dark substance, while in the case of ex vitro rooting, pith cells contained many amyloplasts. The origin of the adventitious roots, in both rooting conditions, was located in the cambial ring. Roots with organized tissue systems emerged from the microshoot stem 10-14 days after the root induction treatments; on day 10 for rooting in vitro, while a 4-day delay was noted in microshoots rooted ex vitro.     

Isolation and characterization of six human hepatic isometallothioneins.

Human brain contains one cationic (pI8.3) and two anionic (pI5.5 and 4.6) forms of glutathione S-transferase. The cationic form (pI8.3) and the less-anionic form (pI5.5) do not correspond to any of the glutathione S-transferases previously characterized in human tissues. Both of these forms are dimers of 26500-Mr subunits; however, immunological and catalytic properties indicate that these two enzyme forms are different from each other. The cationic form (pI8.3) cross-reacts with antibodies raised against cationic glutathione S-transferases of human liver, whereas the anionic form (pI5.5) does not. Additionally, only the cationic form expresses glutathione peroxidase activity. The other anionic form (pI4.6) is a dimer of 24500-Mr and 22500-Mr subunits. Two-dimensional gel electrophoresis demonstrates that there are three types of 26500-Mr subunits, two types of 24500-Mr subunits and two types of 22500-Mr subunits present in the glutathione S-transferases of human brain.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

The charge distribution in the rough endoplasmic reticulum/golgi complex transitional area investigated by microinjection of charged tracers

The distribution of microinjected ferritin, ranging in charge from anionic to highly cationic, has been used to indicate differences in surface charge on the rough endoplasmic reticulum and the Golgi complex of intact cells. Highly cationic ferritins (HCF)(HCF1, pI 7.9-9.1; HCF2, pI 8.5-9.4; and HCF3.pI 9.5-10.1) were mostly bound and caused swelling of the rough endoplasmic reticulum. Cationic ferritin (CF) (pI 7.0-8.0) and anionic ferritin (AF) (pI 4.0-4.4) caused no changes in morphology. The distribution of these ferritins in the cytoplasmic space varied with their charge. Significantly more CF was bound to surfaces than was found in the free cytoplasmic space. Conversely, there was significantly more AF in the free cytoplasmic space than close to surfaces. Therefore, the intracellular surfaces are negatively charged. Comparison of the structures in the secretory pathway showed no differences in ferritin binding to transition vesicles, rough endoplasmic reticulum, Golgi saccules or secretory vesicles. The Golgi complex beads are not distinguished by their charge. It is therefore unlikely that charge differences play a role in regulating membrane-membrane interactions in this region of the secretory pathway.     

Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

甜椒的离体培养及建立再生体系研究

以荷兰进口甜椒的子叶和下胚轴为外植体,接种到附加不同植物生长调节剂的培养基上,筛选出MS+6-BA 4.0 mg/L+NAA 0.1 mg/L为子叶和下胚轴最佳不定芽分化培养基;MS+IBA 0.2 mg/L和MS+IBA 0.5 mg/L为最佳不定根分化培养基。     

Tissue Culture and Plant Regeneration of Jatropha curcas (Euphorbiaceae)

In this paper , plumules, cotyledons, hypocotyls, blades, petioles and stalks of physic nut ( Jatropha curcas L. ) were used as explants, and callus induction and plant regeneration were studied on MS medium contained different concentrations of 6-BA and NAA. The results showed that the MS medium with 5.0 mg/L BA and 1.0 mg/L NAA was the best for callus induction, and with 5.0mg/L BA and 0.1mg/L NAA, for formation of adventitious bud, and with 1.0mg/L BA and 1.0 mg/L NAA, for bud growth, and that 1􊄯2 MS medium with 1.0 mg/L NAA was the best for formation and growth of adventitious root .     

蕨藻红素促进大豆插条不定根的形成

对蕨藻红素影响大豆下胚轴插条不定根形成的研究表明：蕨藻红素促进大豆插条不定根的形成,其最适浓度为0.5μmol·L^-1,最适处理时间为2d。0.5μmol·L^-1蕨藻红素处理大豆插条后,不定根诱导阶段（0～24h）的POD、CAT、IAAox活性较低;而不定根形成生长阶段（24～72h）的CAT和IAAox活性较高,POD活性低于未作处理的。试验推测蕨藻红素促进大豆插条不定根形成的生理基础可能与其影响POD、CAT和IAAox活性有关。     

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.     

Polyamines and Adventitious Root Formation in the Juvenile and Mature Phase of English Ivy

The involvement of polyamines during adventitious root formationwas evaluated using a de-bladed petiole rooting assay for theeasy-to-root juvenile and difficult-to-root mature phase ofEnglish ivy (Hedera helix L.). Auxin (NAA 0.1 mM) stimulatedroot formation in juvenile phase cuttings, but failed to promoterooting in the mature phase. The addition of putrescine, spermineor spennidine (1.0 mM) with or without NAA (0.1 mM) did notaffect the rooting response in either the juvenile or maturephase cuttings. There was a significant increase in endogenouslevels of putrescine and spermidine in NAA-treated cuttings,but the only significant difference between the root formingjuvenile and the non-root forming mature phase cuttings wasan increase in putrescine levels. In NAA-treated juvenile cuttings,the polyamine biosynthesis inhibitor DFMA (1.0 mM) promotedroot formation from 9.2 to 14.5 roots per cutting, while DFMO(1.0 mM) reduced root formation from 9.1 to 1.4 roots per cutting.The promotion of rooting by DFMA was completely reversed byputrescine (1.0 mM), but putrescine, spermine or spermidine(1.0 mM) could not reverse the inhibitory effect of DFMO. NeitherDFMA nor DFMO promoted root formation in mature phase cuttings.DFMA was also added to NAA-treated juvenile petioles at variousstages during the root formation process. DFMA promoted rootingwhen applied during the early stages of root induction (0–3d), but became inhibitory to root formation when applied duringthe organization (6–9 d) or root elongation stages (9–12d). Key words: Hedera helix, organogenesis, root initiation, polyamines, DFMA, DFMO     

小桐子的组织培养和植株再生

以小桐子（Jatropha curcas）的胚芽、子叶、下胚轴、叶柄、叶片和茎段作为外植体,用不同浓度的6-苄基腺嘌呤（6-BA）和α-萘乙酸（NAA）对其进行愈伤组织的诱导和植株再生的研究。结果表明：在MS培养基中加入5．0mg／L6-BA和1．0mg／LNAA对愈伤组织的诱导效果最好;加入5．0mg／L6-BA和0．1mg／LNAA对不定芽的诱导最为有效,加入0．1mg／L6-BA和1．0mg／LNAA有利于芽的生长;加入1．0mg／LNAA的1／2MS培养基对生根最为有利。     

Influence of light conditions on development, apoplastic peroxidase activities and peroxidase isoenzymes in chicory root explants

Cichorium intybus L. (cv. Bea) root explants grown in continuous light had a higher fresh weight, lignin and chlorophyll content than explants grown in darkness. Intermediary values were found when the light conditions were switched after 6 days. Peroxidase activities (EC 1.11.1.7) were measured in the apoplastic fluid with guaiacol, indoleacetic acid (IAA) and syringaldazine as substrates. There was an inverse relationship between specific IAA oxidase activity and explant growth. Specific syringaldazine oxidase activity (SSO) correlated with the lignin content. Analysis by isoelectric focusing (IEF) showed that the apoplastic fluid contained both cationic and anionic peroxidase isoforms, whose expression differed according to culture conditions. Isoform isoelectric point (pI) 7.0 was only detectable when the explants were cultured for 12 days in darkness. When explants were grown for the first 6 days in light, SSO and lignin content were high and the isoforms pI 4.0, 6.6, 7.6 and 8.1 were highly expressed. Conversely, when the first 6 days were in darkness, specific IAA oxidase activity was high and the isoforms pI 4.5, 6.7 and 6.8. were most strongly expressed. The isoperoxidases pI 7.8 and 7.9 were strongly expressed when the explants were cultured for at least 6 days in darkness.     

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.     

Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection

The induction of peroxidases (EC 1.11.1.7) during elicitation of lignification by α-1,4-linked oligogalacturonides in cucumber hypocotyl segments ( Cucumis sativus L. cv, Wisconsin SMR 58) was investigated. The wounding associated with the preparation of hypocotyl segments induced a 19-fold increase in peroxidase activity during the following 72 h. The increase was partially due to an increase in activity of a constitutive peroxidase with a pI of 8.9 and partially due to the expression of new peroxidase isozymes with pIs of 3.8, 5.4, 6.2, 9.1 and 9.4. The oligogalacturonides did not induce any peroxidase activity in addition to the wound-induced activity. These results suggest that either the constitutive peroxidase isozyme (pI 8.9) of intact hypocotyls or some of the wound-induced peroxidases are involved in the oligogalacturonide-induced lignification.
Induction of the peroxidases by wounding was inhibited by cycloheximide. This indicates that they accumulate as a result of de novo protein synthesis. Actinomycin D caused only a modest inhibition of the wound-induction peroxidases, indicating that the process is regulated at the level of translation.
Peroxidase activity increased more rapidly in resistant than in susceptible cucumber hypocotyls after inoculation with the pathogen Cladosporium cucumerinum Ellis & Arthur. The pattern of isozymes which was induced by fungal infection of resistant hypocotyls was similar to the pattern of isozymes induced by wounding. This suggests that similar induction mechanisms may be involved in the two processes.     

Lateral root development and saponin accumulation as affected by IBA or NAA in adventitious root cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis> C.A. Meyer

Summary The effect of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) on lateral root formation was investigated in adventitious root culture of Panax ginseng. Lateral root formation was affected by IBA (24.6 μM) or NAA (9.8 μM). Lateral root primordia emerged from the explant root pericycle after about 7 d of culture when the roots were cultured on Schenk and Hildebrandt (SH) medium supplemented with 24.6 μM IBA or 9.8 μM NAA. However, no changes were observed in the explant root pericycle on auxin-free medium. The IBA treatment was more effective for lateral root induction and root growth compared to NAA. In morphological and histological aspects, the lateral roots formed under IBA treatment developed normally, while NAA-treated roots exhibited abnormal growth. The accumulation of total saponin was greater in roots treated with IBA than with NAA.     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.