Plant Cell, Tissue and Organ Culture 66: 1–7, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Effect of inhibitors of ethylene biosynthesis and signal transduction pathway on the multiplication of in vitro-grown Hancornia speciosa Adaucto B. Pereira-Netto Department of Botany-SCB, Centro Polit´ecnico, University of Parana, C.P. 19031, 81531-970, Curitiba, PR, Brazil (Fax: +55-41-2662042; E-mail:
[email protected]) Received 26 June 2000; accepted in revised form 8 February 2001
Key words: apical dominance, Apocynaceae, axillary shoot formation, clonal propagation, shoot culture
Abstract Initiation and elongation of lateral buds is stimulated in in vitro-grown shoots of Hancornia speciosa (a tropical fruit tree) by high temperature (35 ◦ C), which is associated with the plant’s reduced ability to release ethylene. However, no increase in either lateral shoot elongation or multiplication rate of H. speciosa shoots growing in vitro at nonshoot inducing temperature (31 ◦ C) was associated with the presence of inhibitors of ethylene biosynthesis, such as α-amino isobutyric acid and cobalt ions or (aminooxy) acetic acid. Likewise, no increase in the multiplication rate was associated with aminoethoxyvinylglycine (AVG), another inhibitor of ethylene biosynthesis. In addition, stimulation of lateral shoot elongation in H. speciosa shoots grown at non shoot-inducing temperature was achieved when two inhibitors of the ethylene signal transduction pathway, silver thiosulphate (5–10 µM Ag++ ) and 1methylcyclopropene (90 nl l−1 ) were present in the culture medium or in the culture vessel-headspace, respectively. However, increased multiplication only occurred with the 1-methylcyclopropene-treated shoots. Thus, lateral bud development with 1-methylcyclopropene could be used to enhance H. speciosa multiplication in vitro. Abbreviations: ACC – 1-amino-cyclopropane-1-carboxylic acid; AIB – α-amino isobutyric acid; AOA – (aminooxy) acetic acid; AVG – L-α-(2-aminoethoxyvinyl)-glycine; EBPs – ethylene binding proteins; MR – multiplication rate; 1-MCP – one-methylcyclopropene; STS – silver thiosulphate Introduction Lateral (axillary) shoot proliferation has been shown to be a powerful tool for the enhancement of in vitro multiplication rate for many species (Shekhawat et al., 1993). Usually, lateral bud proliferation can be achieved by the use of cytokinins in the culture medium (Karhu, 1997a, b). Hancornia speciosa (a tropical fruit tree) shoots have strong apical dominance and do not respond to cytokinins for axillary branching. In in vitro-grown H. speciosa, inhibition of ethylene biosynthesis through the use of a high temperature (35 ◦ C) treatment or a treatment with an inhibitor of ethylene biosynthesis, L-α-(2aminoethoxyvinyl)-glycine (AVG) has led to apical dominance breakdown. This thermal induced lateral branch proliferation is related to an inhibited ability of
shoots to release ethylene. However, the high temperature and AVG-induced apical dominance breakdown did not result in enhanced multiplication rates for H. speciosa (Pereira-Netto and McCown, 1999). In this paper, we describe the effects of inhibitors of ethylene biosynthesis and signal transduction pathway on the lateral shoot elongation and multiplication of H. speciosa. Materials and methods Shoot multiplication Single node microcuttings measuring between 10 and 20 mm in length were taken from 30 day-old aseptically-grown shoots of MGB 003 Hancornia speciosa (Apocynaceae) clone and used as explant
2 sources in the experiments. Explants were cultivated in Magenta GA7 (Magenta Corp., Chicago, USA) containers, containing 50 ml of a MS (Murashige and Skoog, 1962) modified basal medium as previously described (Pereira-Netto and McCown, 1999). Culture conditions Cultures were maintained in a controlled environment room using a completely randomized design. Continuous light was provided by cool-white fluorescent tubes giving a photosynthetic photon flux density (PPFD) of 60 µM ol m−2 s−1 at the culture level. Relative humidity was kept at 70 ± 5% and air temperature was maintained constant at 31 ± 0.4 ◦ C. Chemicals AIB (α-amino isobutyric acid), cobalt dichlorate, AOA [(aminooxy) acetic acid], AVG (aminoethoxyvinylglycine), silver nitrate and sodium thiosulphate were obtained from Sigma Chem. Co. (St. Louis, MO). Silver thiosulphate (STS) solution was prepared from separated sodium thiosulphate and silver nitrate stock solutions in a 4:1 molar ratio. Except for cobalt dichlorate, all inhibitors of the ethylene biosynthesis, as well as silver thiosulphate were filter sterilized (0.2 µM -pore size cellulose acetate membrane syringe tip filters, Corning Inc., Corning, NY) and added to the culture media after autoclaving. All of the stock solutions were prepared immediately prior to the beginning of the treatment. One-methylcyclopropene (1MCP) was a gift from Dr. E.C. Sisler (North Carolina State University, Raleigh, NC, USA). One-methylcyclopropene (1-MCP) treatments Single node microcuttings were sealed in 2,000 ml glass vials containing 100 ml culture medium (1,900 ml head space). Known amounts of 1-MCP were filter sterilized as described above and then injected into the vials in order to provide the desired concentration of 1-MCP in the internal headspace. Each 10 days, the vials were vented, re-sealed and 1-MCP was replaced. Statistics A sub-routine (lsmeans/pdiff stderr) of the General Linear Model Procedure (PROC GLM) in SAS (Statistical package SAS, SAS Institute Inc., Cary, USA, 1985) was used for the analysis of the data. Except for the 1-MCP treatments, each treatment consisted
of 5 replicates (1 replicate = 1 GA7 container) with 6 explants per replicate. For the 1-MCP treatments, each treatment consisted of 3 replicates (1 replicate = 1 vial) with 6 explants per replicate. Each experiment was performed at least twice. In this study, the multiplication rate was defined as the number of newly-formed stem segments of 10– 20 mm in length (typical size of the internodes in shoots grown at non branch-inducing temperatures) containing lateral meristems or the apical meristem, after 30 days in culture. Results and discussion In plant tissue, the ethylene production rate is regulated by various physiological and environmental factors (Yu et al., 1980). Ethylene metabolism is controlled through the regulation of 1-aminocyclopropane-1-carboxylic acid (ACC) production, via ACC synthase, conjugation of the ACC to Nmalonyl-ACC (Yang and Hoffman, 1984) or (1γ -L-glutamylamino) cyclopropane-1-carboxylic acid (Martin et al., 1995), conversion of ACC to ethylene by the regulation of ACC oxidase activity, ethylene oxidation to produce CO2 , and (or) incorporation in tissue by conversion to ethylene oxide or ethylene glycol (Yang and Hoffman, 1984; Matthys et al., 1995). α-amino isobutyric acid α-amino isobutyric acid (AIB), a structural analog of ACC, was not able to enhance the multiplication rate or stimulate lateral shoot elongation in in vitro-grown H. speciosa shoots grown at a non shoot-inducing temperature (Figure 1). AIB has been demonstrated to competitively inhibit ACC oxidase and consequently ethylene formation (Vioque and Castellano, 1994). AIB is not converted to ethylene but may be converted to CO2 and malonyl-AIB (Woltering et al., 1995). A possible explanation for the inability of AIB to induce branching in 31 ◦ C-grown shoots is the fact that AIB has been shown to be largely immobile in tissues such as Cymbidium flowers (Woltering et al., 1995). The low mobility of AIB possibly interferes with its transport from the absorption sites to other parts of the shoot. Another possible explanation may be related to the fact that AIB is a weak competitive inhibitor of ACC oxidase in vitro (Ki for AIB is more than 70 times the Km value for ACC) (Vioque and Castellano, 1994). For decapitated shoots of rose (Rosa
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Figure 1. Effect of α-amino isobutyric acid (AIB) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
Figure 3. Effect of (aminooxy) acetic acid (AOA) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
Cobalt ions
Figure 2. Effect of cobalt ions (cobalt dichlorate) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
hybrida), AIB did not effectively inhibit ethylene evolution when used in low concentrations. In addition, AIB became toxic when used in higher concentrations (Gaspar et al., 1989). In this study, it was not established whether or not AIB was able to inhibit ethylene biosynthesis.
Cobalt ions in concentrations ranging from 0.1 to 12.5 µM did not stimulate (p ≥ 0.05) shoot formation or multiplication rate in H. speciosa shoots (Figure 2). CoCl2 inhibits ethylene biosynthesis through the inhibition of the ACC oxidase (Yang and Hoffman, 1984; Biddington, 1992; Vioque and Castellano, 1994). Significant inhibition of ethylene biosynthesis in plant tissue usually requires a concentration of cobalt ions around 10 µM Co++ (Biddington, 1992; Vioque and Castellano, 1994). In apical root sections of maize (Zea mays), a 65% reduction in ethylene evolution was observed after treatment with 10−4 M cobalt nitrate (Mulkey et al., 1982). For Pyrus communis, a 70% inhibition of in vitro ACC oxidase activity was related to the presence of 10 µM Co++ in the reaction mixture (Vioque and Castellano, 1994). The inability of cobalt ions to stimulate branching in H. speciosa shoots could be due to an ineffectiveness of these ions to inhibit ethylene production in H. speciosa. Another possible explanation for the inability of cobalt ions to induce branching in H. speciosa is the fact that, similarly to AIB, cobalt ions cannot be easily translocated in Cymbidium flowers (Woltering et al., 1995). This low mobility of cobalt ions in plant tissues could interfere with its transport from the culture medium to the ethylene producing sites.
4 AOA AOA (0.1 to 100 µM ) had no effect (p ≥ 0.05) on the average number of lateral shoots of in vitro-grown H. speciosa shoots (Figure 3). AOA is a potent inhibitor of pyridoxal phosphate-dependent enzymes such as ACC synthase (Saltveit and Larson, 1983; Biddington, 1992; Keller and Volkenburgh, 1997). In interveinal strips excised from Nicotiana tabacum leaves treated with 10 µM naphtaleneacetic acid, an approximate 90% reduction in ethylene evolution was correlated with the presence of 100 µM AOA (Keller and Volkenburgh, 1997). However, AOA has also been shown to be very toxic for in vitro-grown rose (Rosa hybrida) (Gaspar et al., 1989). In this study, we have found a strong reduction in the multiplication rate for H. speciosa shoots grown in the presence of 100 µM AOA, indicating a toxic effect of this compound in concentrations much lower than 1000 µM . However, we did not establish whether or not AOA was able to inhibit ethylene biosynthesis in H. speciosa. AVG We have previously demonstrated that a 4.5 µM AVG [L-α-(2-aminoethoxyvinyl)-glycine] treatment is able to stimulate (p ≥ 0.01) lateral shoot elongation of in vitro-grown H. speciosa, which is associated with a strong inhibition of ethylene evolution (PereiraNetto and McCown, 1999). Li-Chun and Li (1997) also correlated increased lateral branch elongation in pea (Pisum sativum) with an AVG-mediated reduction in ethylene evolution. For two in vitro-grown apple rootstocks, significant increases in axillary shoot formation and elongation were associated with both the application of ethylene traps inside the flasks and also with the presence of AVG in the culture medium (Lambardi et al., 1997). However, the stimulation of lateral branch elongation in H. speciosa did not result in increased multiplication because the enhanced lateral shoot elongation did not compensate for the inhibition of the main shoot elogation (Figure 4). This inhibition of main shoot elongation is possibly due to phytotoxic effects of AVG, which have also been observed in apple rootstocks, when AVG concentrations exceeded a threshold dose (Lambardi et al., 1997). Inhibitors of the ethylene signal transduction pathway The ethylene signal transduction pathway involves a protein kinase cascade (Kieber, 1997), believed to be initiated by reversible ethylene binding to a
Figure 4. Effect of L-α-(2-aminoethoxyvinyl)-glycine (AVG) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
Figure 5. Effect of silver ions (silver thiosulphate) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
5 ably precedes its interference at ethylene binding sites in EBPs (Veen and Overbeek, 1989; Bleecker and Schaller, 1996; Kieber, 1997). Therefore, a potential STS-driven disturbance of the ethylene signal transduction pathway may be responsible for the branching stimulation associated with the presence of STS in the culture medium at 5, 10 and 20 µM Ag++ . Regardless of the effect of STS on lateral branching stimulation, reduction in the multiplication rate was associated with all of the STS concentrations tested. This reduction in the multiplication rate was associated with strong inhibition of the main branch-elongation, indicating a possible toxic effect of the silver ions when used in concentrations that stimulate lateral branch elongation. One-methylcyclopropene Figure 6. Effect of 1-methylcyclopropene (1-MCP) on the multiplication rate and average number of lateral branches ≥ 10 mm per shoot of H. speciosa. Multiplication rate means refer to individual shoots while average number of lateral branches ≥ 10 mm means refer to the culture vessel. Vertical bars indicate S.E.
transition metal located in ethylene binding proteins (EBPs) (Bleecker and Schaller, 1996). Various EBPs with similar affinity for ethylene but different molecular weight, and different association and dissociation rates, have been found in plants such as Phaseolus vulgaris. These proteins are mainly located in the membranes of the endoplasmic reticulum as highly hydrophobic integral membrane proteins (Matthys et al., 1995). At physiological levels (concentrations) ethylene stimulates EBPs phosphorylation both in vivo and in vitro, an effect that is concentration dependent, specific, and reversible (Hall et al., 1993). Several compounds that bind to EBPs have been shown to reverse plant responses induced by ethylene in various species (Sisler et al., 1996a, b). Silver ions Stimulation of lateral branch elongation in in vitrogrown H. speciosa shoots was associated with the presence of silver thiosulphate (STS) at 5, 10 and 20 µM Ag++ in the culture medium. However, the effect was not significant at p ≥ 0.05 (Figure 5). The silver ion is a very potent inhibitor of ethylene action and has been widely used either in the form of nitrate or the more mobile thiosulphate (Biddington, 1992). Ag++ specifically inhibits the action of ethylene, likely in a non-competitive manner (Sisler, 1991). Dissociation of the anionic silver complex (STS) prob-
One-methylcyclopropene (1-MCP) affected H. speciosa shoot architecture (Figure 6). Ninety nl l−1 of 1-MCP induced a significant (p ≥ 0.01) 9.7 fold increase in the average number of lateral branches ≥ 10 mm. This stimulation of branching was not followed by inhibition of the main branch elongation. The 90 nl l−1 treatment also resulted in a 76% increase (p ≥ 0.01) in the multiplication rate (total), when compared to the control. Since the multiplication rate of the main branch accounted for only 14% of the increased number of new explants (number of newly-formed stem segments of 10–20 mm in length containing lateral meristems or the apical meristem, after 30 days in culture) obtained at 90 nl l−1 1-MCP relative to the control, it can be concluded that the 76% enhancement in the total multiplication rate was due mainly to stimulation of lateral branch elongation. One-MCP is an inhibitor of ethylene action that apparently binds for long periods (many days) to the presumed ethylene receptors, the ethylene binding proteins (EBPs) (Sisler and Serek, 1997). There is a considerable difference in the amount of 1-MCP required to prevent ethylene effects in different plants. One-MCP retards senescence in ornamentals such as Dianthus caryophyllus by a 24 hour exposure at 0.5 nl l−1 . However, a higher dose (40 nl l−1 for 24 h) is required for maximum retardation of pea (Pisum sativum) seedling growth (Sisler and Serek, 1997). One-methylcyclopropene has been demonstrated to reverse several ethylene-induced responses in different species (Sisler et al., 1996a, b; Sisler and Serek, 1997); however, to our knowledge, this is the first time in which 1-methylcyclopropene is reported to induce
6 multiple shoot formation, especially in in vitro-grown plants. The multiplication rates (MRs) found in this experiment are low compared to the MRs found for other experiments in this study in which the culture vessels were not kept sealed. In vitro-grown plant cells and tissues release several toxic volatiles such as ethanol and acetaldehyde, compounds known to inhibit plant cell growth and differentiation (George and Sherrington, 1984; Marino et al., 1995; Armstrong et al., 1997). Sealed vessel conditions cause accumulation of such volatiles which possibly explain the low MRs found for the 1-MCP experiment. Although the MR found for the 1-MCP trial is lower, compared to the other experiments described, axillary branch proliferation was stimulated when shoots were grown in the presence of 1-MCP. Thus, pulse treatments with 1MCP while maintaining the plants in unsealed vessels, might contribute significantly to increase MRs in our system. The results found in this work show that utilizing 1-methylcyclopropene-released axillary bud development is a promising method to enhance the H. speciosa multiplication in vitro and may be useful for other species difficult to micropropagate by shoot culture. These results also show that a potential disruption in the ethylene signal transduction pathway mimics, with advantages, the thermo-induced lateral branch elongation in H. speciosa, indicating that normal growth (unbranched) in H. speciosa is possibly dependent on the plant’s ability to ‘sense’ ethylene.
Acknowledgements To Dr E.C. Sisler (North Carolina State University, Raleigh, NC) for the gift of 1-methyl cyclopropene (1MCP), and to Drs A.B. Bleecker and G. Eric Schaller (University of Wisconsin-Madison, WI) for helpful discussions.
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