Effect of maleic hydrazide on endogenous cytokinin
contents in lateral buds, and its possible role in
¯ower bud formation on the Japanese pear shoot
Akiko Ito
*, Hiroko Hayama, Yoshiki Kashimura, Hirohito Yoshioka
1 Department of Pomology, National Institute of Fruit Tree Science, Ministry of Agriculture, Forestryand Fisheries, Tsukuba, Ibaraki 305-8605, Japan
Accepted 8 May 2000
Abstract
In order to elucidate the role of maleic hydrazide (MH, 1,2-dihydro-3,6-pyridazinedione; coline salt) in increasing ¯ower bud formation on the Japanese pear, the effect of MH on cytokinin contents in the lateral buds of Japanese pear shoots were investigated. Foliar application of MH at 2600 mg lÿ1
increased zeatin, zeatinriboside, and isopentenyladenine levels in lateral buds though isopentenyladenosine concentration decreased. Application of 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of polar auxin transport, also increased the endogenous cytokinin levels in lateral buds. It is supposed that these increases of cytokinin in lateral buds may be involved in the increase of ¯ower bud production on the Japanese pear shoot. The increases of cytokinin induced by these chemicals may be caused via the depletion of the auxin level and/or the activity in shoot tissues.
#2001 Elsevier Science B.V. All rights reserved.
Keywords: Cytokinin; Flower bud production; Japanese pear; Maleic hydrazide;Pyrus pyrifolia; 2,3,5-Triiodobenzoic acid
1. Introduction
Maleic hydrazide (MH, 1,2-dihydro-3,6-pyridazinedione; coline salt) is a growth regulator that is widely used in agriculture, as an inhibitor of sucker
*
Corresponding author. Address: National Institute of Fruit Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan. Tel.:81-298-38-6502; fax:81-298-38-6437.
E-mail address: [email protected] (A. Ito).
1
Present address: Apple Research Center, National Institute of Fruit Tree Science, Morioka, Iwate 020-0123, Japan.
development in tobacco, an inhibitor of lateral bud growth in potato, and as a retardant of shoot growth in grape (Kamuro, 1995). Its mode of action in plants is not clear, though several hypotheses have been proposed and investigated. Previous studies have revealed that, after application to foliage, MH is translocated to meristematic tissues where mitosis is disrupted (Darlington and McLeish, 1951; Hoffman and Parups, 1964; NoodeÂn, 1972); subsequently, MH-treated plants lose or show impaired apical dominance (Naylor and Davis, 1950). Other research has suggested that MH acts as an anti-auxin or a regulator of auxin metabolism (reviewed by Hoffman and Parups, 1964).
In our previous study (Ito et al., 2000), we reported that foliar application of MH increased the number of laterally born ¯ower buds on the Japanese pear shoot. However, the mechanism by which this chemical favors ¯ower production is obscure. Some researchers have reported that cytokinin levels are tied to auxin levels. For example, Bangerth (1994) reported that a decrease in auxin concentration by decapitation increased the cytokinin concentration in the xylem (in bean), and Palni et al. (1988) reported that the stability of zeatinriboside supplied to tobacco pith explants was inversely related to auxin concentration in the incubation medium. In the Japanese pear, when the indole-3-acetic acid level in lateral buds was decreased by shoot inclination at a 458angle, the zeatin level in lateral buds was increased (Ito et al., 1999). These results suggest the possibility that MH may increase cytokinin levels in lateral bud thereby increasing ¯ower bud number. In this study, we examined the effect of MH on ¯ower bud formation and cytokinin contents in lateral buds on Japanese pear shoots.
2. Materials and methods
2.1. Plant material
2.1.1. Maleic hydrazide application
The investigation was carried out in 1998 at the National Institute of Fruit Tree Science, Ibaraki, Japan using approximately 20-year-old fruit-bearing trees of Japanese pear cv. Kosui. Twelve trees of `Kosui' pear growing in an experimental orchard, in a completely randomized design, received one of two treatments: control trees were untreated and treated trees were subjected to MH application. Each group was subdivided into three subgroups consisting of two trees each to allow for replication. Application of MH was made by foliar spraying to run-off at 2600 mg lÿ1
Shoots were sampled from each subgroup atÿ4 (22 June),3 (29 June), 10 (6 July),32 (28 July),61 (26 August), and94 (28 September) days after MH application (DAC), and lateral buds were used for cytokinin analysis.
2.1.2. 2,3,5-Triiodobenzoic acid application
The effect of 2,3,5-triiodobenzoic acid (TIBA), an auxin ef¯ux inhibitor (Lomax et al., 1995), on cytokinin contents was also examined in 1999. TIBAwas purchased from Wako Pure Chemical Industries, Tokyo, Japan. Six trees of the `Kosui' pear, in a completely randomized design, received one of two treatments: control trees were untreated and treated trees were subjected to TIBA application. Trees were arranged in blocks. TIBA was applied on 9 July, by spraying at 300 mg lÿ1 with 0.01% of a surfactant added. Shoots were sampled on10 days after the TIBA treatment (on 19 July), and lateral buds were used for cytokinin analysis.
2.2. Cytokinin extraction and puri®cation
All of the lateral buds (sample fresh weight 7 g) were collected from the harvested shoots, and the buds were analyzed for trans-zeatin (Z), trans -zeatinriboside ([9R]Z), isopentenyladenine (iP), and isopentenyladenosine ([9R]iP) by enzyme-linked immunosorbent assay (ELISA) after puri®cation by high performance liquid chromatography (HPLC). Lateral buds were excised by cutting at the border of the bud and the shoot with a knife after removal of leaves; then the bud scales and internal tissues were collected. Hormonal analysis was performed as described by Ito et al. (1999). In short, samples were homogenized and extracted three times in 30 ml of 80% aqueous acetone containing butyl hydroxy toluene (BHT) at 0.1 g lÿ1
, and subjected to solvent partitioning to obtain a fraction soluble in n-butyl alcohol (n-BuOH). The n-BuOH soluble fraction was dried in vacuo, then dissolved in a small amount of methanol, and loaded on a Pegasil ODS
HPLC column (10 mm i.d.150 mm, Senshu Scienti®c, Japan), which was
equilibrated with 5% acetonitrile and eluted with a gradient of water±acetonitrile containing 0.5% acetic acid. Cytokinin was identi®ed and estimated based on the retention times on HPLC and immuno-reactivities to speci®c antibodies.
Zeatin and [9R]Z were analyzed by ELISA with monoclonal antibody against [9R]Z. Isopentenyladenine and [9R]iP were analyzed with monoclonal antibodies against [9R]iP. These antibodies and cytokinin tracers were purchased from IdeTech, CA, USA.
2.3. Flower number estimation
3. Results and discussion
Since the earliest report by Schoene and Hoffmann (1949), the effect of MH as a growth retardant has been widely studied. The length of the inhibition period appears to be directly proportional to the concentration of the inhibitor used, and the inhibitory effect was shown to last for 2 months at 2000 mg lÿ1when applied to tomato plants (Schoene and Hoffmann, 1949). The mode of action of MH is still not clear. However, it is generally considered that the effectiveness of MH lies in its ability to inhibit cell division. Some researchers concluded that the formation/destruction of IAA is affected by MH. Further, several studies have pointed out that MH interacts with endogenous auxin and acts in opposition to auxin with regard to growth (reviewed by Hoffman and Parups, 1964).
Application of MH was found to in¯uence the cytokinin levels in lateral buds (Fig. 1). The amount of Z, the bioactive form of cytokinin (McGaw and Burch,
1995), in lateral buds showed an increase in MH treated shoots than in control shoots, and the difference became signi®cant just 3 days after treatment (Fig. 1A). The amount of Z was signi®cantly higher in treated shoots than in control shoots at3,10,61, and94 DAC. Similarly, [9R]Z and iP concentrations in lateral buds were increased by MH as evidenced by measurement, except at94 DAC ([9R]Z: Fig. 1B) and at 32 DAC (iP: Fig. 1C), respectively. Conversely, the amount of [9R]iP was more reduced in MH treated shoots than in control shoots at10 DAC and thereafter (Fig. 1D). Isopentenyladenosine is believed to convert to Z via iP or [9R]Z (McGaw and Burch, 1995); thus, these results suggest that MH acts to in¯uence the metabolism of cytokinin in lateral buds rather than to change the transport/distribution of cytokinin to lateral buds from a cytokinin production site elsewhere.
The MH application increased the number of laterally born ¯ower buds on the shoots (Table 1). Several studies have shown that application of synthetic cytokinin increases the number of ¯ower buds in apple trees (McLaughlin and Greene, 1991) and pear trees (Banno et al., 1985; Ito et al., 2000). Thus, the increase of the ¯ower bud number observed in this experiment may be caused by the increase of endogenous cytokinin level in lateral buds.
TIBA acts to speci®cally inhibit polar transport of auxin at the site of auxin ef¯ux (reviewed by Lomax et al., 1995). When TIBA was applied to the Japanese pear shoot, the cytokinin contents in lateral buds were increased at 10 days after the application (Fig. 2). The promotive effects of TIBA on ¯ower bud production of apple reported by Tromp (1972) may also be due to the increase of cytokinin concentrations in lateral buds.
The results of MH application showed the positive relationship between ¯ower bud formation and cytokinin levels in lateral buds. On the other hand, the direct evidence clarifying how cytokinin levels should be controlled by MH is lacking in this experiment. However, several studies have showed that a decrease in auxin concentration may induce an increase in cytokinin concentration in the bud either by enhanced transport from roots or by increased metabolism in the buds (Cline,
Table 1
The effect of MH application on the number of ¯ower buds and lateral buds (vegetative¯ower bud) per Japanese pear shoota
Treatment Flower bud Total lateral bud
Control 2.8 16.5
MH 10.4* 17.2 NSb
a
Numbers of ¯ower buds and lateral buds were counted after the buds burst in the spring following chemical application. Values are means of 20 replicates.
b
NS: nonsigni®cant.
*Signi®cant at
1994 and references therein). Together with the result that TIBA also increased cytokinin concentrations in the lateral bud of Japanese pear shoot, the possibility that the increase of cytokinin by these chemicals may be due to the decrease of auxin content and/or activity in shoot tissue should not be excluded. Thus, the effects of these chemicals on auxin concentration/activity in shoot tissue merits re-examination in order to obtain a clearer picture of interaction of auxin with cytokinin concentration.
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