- Email: [email protected]
Induction of ß-glucosidase activity in maize coleoptiles by blue light illumination
ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 538—545
www.elsevier.de/jplph
Induction of ß-glucosidase activity in maize coleoptiles by blue light illumination Riffat Jabeena, Kosumi Yamadaa,, Hideyuki Shigemoria, Tsuyoshi Hasegawaa, Masakazu Harab, Toru Kuboib, Koji Hasegawaa a
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Department of Environmental Science for Human Life, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
b
Received 23 March 2005; accepted 17 May 2005
KEYWORDS Benzoxazinoid; Blue light; DIMBOA; ß-glucosidase; Phototropism; Phototropismregulating substances; Zea mays
Summary The role of ß-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of ß-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by ß-glucosidase, and its degradation compound 6-methoxybenzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, ßglucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of ß-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviations: DIMBOA, 2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one; DIMBOA-Glc, 2-0-ß-D-glucopyranosyl-4-hydroxy7-methoxy-1, 4-benzoxazin-3-one; GL, D-gluconic acid d-lactone; MBOA, 6-methoxy-benzoxazolinone; TG, 1-thio-D-glucose. Corresponding author. Tel./fax: +81 29 853 6933. E-mail address: [email protected] (K. Yamada).
In phototropism, seedlings of higher plants orient their growth with regard to the direction of light in order to optimize the exposure of their photosynthetic organs (e.g. a leaf blade is oriented perpendicularly to the direction of light). A famous model is the Cholodny–Went theory, in which phototropic curvature is caused by a lateral
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.05.004
ARTICLE IN PRESS Activation of ß-glucosidase by blue light gradient of growth-promoting auxin in the bending organ (Went and Thimann, 1937). However, using physicochemical and immunological methods, some groups have shown that endogenous auxin, indole3-acetic acid (IAA) was evenly distributed in the illuminated and shaded sides of phototropically stimulated organs of different plant species: sunflower (Helianthus annuus) hypocotyls (Bruinsma et al., 1975; Franssen and Bruinsma, 1981; Feyerabend and Weiler, 1988), radish (Raphanus sativus) hypocotyls (Sakoda and Hasegawa, 1989), oat (Avena sativa) coleoptiles (Hasegawa et al., 1989), maize (Zea mays) (Togo and Hasegawa, 1991) and pea (Pisum sativum) (Hasegawa and Yamada, 1992). In contrast to these reports, however, Iino (1991) reported the existence of a lateral gradient in endogenous IAA during phototropism in maize coleoptiles by use of the indolo-apyrone method, supporting the Cholodny–Went theory. However, the spectrometric assay has a shortcoming: high instability of the indolo-a-pyrone derivative causes poor reproducible of measurements, and the specificity of the method may be equivocal (Pickard 1985; Yamamura and Hasegawa, 2001). On the other hand, evidence showing that phototropism is caused by blue light-induced local accumulation of growth inhibitor(s) in the presence of an unchanged, even distribution of auxin, has been presented (Bruinsma et al., 1975; Franssen and Bruinsma, 1981; Hasegawa et al., 1989; Bruinsma and Hasegawa, 1990; Togo and Hasegawa, 1991; Hasegawa and Yamada, 1992; YokotaniTomita et al., 1999; Hasegawa et al., 2001; Yamamura and Hasegawa, 2001; Yamada et al., 2003; Hasegawa et al., 2004a, b; Tamimi, 2004) and constituted the basis of the Bruinsma–Hasegawa theory. As candidates for growth inhibitory substances involved in phototropism, 4-methylthio-3butenyl isothiocyanate and raphanusanins in radish hypocotyls (Hasegawa et al., 2000), uridine in oat coleoptiles (Hasegawa et al., 2001), 8-epixanthatin in sunflower hypocotyls (Yokotani-Tomita et al., 1997), 4-hydroxy-2,3-dimethyl-2-nonen-4-olide from cress (Lepidium sativum) seedlings (Hasegawa et al., 2002), indole-3-acetonitrile from Arabidopsis hypocotyls (Hasegawa et al., 2004a) and 2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 6-methoxy-benzoxazolinone (MBOA) from maize coleoptiles (Hasegawa et al., 1992; Anai et al., 1996; Hasegawa et al., 2004b) have been isolated and identified. In the case of maize coleoptiles, it was indicated that phototropic stimulation rapidly decreased the inactive DIMBOA-Glc, which is a precursor of DIMBOA, and abruptly increased the most active DIMBOA and then the active MBOA in the illumi-
539 nated halves, suggesting that the benzoxazinoids play an important role in the phototropism of maize coleoptiles (Hasegawa et al., 2004b). The aim of the present study is to investigate whether phototropic stimulation can induce the activity of ß-glucosidase, which may activate conversion of the inactive DIMBOA-Glc into the active DIMBOA.
Materials and methods Plant materials and growth conditions Maize (Z. mays L. cv. Canadian Rocky 85, Kaneko seed Co, Japan) seeds were imbibed for 1 day in the dark in running tap water, and were sown on moist vermiculite in a tray, under red light (0.3 mmol m2 s1, lmax: 655 nm) at 25 1C for 1 day. The germinated seeds were then incubated in the dark at 25 1C for one more day. Uniform etiolated seedlings (coleoptiles: 1.5–2 cm long) were transplanted into a row in the seedling case (13.5 6.5 3.5 cm) containing moist vermiculite and were grown 12 h more in the dark at 25 1C (each case with 10 seedlings). In contrast, in case of ßglucosidase inhibitor treatment, uniform etiolated seedlings were selected and transplanted in the tip rack (12.5 8.0 5.5 cm, 96 holes) containing 500 mL distilled water and were grown for 12 h more in the dark at 25 1C. All the manipulation was carried out under a photomorphogenetically inactive intensity of dim green light (0.01 mmol m2 s1).
Light treatments and experimental procedures (a) Phototropic stimulation with unilateral blue light (0.05 mmol m2 s1; lmax: 445 nm) was applied over the whole length of 4-day-old maize seedlings. To examine the effects of ßglucosidase inhibitors on phototropic curvatures, 0.2 and 0.4 mM of D-gluconic acid dlactone (GL) and 1-thio-D-glucose (TG) were incorporated through the roots of maize seedlings, 2 h before the onset of blue-light stimulation. The same volume of distilled water was used for the control. Phototropic curvatures were determined with a protractor at 0, 10, 30, 60 and 90 min after the onset of unilateral blue light illumination. (b) For growth measurement, ion-exchange resin beads (Amberlite XAD-2, ORGANO, Japan) smeared with lanolin, were attached to both sides of coleoptiles, 5 mm from top, at the
ARTICLE IN PRESS 540
R. Jabeen et al. distance of 0.5-1.5 cm downward, just before phototropic stimulation. The seedlings were then unilaterally illuminated with blue light (0.05 mmol m2 s1) for the indicated periods. In case of inhibitor treatment, 0.4 mM of GL was incorporated through the roots of seedlings, 2 h before the onset of blue-light stimulation. Photographs were taken of the dark control and of the illuminated and the shaded sides of the coleoptiles under dim green light. The length between the beads was measured by tracing magnified pictures onto pieces of paper.
Chemicals Standard samples of DIMBOA-Glc and DIMBOA were purified from 7-day-old maize seedlings (5.0–6.0 cm long) as previously described (Nakagawa et al., 1995; Larsen and Christensen, 2000). GL, Glucose oxidase (GOD) and peroxidase (POD) were purchased from Wako Pure Chemicals, Japan, Odianisidine (ODA) from ICN Biomedicals Inc, USA, TG and MBOA from Aldrich Chem. Co, Germany and Coomassie Brilliant Blue (CBB) R-250 from Fluka Chemika, Switzerland. All other chemicals were used of analytical grade.
Determination of DIMBOA-Glc, DIMBOA and MBOA levels After the onset of phototropic stimulation, 6 phototropically stimulated coleoptiles were harvested at 0, 10, 30 and 60 min. Dark control samples were also harvested at the same indicated time periods. Coleoptiles, 0–1.5 cm below the tip of seedlings, were excised and bisected into illuminated and shaded halves with a razor blade under dim green light. Bisected coleoptiles were immediately frozen in liquid N2 and stored at 80 1C until use. Frozen materials were ground in a mortar and homogenized in 8 mL of 80% cold acetone using a homogenizer (ULTRA-TURRAX T25, IKA Laborteck, Germany). The homogenates were centrifuged at 10,000 rpm for 10 min at 4 1C, and supernatants were evaporated to dryness in vacuo at 35 1C. The samples dissolved in MeOH were then subjected to HPLC (ODS-80Ts, 4.6 250 mm, Tosoh, Japan), eluted with 30% CH3CN at 0.8 mL/min flow rate and detection at 280 nm. Endogenous levels of DIMBOA-Glc, DIMBOA and MBOA were calculated from standard curves. The retention time of DIMBOA-Glc, DIMBOA and MBOA were approx. 4.6 min, 7.0 min and 11.6 min, respectively.
Preparation of crude enzyme solution Four-day-old seedlings (coleoptiles: 3.0–3.5 cm long) were illuminated unilaterally with blue light (0.05 mmol m2 s1). Six tips of 2.0–2.5 cm length were excised and bisected into the illuminated and shaded halves with a razor blade under dim green light at the indicated periods. All the coleoptile samples were immediately frozen in liquid N2. The fresh weight of all the samples was measured and then stored at 80 1C until use. Frozen coleoptiles were ground in 6 mL of imidazole-HCl buffer (50 mM, pH 6.2) in a mortar and homogenized for 30 s with a homogenizer. The crude extract was centrifuged at 10,000 rpm for 10 min at 4 1C. The supernatant was filtered by using a centrifugal filter device (Amicon Ultra 4, Mr cut off: 10,000, Millipore, USA) at 7,500 rpm for 10 min at 4 1C. The resulting residue was re-suspended in potassium phosphate buffer (5 mM, pH 7.0; volume used was adjusted according to the g FW). Using this crude enzyme solution, ß-glucosidase activity was determined.
Enzyme assay ß-Glucosidase activity was determined by measuring the production of DIMBOA from DIMBOA-Glc by ß-glucosidase. The crude enzyme solution was incubated with DIMBOA-Glc (0.2 mg) as a substrate for 3 min at 30 1C in an Eppendorf tube (1.5 mL). The resulting solution was then partitioned with an equal volume of EtOAc twice, and the EtOAc layer (DIMBOA fraction) was dried under a centrifuge concentrator (CC-100, TOMY, Japan). The DIMBOA fraction was then dissolved in 50 mL of MeOH, and 10 mL of each sample was subjected to HPLC (ODS80Ts, 4.6 250 mm, Tosoh, Japan) eluted with 20% CH3CN at 1.0 mL/min flow rate and detection at 280 nm. The concentration of DIMBOA was calculated from the standard curve.
Tissue printing Tissue printing technique was performed according to the method by Hara et al. (2001) with some modifications. A polyvinylidene difluoride (PVDF) membrane (Immobilon, pore size 0.45 mm, Millipore, USA) was dipped into MeOH and then into a tissue printing buffer (5 mM potassium phosphate buffer, pH 7.0) for 5 min. In the 4.0-cm-long maize seedlings, the transverse cut was made at the parts of coleoptile. The cut ends were quickly printed onto the membrane for about 15 s. After washing with tissue printing buffer, the membrane was
ARTICLE IN PRESS Activation of ß-glucosidase by blue light
541
incubated in 3 mL of tissue printing buffer containing 1.2 mg/mL GOD, 50 mg/mL POD, 50 mg/mL ODA, and 2.0 mg/mL of DIMBOA-Glc until the signals appeared (approx. 60 min) in the dark at room temperature. These membranes were washed twice with distilled water for 30 min to stop the reaction. The protein on the print was also detected by CBB staining.
Results Time course of the growth of maize coleoptiles in response to blue light The elongation of the illuminated and shaded sides was measured for 90 min after the onset of illumination (Fig. 1). The growth suppression in the illuminated side clearly began to appear within 60 min and continued throughout the course of the experiment. In contrast, the growth rate in the shaded side was nearly equal to that in the dark control. The growth rate in both sides of the dark controls was similar and increased with time.
The lateral distribution of DIMBOA-Glc, DIMBOA and MBOA, between illuminated and shaded halves in maize coleoptiles during phototropic curvature was analyzed using a physicochemical assay (Fig. 2). Endogenous level of DIMBOA-Glc in the illuminated halves decreased for at least 10 min after the onset of phototropic stimulation and then returned to its initial level, while in the shaded halves its level remain nearly the same throughout the experiment (Fig. 2A). In contrast, the DIMBOA level in the illuminated halves significantly increased at 30 min as compared to that of shaded sides and then decreased (Fig. 2B). A slight difference in MBOA level was also found between illuminated and shaded halves during the course of the experiment (Fig. 2C).
Lateral distribution of ß-glucosidase activity in response to blue light The ß-glucosidase activity in both the illuminated and shaded halves (estimated by determining production of DIMBOA from DIMBOA-Glc) in response to phototropic stimulation was determined (Fig. 3). Its activity in the illuminated halves showed a transient increase and reached maximum at 30 min after the onset of illuminations and then decreased thereafter. In contrast, the activity in the shaded halves was almost identical to those in the initial level or lower during the experiment.
0.4
0.3 Increase in length (mm)
Time course of benzoxazinoid levels in response to blue light
0.2
Effect of ß-glucosidase inhibitors on phototropic curvature 0.1
0
0
30
60
90
Time (min)
Figure 1. Time course of the growth of etiolated maize coleoptiles in response to continuous unilateral blue light (0.05 mmol m2 s1). Elongation was measured until 90 min after the onset of phototropic stimulations, in illuminated sides (J), shaded sides (K) and both sides of dark controls (&, ’). Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 6).
When two ß-glucosidase inhibitors, GL, and TG were incorporated into maize seedlings via the roots, the phototropic response of the seedling was significantly inhibited (Fig. 4). Both inhibitors exhibited the decrease in phototropic response in a dose-dependent manner. In the case of GLtreated coleoptiles, a significant decrease in phototropic curvature compared with the control was observed within 10 min and continued throughout the experimental time period (Fig. 4A). On the other hand, decrease in curvature was also observed in TG-treated coleoptiles within 30 min after the onset of blue-light illumination (Fig. 4B). TG exhibited much higher activity than GL during the experimental periods.
ARTICLE IN PRESS 542
R. Jabeen et al. DIMBOA-Glc 160
DIMBOA 140
A
Content (% of control)
140
MBOA 200
B
C
180
120
160
120
100
140
80
120
100 80
100 60
60 40
40
20
20
0
0
80 60 40
0
10
30
60
20 0
10
30
60
0
0
10
30
60
Time (min) Figure 2. Time course of benzoxazinoid levels in response to unilateral blue light. DIMBOA-Glc (A), DIMBOA (B) and MBOA (C) levels were determined in illuminated (J) and shaded (K) halves. Values are given as percentage of control at 0 min in intact coleoptiles. Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 15).
ment compared to non-treated ones, after the onset of blue illumination. The growth rate of both dark controls and the shaded sides with or without inhibitor treatment was similar and increased with time.
260
DIMBOA conc. (nmol/g FW)
240
220
Tissue printing for enzyme activity of ßglucosidase in maize coleoptiles 200
180
0 0
10
30
60
Time (min)
Figure 3. Lateral distribution of ß-glucosidase activity in response to blue light. The activity was estimated by determining production of DIMBOA from DIMBOA-Glc in illuminated (J) and shaded (K) halves. Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 18).
Effect of ß-glucosidase inhibitors on the growth of maize coleoptiles As shown in Fig. 4, both of the ß-glucosidase inhibitors have affected the phototropic curvature. Therefore, the growth in the illuminated and shaded sides with GL treatment was investigated (Fig. 5). A decrease in growth suppression was observed in the illuminated sides with GL treat-
The distribution of ß-glucosidase activity in the tissue of maize coleoptiles was demonstrated by using tissue-printing technique (Fig. 6). In order to detect ß-glucosidase activity on the PVDF membrane, DIMBOA-Glc was administered to the print under the GOD/POD system. If enzyme activity is present on the membrane, DIMBOA-Glc is digested to glucose, which is further oxidized by GOD to produce hydrogen peroxide. Then, the POD polymerizes ODA (without color) using the hydrogen peroxide to form complexed phenolics (brown pigments). The pigment stained specific sites of the membrane where ß-glucosidase molecules were immobilized. For the full reaction, the membrane was dipped into a reaction solution containing GOD, POD, ODA and DIMBOA-Glc (Fig. 6B). Strong signals appeared on the membrane after 60 min at room temperature. As shown in Fig. 6B, signals for ß-glucosidase activity in coleoptile were observed in the peripheral region (including epidermal and cortical cells). No signals were detected on the first leaf (inner part of coleoptile). In contrast, proteins were present along the whole area of the cut surface (Fig. 6C).
ARTICLE IN PRESS Activation of ß-glucosidase by blue light
543 3
16
A
Dark control
14
0.2 mM GL
12
0.4 mM GL
2.5
Increase in length (mm)
10 8
Phototropic curvature (degrees)
6 4 2 0 0 16
10
B
30
60
1
0.5
0.2 mM TG
12
0.4 mM TG
0 0
10 8 6 4 2 0
1.5
90
Dark control
14
2
0
10
30
60
90
10
30 Time (min)
60
90
Figure 5. Effect of ß-glucosidase inhibitor on the growth in maize coleoptiles. 0.4 mM of D-Gluconic acid d-lactone (GL) was incorporated into maize seedlings from the roots, 2 h prior to blue light illumination. Illuminated (D) and shaded (m) sides with inhibitor; illuminated (J) and shaded (K) sides without inhibitor; both sides of dark control with (B, E) or without inhibitor (&, ’) at the indicated time periods.
Time (min) Figure 4. Effect of ß-glucosidase inhibitors on phototropic curvature. D-Gluconic acid d-lactone (GL, A) and 1thio-D-glucose (TG, B) with 0.2 mM (J) and 0.4 mM (D). Solutions were incorporated into maize seedlings via the roots, 2 h prior to blue-light illumination. Measurement of dark controls without inhibitor (&) was also made at the indicated time periods.
Discussion Previously, a transient increase of DIMBOA and MBOA and a decrease of DIMBOA-Glc in response to a blue-light pulse (the first positive phototropism) have been reported in maize coleoptiles (Hasegawa et al., 2004b). As shown in Fig. 2, the kinetic study of these benzoxazinoides in response to a continuous blue light (the second positive phototropism) was also performed and showed a similar result with a low fluence response peaking at 30 min. Together with the results of phototropic bending and the growth rate between the illuminated and shaded sides, a transient up-regulation in both DIMBOA and MBOA in the illuminated side was observed, suggesting the possibility of their direct contribution for phototropic response caused by an
inhibition in the illuminated side. Moreover, the net amount of DIMBOA was much higher than MBOA, though the activity of growth inhibition is similar (data not shown), indicating DIMBOA is a major phototropism-regulating substance in the early response of maize phototropism. In the literature, DIMBOA has been discussed as a possible agent for plant defense (Niemeyer, 1988; Osbourn, 1996; Sicker et al., 2000). ß-Glucosidase, which is a key enzyme of DIMBOA biosynthesis, exhibited a significant up-regulation in response to the blue light illumination (Fig. 3). Although ßglucosidase occurs widely in prokaryotes and eukaryotes, they have substrate specificity. For instance, specific plant ß-glucosidases are involved in the chemical defense system against pathogens and herbivores. A specific ß-glucosidase in maize has been reported to have high affinity for DIMBOAGlc (Babcock and Esen, 1994; Oikawa et al., 1999; Czjzek et al., 2000). Although it is not clear whether the ß-glucosidase described here is highly specific for DIMBOA-Glc, this is the first observation that blue light can induce the activity of ßglucosidase. In a previous report, a kind of specific glycosidase (ß-thioglucosidase, myrosinase), which
ARTICLE IN PRESS 544
R. Jabeen et al.
Figure 6. Tissue printing for enzyme activity of ß-glucosidase in maize coleoptiles. Prints were prepared from the transverse cut ends of the coleoptiles. (A) Control reaction without DIMBOA-Glc; (B) Full reaction with DIMBOA-Glc; (C) CBB staining. Bars represent 1 mm.
has an important role in a defense system in Cruciferae, can also be activated by blue light illumination (Yamada et al., 2003). Released aglycon from glucosinolate by the hydrolysis of myrosinase caused a phototropic curvature in radish, indicating a new role of phytoallexin biosynthesis in plants. As a working hypothesis, inhibition of ß-glucosidase by GL and TG, tight binding competitive inhibitors, was examined. As expected, the response against phototropic stimulation was significantly decreased in the maize coleoptiles with inhibitor treatment (Fig. 4). This result is in good agreement with the significant decrease in growth suppression in the illuminated side with inhibitor treatment (Fig. 5), suggesting that the ß-glucosidase activity is suppressed with the inhibitor treatment, subsequently DIMBOA level does not increase in the illuminated side. Measurement of the benzoxazinoids level and ß-glucosidase activity in response to inhibitor treatment also remain as problems to be investigated. Tissue-printing assay was demonstrated to determine the localization of ß-glucosidase activity in maize coleoptiles. A strong signal of ß-glucosidase activity was detected on the peripheral cell layers including epidermal and cortex regions, which are central regions for plant growth response as well as phototropism (Feyerabend and Weiler, 1988). Therefore, it is likely that the ß-glucosidase involve in DIMBOA biosynthesis can be easily activated in the illuminated side in response to phototropic stimulation. Recently, a mechanism of growth inhibition by DIMBOA has been proposed in oat coleoptiles. The effect of DIMBOA on cell-wall PODs would contribute to its growth inhibitory activity by promoting hydrogen peroxide synthesis and lignification,
which could increase the stiffness at the primary cell-wall level (Gonza ´lez and Rojas, 1999). Furthermore, it was also shown that unilateral application of hydrogen peroxide could induce curvature in wheat coleoptiles, suggesting a close linkage between an oxidative burst and phototropism (Chandrakuntal et al., 2004). Together with the inhibitory effect of DIMBOA (and MBOA) on auxin activity, we conclude that benzoxazinoids, DIMBOA (and MBOA) play important roles in response to phototropic stimulation. Blue light may induce the activity of ß-glucosidase in the illuminated side via signal transduction from blue-light receptor(s), subsequently the up-regulated DIMBOA (and MBOA) would lead to the growth inhibition of epidermal cell layers having a close relation to auxin activity, gene expression, and protein biosynthesis.
References Anai T, Aizawa H, Ohtake N, Kosemura S, Yamamura S, Hasegawa K. A new auxin-inhibiting substance, 4-Cl6,7-dimethoxy-2-benzoxazolinone, from light-grown maize shoots. Phytochemistry 1996;42:273–5. Babcock GD, Esen A. Substrate specificity of maize ßglucosidase. Plant Sci 1994;101:31–9. Bruinsma J, Hasegawa K. A new theory of phototropism: its regulation by a light-induced gradient of auxininhibiting substances. Plant Physiol 1990;79:700–4. Bruinsma J, Karssen CM, Benschop M, Van Dort JB. Hormonal regulation of phototropism in the lightgrown sunflower seedlings, Helianthus annuus L.: Immobility of endogenous indoleacetic acid and inhibition of hypocotyl growth by illuminated cotyledons. J Exp Bot 1975;26:411–8. Chandrakuntal K, Kumar PG, Laloraya M, Laloraya MM. Direct involvement of hydrogen peroxide in curvature
ARTICLE IN PRESS Activation of ß-glucosidase by blue light of wheat coleoptile in blue-light-treated and darkgrown coleoptiles. Biochem Biophys Res Commun 2004;319:1190–6. Czjzek M, Cicek M, Zamboni V, Bevan DR, Henrissat B, Esen A. The mechanism of substrate (aglycone) specificity in b-glucosidases is revealed by crystal structures of mutant maize b-glucosidase –DIMBOA DIMBOAGlc, and -dhurrin complexes. Proc Natl Acad Sci USA 2000;97:13555–60. Feyerabend M, Weiler EW. Immunological estimation of growth regulator distribution in phototropically reacting sunflower seedlings. Plant Physiol 1988;74:185–93. Franssen JM, Bruinsma J. Relationships between xanthoxin, phototropism, and elongation growth in the sunflower seedling (Helianthus annuus L.). Planta 1981;151:365–70. Gonza ´lez LF, Rojas MC. Role of wall peroxidases in oat growth inhibition by DIMBOA. Phytochemistry 1999;50:931–7. Hara M, Eto H, Kuboi T. Tissue printing for myrosinase activity in roots of turnip and Japanese radish and horseradish: a technique for localizing myrosinases. Plant Sci 2001;160:425–31. Hasegawa K, Sakoda M, Bruinsma J. Revision of the theory of phototropism in plants: a new interpretation of a classical experiment. Planta 1989;178:540–4. Hasegawa K, Togo S, Urashima M, Mizutani J, Kosemura S, Yamamura S. An auxin-inhibiting substance from lightgrown maize shoots. Phytochemistry 1992;31:3673–6. Hasegawa K, Yamada K. Even distribution of endogenous indole-3-acetic acid in phototropism of pea epicotyls. J Plant Physiol 1992;139:455–9. Hasegawa T, Yamada K, Kosemura S, Yamamura S, Bruinsma J, Miyamoto K, et al. Isolation and identification of a light-induced growth inhibitor in diffusates from blue light-illuminated oat (Avena sativa L.) coleoptile tips. Plant Growth Regul 2001;33:175–9. Hasegawa T, Yamada K, Kosemura S, Yamamura S, Hasegawa K. Phototropic stimulation induces the conversion of glucosinolate to phototropism regulating substances of radish hypocotyls. Phytochemistry 2000;54:275–9. Hasegawa T, Yamada K, Shigemori H, Goto N, Miyamoto K, Ueda J, et al. Isolation and identification of blue light-induced growth inhibitor from light-grown Arabidopsis shoots. Plant Growth Regul 2004a;44:81–6. Hasegawa T, Yamada K, Shigemori H, Hasegava K, Miyamoto K, Ueda J. Isolation and identification of a growth inhibitor from blue light-illuminated cress seedlings. Plant Growth Regul 2002;37:45–7. Hasegawa T, Yamada K, Shigemori H, Miyamoto K, Ueda J, Hasegawa K. Isolation and identification of phototropism-regulating substances benzoxazinoids from maize coleoptiles. Heterocycles 2004b;63:2707–12. Iino M. Mediation of tropisms by lateral translocation of endogenous indole-3-acetic acid in maize coleoptiles. Plant Cell Environ 1991;14:279–86.
545 Larsen E, Christensen LP. Simple method for large scale isolation of the cyclic arylhydroxamic acid DIMBOA from maize (Zea mays L.). J Agri Food Chem 2000;48:2556–8. Nakagawa E, Amano T, Hirai N, Iwamura H. Non-induced cyclic hydroxamic acids in wheat during juvenile stage of growth. Phytochemistry 1995;38:1349–54. Niemeyer HM. Hydroxamic acids (4-hydroxy-1,4-benzoxazin-3-ones), defense chemicals in the Gramineae. Phytochemistry 1988;27:3349–58. Oikawa A, Ebisui K, Sue M, Ishihara A, Iwamura H. Purification and characterization of a ß-glucosidase specific for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin3-one (DIMBOA) glucoside in maize. Z Naturforsch 1999;54c:181–5. Osbourn AE. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 1996;8:1821–31. Pickard BG. Roles of hormones in phototropism. In: Pharis RP, Reed DM, editors. Encyclopedia of plant physiology. New Series, 11. Berlin: Springer; 1985. p. 365–417. Sakoda M, Hasegawa K. Phototropism in hypocotyls of radish. VI. No exchange indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures. Plant Physiol 1989;76:240–2. Sicker D, Frey M, Schulz M, Gierl A. Role of natural benzoxazinones in the survival strategy of plants. Int Rev Cytol 2000;198:319–46. Tamimi SM. Uridine and the control of phototropism in oat (Avena sativa L.) coleoptiles. Plant Growth Regul 2004;43:173–7. Togo S, Hasegawa K. Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles. Physiol Plant 1991;81:555–7. Went FW, Thimann KV. Phytohormones. McMillan: New York; 1937. Yamada K, Hasegawa T, Minami E, Shibuya N, Kosemura S, Yamamura S, et al. Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation. J Plant Physiol 2003;160:255–9. Yamamura S, Hasegawa K. Chemistry and biology of phototropism-regulating substances in higher plants. Chem Rec 2001;1:362–72. Yokotani-Tomita K, Kato J, Kosemura S, Yamamura S, Kushima M, Kakuta H, et al. Light-induced auxininhibiting substance from sunflower seedlings. Phytochemistry 1997;46:503–6. Yokotani-Tomita K, Kato J, Yamada K, Kosemura S, Yamamura S, Bruinsma J, et al. 8-Epixanthatin, a light-induced growth inhibitor, mediates the phototropic curvature in sunflower (Helianthus annuus) hypocotyls. Physiol Plant 1999;106:326–30.
www.elsevier.de/jplph
Induction of ß-glucosidase activity in maize coleoptiles by blue light illumination Riffat Jabeena, Kosumi Yamadaa,, Hideyuki Shigemoria, Tsuyoshi Hasegawaa, Masakazu Harab, Toru Kuboib, Koji Hasegawaa a
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Department of Environmental Science for Human Life, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
b
Received 23 March 2005; accepted 17 May 2005
KEYWORDS Benzoxazinoid; Blue light; DIMBOA; ß-glucosidase; Phototropism; Phototropismregulating substances; Zea mays
Summary The role of ß-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of ß-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by ß-glucosidase, and its degradation compound 6-methoxybenzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, ßglucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of ß-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviations: DIMBOA, 2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one; DIMBOA-Glc, 2-0-ß-D-glucopyranosyl-4-hydroxy7-methoxy-1, 4-benzoxazin-3-one; GL, D-gluconic acid d-lactone; MBOA, 6-methoxy-benzoxazolinone; TG, 1-thio-D-glucose. Corresponding author. Tel./fax: +81 29 853 6933. E-mail address: [email protected] (K. Yamada).
In phototropism, seedlings of higher plants orient their growth with regard to the direction of light in order to optimize the exposure of their photosynthetic organs (e.g. a leaf blade is oriented perpendicularly to the direction of light). A famous model is the Cholodny–Went theory, in which phototropic curvature is caused by a lateral
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.05.004
ARTICLE IN PRESS Activation of ß-glucosidase by blue light gradient of growth-promoting auxin in the bending organ (Went and Thimann, 1937). However, using physicochemical and immunological methods, some groups have shown that endogenous auxin, indole3-acetic acid (IAA) was evenly distributed in the illuminated and shaded sides of phototropically stimulated organs of different plant species: sunflower (Helianthus annuus) hypocotyls (Bruinsma et al., 1975; Franssen and Bruinsma, 1981; Feyerabend and Weiler, 1988), radish (Raphanus sativus) hypocotyls (Sakoda and Hasegawa, 1989), oat (Avena sativa) coleoptiles (Hasegawa et al., 1989), maize (Zea mays) (Togo and Hasegawa, 1991) and pea (Pisum sativum) (Hasegawa and Yamada, 1992). In contrast to these reports, however, Iino (1991) reported the existence of a lateral gradient in endogenous IAA during phototropism in maize coleoptiles by use of the indolo-apyrone method, supporting the Cholodny–Went theory. However, the spectrometric assay has a shortcoming: high instability of the indolo-a-pyrone derivative causes poor reproducible of measurements, and the specificity of the method may be equivocal (Pickard 1985; Yamamura and Hasegawa, 2001). On the other hand, evidence showing that phototropism is caused by blue light-induced local accumulation of growth inhibitor(s) in the presence of an unchanged, even distribution of auxin, has been presented (Bruinsma et al., 1975; Franssen and Bruinsma, 1981; Hasegawa et al., 1989; Bruinsma and Hasegawa, 1990; Togo and Hasegawa, 1991; Hasegawa and Yamada, 1992; YokotaniTomita et al., 1999; Hasegawa et al., 2001; Yamamura and Hasegawa, 2001; Yamada et al., 2003; Hasegawa et al., 2004a, b; Tamimi, 2004) and constituted the basis of the Bruinsma–Hasegawa theory. As candidates for growth inhibitory substances involved in phototropism, 4-methylthio-3butenyl isothiocyanate and raphanusanins in radish hypocotyls (Hasegawa et al., 2000), uridine in oat coleoptiles (Hasegawa et al., 2001), 8-epixanthatin in sunflower hypocotyls (Yokotani-Tomita et al., 1997), 4-hydroxy-2,3-dimethyl-2-nonen-4-olide from cress (Lepidium sativum) seedlings (Hasegawa et al., 2002), indole-3-acetonitrile from Arabidopsis hypocotyls (Hasegawa et al., 2004a) and 2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 6-methoxy-benzoxazolinone (MBOA) from maize coleoptiles (Hasegawa et al., 1992; Anai et al., 1996; Hasegawa et al., 2004b) have been isolated and identified. In the case of maize coleoptiles, it was indicated that phototropic stimulation rapidly decreased the inactive DIMBOA-Glc, which is a precursor of DIMBOA, and abruptly increased the most active DIMBOA and then the active MBOA in the illumi-
539 nated halves, suggesting that the benzoxazinoids play an important role in the phototropism of maize coleoptiles (Hasegawa et al., 2004b). The aim of the present study is to investigate whether phototropic stimulation can induce the activity of ß-glucosidase, which may activate conversion of the inactive DIMBOA-Glc into the active DIMBOA.
Materials and methods Plant materials and growth conditions Maize (Z. mays L. cv. Canadian Rocky 85, Kaneko seed Co, Japan) seeds were imbibed for 1 day in the dark in running tap water, and were sown on moist vermiculite in a tray, under red light (0.3 mmol m2 s1, lmax: 655 nm) at 25 1C for 1 day. The germinated seeds were then incubated in the dark at 25 1C for one more day. Uniform etiolated seedlings (coleoptiles: 1.5–2 cm long) were transplanted into a row in the seedling case (13.5 6.5 3.5 cm) containing moist vermiculite and were grown 12 h more in the dark at 25 1C (each case with 10 seedlings). In contrast, in case of ßglucosidase inhibitor treatment, uniform etiolated seedlings were selected and transplanted in the tip rack (12.5 8.0 5.5 cm, 96 holes) containing 500 mL distilled water and were grown for 12 h more in the dark at 25 1C. All the manipulation was carried out under a photomorphogenetically inactive intensity of dim green light (0.01 mmol m2 s1).
Light treatments and experimental procedures (a) Phototropic stimulation with unilateral blue light (0.05 mmol m2 s1; lmax: 445 nm) was applied over the whole length of 4-day-old maize seedlings. To examine the effects of ßglucosidase inhibitors on phototropic curvatures, 0.2 and 0.4 mM of D-gluconic acid dlactone (GL) and 1-thio-D-glucose (TG) were incorporated through the roots of maize seedlings, 2 h before the onset of blue-light stimulation. The same volume of distilled water was used for the control. Phototropic curvatures were determined with a protractor at 0, 10, 30, 60 and 90 min after the onset of unilateral blue light illumination. (b) For growth measurement, ion-exchange resin beads (Amberlite XAD-2, ORGANO, Japan) smeared with lanolin, were attached to both sides of coleoptiles, 5 mm from top, at the
ARTICLE IN PRESS 540
R. Jabeen et al. distance of 0.5-1.5 cm downward, just before phototropic stimulation. The seedlings were then unilaterally illuminated with blue light (0.05 mmol m2 s1) for the indicated periods. In case of inhibitor treatment, 0.4 mM of GL was incorporated through the roots of seedlings, 2 h before the onset of blue-light stimulation. Photographs were taken of the dark control and of the illuminated and the shaded sides of the coleoptiles under dim green light. The length between the beads was measured by tracing magnified pictures onto pieces of paper.
Chemicals Standard samples of DIMBOA-Glc and DIMBOA were purified from 7-day-old maize seedlings (5.0–6.0 cm long) as previously described (Nakagawa et al., 1995; Larsen and Christensen, 2000). GL, Glucose oxidase (GOD) and peroxidase (POD) were purchased from Wako Pure Chemicals, Japan, Odianisidine (ODA) from ICN Biomedicals Inc, USA, TG and MBOA from Aldrich Chem. Co, Germany and Coomassie Brilliant Blue (CBB) R-250 from Fluka Chemika, Switzerland. All other chemicals were used of analytical grade.
Determination of DIMBOA-Glc, DIMBOA and MBOA levels After the onset of phototropic stimulation, 6 phototropically stimulated coleoptiles were harvested at 0, 10, 30 and 60 min. Dark control samples were also harvested at the same indicated time periods. Coleoptiles, 0–1.5 cm below the tip of seedlings, were excised and bisected into illuminated and shaded halves with a razor blade under dim green light. Bisected coleoptiles were immediately frozen in liquid N2 and stored at 80 1C until use. Frozen materials were ground in a mortar and homogenized in 8 mL of 80% cold acetone using a homogenizer (ULTRA-TURRAX T25, IKA Laborteck, Germany). The homogenates were centrifuged at 10,000 rpm for 10 min at 4 1C, and supernatants were evaporated to dryness in vacuo at 35 1C. The samples dissolved in MeOH were then subjected to HPLC (ODS-80Ts, 4.6 250 mm, Tosoh, Japan), eluted with 30% CH3CN at 0.8 mL/min flow rate and detection at 280 nm. Endogenous levels of DIMBOA-Glc, DIMBOA and MBOA were calculated from standard curves. The retention time of DIMBOA-Glc, DIMBOA and MBOA were approx. 4.6 min, 7.0 min and 11.6 min, respectively.
Preparation of crude enzyme solution Four-day-old seedlings (coleoptiles: 3.0–3.5 cm long) were illuminated unilaterally with blue light (0.05 mmol m2 s1). Six tips of 2.0–2.5 cm length were excised and bisected into the illuminated and shaded halves with a razor blade under dim green light at the indicated periods. All the coleoptile samples were immediately frozen in liquid N2. The fresh weight of all the samples was measured and then stored at 80 1C until use. Frozen coleoptiles were ground in 6 mL of imidazole-HCl buffer (50 mM, pH 6.2) in a mortar and homogenized for 30 s with a homogenizer. The crude extract was centrifuged at 10,000 rpm for 10 min at 4 1C. The supernatant was filtered by using a centrifugal filter device (Amicon Ultra 4, Mr cut off: 10,000, Millipore, USA) at 7,500 rpm for 10 min at 4 1C. The resulting residue was re-suspended in potassium phosphate buffer (5 mM, pH 7.0; volume used was adjusted according to the g FW). Using this crude enzyme solution, ß-glucosidase activity was determined.
Enzyme assay ß-Glucosidase activity was determined by measuring the production of DIMBOA from DIMBOA-Glc by ß-glucosidase. The crude enzyme solution was incubated with DIMBOA-Glc (0.2 mg) as a substrate for 3 min at 30 1C in an Eppendorf tube (1.5 mL). The resulting solution was then partitioned with an equal volume of EtOAc twice, and the EtOAc layer (DIMBOA fraction) was dried under a centrifuge concentrator (CC-100, TOMY, Japan). The DIMBOA fraction was then dissolved in 50 mL of MeOH, and 10 mL of each sample was subjected to HPLC (ODS80Ts, 4.6 250 mm, Tosoh, Japan) eluted with 20% CH3CN at 1.0 mL/min flow rate and detection at 280 nm. The concentration of DIMBOA was calculated from the standard curve.
Tissue printing Tissue printing technique was performed according to the method by Hara et al. (2001) with some modifications. A polyvinylidene difluoride (PVDF) membrane (Immobilon, pore size 0.45 mm, Millipore, USA) was dipped into MeOH and then into a tissue printing buffer (5 mM potassium phosphate buffer, pH 7.0) for 5 min. In the 4.0-cm-long maize seedlings, the transverse cut was made at the parts of coleoptile. The cut ends were quickly printed onto the membrane for about 15 s. After washing with tissue printing buffer, the membrane was
ARTICLE IN PRESS Activation of ß-glucosidase by blue light
541
incubated in 3 mL of tissue printing buffer containing 1.2 mg/mL GOD, 50 mg/mL POD, 50 mg/mL ODA, and 2.0 mg/mL of DIMBOA-Glc until the signals appeared (approx. 60 min) in the dark at room temperature. These membranes were washed twice with distilled water for 30 min to stop the reaction. The protein on the print was also detected by CBB staining.
Results Time course of the growth of maize coleoptiles in response to blue light The elongation of the illuminated and shaded sides was measured for 90 min after the onset of illumination (Fig. 1). The growth suppression in the illuminated side clearly began to appear within 60 min and continued throughout the course of the experiment. In contrast, the growth rate in the shaded side was nearly equal to that in the dark control. The growth rate in both sides of the dark controls was similar and increased with time.
The lateral distribution of DIMBOA-Glc, DIMBOA and MBOA, between illuminated and shaded halves in maize coleoptiles during phototropic curvature was analyzed using a physicochemical assay (Fig. 2). Endogenous level of DIMBOA-Glc in the illuminated halves decreased for at least 10 min after the onset of phototropic stimulation and then returned to its initial level, while in the shaded halves its level remain nearly the same throughout the experiment (Fig. 2A). In contrast, the DIMBOA level in the illuminated halves significantly increased at 30 min as compared to that of shaded sides and then decreased (Fig. 2B). A slight difference in MBOA level was also found between illuminated and shaded halves during the course of the experiment (Fig. 2C).
Lateral distribution of ß-glucosidase activity in response to blue light The ß-glucosidase activity in both the illuminated and shaded halves (estimated by determining production of DIMBOA from DIMBOA-Glc) in response to phototropic stimulation was determined (Fig. 3). Its activity in the illuminated halves showed a transient increase and reached maximum at 30 min after the onset of illuminations and then decreased thereafter. In contrast, the activity in the shaded halves was almost identical to those in the initial level or lower during the experiment.
0.4
0.3 Increase in length (mm)
Time course of benzoxazinoid levels in response to blue light
0.2
Effect of ß-glucosidase inhibitors on phototropic curvature 0.1
0
0
30
60
90
Time (min)
Figure 1. Time course of the growth of etiolated maize coleoptiles in response to continuous unilateral blue light (0.05 mmol m2 s1). Elongation was measured until 90 min after the onset of phototropic stimulations, in illuminated sides (J), shaded sides (K) and both sides of dark controls (&, ’). Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 6).
When two ß-glucosidase inhibitors, GL, and TG were incorporated into maize seedlings via the roots, the phototropic response of the seedling was significantly inhibited (Fig. 4). Both inhibitors exhibited the decrease in phototropic response in a dose-dependent manner. In the case of GLtreated coleoptiles, a significant decrease in phototropic curvature compared with the control was observed within 10 min and continued throughout the experimental time period (Fig. 4A). On the other hand, decrease in curvature was also observed in TG-treated coleoptiles within 30 min after the onset of blue-light illumination (Fig. 4B). TG exhibited much higher activity than GL during the experimental periods.
ARTICLE IN PRESS 542
R. Jabeen et al. DIMBOA-Glc 160
DIMBOA 140
A
Content (% of control)
140
MBOA 200
B
C
180
120
160
120
100
140
80
120
100 80
100 60
60 40
40
20
20
0
0
80 60 40
0
10
30
60
20 0
10
30
60
0
0
10
30
60
Time (min) Figure 2. Time course of benzoxazinoid levels in response to unilateral blue light. DIMBOA-Glc (A), DIMBOA (B) and MBOA (C) levels were determined in illuminated (J) and shaded (K) halves. Values are given as percentage of control at 0 min in intact coleoptiles. Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 15).
ment compared to non-treated ones, after the onset of blue illumination. The growth rate of both dark controls and the shaded sides with or without inhibitor treatment was similar and increased with time.
260
DIMBOA conc. (nmol/g FW)
240
220
Tissue printing for enzyme activity of ßglucosidase in maize coleoptiles 200
180
0 0
10
30
60
Time (min)
Figure 3. Lateral distribution of ß-glucosidase activity in response to blue light. The activity was estimated by determining production of DIMBOA from DIMBOA-Glc in illuminated (J) and shaded (K) halves. Data represents the mean of three separate experiments. Error bars indicate 7 SE (n ¼ 18).
Effect of ß-glucosidase inhibitors on the growth of maize coleoptiles As shown in Fig. 4, both of the ß-glucosidase inhibitors have affected the phototropic curvature. Therefore, the growth in the illuminated and shaded sides with GL treatment was investigated (Fig. 5). A decrease in growth suppression was observed in the illuminated sides with GL treat-
The distribution of ß-glucosidase activity in the tissue of maize coleoptiles was demonstrated by using tissue-printing technique (Fig. 6). In order to detect ß-glucosidase activity on the PVDF membrane, DIMBOA-Glc was administered to the print under the GOD/POD system. If enzyme activity is present on the membrane, DIMBOA-Glc is digested to glucose, which is further oxidized by GOD to produce hydrogen peroxide. Then, the POD polymerizes ODA (without color) using the hydrogen peroxide to form complexed phenolics (brown pigments). The pigment stained specific sites of the membrane where ß-glucosidase molecules were immobilized. For the full reaction, the membrane was dipped into a reaction solution containing GOD, POD, ODA and DIMBOA-Glc (Fig. 6B). Strong signals appeared on the membrane after 60 min at room temperature. As shown in Fig. 6B, signals for ß-glucosidase activity in coleoptile were observed in the peripheral region (including epidermal and cortical cells). No signals were detected on the first leaf (inner part of coleoptile). In contrast, proteins were present along the whole area of the cut surface (Fig. 6C).
ARTICLE IN PRESS Activation of ß-glucosidase by blue light
543 3
16
A
Dark control
14
0.2 mM GL
12
0.4 mM GL
2.5
Increase in length (mm)
10 8
Phototropic curvature (degrees)
6 4 2 0 0 16
10
B
30
60
1
0.5
0.2 mM TG
12
0.4 mM TG
0 0
10 8 6 4 2 0
1.5
90
Dark control
14
2
0
10
30
60
90
10
30 Time (min)
60
90
Figure 5. Effect of ß-glucosidase inhibitor on the growth in maize coleoptiles. 0.4 mM of D-Gluconic acid d-lactone (GL) was incorporated into maize seedlings from the roots, 2 h prior to blue light illumination. Illuminated (D) and shaded (m) sides with inhibitor; illuminated (J) and shaded (K) sides without inhibitor; both sides of dark control with (B, E) or without inhibitor (&, ’) at the indicated time periods.
Time (min) Figure 4. Effect of ß-glucosidase inhibitors on phototropic curvature. D-Gluconic acid d-lactone (GL, A) and 1thio-D-glucose (TG, B) with 0.2 mM (J) and 0.4 mM (D). Solutions were incorporated into maize seedlings via the roots, 2 h prior to blue-light illumination. Measurement of dark controls without inhibitor (&) was also made at the indicated time periods.
Discussion Previously, a transient increase of DIMBOA and MBOA and a decrease of DIMBOA-Glc in response to a blue-light pulse (the first positive phototropism) have been reported in maize coleoptiles (Hasegawa et al., 2004b). As shown in Fig. 2, the kinetic study of these benzoxazinoides in response to a continuous blue light (the second positive phototropism) was also performed and showed a similar result with a low fluence response peaking at 30 min. Together with the results of phototropic bending and the growth rate between the illuminated and shaded sides, a transient up-regulation in both DIMBOA and MBOA in the illuminated side was observed, suggesting the possibility of their direct contribution for phototropic response caused by an
inhibition in the illuminated side. Moreover, the net amount of DIMBOA was much higher than MBOA, though the activity of growth inhibition is similar (data not shown), indicating DIMBOA is a major phototropism-regulating substance in the early response of maize phototropism. In the literature, DIMBOA has been discussed as a possible agent for plant defense (Niemeyer, 1988; Osbourn, 1996; Sicker et al., 2000). ß-Glucosidase, which is a key enzyme of DIMBOA biosynthesis, exhibited a significant up-regulation in response to the blue light illumination (Fig. 3). Although ßglucosidase occurs widely in prokaryotes and eukaryotes, they have substrate specificity. For instance, specific plant ß-glucosidases are involved in the chemical defense system against pathogens and herbivores. A specific ß-glucosidase in maize has been reported to have high affinity for DIMBOAGlc (Babcock and Esen, 1994; Oikawa et al., 1999; Czjzek et al., 2000). Although it is not clear whether the ß-glucosidase described here is highly specific for DIMBOA-Glc, this is the first observation that blue light can induce the activity of ßglucosidase. In a previous report, a kind of specific glycosidase (ß-thioglucosidase, myrosinase), which
ARTICLE IN PRESS 544
R. Jabeen et al.
Figure 6. Tissue printing for enzyme activity of ß-glucosidase in maize coleoptiles. Prints were prepared from the transverse cut ends of the coleoptiles. (A) Control reaction without DIMBOA-Glc; (B) Full reaction with DIMBOA-Glc; (C) CBB staining. Bars represent 1 mm.
has an important role in a defense system in Cruciferae, can also be activated by blue light illumination (Yamada et al., 2003). Released aglycon from glucosinolate by the hydrolysis of myrosinase caused a phototropic curvature in radish, indicating a new role of phytoallexin biosynthesis in plants. As a working hypothesis, inhibition of ß-glucosidase by GL and TG, tight binding competitive inhibitors, was examined. As expected, the response against phototropic stimulation was significantly decreased in the maize coleoptiles with inhibitor treatment (Fig. 4). This result is in good agreement with the significant decrease in growth suppression in the illuminated side with inhibitor treatment (Fig. 5), suggesting that the ß-glucosidase activity is suppressed with the inhibitor treatment, subsequently DIMBOA level does not increase in the illuminated side. Measurement of the benzoxazinoids level and ß-glucosidase activity in response to inhibitor treatment also remain as problems to be investigated. Tissue-printing assay was demonstrated to determine the localization of ß-glucosidase activity in maize coleoptiles. A strong signal of ß-glucosidase activity was detected on the peripheral cell layers including epidermal and cortex regions, which are central regions for plant growth response as well as phototropism (Feyerabend and Weiler, 1988). Therefore, it is likely that the ß-glucosidase involve in DIMBOA biosynthesis can be easily activated in the illuminated side in response to phototropic stimulation. Recently, a mechanism of growth inhibition by DIMBOA has been proposed in oat coleoptiles. The effect of DIMBOA on cell-wall PODs would contribute to its growth inhibitory activity by promoting hydrogen peroxide synthesis and lignification,
which could increase the stiffness at the primary cell-wall level (Gonza ´lez and Rojas, 1999). Furthermore, it was also shown that unilateral application of hydrogen peroxide could induce curvature in wheat coleoptiles, suggesting a close linkage between an oxidative burst and phototropism (Chandrakuntal et al., 2004). Together with the inhibitory effect of DIMBOA (and MBOA) on auxin activity, we conclude that benzoxazinoids, DIMBOA (and MBOA) play important roles in response to phototropic stimulation. Blue light may induce the activity of ß-glucosidase in the illuminated side via signal transduction from blue-light receptor(s), subsequently the up-regulated DIMBOA (and MBOA) would lead to the growth inhibition of epidermal cell layers having a close relation to auxin activity, gene expression, and protein biosynthesis.
References Anai T, Aizawa H, Ohtake N, Kosemura S, Yamamura S, Hasegawa K. A new auxin-inhibiting substance, 4-Cl6,7-dimethoxy-2-benzoxazolinone, from light-grown maize shoots. Phytochemistry 1996;42:273–5. Babcock GD, Esen A. Substrate specificity of maize ßglucosidase. Plant Sci 1994;101:31–9. Bruinsma J, Hasegawa K. A new theory of phototropism: its regulation by a light-induced gradient of auxininhibiting substances. Plant Physiol 1990;79:700–4. Bruinsma J, Karssen CM, Benschop M, Van Dort JB. Hormonal regulation of phototropism in the lightgrown sunflower seedlings, Helianthus annuus L.: Immobility of endogenous indoleacetic acid and inhibition of hypocotyl growth by illuminated cotyledons. J Exp Bot 1975;26:411–8. Chandrakuntal K, Kumar PG, Laloraya M, Laloraya MM. Direct involvement of hydrogen peroxide in curvature
ARTICLE IN PRESS Activation of ß-glucosidase by blue light of wheat coleoptile in blue-light-treated and darkgrown coleoptiles. Biochem Biophys Res Commun 2004;319:1190–6. Czjzek M, Cicek M, Zamboni V, Bevan DR, Henrissat B, Esen A. The mechanism of substrate (aglycone) specificity in b-glucosidases is revealed by crystal structures of mutant maize b-glucosidase –DIMBOA DIMBOAGlc, and -dhurrin complexes. Proc Natl Acad Sci USA 2000;97:13555–60. Feyerabend M, Weiler EW. Immunological estimation of growth regulator distribution in phototropically reacting sunflower seedlings. Plant Physiol 1988;74:185–93. Franssen JM, Bruinsma J. Relationships between xanthoxin, phototropism, and elongation growth in the sunflower seedling (Helianthus annuus L.). Planta 1981;151:365–70. Gonza ´lez LF, Rojas MC. Role of wall peroxidases in oat growth inhibition by DIMBOA. Phytochemistry 1999;50:931–7. Hara M, Eto H, Kuboi T. Tissue printing for myrosinase activity in roots of turnip and Japanese radish and horseradish: a technique for localizing myrosinases. Plant Sci 2001;160:425–31. Hasegawa K, Sakoda M, Bruinsma J. Revision of the theory of phototropism in plants: a new interpretation of a classical experiment. Planta 1989;178:540–4. Hasegawa K, Togo S, Urashima M, Mizutani J, Kosemura S, Yamamura S. An auxin-inhibiting substance from lightgrown maize shoots. Phytochemistry 1992;31:3673–6. Hasegawa K, Yamada K. Even distribution of endogenous indole-3-acetic acid in phototropism of pea epicotyls. J Plant Physiol 1992;139:455–9. Hasegawa T, Yamada K, Kosemura S, Yamamura S, Bruinsma J, Miyamoto K, et al. Isolation and identification of a light-induced growth inhibitor in diffusates from blue light-illuminated oat (Avena sativa L.) coleoptile tips. Plant Growth Regul 2001;33:175–9. Hasegawa T, Yamada K, Kosemura S, Yamamura S, Hasegawa K. Phototropic stimulation induces the conversion of glucosinolate to phototropism regulating substances of radish hypocotyls. Phytochemistry 2000;54:275–9. Hasegawa T, Yamada K, Shigemori H, Goto N, Miyamoto K, Ueda J, et al. Isolation and identification of blue light-induced growth inhibitor from light-grown Arabidopsis shoots. Plant Growth Regul 2004a;44:81–6. Hasegawa T, Yamada K, Shigemori H, Hasegava K, Miyamoto K, Ueda J. Isolation and identification of a growth inhibitor from blue light-illuminated cress seedlings. Plant Growth Regul 2002;37:45–7. Hasegawa T, Yamada K, Shigemori H, Miyamoto K, Ueda J, Hasegawa K. Isolation and identification of phototropism-regulating substances benzoxazinoids from maize coleoptiles. Heterocycles 2004b;63:2707–12. Iino M. Mediation of tropisms by lateral translocation of endogenous indole-3-acetic acid in maize coleoptiles. Plant Cell Environ 1991;14:279–86.
545 Larsen E, Christensen LP. Simple method for large scale isolation of the cyclic arylhydroxamic acid DIMBOA from maize (Zea mays L.). J Agri Food Chem 2000;48:2556–8. Nakagawa E, Amano T, Hirai N, Iwamura H. Non-induced cyclic hydroxamic acids in wheat during juvenile stage of growth. Phytochemistry 1995;38:1349–54. Niemeyer HM. Hydroxamic acids (4-hydroxy-1,4-benzoxazin-3-ones), defense chemicals in the Gramineae. Phytochemistry 1988;27:3349–58. Oikawa A, Ebisui K, Sue M, Ishihara A, Iwamura H. Purification and characterization of a ß-glucosidase specific for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin3-one (DIMBOA) glucoside in maize. Z Naturforsch 1999;54c:181–5. Osbourn AE. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 1996;8:1821–31. Pickard BG. Roles of hormones in phototropism. In: Pharis RP, Reed DM, editors. Encyclopedia of plant physiology. New Series, 11. Berlin: Springer; 1985. p. 365–417. Sakoda M, Hasegawa K. Phototropism in hypocotyls of radish. VI. No exchange indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures. Plant Physiol 1989;76:240–2. Sicker D, Frey M, Schulz M, Gierl A. Role of natural benzoxazinones in the survival strategy of plants. Int Rev Cytol 2000;198:319–46. Tamimi SM. Uridine and the control of phototropism in oat (Avena sativa L.) coleoptiles. Plant Growth Regul 2004;43:173–7. Togo S, Hasegawa K. Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles. Physiol Plant 1991;81:555–7. Went FW, Thimann KV. Phytohormones. McMillan: New York; 1937. Yamada K, Hasegawa T, Minami E, Shibuya N, Kosemura S, Yamamura S, et al. Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation. J Plant Physiol 2003;160:255–9. Yamamura S, Hasegawa K. Chemistry and biology of phototropism-regulating substances in higher plants. Chem Rec 2001;1:362–72. Yokotani-Tomita K, Kato J, Kosemura S, Yamamura S, Kushima M, Kakuta H, et al. Light-induced auxininhibiting substance from sunflower seedlings. Phytochemistry 1997;46:503–6. Yokotani-Tomita K, Kato J, Yamada K, Kosemura S, Yamamura S, Bruinsma J, et al. 8-Epixanthatin, a light-induced growth inhibitor, mediates the phototropic curvature in sunflower (Helianthus annuus) hypocotyls. Physiol Plant 1999;106:326–30.