1Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan. Email firstname.lastname@example.org
2Department of Organic Chemistry, Faculty of Science, University of Cádiz, Apdo 40, 11520 Puerto Real, Spain. Email email@example.com
It is well known that 6-methoxy-2-benzoxazolinone (MBOA) and its related compounds inhibit the germination and growth of several plant species. However, the physiological mechanism of MBOA on the germination inhibition is not fully understood. MBOA inhibited the germination of cress and lettuce seeds at concentrations greater than 0.03 mM. Induction of α-amylase activity in cress and lettuce seeds was also inhibited by the MBOA at concentrations greater than 0.03 mM. Both inhibitions increased with increasing concentrations of MBOA, and the extent of the germination was positively correlated with the activity of α-amylase in their seeds. α-Amylase is considered to be essential for seed germination because this enzyme principally triggered starch degradation in endosperm of seeds and enable the seeds to germinate and grow. These results suggest that MBOA may inhibit the germination of cress and lettuce seeds by inhibiting the induction of α-amylase activity. It may be one of the possible action mechanisms of MBOA on inhibition of plant germination.
6-Methoxy-2-benzoxazolinone may inhibit the germination of cress and lettuce seeds by inhibiting the induction of α-amylase activity.
α-Amylase, Allelopathy, Germination inhibition, Lactuca sativa, Lepidium sativum, 6-Methoxy-2-benzoxa-zolinone.
MBOA and its related compounds have attracted attention because of their involvements in allelopathic effects (Barnes and Putnam 1987; Niemeyer 1998; Inderjit and Duke 2003; Belz and Hurle 2004). MBOA was reported to inhibit the germination and growth of several plant species (Pérez 1990; Hayashi et al. 1994; Kato-Noguchi et al. 1998; Kato-Noguchi 2000). However, the physiological mechanism of MBOA on the inhibition is not fully understood.
α-Amylase is considered to be essential for seed germination because this enzyme triggers starch degradation in endosperm of seeds and enable the seeds to germinate and grow (Perata et al. 1992; Perata et al. 1997; Vartapetian and Jackson 1997). It might be possible that MBOA inhibits the germination owing to the inhibition of α-amylase activity in the seeds. Thus, the effects of MBOA on germination and α-amylase activity in cress seeds were investigated.
Seeds of cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) were sterilized in a 2 % (w/v) solution of sodium hypochlorite for 15 min and rinsed four times in sterile distilled water. All further manipulations were carried out under sterile conditions. MBOA was dissolved in a small volume of MeOH and added to two sheets of filter paper in a 9-cm Petri dish and dried. The filter paper in the Petri dish was moistened with 4 ml 0.05% (v/v) aqueous Tween 20. On the occasion, sucrose (1 mM) was added into the medium. Fifty seeds of cress and lettuce were arranged on the filter paper in the Petri dish and germinated in the dark at 25 °C for 6 - 48 h. For determination of α-amylase activity, cress and lettuce seeds were harvested, frozen immediately with liquid N2 and freeze-dried.
Extraction and assay of α-amylase
Freeze-dried cress and lettuce seeds (10 seeds for one determination) were ground to a fine powder in a mortar using a pestle. Then, the powder was homogenized with 1.5 ml of ice-cold solution of 100 mM HEPES-KOH (pH 7.5) containing 1 mM EDTA, 5 mM MgCl2, 5 mM DTT, 10 mM NaHSO3 and 50 mM bovine serum albumin. The homogenate was centrifuged at 30,000 g for 30 min, and the supernatant was heated with 3 mM CaCl2 at 75 °C for 15 min to inactivate β-amylase and α-glucosidase (Sun and Henson 1991; Guglielminetti et al. 1995), and used for α-amylase assay.
α-Amylase was assayed by measuring the rate of generation of reducing sugars from soluble starch. Appropriate dilutions of the enzyme preparations were made, and 0.2 ml of the diluted preparations of the enzyme was added to 0.5 ml of 100 mM sodium acetate (pH 6.0) containing 10 mM CaCl2. Reaction was initiated with 0.5 ml 2.5 % (w/v) soluble starch. After incubation at 37 °C for 15 min, the reaction was terminated adding 0.5 ml of 40 mM dinitrosalicylic acid solution containing 400 mM NaOH and 1 M K-Na tartrate, and then placing immediately into a boiling H2O bath for 5 min. After dilution with distilled water, the A530 of the reaction mixture was measured and reducing power evaluated using a standard curve obtained with glucose (Guglielminetti et al. 1995).
Effects of MBOA on germination of cress and lettuce
The effects of exogenously applied MBOA on germination of cress and lettuce were determined. MBOA inhibited the germination of both seeds and their germinations were completely inhibited by 3 mM MBOA. At concentrations greater than 0.03 mM, MBOA inhibited the growth of cress and lettuce radicles and the concentration required for 50 % inhibition on the growth were 0.18 and 0.15 mM for cress and lettuce radicles, respectively. These results suggest that MBOA inhibited the cress and lettuce germination and the inhibition was increased with increasing MBOA concentrations. The growth inhibition by MBOA was recovered by addition of sucrose to the medium, which suggests that the inhibition may not be caused by the toxicity of MBOA itself. The germination inhibition by MBOA was also found in several other plant species (Pérez 1990; Kato-Noguchi 2000), and uptake of MBOA by plant seeds was significantly faster than its precursor hydroxamic acid, 2,4-dihydroxy-7-methoxy- 1,4-benzoxazin-3-one (Pérez 1990).
Effects of MBOA on α-amylase activity in cress and lettuce
MBOA inhibited the activity of α-amylase in cress and lettuce seeds at concentrations greater than 0.03 mM. The concentration required for 50 % inhibition on the activity was 0.16 and 0.12 mM for cress and lettuce, respectively, as interpolated from the concentration-response curves. These values were almost same as the concentrations required 50 % inhibition on the radicle growth of cress and lettuce. MBOA (0.001, 0.03, 0.1, 0.3, 1 or 3 mM) was added into the reaction mixture for α-amylase assay but the activity of α-amylase was not affected by presence of MBOA in the assay mixture, suggesting that MBOA dose not inhibit in vitro α-amylase activity. These results suggest that MBOA inhibited α-amylase induction at the protein level and the inhibition was increased with increasing MBOA concentrations. In addition, concentration-response curves indicate that the length of cress and lettuce radicles were positively correlated with the activity of α-amylase in their seeds.
Figure 1. Effect of MBOA on germination of cress seeds. Cress seeds were incubated with MBOA in the dark at 25°C for 48 h.
Change in α-amylase activity in cress seeds after incubation was determined. The activity in control seeds (0 mM MBOA) was low at the starting time and increased as the process of germination occurred, where radicles of cress seeds emerged around 16 h after incubation. However, MBOA inhibited the increasing of α-amylase activity and the inhibition was greater with increasing MBOA concentrations. At 48 h, the activities in seeds treated with 0.1 and 0.3 mM MBOA, were 58 and 32 % of that in control seeds, respectively, and the activity in seeds treated with 3 mM MBOA remained almost unchanged.
Plant germination is a complex phenomenon, and many kinds of genes and enzymes are known to participate in this event (Thomas 1993; Conley et al. 1999). During germination respiration accelerates to produce metabolic energy and biosynthetic precursors (Perata et al. 1997). Therefore, soluble sugars that can be readily used in respiration must be supplied constantly to maintain respiratory metabolism and germination. However, the amount of readily utilizable soluble sugars in plant seeds is usually very limited, with starch being the main reserve carbohydrate (Saglio et al. 1999; Guglielminetti et al. 2000). α-Amylase was considered to play a major role in degradation of reserve carbohydrate to soluble sugars during germination (Perata et al. 1997; Vartapetian and Jackson 1997). Thus, induction of α-amylase is essential to maintain active respiratory metabolism which allows germination of plant seeds.
MBOA inhibited the germination of cress and lettuce seeds and the induction of α-amylase in the cress and lettuce seeds. α-Amylase activity in the cress and lettuce seeds reflected the extent of their germination. These results suggest that MBOA may inhibit the germination of cress and lettuce seeds by inhibiting the induction of α-amylase. It may be one of the possible mechanisms for MBOA on inhibition of the germination. The result that exogenously applied sucrose overcame the inhibiting effect of MBOA may support this hypothesis.
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