Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination

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1 Rice Science, 2007, 14(4): Copyright 2007, China National Rice Research Institute. Published by Elsevier BV. All rights reserved Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination REN Yan-fang 1, 2, HE Jun-yu 2, WANG Xiao-feng 1 ( 1 College of Life Science, South China Agricultural University, Guangzhou , China; 2 College of Agriculture, Guizhou University, Guiyang , China) Abstract: To investigate the relationships among β-mannanase, β-mannosidase and α-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA 3 on them were surveyed. The activities of β-mannosidase and α-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA 3 could promote the activities of β-mannanase and α-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase. Key words: germination; β-mannanase; β-mannosidase; α-galactosidase; enzyme activity; rice Cell wall disassembly plays significant roles in plant growth and development, including seed germination, embryogenesis, shoot growth, leaf formation, and fruit ripening and so on [1-3]. The endosperm cell walls of many seeds contain mannan polymers, often in the form of galactomannans. These mannan polymers are a form of storage carbohydrate that is mobilized following germination. They may play additional roles: to store water within the seed during germination and early seedling growth in a form of mucilage, thus reducing the susceptibility to drought, or to restrict germination until an appropriate signal is received to weaken the endosperm cell walls in the region around the radicle, to permit the radicle emergence [4]. Analysis of the cell walls of the rice endosperm reveals that the hemicellulose fraction is largely composed of xylose and arabinose (presumably as arabinoxylans), with a complex mixture of other polysaccharides, in which mannose and galactose is predominate, possibly as galacto-mannans. In total, mannose and galactose accounts for about 6% of the total cell wall sugars [5]. Three enzymes are involved in the hydrolysis of galactomannans. They are β-mannanase (EC ) which cleaves the mannan backbone and produces Received: 29 April 2007; Accepted: 20 July 2007 Corresponding author:wang Xiao-feng (xfwang@scau.edu.cn) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 21, No. 3, 2007, Pages oligomannans (normally contain 2-10 mannose residues), β-mannosidase (EC ) which hydrolyses the oligomannans (mannobiose and mannotriose) released by prior β-mannanase activity, and α-galactosidase (EC ) which concomitantly removes the unit galactose side-chains [6-7]. Of these enzymes, β-mannanase has received the most attention, which is widely occurred in seeds [8]. Many reports have been on the changes in the three enzymes activities during and following the dicot seed germination and the relationships between these enzymes and germination. However, the presence of the enzymes in monocot seeds and the relationships among them are rarely reported. Previous reports showed that many hydrolases increase in cereal grains following germination, which are generally associated with the mobilization of the major storage reserves within the endosperm [9]. Moreover, synthesis and secretion of many of these enzymes are induced by GA and suppressed by ABA [9-10]. The effects of GA and ABA on the activities of the three enzymes degrading galactomannans in endosperm cell wall during and following monocot seed germination have not been reported yet. The aim of the present study was to determine the changes in β-mannanase, β-mannosidase, and α-galactosidase activities and the relationships among them, and also the effects of GA and ABA on the three enzymes during and following

2 296 Rice Science, Vol. 14, No. 4, 2007 rice seed germination. MATERIALS AND METHODS Plant materials and germination conditions Dehulled seeds of rice (Oryza sativa L. cv. Taichung 65) were placed on a piece of filter paper in 7-cm Petri dishes moistened with 5 ml of distilled water, 50 µmol/l GA 3 and 100 µmol/l ABA, respectively. The dishes were sealed to prevent evaporation and incubated in a controlled temperature chamber in darkness at 25 C. At 12-hour intervals, the extracts of the three enzymes were derived from intact seeds or seed parts (aleurone layer, scutellum and endosperm). Enzyme extraction Triplicate lots of 5 intact seeds or 5 samples of different seed parts (aleurone layer, scutellum or endosperm) were ground in an ice cold mortar in 300 µl (for 5 intact seeds) or 200 µl (for 5 samples of the seed parts) 0.1 mol/l Hepes-NaOH (ph 8.0) buffer. The extract was centrifuged at g for 10 min at 4 C and the supernatant was used to assay for β-mannanase activity. The pellet was resuspended in 0.1 mol/l Hepes-NaOH (ph 8.0) buffer plus 0.5 mol/l NaCl and mixed. The extract was allowed to stand at room temperature for 15 min, then centrifuged at g for 15 min at 4 C, and the supernatant was used to assay for β-mannosidase. The extraction of α-galactosidase was a little different from the other two enzymes. The buffer was 0.1 mol/l Hepes-NaOH (ph 8.0) plus 10% polyethylenimine (12 µl for 5 intact seeds and 8 µl for 5 seed parts). Then the enzyme was extracted as β-mannanase. β-mannanase, β-mannosidase, and α-galactosidase assays β-mannanase was assayed using a gel-diffusion assay [11-12]. β-mannosidase and α-galactosidase were assayed according to the methods described by Mo and Bewley [13] and Feurtdo et al [14], respectively. Statistical analysis The results were statistically analyzed by SPSS. RESULTS Effects of different treatments on germination rate of rice seeds Dehulled rice seeds of Taichung 65 germinated in ddh 2 O showed the first signs of radicle emergence at about 24 h from the start of imbibition, and 58% of the seed population germinated by 36 h, germination of the population was almost completed by 72 h (Fig. 1). The times that seeds imbibed in 50 µmol/l GA 3 started and completed germination were nearly consistent with that in ddh 2 O. However, the germination rates in 50 µmol/l GA 3 was increased between 12 h and 72 h compared with that in ddh 2 O. Radicle protrusion from 100 µmol/l ABA-imbibed seeds did not commence until 60 h from the start of imbibition, and at 96 h the germination rate did not reach 50% yet. Obviously, seed germination was delayed in 100 µmol/l ABA. Changes in activities of β-mannanase, β-mannosidase, and α-galactosidase from intact rice seeds Activity of β-mannanase in the dehulled rice grains was firstly detected at 48 h from the start of imbibition, and then increased up to 96 h (Fig. 2). However, the activities of β-mannosidase and α-galactosidase were detected in dry and pregerminated seeds, and the activities of both enzymes increased with the imbibition of seeds. Germination rate (%) Fig. 1. The germination rate of rice seeds.

3 REN Yan-fang, et al. Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination 297 β-mannosidase and α-galactosidase activities (nmol/min seed) β-mannosidase α-galactosidase β-mannanase Fig. 2. Changes in activities of β-mannanase, β-mannosidase and α-galactosidase during and following germination of rice seeds. Changes in activities of β-mannanase, β-mannosidase, and α-galactosidase in different rice seed parts To investigate the changes in enzyme activities in different seed parts during and following germination, dehulled grains imbibed for required hours were dissected into aleurone layer, endosperm and scutellum. β-mannanase, β-mannosidase and α-galactosidase were extracted from these seed parts and the enzyme activities were assayed (Fig. 3). Activities of β-mannosidase and α-galactosidase were presented in aleurone layer of dry and pre-germinated seeds. However, activity of β-mannanase just occurred after seed germination. The three enzymes activities increased with rice seed germination, and the increase of β-mannanase activity was prominent. In scutellum and endosperm of rice seeds, the changes in the activities of the three enzymes were similar to those in aleurone layer. Effects of GA 3 and ABA on activities of β-mannanase, β-mannosidase, and α-galactosidase To determine the effects of GA 3 and ABA on the activities of β-mannanase, β-mannosidase and α-galactosidase, the three enzymes were extracted from different parts of seed imbibed with ddh 2 O, 50 µmol/l GA 3 and 100 µmol/l ABA and assayed (Figs. 4-6). The above results had illustrated that GA 3 promoted the rice seed germination and ABA inhibited it (Fig. 1). Here, the results showed that GA 3 and ABA could regulate the activities of β-mannanase, β-mannosidase and α-galactosidase to some degrees. β-mannanase activity (pkat/seed part) β-mannosidase and α-galactosidase activities (nmol/min seed part) A B C α-galactosidase β-mannosidase β-mannanase Fig. 3. Changes in activities of β-mannanase, β-mannosidase and α-galactosidase in aleurone layer (A), scutellum (B) and endosperm (C) of rice seeds during and following germination. In aleurone layer, GA 3 promoted the activities of the three enzymes. ABA just inhibited the activity of β-mannanase and had no effect on the other enzymes. In scutellum and endosperm, activities of β-mannanase and α-galactosidase were increased by GA 3 treatment, but there was no change in activity of β-mannosidase. However, ABA only inhibited the activity of β-mannanase. DISCUSSION Changes in the activities of β-mannanase, β-mannosidase, and α-galactosidase Seed germination is a series of physiological and chemical processes, including degradation of cell wall. β-mannanase activity (pkat/seed part)

4 298 Rice Science, Vol. 14, No. 4, 2007 α-galactosidase activity β-mannosidase activity β-mannanase activity (nmol/min seed part) (nmol/min seed part) (pkat/seed part) α-galactosidase activity β-mannosidase activity β-mannanase activity (nmol/min seed part) (nmol/min seed part) (pkat/seed part) Fig. 4. Changes in the activities of β-mannanase, β-mannosidase and α-galactosidase in aleurone layer of rice seeds during and following germination treated with ddh 2O, 50 µmol/l GA 3 and 100 µmol/l ABA. Fig. 5. Changes in the activities of β-manganese, β-mannosidase and α-galactosidase in scutellum of rice seeds during and following germination treated with ddh 2O, 50 µmol/l GA 3 and 100 µmol/l ABA. Mannan is a major component of hemicelluloses deposited in the cell walls of a variety of higher plant species. Therefore, there is a close relationship between the degradation of mannan and cell wall. Three enzymes are involved in the hydrolysis of galactomannans: β-mannanase, β-mannosidase, and α-galactosidase [6]. Of these enzymes, β-mannanase was a key enzyme [14]. In our study, the activities of the three enzymes could be detected in both intact seeds and different seed parts and increased during and following rice seed germination (Fig. 2 and Fig. 3). This indicates that all the three enzymes play a role during and following rice seed germination. Many reports have showed that β-mannanase exhibits different activity patterns with plant species during and following germination. The enzyme activity is presented prior to radicle protrusion in the micropylar cap of tomato [15] and Datura ferox [16] seeds. However, it occurrs just after seed germination in fenugreek [17], lettuce [6] and legume [8] seeds. Moreover, this enzyme activity generally increases in most of plant species during seed germination. The presence of β-mannanase in certain time might reflect the difference in the physiological roles of the enzyme: before germination the enzyme is thought to be involved in weakening of cell wall prior to radicle emergence, and cell wall weakening would aid in achieving the release of the embryo from its

5 REN Yan-fang, et al. Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination 299 α-galactosidase activity β-mannosidase activity β-mannanase activity (nmol/min seed part) (nmol/min seed part) (pkat/seed part) Fig. 6. Changes in the activities of β-mannanase, β-mannosidase and α-galactosidase in endosperm of rice seeds during and following germination treated with ddh 2O, 50 µmol/l GA 3 and 100 µmol/l ABA. surrounding constraints to permit the completion of germination [18-19] ; whereas after germination the enzyme is associated with mobilization of cell wall mannan reserves during seedling growth [20-21]. In our experiment, this enzyme activity was presented only after rice seed germination. Therefore, we propose that this enzyme plays a role in mobilization of galactomannan reserves and provides nutrient for seedling growth. β-mannosidase is another enzyme degrading galactomannanas co-worked with β-mannanase and is also found in many plant seeds. Our research showed that a slight activity of β-mannosidase could be detected prior to germination and the activity increased to lesser extent with germination. These results were consistent with previous studies on carob, asparagus and Datura ferox seeds [6, 22-23]. β-mannosidase is generally regarded as being dependent upon prior β-mannanase and α-galactosidase activities to provide its oligomeric mannan substrate. In theory, β-mannanase activity should present prior to β-mannosidase. However, β-mannosidase activity was observed prior to β-mannanase during and following rice seed germination (Fig. 2 and Fig. 3). This was consistent with previous reports on these two enzyme activities in tomato seeds. Whether this had physiological significance was unknown. Mo and Bewley proposed that β-mannosidase was capable of acting as an exo-mannanase to degrade cell wall, but the amount of cell wall degradation it could effect in this region at this time was likely to be minor, compared to the later and much larger activity of β-mannanase [13, 24]. Nevertheless, the coincidental and substantial increases in activities of both enzymes following germination suggest the co-operation of both enzymes in the degradation of endosperm cell wall galactomannan. Besides the above two enzymes, α-galactosidase exhibited high activity in dry seed and pregerminated rice seed (Fig. 2 and Fig. 3). It was evident that α-galactosidase was different from β-mannanase with respect to the expression pattern, but exhibited similarities to β-mannosidase. The situation was also consistent with previous reports on tomato [14] and coffee [25] seeds. It was proposed that α-galactosidase action might be a necessary condition to grant access for β-mannanase to the main chain of the galactomannan polymer in certain situations [26]. The enzyme α-galactosidase could debranch galactose from the main chain of the galactomannan. And Obendorf speculated that α-galactosidase could also be linked to the degradation of raffinose family of oligosaccharides and galactosyl cyclitols, and then provided nutrient for early seedling growth [4, 27]. The effects of GA and ABA on the activities of β-mannanase, β-mannosidase, and α-galactosidase GA and ABA are two important hormones regulating seed germination. Our results showed that the two hormones indeed affected rice seed germination

6 300 Rice Science, Vol. 14, No. 4, 2007 (Fig. 1), as well as had a prominent effect on the activities of the three enzymes presented during and following rice seed germination (Figs. 4 to 6). Previously it was shown that GA 3 could induce an increase in β-mannosidase activity in the isolated endosperms of the gib-1 mutant of tomato seed, but it had no evident effect on the activity of α-galactosidase, in that the higher activity had presented in dry seeds [18]. In addition, GA 3 could promote the activity of β-mannanase in lateral endosperm during tomato seed germination [20]. However, α-galactosidase was not sensitive to ABA during tomato seed germination and there was a less substantial decline in the activity and sustained activity of this enzyme [14]. It was proposed that this enzyme had presented in tomato seeds at the early stage of germination and it was possibly not necessary to promote seed germination. Mo and Bewley also confirmed that ABA had no effect on β-mannosidase in tomato seeds [24]. However, it could inhibit β-mannanase activity in the lateral endosperm of tomato seed [20] and endosperm of lettuce seed [28]. Our observations were consistent with the above results. In conclusion, it is apparent that galactomannans are degraded by the three enzymes co-working during seed germination, but they are regulated by hormones to different extents. Hence, we deduce that three enzymes are synthesized or activated probably under the control of hormones. These still need to be further studied. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant No ) and the Guangdong Provincial Natural Science Foundation (Grant No ). REFERENCES 1 Wang S Q. Cell wall. Biology Teaching, 1999, 25(7): 1 2. (in Chinese) 2 Yan J Q. Structure and function of cell wall in higher plant. Bull Biol, 1999, 34(1): (in Chinese) 3 Filichkin S A, Leonard J M, Monteros A, Liu P P, Nonogaki H. A novel endo-β-mannanase gene in tomato LeMAN5 is associated with anther and pollen development. Plant Physiol, 2004, 134: Bewley J D. Seed germination and dormancy. Plant Cell, 1997, 9: Shibuya N, Iwasaki T. Polysaccharides and glycoproteins in the rice endosperm cell wall. Agric Biol Chem, 1978, 42: Reid J S G, Meier H. Enzymatic activities and galactomannan mobilization in germinating seeds of fenugreek (Trigonella foenum-graecum L. Leguminosae): Secretion of α-galactosidase and β-mannosidase by the aleurone layer. Planta, 1973, 112: Edwards M E, Marshall E, Gidley M J, Grant Reid J S. Transfer specificity of detergent-solubilized fenugreek galactomannanan galactosyltransferase. Plant Physiol, 2002, 129: McCleary B V, Matheson N K. Galactomannan structure and β-mannanase and β-mannanosidase activity in germinating legume seeds. Phytochemistry, 1975, 14: Okamoto K, Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds: Amylase formation in the epithelium. Plant Physiol, 1979, 63: Gomez-Cadenas A, Zentella R, Walker-Simmons M K, Ho T H D. Gibberellin/abscisic acid antagonism in barley aleurone cells: Site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules. Plant Cell, 2001, 13: Downie B, Hilhorst H W M, Bewley J D. A new assay for quantifying endo-β-d-mannanase activity using Congo Red dye. Phytochemistry, 1994, 36: Bourgault R, Bewley J D. Gel diffusion assays for endoβ-mannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: A remedy. Analy Biochem, 2002, 300: Mo B, Bewley J D. β-mannosidase (E.C ) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds: Purification, cloning and characterization. Planta, 2002, 215: Feurtdo J A, Banik M, Bewley J D. The cloning and characterization of α-galactosidase present during and following germination of tomato (Lycopersicon esculentum Mill.) seed. J Exp Bot, 2001, 52: Nonogaki H, Matsushima H, Morohashi Y. Galactomannan hydrolyzing activity develops during priming in the micropylar endosperm tip of tomato seeds. Plant Physiol, 1992, 85: Bewley J D. Breaking down the walls a role for endoβ-mannanase in release from seed dormancy? Trends Plant Sci, 1997, 2: Dirk L M A, Griffen A M, Downie B, Bewley J D. Multiple isozymes of endo-β-d-mannanase in dry and imbibed seeds. Phytochemistry, 1995, 40: Groot S P C, Rokicka B K, Vermeer E, Karssen M. Gibberellin-induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protrusion.

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