appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of ...
Bot. Mat. Tokyo 97 : 323 331, 1984
The Botanical Magazine, Tokyo 9 by The Botanical Society of Japan 1984
Hydrolytic E n z y m e Activities in Germinating Spores of Adiantum capillus-veneris L. TOMOKAZU KOSHIBA, TAKAO MINAMIKAWA AND MASAMITSU WADA
Department of Biology, Tokyo Metropolitan University, Fukazawa 2-1, Setagaya-ku, Tokyo 158
Changes in hydrolytic enzyme activities were investigated during spore germination of Adiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imtoibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination. Key words: Adiantum capillus-veneris L. (fern)--Aminopeptidase--Amylase --Spore germination (fern)--Hydrolytie enzymes--Lipase. Seed germination of flowering plants has been investigated from various aspects. As regards the mobilization of seed reserves, it was shown t h a t protein, lipid and starch w h i c h were stored as reserve materials in special tissues of d r y seeds were d e g r a d e d d u r i n g germination a n d t r a n s p o r t e d to an e m b r y o or e m b r y o n i c axis for g r o w t h (Bewley and Black, 1978). I n contrast, fern spores have single-cell structures in w h i c h reserve materials are stored, so t h a t there is no need for the fern spore to t r a n s p o r t the degraded material across cells. This suggests t h a t there m i g h t be great differences between p l a n t seeds and fern spores in the processes of metabolism of reserve materials d u r i n g germination. F e r n spores are good materials for s t u d y of m e t a b o l i s m d u r i n g germination, because spore germination is well k n o w n to be controlled b y light ( F u r u y a , 1983). However, little information is available on Abbreviations : AAPase, alanine aminopeptidase ; LAPase, leucine aminopeptidase ; SDS, sodium dodecylsulfate.
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mobilization of reserve materials during fern spore germination (gaghavan, 1980). Most of the fern spores contained lipids and proteins as reserve materials and these reserves were believed to be degraded spore germination for energy and carbon sources (Raghavan, 1980). Towill and Ikuma (1975) reported that proteins and lipids stored in unimbibed dry spores of Onoclea were hydrolyzed during germination. Metabolism of reserve lipids and related enzyme activities were studied during spore germination of Anemia phyllitidis (Gemmrich, 1979a, b, 1981) and Pteris vittata (Gemmrich, 1980). However, hydrolytic enzymes for other reserve materials have not been studied. We investigated the compositional changes of cell constituents in germinating spores of Adiantum capillus-veneris L. (Minamikawa et al., 1984) and have shown that lipid is stored in dry spores as the main reserve material and that soluble and insoluble carbohydrates also provide carbon and energy sources for the early stage of spore germination. In the present study, changes in hydrolytic enzyme activities which participate in degradation of such reserve materials during dark incubation and following germination are investigated.
Materials and Methods
Plant materials and enzyme extraction Spores of Adiantum caioillus-veneris L. were collected in a greenhouse of the Department of Botany, University of Tokyo in the summer of 1981 and 1982. Culture conditions were the same as described previously in detail (Minamikawa et al., 1984). Two grams of spores were washed with 1% sodium hypochlorite solution containing a few drops of 0.5% SDS and sterilized water, successively. T h e sterilized spores suspended in 50 ml of distilled water were incubated at 25 C for 3 days in the dark, then sterilized again with sodium hypoehlorite (the same concentration as above). The spores were transferred into Petri dishes (5.5-cm in diameter) and they were placed at 25 C under total darkness for dark control or under continuous red light of ca. 1.1 W . m -2 for induction of spore germination. After about 2 days of red light irradiation, rhizoidal cell appeared synchronously and on the next day protonemal cell started to grow. Germination rate was about 50-750/0. By 7 days of red light irradiation the protonemal cell grew with a very low rate of cell division up to about 150-250 ~m. No germination took place when spores were cultured under continuous darkness. At day 3, 6, 8 and 10, spores were collected by centrifugation, washed with distilled water and lyophilized. Germinated spores of day 8 and 10 were separated from ungerminated ones by sieving through a nylon mesh (62 zm). Dry spores and the freeze dried spores or protonemata (100-200 mg) were homogenized with 1/2 weight of Polyclar AT and 7.5-10 volumes of 50 mM Tris-HC1 buffer (pH 7.4) containing 10 mM 2-mercaptoethanol in a cold mortar and pestle. Homogenate was centrifuged at 25,000 • g for 20 rain, and the supernatant solution was used as enzyme solution. All procedures of enzyme extraction were performed at 0-4 C.
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Enzyme assays Lipase activity was assayed by the method of Davies and Chapman (1979) using is-nitrophenyl eaprate and p-nitrophenyl palmitate as substrates. The reaction mixture (500/1l) containing 0.5 mM substrate, 50 mM Tris-HC1 buffer (pH 8.5), 0.5% Triton X-100 and 50/L1 enzyme solution was incubated at 30 C and the reaction was followed photometrically at 410 nm. Amylase activity was assayed as described previously using the dinitrosalicylie acid method (Koshiba and Minamikawa, 1981). Starch phosphorylase activity was measured according to the method of Levi and Preiss (1978) with some modifications. The reaction mixture contained 50mM H E P E S buffer (pH 6.8), 10 mM sodium phosphate (pH 7.4), 0.12 mM NADP, 30/~M glucose-l,6-diphosphate, 1 mg of soluble starch (Merk), 10/lg of P-glucomutase (rabbit muscle, Boehringer Mannheim), 5/zg of glucose-6-phosphate dehydrogenase (Yeast, Boehringer Mannheim) and enzyme solution in 1 ml total volume. The increase in absorbance at 340 nm was recorded at 30 C. Aminopeptidase activity was determined photometrically as described previously (Mitsuhashi et al., 1984), and RNase activity was assayed according to the method of Takaiwa and Tanifuji (1978) using torula yeast RNA as substrate. One unit of all enzyme activities was defined as the activity required to increase one unit in the absorbanee at each wavelengths/hr under the assay conditions. The values of enzyme units were expressed on the basis of g original spore weight but not of protein contents in spores, because this expression is convenient for calculating the enzyme activity per individual germinating spore (1.5 mg spores is ca. 2 x 104 spores, see Nagatani et at., 1983) when protein content is also changing (Minamikawa et al., 1984), and for comparing with the changes in contents of cell constituents in spores shown in the previous paper (Minamikawa et al., 1984). Polyacrylamide gel electrophoresis and detection of enzyme activity on the gel Polyacrylamide gel electrophoresis was performed using 7.5% polyacrylamide gel of 1.5 mm thickness in a pH 9.5 system at 4 C for 4-6 hr. Enzyme solution (35/~l) was loaded on the gel slit with 20% sucrose (w/v). After the electrophoresis, enzyme activities on the gel were detected using soluble starch (for amylase), and L-alanine- or L-leucine-fl-naphtylamide (for AAPase or LAPase) as substrate according to the methods of previous papers (Koshiba and Minamikawa, 1981 ; Mitsuhashi et al., 1984). Results and Discussion
L@ase Changes in lipase activity during dark incubation and germination are shown in Fig 1. Relatively high activity was detected in the extract from dry spores, but the activity decreased during dark incubation. However, when germination was induced by red light, the activity started to increase accompanied by protonemal elongation. Our previous experiments related to changes of intracellular reserve constituents during gemination showed that lipid, the major reserve component, decreased during dark incubation of early imbibition stage and following germination processes
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Fig. 1. Change in lipase activity of Adiantum capillus-veneris L. spores during germination. Spores were allowed to imbibe and cultured at 25 C in the dark (--O--). At day 3, a portion of spores were irradiated by red light (---O--). Values of each points were presented as averages of two experiments. Enzyme extraction and assay were described in Materials and Methods. A, p-nitrophenyl palmitate substrate ; B, p-nitrophenyl eaprate substrate. (Minamikawa et al., 1984). Thus, it is likely that the decrease of total amount of lipid during early dark incubation and spore germination results from the action of lipase. Gemmrich reported, in contrast, that the activities of isocitrate lyase (1979b) and lipase (1982) of A n e m i a phyllitidis were not detected in dry spores b u t increased rapidly during germination under white light until day 7, then decreased. We still do not have a suitable explanation as to why the developmental patterns of lipase were different between A d i a n t u m and Anemia.
Amylolytic activity When the amylase activity was assayed by the dinitrosalicylic acid method, the activity which existed at a relatively high level in dry spores rapidly decreased during dark incubation and germination under red light (Fig. 2A). Zymogram patterns of the amylase activity assayed b y I2-reaction also showed that the intensities of 2 dominant bands (band a and b) and a number of other faint bands decreased during dark incubation, b u t band a increased after spore germination induced b y red light irradiation (Fig. 3). The discrepancy in amylase activities under red light shown b y the two different methods cotrld be explained in the following manner. In the dinitrosalicylic acid method, the activity was shown as total amount of reducing sugar which was released from starch b y total reactions of all amylolytic enzymes and their co-actions. On the other hand, in zymogram patterns on the gel, enzymes were separated individually, so that no co-actions could have occurred. Further, the activity of each enzyme in a zymogram pattern was detected as a decrease of I2reaction to the ~-helical structure of starch. The activities of band a and b were not
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