Preferential Secretion of R-Type ai-Amylase Molecules in Rice. Seed Scutellum at High Temperatures1. Received for publication March 20, 1986 and in revised ...
Plant Physiol. (1986) 82, 880-884 0032-0889/86/82/0880/05/$O 1.00/0
Preferential Secretion of R-Type ai-Amylase Molecules in Rice Seed Scutellum at High Temperatures1 Received for publication March 20, 1986 and in revised form August 14, 1986
TOSHIAKI MITSUI2 AND TAKASHI AKAZAWA*
Research Institute for Biochemical Regulation, School ofAgriculture, Nagoya University, Chikusa, Nagoya 464, Japan accumulate in the Golgi complex (4, 1 1). During this step, the sugar moiety of hemagglutinin was shown to become Endo-3-H Exposure of fresh scutella excised from 4-day-old rice seedlings to resistant and the sugar moiety of G-protein acquired the sialic higher temperatures, (40-420C), drastically reduced the biosynthesis of acid residue. These experimental findings indicate that protein a-amylase as determined by the incorporation of I35Simethionine into the molecules are transported to the trans face of the Golgi cisternae, immunoprecipitable product. However, the intracellular transport and where the terminal glycosylation reactions are known to occur. extracellular secretion of the enzyme molecules were enhanced at high In baby hamster kidney cells infected by VSV at 20°C, it has temperatures, indicating that the biosynthesis and secretion of a-amylase been demonstrated that the trans face Golgi is morphologically are distinguishable in their temperature dependency. At the higher tem- dilated because of the accumulation of G-protein molecules, perature regime (_40°C), the complex-type a-amylase isoform, resistant which was revealed by immunoelectronmicroscopic examination to hydrolytic digestion by endo-B-N-acetylglucosaminidase H (Endo-$- (5). These results suggest that in animal systems the transport of H) was predominantly secreted, whereas at lower temperatures (_15°C), proteins from the trans Golgi to the cell surface may be supthe isoform susceptible to Endo-,B-H attack was the major molecular pressed under certain critical temperatures, and it is intriguing form secreted. to ask whether similar mechanism(s) operates in the biosynthesis of rice seed a-amylase when subjected to high temperatures. In the work presented in this communication, we have attempted to examine the effect of temperature on the biosynthesis and secretion of a-amylase molecules in the scutellar epithelium tissues of rice seedlings. Special attention has been given to the Since the germination of cereal seeds is known to be sensitive structural modification of the oligosaccharide sugar moiety of to environmental temperatures (14), the effect of temperature on the enzyme molecule as affected by high temperature. the secretary process (exocytosis) in higher plant systems has MATERIALS AND METHODS received attention of several investigators in recent years. For example, Fadeel et al. (3) reported that the extracellular discharge Purification of a-Amylase and Preparation of Anti-a-Amylase of a-amylase molecules synthesized from the aleurone layers of IgG. a-Amylase was purified from whole rice (Oryza sativa L. barley seeds exhibits a prominent change between 19 and 21C. cv Kimmaz6) seeds 7-d after germination as reported previously Since the content of total enzyme protein extractable from the (13). The enzyme preparation was used to raise the rabbit antialeurone tissues per se did not change under this temperature a-amylase IgG. The immunochemical specificity of the IgG was range, the above authors speculated that temperature affected tested by double immunodiffusion using an agar block as well as the secretary mechanism. However, in contrast to the low tem- by immunoelectrophoresis. perature stress on the secretion of proteins, there are relatively Preparation of Rice Scutellar Tissues. Rice seeds were sterifew reports dealing with the effect of high temperature on the lized in a 1% NaOCl solution for 15 min, thoroughly rinsed in secretary process. sterile H20, and germinated for 4 d at 30TC in the dark. Scutella, It is generally recognized that low temperature markedly influ- free of endosperm and consisting of scutella proper and epithelial ences the extracellular secretion of proteins in animal cells. There tissues, were freshly dissected from the seedlings under sterile are several reports showing the retardation of the exocytosis of conditions. either secretary proteins or membranous proteins at around Pulse-Label and Pulse-Chase Experiments. In the standard 20C. For example, by shifting down the incubation temperature pulse-label system, 20 pieces of fresh scutella were preincubated from 37 to 20°C either influenza hemagglutinin or G-protein of for 3 h at 30°C in 0.3 ml of 20 mm Tris-HCl (pH 7.0) containing VSV3 infected Media-Darby canine kidney cells were shown to 10 mm CaCl2 and 10 gg/ml each of penicillin G and streptomycin (buffer A). They were then incubated in 0.3 ml of buffer A 'This is paper No. 21 in the series "Enzymic Mechanism of Starch containing either [35S]methionine (20 ,uCi), [3H1mannose (100 Breakdown in Germinating Rice Seeds." The research was financially ,uCi), or [3H]fucose (100 MCi), for 3 h at different temperatures ABSTRACT
aided by grants from the Japanese Ministry of Education (Mombusho) and Mitsubishi Foundation (Tokyo). 2 Holder of Fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists, 1986,4-1986,6. Present Address: Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-21, Japan.
(10-420C).
In the case of pulse-chase experiments, 20 scutella were incubated for 1 h at 30°C in 0.3 ml of buffer A (pH 7.0) containing [35S]methionine (50 ,Ci). Afterward, they were chased in 0.3 ml of buffer A (pH 7.0) containing 1 mm nonradioactive methionine for 3 to 6 h at different temperatures (1 2-40°C). After selected time periods of pulse or pulse-chase, as indicated 'Abbreviations: VSV, vesicular stomatitis virus; Endo-,B-H, endo-f,in each experimental system, radioactivity incorporated into aN-acetylglucosaminidase H; hsp, heat shock protein. 880
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FIG. 1. Effect of temperature on biosynthesis, intracellular transport and extracellular secretion of a-amylase. Twenty segments of freshly dissected scutella of rice seedlings at the 4-d germination stage were placed in 0.3 ml of buffer A (pH 7.0), and incubated for various periods at the indicated temperatures (see text). A, Incorporation of [35S]methionine into both intracellular and extracellular fractions. Total radioactivity in the immunoprecipitable enzyme molecules produced at 30'C was arbitrarily defined as 10 units. The incorporation of [35S]methionine into a-amylase was constant up to 3 h incubation, and the data are mean values of triplicate experiments. B, Incorporation of [35S]methionine into the total protein fraction at various incubation temperatures was examined by fluorography. From the densitometric analysis, percent contribution of 70 kD hsp in the total protein component is shown in the figure. C, Experimental protocols for the pulse-chase labeling to determine the intracellular transport and extracellular secretion of [a35S]amylase molecules at various temperatures are given in the upper part. Time-dependent secretion of a-amylase molecules measured at various incubation temperatures is presented in bottom. In each experiment, the total amount of [a35S]amylase in intracellular plus extracellular fractions after chase was arbitrarily defined as 1 unit.
amylase in either intracellular (tissue extracts) or extracellular (incubation media) fractions was determined. To prepare the tissue extracts, scutella were homogenized in a small mortar using 0.5 ml of buffer A containing 0.1% (v/v) Triton X-100. Radioactivities incorporated into the hot TCA-insoluble fraction of either scutellar extracts or incubation media were measured in a liquid scintillation spectrometer (Aloka liquid scintillation system LSC-700). Each of the tissue extracts or samples of incubation media were then subjected to immunoprecipitation, SDS-PAGE and fluorography as described below. To examine the incorporation of [35S]methionine into the total protein fraction at various incubation temperatures, aliquots of the scutellar
extracts prepared in the pulse-labeling experiments were directly applied to SDS-PAGE and fluorography. Immunoprecipitation, SDS-PAGE and Fluorography. Experimental procedures employed were identical to those described previously (12). Aliquots of the scutellar extracts (100 41) and incubation media (100 gl) were made to 1% (w/v) with SDS, heated at I0OC for 2 min, and finally made to 2% with Triton X-100. After addition of the buffer solution (0.5 ml) containing 20 mM Tris-HCl (pH 7.5), 0.1% Triton X-100, and 0.14 M NaCl to the mixture, 25 ug of anti-a-amylase IgG was added and the samples were incubated for 1 h at 30TC. Protein A-Sepharose (2.5 mg dry gel/tube) was added and the whole mixture was
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1 /T X1 03(K-1) FIG. 2. Arrhenius plot analysis of biosynthesis and secretion of aamylase. Data shown in Figure 1 were converted to the Arrhenius plots. A, Biosynthesis of a-amylase (from Fig. lA) and 70 kD hsp (from Fig. B). B, Initial velocity (v) of extracellular secretion of a-amylase (from
Fig. IC).
vigorously shaken for 2 h at 30°C, and then the slurry was poured onto a small column to retain the Protein A-Sepharose-immu-
noprecipitate complex. The immunoprecipitates were eluted from the column with 50 gl of 1% SDS-solution. a-Amylase molecules dissociated from IgG by heating at 100°C for 2 min were subjected to the radioactivity measurements. The immunoprecipitates were analyzed by SDS-PAGE following the procedure of Laemmli (9). After electrophoresis, the gels were used for fluorography using Enhance (New England Nuclear) and exposed to a sensitized (H2 gas-treated and preflashed) x-ray film (Fuji Rx) at -80°C (10). Fluorograms were then analyzed by densitometry using a Shimadzu CS-9 10 dual wavelength scanner as described earlier ( 12). Treatments of a-Amylase and Cellular Glycoproteins with Endo-fl-H. The hydrolytic digestion of the oligosaccharide chains in a-amylase molecules and cellular glycoproteins with Endo-,BH was carried out as previously reported (8, 12, 16). Tissue extracts and incubation media were adjusted to pH 5.0 with 0.1 M citrate-phosphate buffer or 0.1 M acetate buffer, and after adding Endo-,3-H (-'20 milliunits/ml), the whole mixture was incubated for 3 h at 30°C. After incubation, radioactivity in the hot TCA-insoluble fraction of the reaction mixture was measured. The reaction mixtures containing [a35S]amylase molecules were neutralized and then subjected to immunoprecipitation, SDS-PAGE and fluorography. Materials. The following commercial products were used. [35S] methionine ( 1330 Ci/mmol), -[5-3H]mannose (28.1 Ci/mmol), and L-[5,6-3H]fucose (56 Ci/mmol) from the Radiochemical Center, Amersham; penicillin G and streptomycin from Meiji Seika Co. Ltd; Endo-,3-H was a kind gift from Dr. T. Okuyama (Seikagaku Kogyo, Tokyo).
Plant Physiol. Vol. 82, 1986
RESULTS Effect of Temperature on Biosynthesis and Intracellular Transport of a-Amylase. The biosynthesis and intracellular transport of a-amylase in the scutellar epithelium of rice seedlings under various temperatures (10-42C) was studied using pulse-label and pulse-chase techniques. Figure IA shows that maximal incorporation of [35S]methionine into the immunoprecipitable total a-amylase (intracellular plus extracellular) occurred at 35°C, and at higher temperatures, the level of incorporation declined abruptly. Figure lB shows the fluorogram of 35S-labeled total protein molecules extractable from the scutellar tissues. It is clear that a protein, Mr about 70,000 became prominently radioactive above 37C. Labeling of the 70 kD protein paralleled the decline of the formation of other protein molecules, and at 42°C, accounted for 20% of the total protein component. We designate this unique protein as hsp, because its synthetic pattern and molecular size appears to be similar to hsp in other systems (1, 2, 7). To examine the effect of environmental temperature on the intracellular transport and secretion of a-amylase, pulse-chase experiments were conducted following the experimental protocol as given in the upper part of Figure IC. The immunoprecipitable a-amylase labeled with [35S]methionine in the extracellular fractions was quantified and results presented in Figure IC. In contrast to the pattern of biosynthesis (cf Fig. IA), it is evident that a-amylase secretion is enhanced by elevating temperature up to 40C. The rate of secretion at each temperature has been calculated from the slope in initial velocity (v) and the following values were obtained taking v at 30°C as 10; v (40°C) = 14.7; v (30°C) = 10; v (22°C) = 7.4; V(15°C) = 2.9; v (12°C) = 2.3. The data of Figure 1, A and B, were used to construct the Arrhenius plots which are shown in Figure 2A. Two inflection points at 13 and 37°C are apparent in the biosynthesis of aamylase. Interestingly, whereas the biosynthesis of 70 kD hsp continues to increase above 37C, the production of a-amylase starts to decline sharply above this temperature. In contrast, the rate of a-amylase secretion only shows one inflection point (22°C) in the temperature range of 12 to 40°C (Fig. 2B). These results indicate that the temperature dependency of a-amylase biosynthesis is separate from that of the intracellular transport and secretion. Effect of Temperature on the Formation of a-Amylase Isoforms. Since our previous studies (12) have shown that two molecular forms of a-amylase are produced in the rice seedlings, we then examined the temperature effect on the formation of Rand S-type a-amylase molecules. Scutellar tissues were incubated for 3 h with [35S]methionine in the presence of 10 mm Ca2l or 0.1 mm EGTA at various temperatures. The extracellularly secreted or intracellularly accumulated [a35S]amylase molecules were digested by Endo-fl-H, and each of R- and S-type enzyme species were analyzed from the densitometric tracing of the fluorograms (12). The results presented in Figure 3A show the temperature-dependent shift of S-type a-amylase to R-type enzyme; above 40°C nearly all the enzyme molecules secreted are R-type, whereas at low temperature (4°C, 15°C) the dominant species are S-type. The temperature effect on the intracellular accumulation of a-amylase clearly shows that in the absence of Ca2" (+0.1 mM EGTA), the formation of R-type a-amylase is scarcely detectable at both 30°C and 42°C (Fig. 3C). The addition of 10 mM Ca2` promotes the production of R-type enzyme molecules, although the net enzyme formation is reduced by elevating temperature (Fig. 3B). These results indicate that the temperature dependent shift of the S-type a-amylase to the Rtype requires Ca2+. Effect of Temperature on the Biosynthesis of Nonsecretory Glycoprotein. There are many nonsecretory glycoproteins other than a-amylase in the rice scutellar tissues, though they are not
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FIG. 3. Effect of temperature on terminal glycosylation of a-amylase molecules. The basic experimental protocols for radiolabeling of a-amylase molecules and separation of R- and S-type a-amylase species by Endo-f,-H digestion are described in the text. A, Distribution pattern of R- and Stype [a35S]amylase molecules in the extracellular fractions at various incubation temperatures. The pulse-labeling reaction mixture contained 10 mM Ca2", and the incubation was performed for 3 h. B, Distribution pattern of R- and S- type [a35S]amylase molecules intracellularly accumulated at 4 different incubation temperatures. The reaction mixtures contained 10 mm Ca2. C, Absence of R-type [a35S]amylase in the intracellular fractions synthesized without Ca2" at two different incubation temperatures (30 and 42°C). All other experimental conditions were the same as those in Figure 3, A and B, except for adding 0.1 mM EGTA to the reaction mixtures.
884
MITSUI AND AKAZAWA
Plant Physiol. Vol. 82, 1986
accelerated accumulation of a-amylase at the lower temperature regime (1 5-20°C). It has been reported that the secretion of aScutella were incubated with [3H]mannose or [3H]fucose for 3 h at amylase from barley seed aleurones is markedly suppressed below two different temperatures (15 or 40°C). After removing incubation 20°C (3). It is likely that this lower threshold temperature is media, the whole tissues were homogenized with 0.1% Triton X-100 critically linked to the transport of a-amylase molecules. We have previously reported that the secretion of the R-type solution. Aliquots of the tissue extracts were subjected to the Endo-,B-H a-amylase molecules is stimulated by Ca2", whereas that of the digestion as described in the text. Radioactivity in the hot TCA-insoluble fraction of either Endo-fl-H treated or nontreated tissue extracts were S-type molecules is indifferent to the external Ca2" level (12). In the present investigation, it has been found that above 40°C the measured. R-type enzyme molecules are preferentially secreted in the presRadioactivity in Proteins ence of Ca2+, whereas below 15°C the dominant species secreted are the S-type (Fig. 3). From these results, it can be inferred that at Temperature: Substrate the secretory process of a-amylase isoforms in the rice seedlings, 150C 400C i.e. R- and S-type, is under the control of environmental factors, dpm such as temperature and Ca2'. Thus experimental manipulation causing the preferential secretion of R-type a-amylase molecules [3H]Mannose separate from the S-type may lead to an understanding of the 0.77 x 106 1.14 x 106 (A) Control precise nature of secretory process in plant cells. The modifica0.72 x 106 0.29 x 106 (B) Endo-fl-H treated tion of the oligosaccharide chain making up the R-type a-amylase 0.38 0.63 (B) /(A) species was shown to be profoundly affected by the environmen[3H]Fucose tal temperature, and a similar temperature effect appears to 0.95 x 106 0.40 x 106 (C) Control operate in the sugar modification of the nonsecretory glycopro0.32 x 106 0.73 x 106 (D) Endo-,B-H treated tein molecules (Table I). There have been reports of structural 0.77 0.80 (D) /(C) alterations in the sugar moiety of glycoproteins induced by [3H]Fucose/[3H]Mannose environmental stresses. For example, structural changes in the 0.83 0.52 (C) /(A) oligosaccharide chain of glycoprotein molecules of the sindbis coat in infected chick embryo fibroblasts have been revirus as well structurally characterized. For the purpose of studying that are due to altered host-growth (6). Also the oligosacported the effect of temperatures on their biosynthesis, we examined the charide molecules linked to Asn-N in cell surface glycoproteins incorporation of [3H]mannose and [3H]fucose into protein frac- in cancer cells are more highly branched compared to those tions and their Endo-fl-H digestibility. Scutellar tissues were existing in normal cells (15). It is thus conceivable that the incubated with [3H]mannose or [3H]fucose for 3 h at 40 and structure of the oligosaccharide chain in the glycoprotein mole15°C. Subsequently, the whole tissues were homogenized with cule is delicately altered by environmental conditions. 0.1% Triton X- 100, and the radiolabeling of the hot 10% TCAinsoluble fractions was determined. As can be seen from Table LITERATURE CITED I, the ratio of the radioactivities, [3H]fucose/[3H]mannose, of the BONNER 1979 The induction of gene activity in Drosophila M, JJ 1. ASHBURNER sample at 40C was greater than that at 1 5C. Further, Endo-,Bby heat shock. Cell 17: 241-254 H digestibility of the labeled total proteins was examined, and 2. COOPER P, T-H D Ho 1983 Heat shock proteins in maize. Plant physiol 71: the hydrolytic susceptibility of [3H]mannose labeled fractions 2 15-222 A, BA MOLL, RL JONES 1980 Effect of temperature on the synthesis produced at 1 5°C was found to be larger than that of the sample 3. FADEEL and secretion of a-amylase in barley aleurone layers. Plant Physiol 66: 466produced at 40C [(B)/(A)]. On the other hand, as to the [3H] 470 fucose labeled glycoprotein molecules, the samples produced at 4. FULLER SD, R BRAVO, K SIMONS 1985 An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share 40C and 15C were resistant to Endo-,B-H [(D)/(C)]. These the same late Golgi compartments. EMBO J 4: 297-307 results show that the glycosylation reactions involved in the 5. GRIFFITHS G, S PFEIFFER, K SIMoNs, K MATLIN 1985 Exit of newly synthesized formation of nonsecretory glycoproteins in the rice seedlings are membrane proteins from the trans cisternae of the Golgi complex to the also subject to the environmental temperature stress. plasma membrane. J Cell Biol 101: 949-964 Table 1. Effect of Temperature on Terminal Glycosylation of Total
Cellular Glvcoproteins
DISCUSSION In the present study we have examined the influence of environmental temperature on the biosynthesis and secretion of aamylase molecules in rice scutellar epithelium. Results presented in Figure 1 show that in a temperature range where the synthesis of hsp is prominent, the formation of a-amylase molecules is abruptly suppressed but their intracellular transport and secretion are scarcely affected by the heat stress. Overall observation supports the notion that the biosynthesis and secretion of aamylase molecules are distinguishable in temperature-dependency, presumably governed by distinct mechanisms. By studying the temperature effect on the germination potential of rice seeds, and based on the Arrhenius plot analysis, Nishiyama (14) has reported the presence of an inflection temperature at 1 7C. From the results in Figure 2, it can be seen that both biosynthesis and secretion of a-amylase molecules in the rice scutellar tissues are retarded by lowering the environmental temperature, and that two inflection points, 13 and 22°C, are present. Interestingly, as can be seen from Figure 3B, the intracellular accumulation of [a35S]amylase molecules at 15 or 20°C is more conspicuous compared to that occurring at 30°C; these results indicate an
6. HAKIMI J, PH ATKINSON 1980 Growth-dependent alterations in oligomannosyl glycoproteins expressed in sindbis virus glycoproteins. Biochemistry 19: 56 19-5624 7. KEY JL, CY LIN, YW CHEN 1981 Heat shock proteins of higher plants. Proc Natl Acad Sci USA 78: 3526-3530 8. KoIDE N, T MURAMATSU 1974 Endo-,5-N-acetylglucosaminidase acting on carbohydrate moieties of glycoproteins: purification and properties of the enzyme from Diplococcus pneumoniae. J Biol Chem 249: 4897-4904 9. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227: 680-685 10. LASKEY RA, AD MILLS 1975 Quantitative film detection 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56: 335-341 11. MATLIN KS, K SIMONS 1983 Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34: 233-243 12. MITSUI T, T AKAZAWA, JT CHRISTELLER, AM TARTAKOFF 1985 Biosynthesis of rice seed a-amylase: two pathways of amylase secretion by the scutellum. Arch Biochem Biophys 241: 315-328 13. MIYATA S, K OKAMATO, A WATANABE, T AKAZAWA 1981 Enzymic mechanism of starch breakdown in germinating rice seeds. 10. In vivo and in vitro synthesis of a-amylase in rice scutellum. Plant Physiol 68: 1314-1318 14. NISHIYAMA I 1975 A break on the Arrhenius plot of germination activity in rice seeds. Plant Cell Physiol 16: 533-536 15. SANTER UV, F GILBERT, MC GLICK 1984 Change in glycosylation of membrane proteins after transfection of NIH 3T3 with human tumor DNA. Cancer Res 44: 3730-3735 16. TARENTINO AL, TH PLUMMER JR, F MALEY 1974 The release of intact oligosaccharides from specific glycoproteins by endo-fl-N-acetylglucosaminidase H. J Biol Chem 249: 8 18-824
