Physicochemical and Serological Characterization of Rice a … · Plant Physiol. (1 996) 1 1 O: 1395-1 404 Physicochemical and Serological Characterization of Rice a-Amylase lsoforms

Plant Physiol. (1 996) 1 1 O: 1395-1 404

Physicochemical and Serological Characterization of Rice a-Amylase lsoforms and Identification of Their

Corresponding Cenes'

Toshiaki Mitsui*, Junji Yamaguchi, and Takashi Akazawa

Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-21, japan (T.M.); Nagoya University BioScience Center, Chikusa, Nagoya 464-01, Japan (J.Y.); and Suzuka

lnternational University, Suzuka 51 0-02, Japan (T.A.)

We have identified, purified, and characterized 1 O a-amylase isoforms from suspension-cultured rice (Oryza safiva L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, C , H, I, and 1. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, C , H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-C49) inhibited the activities of isoforms E, C, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and 2. Judging from their physicochemical and serological properties, we classified the rice a-amylase isoforms into two major classes, class I (A, B, Y, and Z) and class II (E, F, C , H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, C , H, I, and J). Partia1 amino acid sequences for isoforms A, E, C, and H were also determined. In addition, the recombinant a-amylases expressed by plasmid pEno/lO3 containing the rice a-amylase gene RAmylA in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmylA, isoforms C and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.

The amylolytic breakdown of reserve starch in the endosperm of cereal seed is a prerequisite step for seed germination and subsequent seedling growth in terms of both energy production and provision of carbon skeletons of new cellular components. a-Amylase (EC 3.2.1.1) plays an important role by catalyzing the hydrolytic cleavage of interna1 a-1,4-glucan bonds of starch. It is now well estab- lished that a-amylase is initially expressed in the scutellar epithelial tissue of starchy seeds and then later expressed at high levels in the aleurone layer. The a-amylase synthesized in both tissues has been shown to be secreted subsequently into the starchy endosperm (Akazawa et al., 1988; Fincher, 1989; Itoh et al., 1995).

This research was supported in part by Iijima Memorial Food

* Corresponding author; e-mail [email protected]; fax Science Foundation (to T.M.).

81-25-263-1659.

Multiple isoforms of a-amylase are present in cereal seeds. In barley, isoforms can be separated into two groups having distinct pIs, high pI (5.9-6.6) and low pI (4.5-5.5) (Jacobsen and Higgins, 1982). These two groups differ in several other characteristics, including sensitivity to Ca2+, sulfhydryl reagents, and heavy metals; pH stability; serological properties; and GA, response. Isoforms within a group are similar to each other. A number of a-amylase cDNA clones have been isolated from barley aleurone and subsequently sequenced (Rogers and Milliman, 1983; Chandler et al., 1984; Huang et al., 1984; Deikman and Jones, 1985; Rogers, 1985). Southern blot analyses of genomic DNA have demonstrated that these genes fall into two classes; about three belong to the low-pI class and seven or eight belong to the high-pI class (Muthukrishnan et al., 1984; Chandler et al., 1984; Rogers and Milliman, 1984; Rogers, 1985; Khursheed and Rogers, 1988). Studies of chromosome addition lines at the DNA and protein levels have shown that loci for the low- and high-pI genes are present on chromosomes 1 and 6, respectively (Brown and Jacobsen, 1982; Muthukrishnan et al., 1984). The a-amylase isozyme pattern of germinated wheat grains is more complex than that of barley (Gale et al., 1983). Three classes of a-amylase genes are present (Baulcombe et al., 1987), and these map to different chromosomes, with a-Amyl genes on group 6, a-Amy2 genes on group 7, and a-Amy3 genes on group 5 chromosomes (Baulcombe et al., 1987; Huttly et al., 1988).

Using the IEF gel technique, several a-amylase isoforms have been detected in rice (Oryza sativa L.) (Tanaka et al., 1970; Daussant et al., 1983; Mitsui et al., 1993). In germinating rice seeds, three a-amylase isoforms, A, B, and D, have been identified and characterized (Okamoto and Aka- zawa, 1978; Daussant et al., 1983). Isoforms A and B could not be distinguished immunochemically, but isoform D differed in antigenicity from isoforms A + B (Daussant et al., 1983). Isoforms A and B were also shown to be typical secretory glycoproteins bearing Asn-linked oligosaccharide chains (Miyata and Akazawa, 1982, 1983; Mitsui et al., 1985; Mitsui and Akazawa, 1986; Hayashi et al., 1990; Lecommandeur et al., 1990). The Asn-linked oligosaccha-

Abbreviations: CBB, Coomassie brilliant blue; MS, Murashige- Skoog; RPMI, Roswell Park Memorial Institute.

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1396 Mitsui et al. Plant Physiol. Vol. 11 O, 1996

ride chains were later shown to be heterogeneous (Hayashi et al., 1990), but this did not affect the relative mobilities of the enzyme on IEF gels (Mitsui and Akazawa, 1986). Re- cently, it was observed that a-amylases from suspension- cultured rice cells form a more complex isoform pattern than those from germinating seeds (Simmons et al., 1991; Mitsui et al., 1993). a-Amylase isoform H, exclusively expressed in suspension-cultured rice cells, has been purified and characterized. These results indicated that the enzymatic and serological properties of isoform H were markedly different from those of isoform A + B dominantly expressed in germinated grains (Mitsui et al., 1993).

A number of cDNA and genomic DNA clones of rice a-amylase genes have been isolated and characterized (Huang et al., 1990a, 1990b, 199213; Yu et al., 1991, 1992). Rodriguez and associates reported that there are at least 10 genes for a-amylase in rice, occurring in three subfamilies, i.e. RAmyl(A, B, C), XAmy2(A), and RAmy3(A, B, C, D, E, F ) (Huang et al., 1990a; Thomas and Rodriguez, 1994). Chro- mosomal mapping of these genes using trisomics indicated that RAmyZA and XAmyZC are located on chromosome 2, RAmylB is located on chromosome 1, XAmy2A is located on chromosome 6, XAmySA, 3B, and 3C are located on chromosome 9, and RAmy3D and RAmy3E are located on chromosome 8 (Ranjhan et al., 1991). Furthermore, when the DNA sequences of 17 genes were analyzed, the a-amylase genes in cereals were divided into two major classes, AmyA and AmyB (Huang et al., 1992b). The AmyA class was further subdivided into Amyl and Amy2 subfamilies, and the AmyB class included the Amy3 subfamily.

In the present study, we attempted to purify 10 a-amylase isoforms from rice cells and examined their physicochemical and serological properties. On the basis of these analyses, rice a-amylase isoforms could be classified into two major classes and four subgroups. Furthermore, we attempted to identify the genes corresponding to these a-amylase isoforms by partia1 amino acid sequencing of the protein molecules and analyzing the expression products of a cloned rice a-amylase gene.

MATERIALS A N D METHODS

Materials

Reagents and chemicals were purchased from the following commercial sources: Ampholines, Pharmacia-LKB; RPMI 1640 and fetal bovine serum, Flow (Irvine, Scotland); 2,6,10,14-tetramethylpentadecane (pristane), hypoxanthine-aminopterin-thymidine, and hypoxanthine-thymidine, Sigma; alkaline phosphatase-conjugated goat anti- rabbit IgG, EY Laboratories (San Mateo, CA); mouse antibody isotyping kit, GIBCO-BRL; western blot chemilu- minescence reagent, DuPont; peroxidase-conjugated ‘goat anti-mouse IgG, Bio-Rad; Freund’s adjuvant (complete and incomplete), Staphylococcus aureus protease V8, and o-phen- ylenediamine, Wako (Osaka, Japan); myeloma cell line (X63Ag8.653), Dainihonseiyaku (Osaka, Japan); and 2,4-D, Tokyokasei (Tokyo, Japan). Saccharomyces cerevisiae strain LL20 (pEno/l03) was kindly provided by Dr. M. Ter- ashima (Department of Chemical Engineering, Kyoto Uni- versity, Japan).

Rice Cell Culture and Seed Cermination

The procedure for cell culture of rice (Oryza sativa L. cv Nipponkai) was identical with the method described previously (Igaue et al., 1973; Mitsui et al., 1993). About 2 g (fresh weight) of cells were grown in a 500-mL Sakaguchi flask containing 120 mL of MS medium (Murashige and Skoog, 1962) on a reciproca1 shaker operated ai 110 strokes/min with a 70-mm amplitude at 28°C in darkness.

Rice seeds (cv Nipponkai, Kimmaze, or Nipponbare) were soaked in 1% NaClO solution for 15 min, and after thorough washing in distilled water, seeds were germinated on filter paper moistened with water in a plastic tray in a dark chamber at 30°C. Seeds incubated for 5 d were used for prepaxing seed a-amylase enzymes.

Assays

A standard tr-amylase assay was performed as described previously (Mitsui et al., 1993). Protein content was determined by the dye-binding procedure of Bradford (1976) with bovine y-globulin as a standard.

Enzyme Purification

a-Amylase isoform A + B, which is a mixture of isoforms A and B, was purified from germinating rice seeds according to the procedure reported by Miyata et al. (1981). Purification of isoforms A, E, F, G, H, I, J, Y, and Z , expressed in suspension-cultured rice cells, was carried out as described previously (Mitsui et al., 1993).

Production of Polyclonal and Monoclonal Antibodies

BALB/c mice were immunized by intraperitoneal injection of 20 pg of purified a-amylase isoform E or H with complete Freund’s adjuvant. Four weeks later, 20 pg of the enzyme protein with incomplete adjuvant were injected intraperitoneally. After the second injection, one intraperitoneal booster injection of 20 pg of the enzyme protein without adjuv,ant was given 4 d prior to fusion. This final injection coulcl be administered up to 4 months after the initial injection. The spleen was removed after the whole blood was collected. The serum was used as polyclonal antibodies. Spleen cells were fused to a myeloma cell line, X63Ag8.653 (Kearney et al., 1979), according to the standard method with the modification described by Araki and Ikebe (1991) and Higashihara et al. (1989). The fused cells were resuspended in RPMI medium with 15% (v/v) fetal bovine serum and then were distributed into 96-well t-lat- bottom polystyrene tissue plates (Iwaki, Chiba, Japan) at a density of 5 X 105 cells/well. Selection with hypoxanthine- aminopterin-thymidine medium was begun 24 h after fusion. Throughout the cloning procedures, the cells were cultured in RPMI medium supplemented with 15% fetal bovine serum, 0.225% (w/v) NaHCO,, 2 mM L-Gln, 60 mg L-’ kanamycin sulfate, and 50 mg L-l gentamicin sulfate. Screening of antibody-producing cells was carried out using an ELISA a described below, and then the hybridoma cells were recloned twice by limiting dilution using BALB/c mouse splenocytes as a feeder layer. An estab-

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Characterization of Rice a-Amylase Isoforms 1397

lished hybridoma clone was cultured in RPMI mediumwith 15% fetal bovine serum or injected intraperitoneallyinto pristane-primed BALB/c mice. The cultured superna-tant and the ascitic fluid were used as the monoclonalantibody source.

Production of polyclonal antibodies against a-amylaseisoform A + B and H using rabbits and IgG purificationwere as described previously (Mitsui et al., 1993).

Purification of Monoclonal Antibodies

IgG class antibodies were purified by a protein G-Sepha-rose column (Pharmacia-LKB) from the monoclonal anti-body source according to the manufacturer's protocol. Theisotype of monoclonal antibodies was determined by usinga mouse antibody isotyping kit (GIBCO-BRL). All mono-clonal antibodies used in this study were found to contain

heavy chain and K light chain (data not shown).

Peptide Mapping and Amino Acid Sequencing ofIsoforms of a-Amylase

Purified a-amylase isoforms were subjected to SDS-PAGE (Laemmli, 1970), and the protein bands visualizedby CBB staining were cut out. The samples (approximately100 pmol) were then subjected to partial digestion with V8protease (5 pmol), according to the method described byCleveland et al. (1977). The digested peptides separated bySDS-PAGE were transferred to a ProBlott membrane (Ap-plied Biosystems), as described by the manufacturer, andstained with 0.1% (w/v) CBB R-250 in 40% (v/v) methanoland 10% (v/v) acetic acid for 5 min. Then, after destainingin 50% methanol, each peptide band was excised and sub-jected to analysis in a protein microsequence analyzer(model 476A; Applied Biosystems).

IEF-PAGE and Zymograms

a-Amylase samples were subjected to IEF in polyacryl-amide gels (pH 4-7) and stained to visualize activity fol-lowing the procedure reported previously (Mitsui andAkazawa, 1986).

Immunoblots and ELISA

Purified a-amylase isoforms were subjected to SDS-PAGE and western blotting (Towbin et al., 1979; Mitsui etal., 1993). The a-amylase protein molecules blotted on anitrocellulose sheet were reacted with specific antibodies,followed by detection with alkaline phosphatase-conju-gated anti-rabbit IgG for rabbit antibodies or peroxidase-conjugated anti-mouse IgG for mouse antibodies. The de-tection of alkaline phosphatase activity was carried out asdescribed by Blake et al. (1984), and that of peroxidase wasperformed according to the method of Wood and Sarinana(1975), modified by Mitsui et al. (1990), or using a chemi-luminescence reagent (DuPont).

The ELISA procedure was identical with that of Higashi-hara et al. (1989).

Preparation of Recombinant Rice a-Amylases Expressed byYeast Strain LL20

To prepare the recombinant a-amylases expressed byplasmid pEno/103, which contains the rice a-amylasecDNA fragment, OS103 (O'Neill et al., 1990), S. cerevisiaestrain LL20 was cultured according to the procedure de-scribed by Kumagai et al. (1990). Recombinant rice a-amy-lases in the culture medium were precipitated with 90%(v/v) ethanol; lyophilized; dissolved in a buffered solutionconsisting of 100 mM Hepes-KOH (pH 7.6), 10 niM CaCl2,and 0.1% (w/v) Triton X-100; and analyzed on zymogramsand by immunoblotting.

RESULTS

Purification of Rice a-Amylase Isoforms

We identified 10 a-amylase isoforms (A, B, E, F, G, H, I,J, Y, and Z) capable of hydrolyzing /3-limit dextrin and withdistinct pis in suspension-cultured cells of rice (cv Nippon-kai) (Fig. 1). When cells were cultured using 1.8 g of cellfresh weight per 120 mL of MS medium containing 1.5%(w/v) Sue at 28°C for 7 d, all 10 isoforms were secreted intothe medium. However, only isoforms A, B, Y, and Z weredetected when the initial cell content was reduced from 1.8to 0.6 g (Fig. 1). Appropriate culture conditions were usedfor purification of each a-amylase isoform.

a-Amylase isoforms A, G, H, I, and J were highly puri-fied by electrofocusing in an ampholine column (Fig. 2).These isoforms had identical mobilities on SDS gels (Fig. 3,estimated Mr 44,000) and their pis were estimated to be 4.6,5.4, 5.7, 6.3, and 6.6, respectively. Optimum temperatures

1 2pH 7

PH4

Figure 1. Zymograms of a-amylases secreted from rice cells culturedin different conditions. Rice cells were cultured for 7 d in MSmedium containing 1.5% Sue. Lane 1, Initial cell content, 1.8 g freshweight/120 mL; lane 2, 0.6 g fresh weight/120 mL. Culture mediaobtained from two different conditions were subjected to IEF-PACEand activity staining.

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1398 Mitsui et al. Plant Physiol. Vol. 110, 1996

pH7

pH4

PH7

pH4

pH7

pH4( 2 3 4 5 6 7

Figure 2. Zymograms of the purified a-amylase isoforms A, G, H, I,J, Y, and Z. Enzyme preparations were subjected to IEF-PAGE andactivity staining, a, Lane 1, Crude enzymes prepared from grains ofrice seedlings; lane 2, crude enzymes from the culture medium ofrice cells; lane 3, isoform A; lane 4, isoform C; lane 5, isoform H;lane 6, isoform I; lane 7, isoform ). b, Lane 1, isoform A; lane 2,isoform Y. c, Lane 1, isoform A; lane 2, isoform Z.

for enzymatic activity of isoforms A (70°C) and H (37°C)were distinctly different (Fig. 4), whereas isoforms G and Ihad an optimum temperature similar to that of H (data notshown). a-Amylase isoforms Y and Z were partially puri-fied. Neither enzyme preparation had significant levels ofother a-amylases (Fig. 2, b and c), although both containedother proteins (data not shown). The pi values of isoformsY and Z were determined to be 4.35 and 4.45, respectively.They had the same electrophoretic mobility and optimumtemperature as isoform A. a-Amylase isoform E hadunique physicochemical properties. The pi was estimated

kD

66 —

45-* »«

29 —24 —

2O—

14-

HFigure 3. SDS-PAGE analysis of a-amylase isoforms A, E, and H. Thepurified isoforms A, E, and H were subjected to SDS-PAGE, andproteins were visualized by CBB staining. M, of isoform E wasestimated to be 42,000. Bovine albumin (66 kD), egg albumin (45kD), glyceraldehyde-3-phosphate dehydrogenase (36 kD), carbonicanhydrase (29 kD), trypsinogen (24 kD), trypsin inhibitor (20 kD),and a-lactalbumin (14 kD) were used as molecular mass standards.A faint band below isoform H is a degradation product from isoformH (data not shown). Arrows indicate a-amylase bands (lower, isoformE; upper, isoform A and H).

140

120

£ 100x•I 80t!<a> 60>1 40(A

20

20 40 60 80Temperature (°C)

100

Figure 4. Effects of temperature on the enzymatic activities of iso-forms A, E, and H. Enzyme assays were carried out at differenttemperatures as indicated. The maximum activity of each isoform atoptimum temperature (5-8 units) was normalized to 100%.

to be 5.0, and its Mr (42,000) was smaller than that of theother isoforms (44,000) (Figs. 3 and 5). The optimum tem-perature for activity of isoform E under standard assayconditions was 26°C (Fig. 4). a-Amylase isoform F had a piof 5.1 and was partially purified; its Mr was estimated to be44,000 (Fig. 5).

Physicochemical properties of a-amylase isoforms aresummarized in Table I.

Serological Characterization of a-Amylase Isoforms

In contrast to a-amylase isoforms A and B, which areabundantly produced in the scutella and endosperms ofyoung rice seedlings, isoforms E and H were found to beexclusively expressed in suspension-cultured cells (Figs. 1and 2). The a-amylase isoforms A + B, E, and H exhibitedcharacteristic properties (Table I). Therefore, a series ofpolyclonal and monoclonal antibodies were preparedagainst them to examine serological relationships betweenthe 10 a-amylase isoforms.

Figure 5 shows the results of immunoblotting experi-ments using three polyclonal antibodies and five monoclo-nal antibodies. Polyclonal anti-a-amylase isoforms A + B(anti-AB) antibodies (Daussant et al., 1983; Mitsui et al.,1993) bound to the isoforms A and A + B but had little orno activity against isoforms E, F, G, H, I, and J (Fig. 5a).Polyclonal anti-a-amylase isoform H (anti-H) antibodies(Mitsui et al., 1993) detected isoforms F (upper band of theE + F doublet), G, H, I, and J but hardly recognizedisoforms A, B, and E. Isoforms Y and Z were recognized bythe anti-AB antibodies but not by anti-H antibodies (Fig.5b). Polyclonal anti-a-amylase isoform E (anti-E) antibod-ies were preferentially bound to isoform E (Fig. 5d). Onemonoclonal anti-H antibody, H-B35, was similar to poly-clonal anti-H and recognized isoforms F, G, H, I, and J butnot isoforms A, B, and E. The other monoclonal anti-Hantibody, H-G49, recognized all of these isoforms (Fig. 5a).Monoclonal anti-E antibodies, E-24, E-53, and E-54, recog-nized all isoforms tested (A, E, F, and H) (Fig. 5c).

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Characterization of Rice a-Amylase Isoforms 1399

©ABS

Poly antl-AB

Poly antl-H

A E - F G H I J A- l

—— Mono E-24

Mono E-53

A E E-F H

A E E-F H

Mono H-G49

A E E-F H

A E E-F H

A E-F C H I J A - i

®

A E-F HPoly antl-AB

Poly antl-H

A A-Y A-Z 1-1 V ZPoly antl-E

A E-F H

Figure 5. Immunoblots of three polyclonal andfive monoclonal antibodies against a-amylaseisoforms. a, Approximately 0.3 to 1 jig of eachisoform, A, E • F (mixture of isoforms E and F), C,H, I, J, and A • B (mixture of isoforms A and B),was subjected to SDS-PACE and immunoblot-ting with polyclonal anti-a-amylase A • B (Polyanti-AB), polyclonal anti-a-amylase H (Poly an-ti-H), monoclonal anti-H H-B35 (Mono H-B35),and H-G49 (Mono H-G49) antibodies, respec-tively, as described in "Materials and Methods."Protein bands on the nitrocellulose filter werevisualized by Amido black staining (ABS). Faintbands below isoforms G and H are degradationproducts (data not shown), b, Top, Isoforms A(lane 1, 0.55 jig; lane 2, 0.28 jig; lane 3, 0.14jig; lane 4, 0.07 jig; lane 5, 0.04 jig; lane 6,0.02 ^g), Y (4.5 ^g), and 2. (4 jig) were sub-jected to SDS-PAGE and immunoblotting withpolyclonal anti-AB antibodies. Middle, IsoformsA (0.14 jig), Y (4.5 jig), Z (4 jig), A • Y (mixtureof A and Y), A • Z (mixture of A and Z), and Y •Z (mixture of Y and Z) were subjected to SDS-PAGE and immunoblotting with the anti-AB.Bottom, Isoforms H (lane 1, 0.5 jig; lane 2, 0.25jig; lane 3, 0.12 jig; lane 4, 0.06 jig; lane 5,0.03 jig; lane 6, 0.01 jig), Y (4.5 jig), and Z (4jig) were subjected to SDS-PAGE and immuno-blotting with polyclonal anti-H antibodies, c,One microgram of each isoform, A, E, E • F, andH, was subjected to SDS-PAGE and immuno-blotting with monoclonal anti-E antibodies E-24,E-53, and E-54, respectively. In this experiment,immunodetection was carried out using achemiluminescence reagent according to themanufacturer's instructions (DuPont). d, Onemicrogram of each isoform, A, E • F, and H, wassubjected to SDS-PAGE and immunoblottingwith polyclonal anti-a-amylase E antibodies(Poly anti-E). Arrows indicate a-amylase bands.Lower of double arrows shows isoform E,whereas upper arrow shows other isoforms.

When the inhibitory effects of H-B35 and H-G49 on theenzymic activity of isoform H were examined, it was foundthat H-G49 inhibited enzyme activity stoichiometrically,whereas H-B35 had no effect (Fig. 6a). This result indicatesthat H-G49 may recognize the active site domain of isoformH or may interfere with substrate binding, whereas H-B35binds to some other epitope common to isoforms F, G, H,I, and J. Furthermore, H-G49 inhibited the activities ofisoforms E, G, I, and J as effectively as isoform H but didnot inhibit isoforms A and B (Fig. 6b). This result indicatesthat the active site structure in isoform molecules E, G, H,I, and J is conserved and differs from that in isoforms Aand B.

As shown in Figure 6c, the enzymatic activity of isoformE was effectively and stoichiometrically inhibited by E-24but not by E-53 and E-54. Furthermore, the inhibitory ef-fects of E-24 toward the other isoforms were similar tothose of H-G49 (Fig. 6d), indicating similarities in the activesite of isoform E to those of isoforms G, H, I, and J, despite

the observation that other domains of isoform E weredistinguishable from those isoforms (Fig. 5). Results ofbinding and inhibition experiments using E-53 and E-54suggested that these antibodies recognize conserved do-mains in rice a-amylase isoforms that are distinct from theactive site.

The activities of isoforms Y and Z were not inhibited bythe anti-AB antibodies (data not shown), although the an-tibodies recognize these enzymes (Fig. 5b). Neither mono-clonal anti-H (H-G49) nor anti-E (E-24) antibodies pre-vented these enzyme activities (data not shown). Theseresults indicate that isoforms Y and Z have an active siteserologically distinguished from those of isoforms A, B, E,F, G, H, I, and J, although there are other, common epitopesamong the isoforms A, B, Y, and Z.

The serological properties of isoforms, A, B, E, F, G, H, I,J, Y, and Z are summarized in Table II. Judging from theseand their physicochemical properties (Table I), we definetwo major classes (I and II) and four subgroups (1, 2, 3, and

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Table 1. Physicochemical properties of oc-amylase isoformsa-Amylase Isoform

YZA + Ba

AEFGHa

1J

M,

44,00044,00044,00044,00042,00044,00044,00044,00044,00044,000

pi Optimum Temperature

4.354.45

4.6-4.74.65.05.15.45.76.36.6

aData obtained from the previous report (Mitsui et al.,Not determined.

°C7070707026

N.D."373737

N.D.

1993). bN.D.,

4). Class I includes group 1 (A and B) and group 2 (Y andZ), and class II includes group 3 (E) and group 4 (F, G, H,I, and J).

Identification of a-Amylase Genes with Isoforms

Peptide mapping of rice a-amylase isoforms was carriedout according to the method described by Cleveland et al.(1977). As shown in Figure 7, peptide-mapping patterns ofa-amylase isoforms A, E, and H were quite different (Fig. 7,left), whereas similar patterns were observed between iso-forms A and B (Fig. 7, middle), and between isoforms Gand H (Fig. 7, right). Peptides obtained by these partialprotease digestions were used to determine their aminoacid sequences. We obtained sequence data for isoforms A,E, G, and H (Fig. 8). These results indicated that isoforms Aand E are the gene product of RAmylA and RAmy3E,respectively, and that both isoforms G and H are encoded

kD66 -

45 -36 -

29-24 -

20-

14 -~14 —

H B HFigure 7. Peptide maps of a-amylase isoforms A, B, E, C, and H. Left,The isoforms A (10 jug), E (3 jug), and H (10 jug) were digested with0.2 jug of V8 protease. Middle, The isoforms A (4 /ug) and B (2 jig)were digested with 0.2 /ug of the protease. Right, The isoforms C (6/Ag) and H (6 jig) were digested with 0.2 jug of the protease. Theprotein amount of each isoform was estimated by CBB staining onSDS gels. The other experimental details of peptide mapping aredescribed in "Materials and Methods."

a-Amylase A

RAmylARAmy2A

RAmy3B

RAmySD

RAmySE

a-Amylase E

RAmylA

RAmy2ARAmySARAmySBRAmySC

RAmySD

RAmySE

Amyc2

CSamy-c

-VGGAN-NGTAF NGG-YNFLMG

D/RVGGANSNGTAF E/NGGWYNFLMG

D/WVDRVGGTAS&G Q/SGGWYNLLMG

E/LVNWAQAVGGPA K/QGGWYNFLHG

E/LVNWVHAVGGPA Q/QGGWYNMLKG

E/GVGKPATAFDFT K/QGGWYNFLHE

LVNWVEGVGK GGTPDG-LDWGP

L/LVNWVDRVGG E/GGTPDSRLDWGP

A/LVDWVDRVGG E/GGTPDGRLDWGP

E/LVBWVKQVGG K/GGGPRGCLDWGP

E/LVNWAQAVGG E/GGTPDSRLDWGP

E/LVNWAQAVGG E/GGTPDSRLDWGP

E/LVNWVNAVGG E/GGTPDRLDWGPG

E/ LVNWVEGVGK E/GGTPDGRLDWGP

A/LVDWVDRVGG E/GGTPDGRLDWGP

E/LVNWVNRVGG

a-AmylaseG! LVNWVNAVGG IWNSLSYNG GGTPD-LD

a-Amylase H

RAmylARAmy2A

RAmySB

RAmySD

RAmySE

j LVNWVNAVGG IWNSLSYNG GGTPDRLD

E/LVNWVDRVGG E/IWTSMANGG E/GGTPDSRL

A/LVDWVDRVGG B/LWDSMAIGG E/GCTPDGRL

E/LviWAGAVGG E/IWSNMRYDG E/GGTPDSRL

E/LVNWVNAVGG E/ IWNSLSYNG E/ GGTPDRLD

E/LVNWVEGVGK E/IWSSLIYNG E/GGTPDGRL

Figure 8. Correlation of partial amino acid sequences of a-amylaseisoforms A, E, G, and H with those deduced from various genes. Thepurified isoforms were subjected to amino acid sequencing (see"Materials and Methods"). Amino-terminal amino acid sequences ofpeptides of the isoforms digested with V8 protease are shown in theupper parts of panels. DNA sequence information was obtained fromthe following sources: RAmylA (Huang et al., 1990b), RAmylA(Huangetal., 1992a), RAmy3A (Sutliff et al., 1991), RAmySB (Sutliffet al., 1991), RAmySC (Sutliff et al., 1991), RAmySD (Huang et al.,1990a), RAmySE (Huang et al., 1990a), Amyc2 (from GenBank database accession No. X64619), OSamy-c (Kirn and Wu, 1992). Barsrefer to residues that were not identified by sequencing. Shaded areasindicate the identity between determined and deduced amino acidresidues.

by one gene, RAmySD. Kumagai et al. (1990) reported thatthe recombinant a-amylase expressed by plasmid pEno/103 (which contains the cDNA fragment of RAmylA,OS103; O'Neill et al., 1990) was similar in most respects tothe native enzyme from germinating rice seeds. To furthercharacterize the expression products of RAmylA, the re-combinant a-amylase expressed by plasmid pEno/103 inyeast was analyzed in our experimental system. As shownin Figure 9, both isoforms A and B are produced by thegene RAmylA.

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Characterization of Rice a-Amylase lsoforms 1401

I I I I

O 0.5 1 1.5 2 2.5 Molar Ratio of IgG/Amylase H

o 125

E-54

0- O 2 4 6

Molar Ratio of IgC/Amylase E

125 1-

A+B

O 0.25 0.5 0.75 1 1.25 Molar Ratio of IgG (H-G49)/Amylase

O 2 4 6 8 Molar Ratio of IgG (E-24)/Amylase

Figure 6. lnhibitory effects of monoclonal antibodies H-B35, H-C49, E-24, E-53, and E-54 on a-amylase activity. a, Effects of H-B35 and H- C49 on the activity of isoform H. The enzyme protein (1.9 pg) and various amounts of the monoclonal antibodies (0-1 2.7 pg) were preincubated for 5 min at 37"C, and then the enzyme activity was measured in the standard assay system as described in "Materials and Methods." b, Effects of H-G49 on the activities of the isoforms A + B (W), E (O), G (A), H (O), I (O), and 1 (V). Experimental details were as described for a, except for isoform E, where 0.5 pg of the enzyme protein and various amounts of the monoclonal antibodies were preincubated for 5 min at 2 6 T , and then enzyme activity was measured at 26°C in the standard assay system. c, Effects of E-24, E-53, and E-54 on the activity of isoform E. The enzyme protein (0.5 pg) and various amounts of the monoclonal antibodies (0-10.7 pg) were preincubated for 5 min at 2 6 T , and then enzyme activity was measured at 26°C. d, Effects of E-24 on the activity of isoforms A (W), E (O), G (A), H (O), I (O), and J (V). Experimental details for isoform E were as described for c, whereas those of other isoforms were as described for a and b except using the antibody E-24.

The relationship of our a-amylase isoforms to cloned The gene family contained more than 10 a-amylase genes (Huang et al., 1990a; Yu et al., 1992), and these genes are located on five different chromosomes (Ranjhan et al., 1991). A homology and phylogenetic relationship for those genes was also constructed (Huang et al., 1992a). In contrast to the wealth of information regarding gene number and structure, character ization of a-amylase isoforms

genes is shown in Figure 10.

DI SCUSSION

Recent molecular genetic approaches have demonstrated that a multigene family in rice comprise a-amylase genes.

Table II. Serological properties of a-amylase isoforms

determined. +, Strongly recognized; +I-, weakly recognized; -I+, scarcely recognired; -, unrecognized; (+), inhibited; (-), not inhibited; n.d., not

a-Amylase in Crains of a-Amylase in Rice Cells

Rice Seedlings A + B A E F C H I I Y Z Antibody

Poly anti-AB + + -/+ -/+ -/+ -/+ -/+ -/+ + + (inhibition) (+)" (+) n.d. n.d. ( - )a (-)a n.d. n.d. (-) (-) Poly anti-H -I+ Poly anti-E +/-b +/- + +/- n.d. +/- n.d. n.d. n.d. n.d.

- + + + + + n.d. n.d. Mono H-635 - -

Mono H-G49 + + + + + + + + n.d. n.d.

Mono E-24 +b + + + n.d. + n.d. n.d. n.d. n.d.

Mono E-53 +b + + + n.d. + n.d. n.d. n.d. n.d. Mono E-54 +' + + + n.d. + n.d. n.d. n.d. n.d.

- - + + + + + - -

(inhibition) (-) (-1' ( + I n.d. (+) (+) (+) (+) (-1 (-)

(inhibition) (-)b (-) (+) n.d. (+) (+) (+) (+) (-) (-1

"These inhibitory effects toward a-amylase A + B, C, and H are from a previous report (Mitsui et al., 1993). 'Data not shown in the text.

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1: Endosperm

2: pEno 103

3: Endosperm

5 4:pEno103

pH4.4 pH5.5

Figure 9. Zymogram and immunoblolting patterns of the recombi-nant a-amylases produced by S. cerevisiae strain LL20 (pEno/103).a-Amylases prepared from endosperms of rice seedlings (cv Nippon-bare) and culture media of tl_20 were subjected to IEF-PAGE, fol-lowed by activity staining (lanes 1 and 2) or immunoblotting withpolyclonal anti-AB antibodies (lanes 3 and 4). Arrowheads A and Bindicate isoform A and B, respectively.

encoded by these genes on the protein level has beenlacking.

In our present study, 10 a-amylase isoforms, A, B, E, F,G, H, I, J, Y, and Z, with different pi values, have beenpurified and characterized from suspension-cultured cellsof rice (O. saliva L. cv Nipponkai). These rice a-amylaseisoforms exhibited distinguishable optimum temperaturesand molecular sizes. In addition, we prepared and charac-terized three polyclonal and five monoclonal antibodiesagainst three isoforms that have characteristic physico-chemical properties. Judging from their physicochemicaland serological properties, we ultimately classified thesea-amylase isoforms into two major classes and four sub-groups as shown in Figure 10. Furthermore, we determinedthe partial amino acid sequences of four a-amylase iso-forms dominantly expressed in rice cells (Fig. 8) and foundthat these major isoforms are encoded by three genes,RAmylA, RAmySD, and RAmy3E, identified previously(Simmons et al., 1991; Thomas and Rodriguez, 1994). Theuse of antibodies to probe structural and functional do-mains of the different enzyme molecules should be a pow-erful tool for future studies of rice a-amylase proteins. Cell

biochemical studies using these specific antibodies to clar-ify the regulatory mechanism of protein biosynthesis andintracellular transport of each a-amylase isoform groupin rice cells are now in progress in our laboratory.

The present study of a-amylase isoforms provides infor-mation concerning posttranslational modification ofa-amylase protein molecules in rice. Some a-amylase iso-forms within a subgroup were found to be encoded by onegene (Figs. 8 and 9). Previously, it was demonstrated thata-amylase isoforms A and B bear heterogeneous Asn-linked oligosaccharide chains (Hayashi et al., 1990) result-ing from their modification in the Golgi complex. (Mitsui etal., 1985). However, Asn-linked oligosaccharide heteroge-neity could not explain differences in pi. Sticher and Jones(1991) proposed that posttranslational modifications by adeamidase gives rise to multiple forms of HAMY1 (low pi)a-amylase in barley aleurone. Alternatively, Segaard et al.(1991) reported that C-terminal processing of low-pi barleya-amylase results in multiple forms in malt, aleurone pro-toplasts, and transformed yeast. Our data indicate thatposttranslational protein modification, which alters the pivalue of a-amylase molecules, also occurs in rice cells.

The inhibition experiments, in which anti-AB, H-G49,and E-24 antibodies were used, demonstrated that group 1,group 2, and group 4 a-amylase isoforms have three dis-tinctly different structural configurations of their active sitedomains, whereas those of group 3 and group 4 isoformsare indistinguishable (summarized in Table II). Group 1isoforms are predominantly expressed in the scutellar andendosperm tissues (Fig. 2; Okamoto and Akazawa, 1978;Miyata and Akazawa, 1982). Group 2 isoforms are detectedas the most abundant enzyme in green shoot tissues ofyoung seedlings of rice (T. Mitsui and Y. Kobayashi, un-published data). Group 4 isoforms, or mRNA of RAmy3D,accumulated in embryos when the tissues isolated fromdry seed were germinated in the absence of sugar (Karrerand Rodriguez, 1992; J. Yamaguchi and T. Mitsui, unpub-lished data). Thus, three isoform groups that have immu-nologically distinct active site domains are preferentially

Figure 10. Classification of a-amylase isoformsexpressed in rice suspension-cultured cells andtheir corresponding genes.

RAmylA

RAmySE

RAmySD

Class-1

Class-II

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Characterization of Rice a-Amylase lsoforms 1403

expressed in the different tissues. This may indicate distinct roles in starch breakdown in each tissue. In this re- spect, the identification of the gene(s) encoding the group 2 isoforms Y and Z is needed to clarify the role of group 2 proteins in green shoot tissues.

A novel isoform E (Table I), with an apparent smaller molecular size and a lower optimum temperature in comparison with the other nine isoforms, is worthy of further de- scription. The presence of two molecular sizes of rice a-amylases was reported by Chiba et al. (1990) and Chen et al. (1994). The optimum temperature of a-amylase I11 (M,. 40,000, p15.8) was similar to that of isoform E, and the partia1 amino acid sequence analysis of this enzyme suggested that it may be encoded by the genetic locus RAmy3E (Chiba et al., 1991), indicating that a-amylase 111 likely belongs to group 3, which includes the isoform E. Chen et al. (1994) identified 44- and 46-kD a-amylase isoforms in rice cells. The smaller molecular size a-amylases (44 kD) have been shown to be expressed constitutively, even if the cells are deprived of SUC (Chen et al., 1994). The RAmy3E (aAmy8) gene, which encodes the protein molecule of isoform E, has been found to be markedly suppressed by metabolic sugar (Yu et al., 1991; Huang et al., 1993). Although no detailed information is available for the structural and catalytic properties of the 44-kD isoform, it may be different from isoform E and a-amylase 111. Further characterization of catalytic properties and tissue distribution of isoform E are required to clarify its physiological role.

ACKNOWLEDCMENTS

We wish to thank Dr. E. Blumwald (University of Toronto, Canada) and Dr. J.C. Rogers (University of Missouri, Columbia) for their kind suggestions and comments. We also thank N. Geshi (Nagoya University, Japan) and N. Suzuki, H. Goto, Y. Kobayashi, and T. Takamuki (Niigata University, Japan) for their excellent technical assistance.

Received August 14, 1995; accepted December 4, 1995. Copyright Clearance Center: 0032-0889/96/110/1395/10.

LITERATURE ClTED

Akazawa T, Mitsui T, Hayashi M (1988) Recent progress in a-amylase biosynthesis. In J Preiss, ed, The Biochemistry of Plants, Vol 14. Academic Press, New York, pp 465-492

Araki Y, Ikebe M (1991) Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recog- nizes the calmodulin-binding region. Biochem J 275: 679-684

Baulcombe DC, Huttly AK, Martienssen RA, Barker RF, Jarvis MG (1987) A novel wheat a-amylase gene (a-Anzy3). Mo1 Gen Genet 209: 33-40

Blake MS, Johnston KH, Russel-Jones GJ, Gotshlich EC (1984) A rapid, sensitive method for detection of alkaline phosphatase conjugated anti-antibody on western blots. Anal Biochem 136:

Bradford MM (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254

Brown AHD, Jacobsen JV (1982) Genetic basis and natural vari- ation of a-amylase isozymes in barley. Genet Res 40: 315-324

Chandler PM, Zwar JA, Jacobsen JV, Higgins TJV, Inglis AS (1984) The effects of gibberellic acid and abscisic acid on a-amylase mRNA levels in barley aleurone layers studies using an a-amylase cDNA clone. Plant Mo1 Biol 3: 407418

175-179

Chen M-H, Liu L-F, Chen YR, Wu H-K, Yu S-M (1994) Expression of a-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant

Chiba Y, Nieda Y, Nakajoma T, Ichishima E (1991) Unique enzymatic properties of a-amylase-111 from suspension-cultured rice cells. Agric Biol Chem 55: 901-902

Chiba Y, Ui M, Kato Y, Nakajoma T, Ichishima E (1990) Two groups of intracellular a-amylase isoenzymes from cultured rice cells. Phytochemistry 29: 2075-2078

Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102- 1106

Daussant J, Miyata S, Mitsui T, Akazawa T (1983) Enzymic mechanism of starch breakdown in germinating rice seeds. 15. Immunochemical study on multiple forms of amylase. Plant Physiol 71: 88-95

Deikman J, Jones RL (1985) Control of a-amylase mRNA accu- mulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol 78: 192-198

Fincher GB (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu Rev Plant Physiol Plant Mo1 Biol 40: 305-346

Gale MD, Law CN, Chojecki AJ, Kempton RA (1983) Genetic control of a-amylase production in wheat. Theor Appl Genet 64:

Hayashi M, Tsuru A, Mitsui T, Takahashi N, Hanzawa H, Arata Y, Akazawa T (1990) Structure and biosynthesis of the xylose- containing carbohydrate moiety of rice a-amylase. Eur J Bio- chem191:287-295

Higashihara M, Frado L-LY, Craig R, Ikebe M (1989) Inhibition of conformational change in smooth muscle myosin by a monoclo- na1 antibody against the 17 kDa light chain. J Biol Chem 264:

Huang N, Chandler J, Thomas BR, Koizumi N, Rodriguez RL (1993) Metabolic regulation of a-amylase gene expression in transgenic cell cultures of rice (Oryza sntiva L.). Plant Mo1 Biol23: 737-747

Huang N, Koizumi N, Reinl S, Rodriguez RL (1990a) Structural organization and differential expression of rice a-amylase genes. Nucleic Acids Res 18: 7007-7013

Huang N, Reinl SJ, Rodriguez RL (1992a) XAmyZA; a novel a- amylase-containing gene in rice. Gene 111: 223-228

Huang N, Stebbins GL, Rodriguez RL (1992b) Classification and evolution of a-amylase genes in plants. Proc Natl Acad Sci USA

Huang N, Sutliff TD, Litts JC, Rodriguez RL (1990b) Classifica- tion and characterization of the rice a-amylase multigene family. Plant Mo1 Biol 1 4 655-668

Huang J-K, Swegle M, Dandekar AM, Muthukrishnan S (1984) Expression and regulation of a-amylase gene family in barley aleurones. J Mo1 Appl Genet 2: 579-588

Huttly AK, Martienssen RA, Baulcombe DC (1988) Sequence heterogeneity and differential expression of the a-Amy2 gene family in wheat. Mo1 Gen Genet 214: 232-240

Igaue I, Nishino M, Kurasawa F (1973) Occurrence of acid phosphatase isozymes repressible by inorganic phosphate in rice plant cell cultures. Agric Biol Chem 37: 941-943

Itoh K, Yamaguchi J, Huang N, Rodriguez RL, Akazawa T, Shimamoto K (1995) Developmental and hormonal regulation of rice a-amylase (RAmylA)-gusA fusion genes in transgenic rice seeds. Plant Physiol 107: 25-31

Jacobsen JV, Higgins TJ (1982) Characterization of the a-amylases synthesized by aleurone layers of Himalaya barley in response to gibberellic acid. Plant Physiol 70: 1647-1653

Karrer EE, Rodriguez RL (1992) Metabolic regulation of rice a-amylase and sucrose synthase genes ia planta. Plant J 2 517-523

Kearney JF, Radbruch A, Liesegang B, Rajewsky K (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123: 1548-1550

J 6: 625-636

309-316

5218-5225

89: 7526-7530

Dow




Khursheed B, Rogers JC (1988) Barley a-amylase genes. Quanti- tative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells. J Biol Chem 263: 18953-18960

Kim J-K, Wu R (1992) Nucleotide sequence of a high-pI rice (Oryza sativa) a-amylase gene. Plant Mo1 Biol 18: 399-402

Kumagai MH, Shah M, Terashima M, Vrkljan Z, Whitaker JR, Rodriguez RL (1990) Expression and secretion of rice a-amylase by Saccharomyces cerevisiae. Gene 94: 209-216

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685

Lecommandeur D, Sirou Y, Lauriere C (1990) Glycan research on barley, maize, oats, and sorghum grain a-amylases: comparison with rice a-amylase. Arch Biochem Biophys 278: 245-250

Mitsui T, Akazawa T (1986) Secondary modification of carbohydrate chains in a-amylase molecules synthesized in rice scutellum. Physiol Veg 24: 629-638

Mitsui T, Akazawa T, Christeller JT, Tartakoff AM (1985) Bio- synthesis of rice seed a-amylase: two pathways of amylase secretion by the scutellum. Arch Biochem Biophys 241: 315-328

Mitsui T, Kimura S , Igaue I (1990) Isolation and characterization of Golgi membranes from suspension-cultured cells of rice (Oryza sativa L.). Plant Cell Physiol 31: 15-25

Mitsui T, Ueki Y, Igaue I (1993) Biosynthesis and secretion of a-amylase by rice suspension-cultured cells: purification and characterization of a-amylase isozyme H. Plant Physiol Biochem

Miyata S , Akazawa T (1982) Enzymic mechanism of starch breakdown in germinating rice seeds. 12. Biosynthesis of a-amylase in relation to protein glycosylation. Plant Physiol 70: 147-153

Miyata S, Akazawa T (1983) Biosynthesis of rice seed a-amylase: proteolytic processing and glycosylation of precursor polypep- tides by microsomes. J Cell Biol 96: 802-806

Miyata S, Okamoto K, Watanabe A, Akazawa T (1981) Enzymic mechanism of starch breakdown in germinating rice seeds. 10. In vivo and in vitro synthesis of a-amylase in rice seed scutellum. Plant Physiol 68: 1314-1318

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Plant Physioll5 473-479

Muthukrishnan S , Gil1 BS, Swegle M, Chandra GR (1984) Struc- tural genes for a-amylases are located on barley chromosomes 1 and 6. J Biol Chem 259: 13637-13639

Okamoto K, Akazawa T (1978) Purification of a-amylase and p-amylase from endosperm tissues of germinating rice seeds. Agric Biol Chem 42: 1379-1384

31: 863-874

O'Neill SD, Kuinagai MH, Majumdar A, Huang N, Sutliff TD, Rodriguez RL (1990) The a-amylase genes in Oryza sativa: characterization of cDNA clones and mRNA expression during germination. Mo1 Gen Genet 221: 235-244

Ranjhan S, Litts JC, Foolad MR, Rodriguez RL (1991) Chrorno- soma1 localization and genomic organization of a-amylase genes in rice (Oryza :;ativa L.). Theor Appl Genet 8 2 481438

Rogers JC (1985) Two barley a-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260: 3731-3733

Rogers JC, Milliman C (1983) Isolation and sequence analysis of a barley a-amylase cDNA clone. J Biol Chem 258: 8169-8174

Rogers JC, Milliman C (1984) Coordinate increase in major tran- scripts from the high pI a-amylase multigene family in barley aleurone cells stimulated with gibberellic acid. J Biol Chem 259:

Simmons CR, Huang N, Cao Y, Rodriguez RL (1.991) Synthesis and secretion of a-amylase by rice callus: evidence for differen- tia1 gene expression. Biotechnol Bioeng 38: 545-551

Segaard M, Olsen FL, Svensson B (1991) C-terminal processing of barley a-amylase 1 in malt, aleurone protoplasts, and yeast. Proc Natl Acad Sci USA 88: 8140-8144

Sticher L, Jones RL (1991) Isolation and partia1 characterization. of a factor from Eiarley aleurone that modifies a-amylase in vitro. Plant Physiol 97: 936-942

Sutliff TD, Huang N, Litts JC, Rodriguez RL (1991) Character- ization of an a-amylase multigene cluster in rice. Plant Mo1 Biol

Tanaka Y, Ito T, Akazawa T (1970) Enzymic mechanism of starch breakdown in germinating rice seeds. 111. a-Amylase isozymes. Plant Physiol46 650-654

Thomas BR, Rodriguez RL (1994) Metabolite signals regulate gene expression and source/sink relations in cereal seedlings. Plant Physiol 106: 1235-1239

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 7 6 4350-4354

Wood JG, Sarinana FO (1975) The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase. Ana1 Biochem 69: 320-322

Yu S-M, Kuo Y-H, Sheu G, Sheu Y-J, Liu L-F (1991) Metabolic derepression of a-amylase gene expression in suspension-cultured cells of rice. J Biol Chem 266: 21131-21137

Yu S-M, Tzou W-S, Lo W-S, Kuo Y-H, Lee H-T, Wu R (1992) Regulation of a-amylase-encoding gene expression in germinating seeds and cultured cells of rice. Gene 122: 247-253

12234-12240

16: 579-591

Dow



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Physicochemical and Serological Characterization of Rice a … · Plant Physiol. (1 996) 1 1 O: 1395-1 404 Physicochemical and Serological Characterization of Rice a-Amylase lsoforms