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Genetic analysis of high molecular weight proteins in rice (Oryza sativa L.) endosperm.

Rice endosperm storage proteins have been extensively studied by means of electrophoretic methods to determine ways of improving the nutritional value and eating quality especially (Kumamaru et al., 1988; Iida et al., 1993). Such proteins are incorporated into two types of protein bodies: PB-I, which contains prolamin (mol. wt. [is less than] 16 kDa), and PB-II, which contains glutelin (mol. wt. [is less than] 39 kDa) (Tanaka et al., 1980; Yamagata and Tanaka, 1986). Glutelin, the main storage protein, consists of several subunits, and their expression patterns differ among cultivars (Chen et al., 1987; Sarker and Bose, 1984). Rice storage proteins in endosperms have been classified into four types (A, B, C, and D) based on the characteristics of the glutelin subunits (Chen et al., 1989; Kagawa et al., 1988). Recently, Saruyama and Shinbashi (1992) investigated the inheritance of a protein of 33 kDa in the [F.sub.1] generation of a cross between japonica and indica cultivars using 2D-PAGE. Furthermore, a high molecular weight protein of 60 kDa, known as Wx protein that controls different amounts of the gene product as well as amylose content, has been identified (Sano, 1984; Sano et al., 1991). However, in contrast with the intensive studies on the main storage proteins (glutelin, globulin, and prolamin), information on the high molecular weight protein ranging from 40 to 90 kDa is lacking.

The objectives of this study were to obtain basic information on genetic characteristics of the high molecular weight proteins in endosperms of japonica and indica cultivars.


Plant Material

Six rice cultivars were grown in a greenhouse at Yamagata University in 1992. Three japonica cultivars including Taichung 65, Sasanishiki, and Calrose, and 3 indica cultivars including BlueBelle, Tadukan, and Taichung native 1 were selected for the present experiments based on their flowering times. Crosses between japonica and indica cultivars were conducted on 5 to 10 August 1992. The cross combinations were as follows: Taichung 65 X BlueBelle, Sasanishiki X Tadukan, and Calrose X Taichung native 1.

The [F.sub.1] seeds were sown in mid-October in the same year for generation advancement, and the [F.sub.2] seeds were harvested at the end of March in 1993. Milled grains of the [F2.sub.2] generation and parents were used for 2D-PAGE.

Extraction of Endosperm Protein

For each sample of milled grains, a single endosperm of 0.01 g was ground with mortar and pestle in 0.4 mL of extraction buffer containing 8.5 M urea, 20 mL [L.sup.-1] nonidet P-40, 50 mL [L.sup.-1] 2-mercaptoethanol, and 20 mL [L.sup.-1] ampholine (pH 3.5-10.0, Pharmacia LKB Biotech, Upsala, Sweden). The homogenate was centrifuged at 15 000 X g for 20 min, and the supernatant was subjected to 2D-PAGE.

Two-Dimensional Gel Electrophoresis

Endosperm proteins were analyzed by 2D-PAGE according to the protocol of O'Farrell (1975). The isoelectic focusing (IEF) gel solution consisted of 8 M urea, 20 mL L` nonidet P-40, 40 g [L.sup.-1] acrylamide, 2 g [L.sup.-1] N',N'-bis-methylene-acrylamide, and 50 mL [L.sup.-1] ampholine (pH 3.5-10.0, Pharmacia LKB Boitech. Upsala, Sweden). The IEF solution was polymerized by the addition of 0.1 g [L.sup.-1] ammonium persulfate and 0.6 mL [L.sup.-1] N,N,N',N'tetramethylethylenediamine. The solution was loaded into the gel tube (1.5 by 125 mm). The polymerized gel was overlaid with protective buffer containing 5 M urea, 20 ml [L.sup.-1] ampholine, and 20 mL L` nonidet P-40. Each sample of 20 [micro]L was then overlaid onto the IEF gel. The upper and lower reservoirs were filled with 0.02 M NaOH and 0.85 mL [L.sup.-1] phosphoric acid, respectively. The gel was then run for 16 h at 700 V (Dunbar, 1987).

Prior to the second dimension electrophoresis, the gels were equilibrated in an isogel equilibration buffer containing 6.25 mM Tris-HCl (pH 6.8), 20 g [L.sup.-1] sodium dodecyl sulfate, and 50 mL [L.sub.-1] mercaptoethanol by shaking twice for 20 min at 40 [degrees] C. The second dimension electrophoresis was carried out with a separation gel of 140 g [L.sup.-1] acrylamide and stacking gel of 40 g [L.sup.-1] acrylamide according to the procedure of Laemmli (1970). Calibration kits of standard proteins for isoelectrofocusing and molecular weight markers (Pharmacia LKB Biotech, Uppsala, Sweden) were used.

Proteins on the gel were detected by 2.5 g [L.sup.-1] coomassie brilliant blue R-250, and double-stained with silver according to the method of Oakley et al. (1980). Gels were dried and scanned on a Cannon IX-4015 scanner (Cannon Co. Ltd., Tokyo, Japan) using the Image 1.44 program (NIH, Washington, DC) to determine the density of each spot. Nine gels of each parent were analyzed to obtain the mean and standard deviation of parents.


In the cross combination of Taichung 65 X BlueBelle, protein spots D, E, F, and G with molecular weights of about 69, 64, 57, and 54 kDa, respectively, were detected in [F.sub.2] endosperms. These spots were predominantly present in cv. Taichung 65. However, protein spot M with a molecular weight of about 40 kDa was also detected in [F.sub.2] endosperms. This spot was predominantly present in cv. BlueBelle (Fig. 1).


In the cross combination of Sasanishiki x Tadukan, protein spots I, K, and L with the molecular weights of about 52, 44, and 42 kDa, respectively, were detected in [F.sub.2] endosperms. These proteins occurred in cv. Tadukan. Protein spots F, G, and H with molecular weights of about 57, 54, and 52 kDa, respectively, were also detected in [F.sub.2] endosperms. These proteins were present in cv. Sasanishiki (Fig. 2).


In the cross combination of Calrose x Taichung native 1, protein spots H, K, and L with molecular weights of about 52, 44, and 42 kDa, respectively, were detected in [F.sub.2] endosperms. These protein subunits were predominantly present in cv. Taichung native 1. Protein spot I with a molecular weight of about 52 kDa was also detected in [F.sub.2] endosperms, and was predominantly expressed in cv. Calrose (Fig. 3).


Protein spots K and L were commonly expressed in the indica cultivars, Tadukan and Taichung native 1. However, protein spot M was expressed only in cv. BlueBelle. Protein spot F was commonly present in cv. Taichung 65 and Sasanishiki, but was absent in cv. Calrose.

Protein spots on the gel exhibited different color densities between endosperms of parents and [F.sub.2] seeds. Four different types of triploid endosperm were distinguished. The dose effects of genes on expression of the protein spots and their phenotypic frequency distribution are shown in Table 1. The frequency distributions that correspond to paternal and maternal genotypes in [F.sub.2] seed populations were determined with reference to mean [+ or -] standard deviation of parents. The nine protein spots that were detected in [F.sub.2] seeds fit a segregation ratio of 1:1:1:1 (P [is greater than] 0.5) resulting from double fertilization.



Qualitative studies on endosperm proteins have mainly focused on the major storage proteins such as glutelin. The glutelin [Alpha]-subunit group consists of three subunits, but the [Alpha]-3 subunit was absent in many indica cultivars (Kagawa et al., 1988). A large difference in storage protein composition was identified between ordinary (gamma-ray-induced or natural) and methylnitrosourea-induced mutants (Kumamaru et al., 1988). Cultivar variations were identified electrophoretically only in minor bands (Chen et al., 1987).

Variations among different cultivars in the intensities of protein spots that correspond to the amylose synthesis enzymes, [Wx.sup.a] and [Wx.sup.b] (both about 60 kDa), have been reported (Sano, 1984). Of special interest is the fact that [Wx.sup.b] may be similar to the F spot in the present experiments. Other than studies on [WX.sup.a] and [WX.sup.b], information on quantitative characteristics of proteins in the high molecular weight domain is limited. However, the present results show that there are many proteins in this region.

The general genotype and segregation ratio in endosperm due to double fertilization are denoted as AAA:AAa:Aaa:aaa = 1:1:1:1. The breeding behavior of the nine protein spots were not polypeptide-residues in the processes of translocation and/or decomposition. Thus, these proteins appear to be functional and to be inherited like the amylose syntheses [WX.sup.a] and [WX.sup.b] (Sano, 1984; Sano et al., 1991).

A precursor peptide of glutelin subunits with the same molecular weight as the F protein spot (57 kDa) was reported in immature rice seeds (Sarker et al., 1986). However, the F protein spot differs in isolectric point from the precursor of the glutelin subunits.

Many storage substances such as starches, proteins, and lipids are synthesized in the endosperm, and many enzymes involved in the synthesis of these storage substances have still not been identified. Therefore, proteins in the high molecular weight domain may be enzymes involved in the biosynthesis of many storage substances. In order to ensure characteristics of the enzymes in the high molecular weight domain, physiological and molecular biological studies are required. Furthermore, studies on genetic variations in proteins in the high molecular weight domain, genetic mapping of their corresponding genes, and biochemical characterization of the encoded proteins also remain to be examined.

Abbreviations: IEF, isoelectric focusing; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis.


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Lab. of Plant Breeding, Faculty of Agriculture, Yamagata Univ., Tsuruoka 997, Japan. Received 12 Jan. 1996. Takeo Sasahara, Corresponding author (E-mail:
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Author:Sadimantara, Gusti Ray; Abe, Toshinori; Sasahara, Takeo
Publication:Crop Science
Date:Jul 1, 1997
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