Plant Physiol. (1999) 119: 353-362

Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b₅ Reductase from Arabidopsis

Masako Fukuchi-Mizutani^*, Masaharu Mizutani, Yoshikazu Tanaka, Takaaki Kusumi, and Daisaku Ohta

Institute for Fundamental Research, Suntory Limited, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0024, Japan (M.F.-M., Y.T., T.K.); Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan (M.M.); and College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan (D.O.)

	ABSTRACT

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b₅ reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b₅, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b₅ reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b₅ reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b₅ reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b₅ (AtB5-A), whereas no Cyt b₅ reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b₅ with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase can reduce Cyt b₅ and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.

	INTRODUCTION

The ER membrane of eukaryotic cells contains two electron-transfer systems: one is the NADH-dependent system containing NADH-Cyt b₅ reductase and Cyt b₅, and the other is the NADPH-dependent system containing NADPH-Cyt P450 reductase.

NADH-Cyt b₅ reductase is a membrane-bound flavoprotein containing a single FAD as a prosthetic group. It transfers electrons from NADH to Cyt b₅, which is another membrane protein containing a single heme group. In higher plants Cyt b₅ has been shown to function as an intermediate electron donor in the desaturation of fatty acids of the microsomal membranes from developing safflower cotyledons (Smith et al., 1990; Kearns et al., 1991), in the C5(6) desaturation of sterol precursors in maize (Rahier et al., 1997), and in the hydroxylation of oleate in castor bean seeds (Smith et al., 1992). It is therefore generally accepted that NADH-Cyt b₅ reductase in higher plants is the major electron-transfer component involved in these lipid-modification reactions and that it transfers reducing equivalents from NADH to Cyt b₅. The cDNAs encoding Cyt b₅ have been isolated from several plant species (Kearns et al., 1992; Smith et al., 1994b; Napier et al., 1995), whereas a cDNA encoding NADH-Cyt b₅ reductase has not yet been isolated from higher plants. Thus, no direct evidence has been presented so far from reconstitution assay systems containing NADH-Cyt b₅ reductase, Cyt b₅, and the fatty acid desaturases.

Slack et al. (1976) showed in pea and maize leaves that both NADH and NADPH could stimulate microsomal oleate desaturation. Smith et al. (1990) demonstrated with the microsomes of developing safflower cotyledons that Cyt b₅ reduction was observed by the addition of NADH (and also NADPH, but to a lesser extent). Furthermore, they showed that microsomal fatty acid desaturation activity was supported by both NADH and NADPH. Taton and Rahier (1996) also reported that both NADH and NADPH served as the electron donors for the C5(6) desaturation of sterol precursors in maize microsomes. These results suggested the involvement of a NADPH-dependent electron transfer as well as the NADH-dependent transfer in the desaturation reactions. Thus, one possibility is that NADH-Cyt b₅ reductase in higher plants may accept the electrons from both NADH and NADPH to reduce Cyt b₅. The other possibility is that, as reported in mammalian systems (Enoch and Strittmatter, 1979), NADH and NADPH may be used for the reduction of Cyt b₅ through two distinct reductases, NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase, respectively.

To address these questions, we characterized these two microsomal electron-transfer chains in higher plants by reconstituting in vitro the NADH- and NADPH-dependent electron-transfer chains. First we isolated cDNAs encoding NADH-Cyt b₅ reductase and two Cyt b₅ isoforms of Arabidopsis and demonstrated, using the recombinantly expressed NADH-Cyt b₅ reductase and Cyt b₅ proteins, that NADH but not NADPH was specifically utilized for the reduction of Cyt b₅ by the Arabidopsis NADH-Cyt b₅ reductase. We also confirmed that the recombinant Arabidopsis NADPH-Cyt P450 reductase prepared previously (Mizutani and Ohta, 1998) has a clear specificity toward NADPH in the reduction of Cyt b₅. To our knowledge, this is the first reconstitution study to show that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase are able to reduce Cyt b₅ with strict specificity in the utilization of NADH and NADPH, respectively. We discuss the possible physiological significance of the distinction and functional overlap between the NADH- and NADPH-dependent microsomal electron-transfer systems in higher plants.

	MATERIALS AND METHODS

Plant Materials

Isolation of the NADH-Cyt b₅ Reductase cDNA

Isolation of Cyt b₅ cDNAs

Isolation of the AtCBR Gene

DNA and RNA Analysis

Total RNA was isolated as described by Lagrimini et al. (1987), and 5 µg of total RNA was analyzed by northern hybridization with the full length of cDNAs of AtCBR, AtB5-A, and AtB5-B labeled with [-³²P]dCTP. The hybridization signals were detected using an imaging analyzer (BAS2000, Fuji Film, Tokyo, Japan).

Heterologous Expression of the AtCBR Protein in Insect Cells

The expressed AtCBR protein was purified from the infected Sf21 cells. The infected cells were sonicated and centrifuged at 100,000g for 1 h. The pellet was homogenized with buffer A containing 20 mM potassium phosphate, pH 7.25, 20% glycerol, and 1 mM DTT, and proteins were solubilized in buffer B containing the same constituents as buffer A plus 1% Emulgen 913 (Kao Atlas, Tokyo, Japan). After centrifugation at 100,000g for 1 h, the supernatant was applied to a 5-AMP Sepharose column (1 × 7 cm) equilibrated with buffer B, and the protein was eluted from the column with 10 mM potassium phosphate buffer, pH 7.25, containing 20% glycerol, 1 mM EDTA, 0.1 mM DTT, and 0.5 mM NAD.

Heterologous Expression of the AtB5-A Protein in Escherichia coli

Assay Methods

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1

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K_m values of the recombinant NADH-Cyt b₅ reductase or NADPH-Cyt P450 reductase were determined for the electron-donor substrate NADH or NADPH, respectively. For the measurement of the K_m values, the concentration of NADH or NADPH was varied from 0.1 to 100 µM in the reaction mixture, and the Cyt b₅ reduction was measured as described above. Data were transformed and plotted as Lineweaver-Burk graphs to allow calculation of K_m values.

	RESULTS

Cloning of Arabidopsis NADH-Cyt b₅ Reductase cDNA

Figure 1 shows the alignment of the amino acid sequence deduced from the AtCBR cDNA and those of the NADH-Cyt b₅ reductases from yeast and human. The deduced primary structure of the AtCBR protein is homologous to the NADH-Cyt b₅ reductases of mammals and yeast: 40% identical to human (Yubisui et al., 1984) and yeast microsomal NADH-Cyt b₅ reductases (Csukai et al., 1994) and 38% identical to yeast mitochondrial NADH-Cyt b₅ reductase (Hahne et al., 1994). The regions highly homologous to those of yeast and mammalian NADH-Cyt b₅ reductases were found principally in the presumed FAD- and NADH-binding regions (Nishida et al., 1995). The AtCBR protein sequence also contained a region significantly similar to the flavin-binding domain of the Arabidopsis nitrate reductase (Crawford et al., 1988; data not shown). These observations suggested that the AtCBR cDNA encodes a NADH-Cyt b₅ reductase of Arabidopsis.

View larger version (87K): [in this window] [in a new window]   Figure 1. The multiple alignment of amino acid sequences of AtCBR with those of NADH-Cyt b5 reductase from human (Yubisui et al., 1984) and ER (Csukai et al., 1994) and mitochondria (MT) (Hahne et al., 1994) of yeast. Shading indicates the conserved amino acid residues among the aligned sequences. The arrowheads and numbers over the AtCBR sequence indicate positions where introns are inserted in the AtCBR gene. The sequence of the AtCBR cDNA and the AtCBR gene were deposited in the databank under accession nos. AB007799 and AB007800, respectively.

The AtCBR protein does not have a typical ER retention signal (KXKXX or KKXX) at the C terminus (Jackson et al., 1990). The AtCBR protein, however, contained an N-terminal hydrophobic stretch with approximately 30 amino acids and a few charged residues flanking the hydrophobic stretch, Asp-2, Glu-4, and continuous Lys-16 to Arg-19. These structural properties are similar to those observed in the signal-anchor sequences of microsomal Cyt P450s, which are suggested to be major determinants of targeting to the ER and transmembrane orientation on the ER surface of newly synthesized Cyt P450s (Beltzer et al., 1991).

In mammalian tissues NADH-Cyt b₅ reductase is expressed as N-myristoylated and non-myristoylated forms encoded by a single gene (Meldolesi et al., 1980; Pietrini et al., 1988; Borgese et al., 1990, 1993). N-myristoylation is the cotranslational attachment of myristic acid to the Nterminal Gly of target proteins. The first five amino acid residues conform to a loose consensus sequence including the essential second Gly residue (Johnson et al., 1994; Casey, 1995). The predicted amino acid sequence in the AtCBR, however, contains no N-terminal consensus sequences for N-myristoylation, which is responsible for the targeting of the mammalian NADH-Cyt b₅ reductase protein to mitochondrial outer membranes (Borgese et al., 1996). Thus, together with its N-terminal properties, which are similar to the microsomal Cyt P450s described above, it is more likely that the AtCBR protein is localized at the ER membrane, as reported for the non-myristoylated NADH-Cyt b₅ reductase isoform in mammalian cells (Borgese et al., 1996).

Characterization of AtCBR Gene Organization

View larger version (18K): [in this window] [in a new window]   Figure 2. Gene organization of the AtCBR gene. A, AtCBR gene organization. Open boxes with numbers show the exons, and bars between open boxes show introns. B, Southern analysis of the AtCBR gene. One microgram of genomic DNA was digested with the indicated restriction enzymes and probed with [-32P]dCTP-labeled AtCBR cDNA. E, EcoRI; B, BamHI; X, XhoI.

Southern analysis was performed to estimate the copy number of the AtCBR gene in the Arabidopsis genome (Fig. 2B). Genomic DNA was digested with each of the three restriction enzymes, EcoRI, BamHI, and XhoI, and probed with the full length of AtCBR cDNA. The AtCBR gene contained two EcoRI recognition sites, one in the first intron and the other in the third intron, and one BamHI site, whereas XhoI had no recognition site in the AtCBR gene. EcoRI digestion produced three hybridization signals, including a band at 0.6 kb, which was consistent with the size expected for the region between the two EcoRI sites in the AtCBR gene. Two hybridization signals were observed in the digestion with BamHI, whereas a single band was detected in the digestion with XhoI. Hybridization under low-stringency conditions gave the same results as those observed under high-stringency conditions. These hybridization patterns were consistent with the restriction map of the AtCBR gene (Fig. 2A), indicating that AtCBR exists as a single-copy gene in the Arabidopsis genome.

As described above, mammalian tissues contain both mitochondrial and ER forms of the NADH-Cyt b₅ reductases, which are encoded by a single gene (Pietrini et al., 1988), and cotranslational N-myristoylation of a NADH-Cyt b₅ reductase precursor protein is necessary for targeting of the NADH-Cyt b₅ reductase to the mitochondrial outer membrane (Borgese et al., 1996). In contrast to mammals, yeast contains two independent genes for NADH-Cyt b₅ reductase isoforms targeted to either the ER or the mitochondrial outer membrane (Csukai et al., 1994; Hahne et al., 1994). Although no additional NADH-Cyt b₅ reductase genes in Arabidopsis were revealed by the genomic Southern hybridization analysis, we cannot rule out the possibility that, as in the yeast system, Arabidopsis contains another NADH-Cyt b₅ reductase gene encoding a mitochondrial isoform.

Isolation of Two Cyt b₅ cDNAs

View larger version (75K): [in this window] [in a new window]   Figure 3. Multiple alignment of the amino acid sequences of AtB5-A and AtB5-B with those of Cyt b5 from cauliflower (Brassica) (Kearns et al., 1992), tobacco (Smith et al., 1994a, 1994b), tobacco seeds (Napier et al., 1995), and rice (Smith et al., 1994a, 1994b). Shading indicates the conserved amino acid sequences among the aligned sequences.

Expression Patterns of the AtCBR and AtB5 Genes in Arabidopsis

View larger version (49K): [in this window] [in a new window]   Figure 4. Tissue-specific expression of the AtCBR, AtB5-A, and AtB5-B genes. Total RNA was isolated from the roots and the leaves of 3-week-old plants, from the inflorescence stems and flowers of 4-week-old plants, and from the siliques of 5-week-old plants. Plants were grown under continuous light.

Tobacco contains two isoforms of Cyt b₅: one is specifically expressed in the developing seed and the other is expressed in the whole plant (Smith et al., 1994b; Napier et al., 1995). Considering the essential roles of NADH-Cyt b₅ reductase and Cyt b₅ in fatty acid biosynthesis, the low expression levels of the AtB5 genes in the silique containing developing seeds suggests that Arabidopsis may have an additional seed-specific Cyt b₅ isoform that is predominantly involved in the biosynthesis of storage lipids.

Characterization of Recombinant NADH-Cyt b₅ Reductase Proteins

View larger version (29K): [in this window] [in a new window]   Figure 5. Heterologous expression of the recombinant AtCBR protein in insect cells. A, SDS-PAGE was performed using a 16% polyacrylamide slab gel, and proteins were visualized by staining with Coomassie brilliant blue R-250. Lane 1, Molecular mass marker proteins; lane 2, 100,000g precipitate of mock-infected Sf21 cells; lane 3, 100,000g precipitate of the Sf21 cells infected with the recombinant virus containing the full-length AtCBR cDNA; lane 4, 100,000g supernatant of the AtCBR-expressing Sf21 cells; lane 5, solubilized fractions of 100,000g precipitate of the AtCBR-expressing Sf21 cells; lane 6, the purified recombinant AtCBR. B, Absolute absorption spectrum of the purified recombinant AtCBR in its oxidized form (solid line) and reduced by the addition of 100 µM NADH (dashed line).

Reduction of Cyt b₅

Most of the recombinant Cyt b₅ protein was recovered in the membrane fraction in E. coli lysate, implying that the recombinant Cyt b₅ could be interacting with the bacterial membrane, probably via the C-terminal hydrophobic anchor sequence. This recombinantly expressed Cyt b₅ was solubilized in 1% Triton X-100 and affinity purified with the aid of the N-terminal His tag using a Ni-nitrilotriacetic acid agarose column. The recombinant Cyt b₅ showed an absorption maximum at 413 nm (Fig. 6), and the dithionite-reduced form exhibited prominent peaks at 424, 526, and 557 nm (data not shown). These spectral characteristics are typical of the native Cyt b₅ proteins purified from other organisms (Bonnerot et al., 1985).

View larger version (16K): [in this window] [in a new window]   Figure 6. Absolute absorption spectra of the recombinant AtB5-A protein. Solid line, Oxidized AtB5-A protein; dashed line, the AtB5-A protein reduced by the recombinant AtCBR with 100 µM NADH.

When the oxidized form of the recombinant Cyt b₅ was incubated with the recombinant AtCBR protein and 100 µM NADH, it was rapidly reduced and showed an absolute absorption spectrum similar to that of the dithionite-reduced form (Fig. 6). Thus, the recombinant AtCBR protein was functionally active as a NADH-Cyt b₅ reductase. The reduction of the recombinant Cyt b₅ by AtCBR was NADH dependent, with the K_m value for NADH of 1.5 µM (data not shown). On the other hand, no reduction of Cyt b₅ was observed in the presence of the AtCBR protein and NADPH, demonstrating that the AtCBR did not transfer the reducing equivalents from NADPH to Cyt b₅ (Fig. 7A).

View larger version (10K): [in this window] [in a new window]   Figure 7. NADH- and NADPH-dependent reduction of Cyt b5. Recombinant Cyt b5 (AtB5-A) was reduced at 25°C in a reaction mixture containing 10 mM potassium phosphate buffer, pH 7.25, 1.5 µM Cyt b5, either NADH or NADPH (100 µM), and a catalytic amount of AtCBR (A) or NADPH-Cyt P450 reductase (B). The reaction was initiated by addition of either NADH (solid line, 100 µM) or NADPH (dashed line, 100 µM) as an electron donor.

It has been reported that in the microsomal fractions of higher plants Cyt b₅ is reduced by the addition of not only NADH but also NADPH (Slack et al., 1976; Smith et al., 1990), suggesting, as reported in a mammalian system (Enoch and Strittmatter, 1979), the involvement of NADPH-Cyt P450 reductase in the NADPH-dependent reduction of Cyt b₅. We previously isolated two Arabidopsis cDNAs encoding NADPH-Cyt P450 reductase and obtained the recombinantly expressed NADPH-Cyt P450 reductase proteins (Mizutani and Ohta, 1998). The NADPH-Cyt P450 reductases were able to reduce the recombinant Cyt b₅ protein with NADPH (the K_m value for NADPH = 2 µM, data not shown) but not with NADH (Fig. 7B). The two NADPH-Cyt P450 reductase isoforms in Arabidopsis (Mizutani and Ohta, 1998) showed the same properties in the reduction of the Arabidopsis Cyt b₅ encoded in AtB5-A cDNA (data not shown).

Even after a longer incubation (up to 30 min) or incubation with a higher concentration of pyridine nucleotide (1 mM), no significant reduction of Cyt b₅ by NADH-Cyt b₅ reductase with NADPH or by NADPH-Cyt P450 reductase with NADH was observed (data not shown).

	DISCUSSION

We isolated a cDNA encoding NADH-Cyt b₅ reductase (AtCBR) and two cDNAs for Cyt b₅ isoforms (AtB5-A and AtB5-B) from Arabidopsis and recombinantly expressed the NADH-Cyt b₅ reductase protein and the Cyt b₅ protein encoded in AtB5-A. The recombinant NADH-Cyt b₅ reductase and Cyt b₅ were used to study the microsomal NADH-dependent electron transfer of higher plants, which is involved in the desaturation and hydroxylation of fatty acids and in the desaturation of sterol precursors (Smith et al., 1990, 1992; Kearns et al., 1991; Rahier et al., 1997).

It is widely accepted from studies of the mammalian microsomal electron-transfer system that Cyt b₅, which is usually reduced by NADH-Cyt b₅ reductase in the presence of NADH, can also be reduced by NADPH-Cyt P450 reductase, with NADPH as the electron donor (Enoch and Strittmatter, 1979). Cyt b₅ reduced by NADH-Cyt b₅ reductase or by NADPH-Cyt P450 reductase can donate its reducing equivalents to a series of lipid-modification reactions such as desaturation and hydroxylation. In addition, the reduced Cyt b₅ can provide the second electron for some of the Cyt P450-dependent monooxygenase reactions (Enoch and Strittmatter, 1979; Imai, 1981; Ruckpaul et al., 1989). Thus, both NADH and NADPH can serve as the electron donors via the two different reductases for the various microsomal terminal electron acceptors, such as the fatty acid desaturases and Cyt P450 monooxygenases.

Several studies of the electron-transfer system in higher plants at the microsomal level have also been reported. In a microsomal preparation from developing safflower cotyledons, Cyt b₅ was fully reduced by the addition of NADH and partially reduced by the addition of NADPH (13% of the reduction by NADH; Smith et al., 1990). On the other hand, the 12 desaturase activity in the microsomal preparation was observed in the presence of either NADH or NADPH as the electron donor (Smith et al., 1990). It has also been reported that with the microsomal fraction of maize either NADH or NADPH can support the C5(6) desaturation of sterol precursors (Taton and Rahier, 1996; Rahier et al., 1997). These observations suggested, as reported in a mammalian system (Enoch and Strittmatter, 1979), the involvement of NADPH-Cyt P450 reductase in the NADPH-dependent reduction of Cyt b₅, which is implicated in the NADPH-dependent activities of the desaturation of fatty acids and sterol precursors. However, it has not been elucidated whether plant NADPH-Cyt P450 reductase can directly reduce Cyt b₅, or what specificities, if any, exist between NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase in terms of the utilization of NAD(P)H for the reduction of Cyt b₅.

This ambiguity was solved with our in vitro reconstitution studies using the recombinantly expressed proteins involved in microsomal electron-transfer systems (Fig. 7). The Arabidopsis NADH-Cyt b₅ reductase was able to reduce Cyt b₅ in the presence of NADH, but not in the presence of NADPH. Arabidopsis NADPH-Cyt P450 reductase reduced Cyt b₅ with NADPH, but NADH was not accepted as the electron donor by the NADPH-Cyt P450 reductase. These results indicate that NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase have strict substrate specificities toward NADH and NADPH, respectively. Nonetheless, both reductases are capable of reducing Cyt b₅, which is also involved in a wide range of microsomal enzymatic activities.

Our results demonstrated that both reductases have the ability to reduce Cyt b₅ in vitro. The next question is how the reducing equivalents from NAD(P)H are transferred through these two reductases to the microsomal terminal electron acceptors in vivo. It is possible that the fatty acid and sterol desaturases of the ER may accept their reducing equivalents from both NADH and NADPH in vivo (Fig. 8). Although it has been suggested that only NADH-Cyt b₅ reductase and Cyt b₅ are predominantly involved in the desaturation of microsomal fatty acids and sterol precursors, the NADPH-dependent electron transfer through NADPH-Cyt P450 reductase could also participate in these reactions in microsomal membranes of higher plants. We also cannot rule out the possibility that the desaturases may bypass Cyt b₅ and accept electrons directly from NADPH-Cyt P450 reductase.

View larger version (9K): [in this window] [in a new window]   Figure 8. Microsomal electron-transfer systems in higher plants. Solid black line, Electron transfers confirmed by in vitro assay; dashed gray line, possible electron transfers to be investigated further.

As described above, NADPH can be utilized via NADPH-Cyt P450 reductase as the electron donor for those reactions, which are generally NADH dependent. The reducing equivalents from NADH apparently also participate in some Cyt P450-dependent reactions in higher plants; Cyt P450-dependent hydroxylations of lauric acid and monoterpenes were stimulated by the addition of NADH (Benveniste et al., 1982; Karp et al., 1990). Therefore, together with the possible involvement of NADPH and NADH in some microsomal desaturase reactions and Cyt P450-dependent reactions, it is conceivable that the NADH- and NADPH-dependent microsomal electron-transfer chains in higher plants are not completely independent but occasionally cross over and complement each other in a wide variety of reactions, such as the desaturations of fatty acids and sterols and Cyt P450-dependent hydroxylations (Fig. 8). It has been reported that a mutant in yeast deficient in either NADH-Cyt b₅ reductase or NADPH-Cyt P450 reductase was able to grow under normal conditions (Sutter and Loper, 1989; Csukai et al., 1994; Truan et al., 1994), but the disruption of both of the reductases was lethal (Csukai et al., 1994). These observations provide further evidence for the possible complementation of the NADH- and NADPH-dependent electron-transfer chains in vivo.

Another important question remains to be answered regarding the physiological significance of the NADPH-dependent lipid-desaturation activities. This will be clarified through studies with reconstitution systems containing the fatty acid desaturases with other NADH- and NADPH-dependent electron-transfer components. Reconstitution studies with these electron-transfer components should also focus on the characterization of the microsomal fatty acid desaturases in higher plants, including the fatty acid desaturase homologs with unknown enzymatic activities from rose and Arabidopsis (Fukuchi-Mizutani et al., 1995, 1998). These biochemical studies, along with molecular-genetics studies such as mutant and transgenic analyses, will constitute important advances toward understanding microsomal lipid metabolism in higher plants.

	FOOTNOTES

   Received April 13, 1998; accepted October 15, 1998.

	ABBREVIATIONS

Abbreviations: EST, expressed sequence tag.

	ACKNOWLEDGMENTS

The authors thank Mmes. Yoriyo Takeuchi and Matsuyo Okamoto for their technical support.

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