In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine nonaccumulating plant. Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, and Arabidopsis thaliana. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDHdeleted E. coli mutant. Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine. The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMOlike gene was expressed in E. coli, the protein was detected but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus sp. PCC7942, and Arabidopsis conferred resistance to abiotic stress. These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively.Many bacteria, plants, and animals accumulate glycine betaine (betaine) under water or salt stress conditions (1-3). In these organisms, it was shown that betaine is synthesized in two steps, choline 3 betaine aldehyde 3 glycine betaine. The enzyme involved in the second step seems to be the same in plants, animals, and bacteria, namely, NAD ϩ -dependent betaine aldehyde dehydrogenase (BADH) 1 (4 -6). By contrast, different enzymes are involved in the first step. In plants, the first step is catalyzed by a novel Rieske-type iron-sulfur enzyme choline monooxygenase (CMO) (7,8). CMO is not found in animals or bacteria. In these organisms, the first step is catalyzed by membrane-bound choline dehydrogenase (CDH) or soluble choline oxidase (COX) (9 -11). CMO is not well known, having so far been found only in Chenopodiaceae (spinach and sugar beet) and Amaranthaceae (7, 12, 13), and not detected even in some betaine-accumulating plants such as mangrove (14). CMO purified from spinach leaves are shown to be soluble and insensitive to carbon monooxide, contains a Rieske-type iron-sulfur center, and consists of homo-dimer or -trimer of subunit M r 42,000 (7,8). These properties are completely unrelated to CDH, COX, and cytochrome P-450-type monooxygenases. The activity of purified CMO is extremely low (393 picokatal mg Ϫ1 ) probably due to the labile property of the enzyme (7), which hampers further characterization.For functional characterization of CMO, it would be useful to express it in E. coli, which has not yet been reported. In this study, we examined whether CMO could function in E. coli and cyanobacteria. E. coli contains a bet gene cluster consisting of betA, betB, betT, and betI, which encode CDH, BADH, choline ...

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To characterize the low-molecular-weight glutenin subunit (LMW-GS), we developed specific PCR primer sets to distinguish 12 groups of LMW-GS genes of Norin 61 and to decide their loci with nullisomic-tetrasomic lines of Chinese Spring. Three, two, and ten groups were assigned to Glu-A3, Glu-B3, and Glu-D3 loci, respectively. To identify the proteins containing the corresponding amino acid sequences, we determined the N-terminal amino acid sequence of 12 spots of LMW-GSs of Norin 61 separated by two-dimensional gel electrophoresis (2DE). The N-terminal sequences of the LMW-GS spots showed that 10 of 12 groups of LMW-GSs were expressed as protein products, which included LMW-i, LMW-m, and LMW-s types. Four spots were encoded by Glu-A3 (LMW-i). Three spots were encoded by Glu-B3 (LMW-m and LMW-s). Five spots were encoded by Glu-D3 (LMW-m and LMW-s). A minor spot of LMW-m seemed to be encoded by the same Glu-B3 gene as a major spot of LMW-s, but processed at a different site. Comparing among various cultivars, there were polymorphic and non-polymorphic LMW-GSs. Glu-A3 was highly polymorphic, i.e., the a, b, and c alleles showed one spot, the d allele showed four spots, and the e allele had no spot. Insignia used as one of the Glu-A3 null standard cultivars had a LMW-GS encoded by Glu-A3. We also found that Cheyenne had a new Glu-D3 allele. Classification of LMW-GS by a combination of PCR and 2DE will be useful to identify individual LMW-GSs and to study their contribution to flour quality.

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Using various rice flours prepared by different milling methods, the relationship between rice flour properties and specific loaf volumes of one-loaf bread made from rice flour with wheat vital gluten were studied. Damaged starch content of rice flour varied from 1.0% to 22.1%. A significant negative correlation was verified between damaged starch content and specific loaf volume of one-loaf bread. Rice flour with low damaged starch content mainly consisted of compound starch granules, aggregated polyhedral single starch granules and smooth surface cells surrounded by the cell wall. The structures of starch granules and cells were maintained in rice flour with low damaged starch content. Rice flour with high damaged starch content consisted of only fine irregular particles without the apparent rice starch structure, or contained fractured large and small cells with rough surface. Although there was not significant correlation between specific loaf volume and median particle size, rice flours which were successful at making bread with high specific loaf volume commonly showed a peak centered around 60 µm with a smaller amount of larger size particles in the particle distribution profile. Thus, the flour particle size distribution appears to affect the specific loaf volume of one-loaf bread. Both the damaged starch content and the profile of particle size distribution were important for high specific loaf volume of one-loaf bread. Less damage to starch and cell structures while lowering the particle size during milling process is critical in obtaining better flour for rice one-loaf bread making.