Institute of Agricultural Biotechnology

Esterase organ-specific pattern revisited

--Emil E. Khavkin and M. V. Zabrodina

Previous studies of somaclones (MNL 64:91, 1990; 65:88, 1991) led to the suggestion that several, presumably organ-specific, isozyme loci might vary more often than those expressed at all developmental stages. To test this assumption, we correlated our isozyme patterns obtained by neutral (modified Taber and Sherman) and alkaline (Davis) vertical slab PAGE to esterase loci assigned earlier upon starch gel electrophoresis.

Two cathodic esterases slowly migrating in the neutral gel (Fig. 1) were apparently the E3 and E1 isozymes described by Schwartz, D (PNAS 51:602, 1964). The former locus was prominent in the endosperm, weakly expressed in the scutellum and obviously absent from the embryo axis. Several public inbreds employed in this study had slow (Tx601) and fast (predominant) forms of E3 esterase. E1 esterase was most active in the endosperm, quite discernible in the axis and absent from the scutellum. There were two additional lightly (perhaps non-specifically) stained diffuse zones in cathodic zymograms: one immediately preceding E1 was found in leaves while another, with higher mobility, was characteristic of roots. These zones were not identified. The slowest zone in the alkaline gel system (Fig. 2), found only in the endosperm, might be formed by fused E1 plus E3 bands.

Several specific loci could be easily identified among anodic esterases, especially in neutral gels. The most polymorphic, the E4 series described by Harris (MNL 40:53, 1966) and Macdonald and Brewbaker (Hawaii Agr. Exp. Sta. Tech. Bull. No. 89, 1975), was very active in roots (up to 7 bands were excellently resolved in several inbreds), evident in other embryo tissues and green leaves, but completely absent from the endosperm. The quantitative changes in E4 expression in embryo tissues might at least account for some of the previously described effects of somaclonal variability. In roots at least five E4 alleles were found (in the order of their decreasing mobility towards anode): 1) A188; 2) A239; 3) B14, C103, Tx601, Va35 and W64A; 4) B73 and Oh43; and 5) B37.

Figure 1. (left) Cathodic and anodic esterases in the neutral (modified Taber and Sherman) PAGE system. E1 to E10 are esterase loci and 10 is band 10 as described by Macdonald and Brewbaker (1975). EN, endosperm; SC, scutellum; EA, embryo axis; PR, primary root; GL, green leaf. Arrowheads show qualitative and quantitative organ-specific changes of esterase patterns.

Figure 2. (right) Anodic esterases in the alkaline (Davis) PAGE system. ME, mesocotyl; CO, coleoptile; EL, etiolated leaf; LB, leaf base. Other symbols as in Figure 1.

Allelic forms of E9 esterase were readily discerned in neutral gels with fast isozyme in Va35 (and maybe also in Tx601) and one or two slower allelomorphs in other inbreds. The E8 band was easily identified in alkaline gels by its position and intensive staining in all tissues, however, three allelic forms corresponding to those reported by Stuber and Goodman (USDA Agr. Res. Serv., Southern Ser., No. 16, 1983) were difficult to discriminate by their mobility. In neutral gels, E9 and E8 bands were preceded by their "twins". In several inbreds these twins were as prominent as E9 and E8 isozymes, except in the endosperm and leaves. As these bands coincided with zones singled out as presumable targets of somaclonal variation, they need further clarification. At present they might be cautiously related to the E5 locus and 42 and 43 bands described by Macdonald and Brewbaker (1975). In alkaline gels one of these bands preceded the E9 isozyme while two other zones were more compact and adjacent to the E8 major isozyme.

Somaclonal variations were also manifest in the interval between the E4 and E8 zones, and here several minor and apparently organ-specific bands were observed. A well-resolvable series of at least four bands in alkaline and neutral gels was characteristic of endosperm and scutellum and absent from axial tissues. It might belong to the E10 locus described by Macdonald and Brewbaker (1975), which has at least two alleles. Some bands were present only in the scutellum, while most of the electromorphs in this zone were absent from the endosperm and found, with varying staining activity, in some or all embryo tissues. Staining activity of some of the leaf-specific bands depended on greening.

Two additional organ-specific zones migrated ahead of the E8 band in the neutral gel. One of them, a diffuse band that was absent from endosperm and root but present in scutellum and shoot, might be related to the E6 esterase of Macdonald and Brewbaker (1975). A fast allelomorph was predominant and a slow variant was found in A239. Another isozyme twice as mobile as the E8 band apparently corresponded to band 10 as described by these authors in partially senescent roots. The inbreds greatly differed by activity and mobility of this isozyme: there were fast (Tx601 and WF9), intermediate (predominant) and slow (A239) variants.

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