PNL Volume 17

1985

RESEARCH REPORTS

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF 3 BETA-GALACTOS1DASE

ISOZYMES IN PEA LEAVES

Weeden, N. F.

NYS Agricultural Experiment Station, Geneva, NY USA

Enzymes catalyzing the hydrolysis of beta-D-galactose units from

the non-reducing ends of B-galactosides may be conveniently visualized

after electrophoresis on starch gels by flooding the gel with a solution

containing 4-methylumbelliferyl galactoside (Sigma) and observing the

fluorescence of the 4-methyl umbelliferone product under long-wave

ultraviolet light (1). Analysis of pea leaf extracts revealed three

zones of beta-galactosidase activity (Fig. 1). One zone migrated

anodally on a pH 8.1 gel while the other two migrated cathodally on a pH

6.1 gel (extraction buffers and gel systems were as described in [2]).

Sharp, well defined bands of activity were produced by the anodally

migrating enzyme, which showed maximum activity at alkaline pH (assay

solution: 0.1 M Tris pH 8.5 containing 2 mg 4-methylumbelliferyl-beta-D-

galactoside which had been dissolved in 1 ml acetone). The cathodal

enzymes exhibited an acid pH optimum (assay solution 0.1 M sodium

citrate pH 4.5 containing 2 mg substrate as described above) and formed

broader zones of activity which were often very faint (Fig. 1). Young

leaves proved to be the most convenient tissue to sample for beta-galac-

tosidase activity; however, seed and root extracts also gave similar

phenotypes.

PNL Volume 17 1985

RESEARCH REPORTS 7 7

A survey of inbred lines provided by Dr. G. A. Marx and several

P.I. accessions revealed polymorphism for all three zones of beta-galac-

tosidase activity. Variants of the anodal enzyme were rare and identi-

fied by a different electrophoretic mobility. In contrast, variants in

the cathodal regions were relatively common and often exhibited a dif-

ference in level of activity (Fig. 1).

In order to determine if the three zones of activity represented

products of different loci, appropriate crosses were made and the F₂

populations analyzed for segregation within each zone. Certain of the

variants showed so little activity that they were treated as "null"

alleles. In these cases the expected ratio was 3:1, for the heterozy-

gotes were difficult to distinguish from the homozygous "active" pheno-

type and thus were grouped together as "galactosidase plus". For cases

in which the mobility of the variants differed, three classes of progeny

could be distinguished, and the expected ratio was 1:2:1.

The segregation observed within individual zones is summarized in

Table 1. The variants within a zone appeared to be products of dif-

ferent alleles, for normal segregation ratios were obtained in all

cases. That each zone of activity represented a distinct locus was

demonstrated by joint segregation analysis presented in Table 2.

Although the size of the populations listed in Table 2 was too small to

eliminate the possibility of loose linkage between loci, the significant

level of recombination exhibited between each pair of loci establishes

the presence of three distinct beta-galactosidase loci in pea. The loci

have been designated Gal-1, Gal-2, and Gal-3 in order of relative

mobility of their isozymes in the anodal direction. Thus Gal-1

specifies the anodal forms, Gal-2 the cathodal enzyme closest to the

origin, and Gal-3 the most cathodal isozyme.

1. Vallejos, E. E. 1983. In S. D. Taksley and T. J. Orton (eds.).

Isozymes in Plant Genetics and Breeding, Part. A. Elsevier,

Amsterdam, pp. 469-516.



78	PNL Volume 17	1985	RESEARCH REPORTS





Fig. 1. Zymogram of beta-galactosidase phenotypes observed after electro- phoresis on horizontal starch gels. Pea lines mentioned in text are designated in the figure by their final 3-digit code. Width of band in the cathodal region reflects relative intensity of respective isozyme band.