Our interest in corn pollen is outlined above. Employing microelectrophoretic protein resolution and computerized digital densitometry (contributions 4 and 5), it is possible to analyze extractable protein of anthetic corn pollen. Pollen from a number of cultivars (grown under identical field conditions), was collected according to the schedule: Freshly dehiscent tassel bagged (6 hr maximum); bag carefully removed (1 hr); sieve pollen free of debris through 125 µ mesh (30 min); transfer 10 mg of protein to 5 ml glass test tube; freeze pollen in dry-ice-acetone bath (5 min); lyophilize (24 hr); test tube stoppered, coded and stored at -200 C.
The electrophoretic separation of protein from plant tissue extracts is complicated by other macro-ions indigenous to the plant. Our method of extraction attempts to minimize the presence of such compounds (e.g. aromatic hydrocarbons, starch, fatty acids):
Buffer A consists of: 9 M urea, 3% NP-40 (v/v), 5% 2-mercaptoethanol (v/v), 2.5% carrier ampholytes (pH 3-10, v/v), 3% glycerol (v/v), 1% sodium dodecyl sulphate (w/v), and 1% polyvinyl pyrolidone (w/v). Buffer B is formulated of: 9.0 M urea, 1% NP-40 (v/v), 5% 2-mercaptoethanol (v/v), 2% carrier ampholytes (pH 3-10, v/v), and 3% glycerol (v/v). Buffer C is made by combining: 8 M urea, 20 mM TRIS pH 7.0, 2.5% 2-mercaptoethanol (v/v), 10 mM EDTA, 2% sodium dodecyl sulphate (w/v), and 3% glycerol. The tracer dye solution is .01% Bromophenol blue (w/v), .01% xylene cyanole FF (w/v), in 15% glycerol.
Extracts of two inbred lines (A and B) were selected for electrophoretic protein comparison. Two replicate sets of four gels were used to obtain electrophoretic patterns by isoelectric focusing, molecular weight and two-dimensional separation methods (see contribution 4).
The patterns observed by isoelectric precipitation of lines A and B are nearly identical. Protein is resolved throughout the pH range of 3-10. The largest proportion of protein precipitation is localized in three pH ranges (3-4, 7-8 and 9.5-10). Three variations were identified in the patterns of A and B: two protein bands (pI 3.4 and 4.7) were present in A but not B; one protein band (pI 8.4) was absent in A but not B; line A was resolved into 33 bands, line B into 32 bands.
Molecular weight resolution of lines A and B is carried out over a range of 14 K to 200 K daltons. Most of the proteins are resolved above 60 K daltons. The comparison of line A and B protein band patterns showed that: three protein bands (65 K, 78 K and 130 K daltons molecular weight) are detectable in line A but not B; line B possesses two protein bands (43 K and 200 K daltons molecular weight) which are not found in the pattern of line A; and lines A and B could be resolved into 19 and 17 bands respectively.
Two-dimensional electropherograms of lines A and B showed greater complexity in the patterns and number of protein spots resolved (136 and 135 respectively). In general the resolved patterns of A and B are similar with the following exceptions: three protein species (pI and molecular weight coordinate pairs of 3.3, 130 K daltons, 3.2 and 200 K daltons, 7.0 and 81 K daltons respectively) are present in A but not B; and three protein species (pI and molecular weight coordinate pairs of 8.1 and 40 K daltons, 8.1 and 43 K daltons, 7.9 and 65 K daltons respectively) are present in B but not A.
These results demonstrate the feasibility of characterizing proteins extracted from pollen. We have as yet an incomplete library of such protein landscapes; nevertheless, we have observed differences in the numbers and coordinates of several bands among several stocks. Since these methods can be used with small amounts of tissue, it is possible to study 'landscapes' as a function of growth and differentiation in the sporophyte.
We have found that the pollen from most of the inbreds we have studied: (1) yields repeatable landscapes; and (2) differs at one or more coordinates. Such methods as we report above may provide high resolution identification of stocks, hybrids, seed purity and may be useful in large scale breeding programs.
W. G. Hughes and D. B. Walden
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