Gametophytic transcription for acid phosphatases in pollen of Cucurbita species hybrids

A microslab isoelectric focusing gel is demonstrated, using single pollen grains from F, hybrids of Cucurbita species. When zymograms of these single pollen grains were stained to indicate the presence of acid phosphatase zones, it was seen that at least 11 out of 37, or 29.7 percent of such zones are controlled by loci that are transcribed in the microgametophyte. These results suggest that gametophytically transcribed loci may be fairly common and also that their investigation is now technically unchallenging. IT HAS LONG BEEN suggested that pollen, with its haploid genotype and very large population sizes, might undergo selective pressures that greatly exceed those found in other parts of the angiosperm life cycle. This possibility is a double-edged one, representing, on one hand, a vulnerability in which "a gene which greatly accelerates pollen tube growth will spread through a species even if it causes moderately disadvantageous changes in the adult plant. " It is perhaps this that prompted some investigators to suggest the number of genes actually transcribed in the pollen is probably quite small. Conversely, a gene that benefits the mature plant, if it benefits also the gametophyte, could be increased rapidly by pollen tube selection. Finally it must be considered that if genes are not transcribed in the pollen, then both deleterious and beneficial effects of pollen tube selection are precluded. Evidence that genes are transcribed in pollen comes from several sources; but many of these rely on specialized phenomena such as selfincompatibility or uniquely convenient biochemical differences such as the presence or absence of alcohol dehydrogenases or waxy versus nonwaxy starch. These phenomena have provided valuable insights into the biology of pollen but, at the same time, they raise a critical question. Does the rarity of such pollen makers indicate that much of the pollen genome is indeed inactive, or is it that we have not yet learned to detect other gametophytically transcribed loci? Mascarenhas has The authors are affiliated, respectively, with the Department of Botany, University of Massachusetts, Amherst, MA 01003; the Department of Seed and Vegetable Research, Cornell University, Geneva, NY 14456; the Department of Biochemistry, National Institute of Genetics, Mishima, 444 |apan, and the Department of Agronomy, University of California, Davis, CA 95616. This research was supported in part by NSF grant DEB 79-03685 and USDA/SEA grant 5901-0410-9-0365-0. © 1981, American Genetic Association. demonstrated that there is extensive protein synthesis in pollen but he points out the possibility that some of this could be the translation of longlived messenger transcribed by sporophytic tissues. A resolution of this issue may be provided by electrophoretic studies of pollen, using either of two approaches. The first of these, recently employed by Weeden and Gottlieb is to examine dior multi-meric enzymes. If pollen from segregating sporophytes exhibits homomultimers and, unlike the sporophytes, lacks heteromultimers, then the locus in question must be transcribed post-meiotically. Products of segregation would then be capable of responding to selection. An alternative electrophoretic test for postmeiotic expression involves the analysis of individual pollen grains. In this case, postmeiotic transcription is indicated by segregation of isozymes in pollen from heterozygous individuals. It is the latter method that is described in the present paper. Specifically, the method employs a microslab gel and isoelectric focusing. Materials and Methods To form a gel, a sheet of GelBond film (Bioproducts, Rockland, Maine 04841) was placed, hydrophobic side up, on a sheet of glass or other smooth surface. Two strips of plastic, each approximately 0.5 X 4.0 X 80.0 mm, were then placed on the GelBond film, parallel to each other and about 20.0 mm apart. On top of these plastic strips was placed a microscope slide, treated with Silane A174 (Pharmacia) to promote gel adhesion. The space between the silanized slide and the gelbond film was then filled with the polyacrylamide solution: 180 jtl of acrylamide and bisacrylamide stock (4.75 g acryl. + 0.25 g bis. in 10 ml H2O), 30 fi\ TEMED (0.20 ml TEMED in 10 ml H2O), 180 fi\ ammonium persulfate solution (0.025 g/10 ml H2O) 90 fi\ Serva ampholytes (pH 4-9), and 0.750 ml H2O. (This mixture is slightly modified from Ruchel.) Once polymerization has taken place, the gels may be used at once. This is done by lifting and turning over the gel form and either sliding or peeling the GelBond film off from the gel. Free liquid on the gel surface should be removed by touching a piece of absorbent paper to the lower edge of the upturned gel. Single pollen grains are placed within 2-3 mm of one edge of the gel, moistened with an aqueous solution containing 0.01 percent of ascorbic acid, 0.01 percent of cysteine, 0.2 M urea and 1.0 percent Serva ampholyte (pH 2-4). Within a few seconds, the grains increase diameter by approximately 30 percent and then are crushed with fine forceps. (This operation is facilitated by bringing forceps to a very sharp point with a fine grained abrasive stone.) The gel is then placed on a rubber or other nonslip surface, while strips of filter paper (8.0 X 70.0 mm) are moistened with 0.025 M aspartic acid and 0.025 M glutamic acid in water or 0.025 M arginine and 0.025 M lysine in 2.0 M ethylenediamine (see Radola). The aspartic-glutamic strip is placed at one edge of the gel and the arginine-lysine strip to • > < PLATINUM WIRE