The Journal of Heredity 72: 353-354. 1981.

Gametophytic transcription for

acid phosphatases in pollen of

Cucurbita species hybrids

David L. Mulcahy, Richard W. Robinson,

Masaaki lhara, and Richard Kesseli

ABSTRACT: A microslab isoelectric focusing gel isdemonstrated, using single pollen grains from F, hybridsof Cucurbita species. When zymograms of these singlepollen grains were stained to indicate the presence ofacid phosphatase zones, it was seen that at least 11 outof 37, or 29.7 percent of such zones are controlled byloci that are transcribed in the microgametophyte. Theseresults suggest that gametophytically transcribed locimay be fairly common and also that their investigationis now technically unchallenging.

IT HAS LONG BEEN suggested that pollen, withits haploid genotype and very large populationsizes, might undergo selective pressures thatgreatly exceed those found in other parts ofthe angiosperm life cycle. This possibility is adouble-edged one, representing, on one hand,a vulnerability in which "a gene which greatlyaccelerates pollen tube growth will spreadthrough a species even if it causes moderatelydisadvantageous changes in the adult plant."1

It is perhaps this that prompted some investi-gators2 to suggest the number of genes actuallytranscribed in the pollen is probably quitesmall. Conversely, a gene that benefits themature plant, if it benefits also the gameto-phyte, could be increased rapidly by pollentube selection. Finally it must be consideredthat if genes are not transcribed in the pollen,then both deleterious and beneficial effects ofpollen tube selection are precluded. Evidencethat genes are transcribed in pollen comesfrom several sources; but many of these relyon specialized phenomena such as selfin-compatibility or uniquely convenient bio-chemical differences such as the presence orabsence of alcohol dehydrogenases or waxyversus nonwaxy starch. These phenomenahave provided valuable insights into thebiology of pollen but, at the same time, theyraise a critical question. Does the rarity of suchpollen makers indicate that much of the pollengenome is indeed inactive, or is it that we havenot yet learned to detect other gametophyti-cally transcribed loci? Mascarenhas3 has

The authors are affiliated, respectively, with theDepartment of Botany, University of Massachusetts,Amherst, MA 01003; the Department of Seed andVegetable Research, Cornell University, Geneva,NY 14456; the Department of Biochemistry, NationalInstitute of Genetics, Mishima, 444 |apan, and theDepartment of Agronomy, University of California,Davis, CA 95616. This research was supported inpart by NSF grant DEB 79-03685 and USDA/SEAgrant 5901-0410-9-0365-0.© 1981, American Genetic Association.

demonstrated that there is extensive proteinsynthesis in pollen but he points out the pos-sibility that some of this could be the transla-tion of longlived messenger transcribed bysporophytic tissues.

A resolution of this issue may be providedby electrophoretic studies of pollen, using ei-ther of two approaches. The first of these, re-cently employed by Weeden and Gottlieb8 isto examine di- or multi-meric enzymes. Ifpollen from segregating sporophytes exhibitshomomultimers and, unlike the sporophytes,lacks heteromultimers, then the locus inquestion must be transcribed post-meiotically.Products of segregation would then be capableof responding to selection.

An alternative electrophoretic test forpostmeiotic expression involves the analysisof individual pollen grains. In this case, post-meiotic transcription is indicated by segrega-tion of isozymes in pollen from heterozygousindividuals. It is the latter method that is de-scribed in the present paper. Specifically, themethod employs a microslab gel and isoelec-tric focusing.

Materials and Methods

To form a gel, a sheet of GelBond film(Bioproducts, Rockland, Maine 04841) wasplaced, hydrophobic side up, on a sheet ofglass or other smooth surface. Two strips ofplastic, each approximately 0.5 X 4.0 X 80.0mm, were then placed on the GelBond film,parallel to each other and about 20.0 mmapart. On top of these plastic strips was placeda microscope slide, treated with Silane A174(Pharmacia) to promote gel adhesion. Thespace between the silanized slide and thegelbond film was then filled with the polyac-rylamide solution: 180 jtl of acrylamide andbisacrylamide stock (4.75 g acryl. + 0.25 g bis.in 10 ml H2O), 30 fi\ TEMED (0.20 ml TEMEDin 10 ml H2O), 180 fi\ ammonium persulfatesolution (0.025 g/10 ml H2O) 90 fi\ Serva am-pholytes (pH 4-9), and 0.750 ml H2O. (Thismixture is slightly modified from Ruchel7.)

Once polymerization has taken place, thegels may be used at once. This is done by lift-ing and turning over the gel form and eithersliding or peeling the GelBond film off fromthe gel. Free liquid on the gel surface shouldbe removed by touching a piece of absorbentpaper to the lower edge of the upturned gel.

Single pollen grains are placed within 2-3mm of one edge of the gel, moistened with anaqueous solution containing 0.01 percent ofascorbic acid, 0.01 percent of cysteine, 0.2 Murea and 1.0 percent Serva ampholyte (pH2-4). Within a few seconds, the grains increasediameter by approximately 30 percent andthen are crushed with fine forceps. (This op-eration is facilitated by bringing forceps to avery sharp point with a fine grained abrasivestone.) The gel is then placed on a rubber orother nonslip surface, while strips of filterpaper (8.0 X 70.0 mm) are moistened with0.025 M aspartic acid and 0.025 M glutamicacid in water or 0.025 M arginine and 0.025 Mlysine in 2.0 M ethylenediamine (see Radola6).The aspartic-glutamic strip is placed at oneedge of the gel and the arginine-lysine strip to

• > <PLATINUM WIRE

AlCROSLIDt

FIGURE 1 Electrophoresis apparatus. The mi-croscope slide is 2.5 cm wide. See text for details.

the other. (Strips should be touching but notatop the gel.) The anode is connected to thefirst strip and the cathode to the other (seeFigure 1). Electrophoresis is started at 100 voltsand after 2-3 minutes, changed to constantpower (approximately 0.5 watts). All runs areconducted at room temperature. Within 20-40minutes, voltage increases as the conductivityof the focusing gel declines. The end of the runis signaled by a rapid increase of voltage to themaximum capability of the power supply (500or 1000 v). No advantage is gained by using the1000 v setting. After focusing, the gel is placedin a standard stain for acid phosphatases (fastgarnet GBC and alpha-naphthol phosphate in0.2 M acetate buffer, pH 5.0) for approxi-mately 1 hour at room temperature, less at 37C. At the end of this time (45-60 minutes) thestaining solution is exhausted and thus re-placed with another freshly prepared fastgarnet and alpha naphthol phosphate solution.At the end of a second 45 to 60-minute stain,gels can then be cleaned with cotton swabs,photographed and, if desired, stained forgeneral proteins using the simplified silvermethod of Oakley et al.5 (It is necessary to usethe pre-wash that Oakley et al. suggested for10 percent acrylamide gels in order to removeampholytes from the slab before staining.)

The above method is technically less de-manding than the microcolumn system pre-viously described4 and the slab configurationpermits precise comparison and interpretationof samples. Also, the isoelectric focusingseems to provide a greater degree of repro-ducibility between separate gels than do othersystems.

Results and Discussion

When single pollen grains from the F t hy-brid, Cucurbita moschata X C. palmeri, aresubjected to isoelectric focusing and thenstained for acid phosphatase activity, eightzones of staining are seen (Figure 2). Of theseeight, six are found in all of the samples. Theremaining two zones are apparently deter-mined by a single segregating locus since eachis found in approximately one of the samples.It may thus be concluded with confidence thatat least two of the eight acid phosphatasezones in the pollen of this hybrid are under thecontrol of the gametophytic genotype. As isshown, also in Figure 2, there is no evidenceof segregation in any of the eight acid phos-phatase zones produced from pollen of the Fihybrid, C. maxima X C. moschata. Corre-sponding data for the F1 between C. sororia XC. moschata (Figure 2) are four segregating out

Notes September / October 1981 353

PH

PH'

cm

4 origin

moschata X

C. paimeri

C_. maxima X C_. sororia X

C. moschata C moschata

FIGURE 2 Zymograms of single pollen grains from three interspecific hybrids of Cucurbita. Both the C.moschata X C. paimeri and C. sororia X C. moschata exhibit segregation in the pH range of 5.5. The latterhybrid is segregating also at pH 6.5. The pollen from C. maxima X C. moschata hybrid shows no segregationfor acid phosphatases in this gel.

of a total of seven. For C. texana X C. palmata(not illustrated), three out of seven, and for C.pepo X C. texana (not illustrated), two out ofseven. In all, 11 out of the 37, i.e., 29.7 percentof the acid phosphatase zones observed inthese gels were definitely the products ofgametophytic transcription. Furthermore, itmust be considered that this method indicates

gametophytic transcription for heterozygousloci only. Without further information, it is notpossible to determine the origin of the 26 otheracid phosphatase zones. Some or all may bethe products of sporophytic transcription, orthey may be products of gametophyticallytranscribed loci that are not segregating in thismaterial. These results thus provide a con-

servative estimate of gametophytic tran-scription. Nonetheless, they suggest that suchtranscription may be quite extensive. Theyalso demonstrate that it is now possible toundertake a detailed investigation of game-tophytic genetic activity.

References

1. HALDANE, ). B. S. The Causes of Evolution.Longmans, Green, and Co., London. 1932.

2. HESLOP-HARRISON, J. Summary and perspec-tives in higher plants as monitors of environ-mental mutagens. Environ. Health Perspect.27:197-206. 1978.

3. MASCARENHAS, J. P. The biochemistry of an-giosperm pollen development. Bot. Rev. 41:259-314.1975.

4. MULCAHY, D. L., G. B. MULCAHY, and R. W.ROBINSON. Evidence for postmeiotic geneticactivity in pollen of Cucurbita species. ]. Hered.70:365-368.1979.

5. OAKLEY, B. R., D. R. KIRSCH, and N. R. MORRIS.A simplified ultrasensitive silver stain for de-tecting proteins in polyacrylamide gels. Anal.Biochem. 105:361-363.1980.

6. RADOLA, B. J. Ultrathin-layer isoelectric focusingin polyacrylamide gels. Electrophoresis 1:43-56.1980.

7. RUCHEL, R. Two-dimensional micro-separationtechnique for proteins and peptides, combiningisoelectric focusing and gradient gel electro-phoresis. J. Chromatog. 132:451-468.1977.

8. WEEDEN, N. F. and L. D. GOTTLIEB. Distin-guishing allozymes and isozymes of phospho-glucoisomerases by electrophoretic comparisonsof pollen and somatic tissues. Biochem. Genet.17:287-296. 1979.

The Journal of Heredity 72: 354-355. 1981.

A compensating trisomic in

pearl millet

R. S. Saini and J. L. Mlnocha

ABSTRACT: A compensating trisomic was isolated fromthe progeny of a multiple interchange trisomic in pearlmillet, Pennisetum typhoides. It showed the character-istic morphological features of trisomy and formed achain-of-seven chromosomes with the absence of aring-of-four-or-six chromosomes.

ANEUPLOIDS, particularly trisomics, havebeen extensively used in genetic analyses andmapping of chromosomes of diploid plantspecies1'3. In pearl millet, Pennisetum ty-

The authors are, respectively, scientist (S-l), In-dian Council of Agricultural Research, New Delhi;and senior geneticist, Punjab Agricultural Univer-sity, Ludhiana, India. This study is based on researchconducted by the senior author in partial fulfillmentof the requirements for the Ph.D. degree. Pleaseaddress reprint requests to Dr. Minocha.© 1981, American Genetic Association.

phoides (2n = 14), an important cereal ofsemiarid regions, a complete series of primarytrisomics, a number of interchange stocks andtertiary trisomics are available267. Compen-sating trisomics in which one missing chro-mosome is compensated for by two modifiedchromosomes, have not been reported in pearlmillet. However, such trisomics have beensuccessfully used for locating genes on par-ticular arms of chromosomes in the tomato4.The present communication is the first reporton the origin, isolation, and cytological be-havior of a compensating trisomic in pearlmillet.

A compensating trisomic was isolated fromthe progeny of a multiple interchange tri-somic. The origin of multiple interchangetrisomics was described by Minocha andBrar5. The expected types of aneuploidsarising from the progeny of multiple inter-change trisomics are shown in Figure 2, where'n' designates the haploid chromosome com-plement with standard or interchangedchromosomes. Chromosome composition ofthe extra dose in the gametes and the zygotesis also given. For simplicity, the male gametesare shown with only the standard chromo-some complement. One of the five combina-tions (square 3) involving the union of a nor-mal male gamete (n chromosome number)

with a female gamete (n + 1 chromosomenumber) possessing two modified chromo-somes in place of normal chromosome (1S.1L)results in the compensating trisomic. This wasconfirmed cytologically by the presence of achain configuration of seven chromosomes(lvn + 4n) and the absence of a ring-of-four-or-six chromosomes (Figure 1A). The meioticanalysis of the compensating trisomic showeda chain of seven chromosomes in 14.4 percentof the cells. The other chromosome associa-

B

FIGURE 1 A—diakinesis in a compensating tri-somic showing a chain of 7 chromosomes. B—ana-phase I with 8-7 chromosome distribution in thecompensating trisomic.

354 The Journal of Heredity Notes