Biochimica et Biophysica Acre, 1089(1991)41!-413
411
© 1991 ElsevierSciencePublishersB.V. 0167-4781/91/$03.50 ADONIS 0167478191001864 BBAEXP 90250
Short Sequence-Paper
Pollen specific c D N A clones from Zea mays H.J. Rogers i, R.L. Allen 2, W.D.O. Hamilton 3 and D.M. Lonsdale 2 7 Royal Holloway and Bedford New College, Egham Hill, Egham (U.K.), 2 IPSR Cambridge Laboratory, lohn lnnes Centre for Plant Science Research, Norwich (U.K~) and 3 Applied Plant Technology Laboratory, Agricultural Genetics Company Ltd., Babraham, Camlm'dge (U.K.)
(Received5 February 1991) (Revisedmanuscript7 May 1991) Key words: Pollen; cDNA: Polygalacturonase;(Maize) We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-r/pening polygalacturonase and to a pollen-specific polygalacturonase from Oenothem. The other three clones have no significant homologies to any reported sequence.
The male gametophyte (pollen) is a terminally specialised ,issue and its development requires a unique pattern of gene expression involving both sporophytic and gametophytic genes. The cytological differentiation occurring during this process is accompanied by biochemical changes which involve synthesis of complex polysaccharides, pigments and storage products [1]. It has also been shown that stored messages are present in the pollen grain which encode proteins important for pollen maturation, pollen germination and early pollen tube growth [2]. Both biochemical evidence relating to isozyme studies [3-5], and reassociation kinetics experiments [6] indicate that there is a class of genes which are uniquely expressed in pollen. It therefore seemed possible to isolate pollen-specific messages by differential screening. A library of 1000 cDNA clones was constructed from maize pollen using dG tailed pUC9 vector (Pharmacia). Colony lifts from the library were screened using cDNA probes made from poly(A) ÷ RNA isolated from pollen and a vegetative tissue: the coleoptile [7]. 40 cDNA clones were found by this method to be differentially expressed. However, when the DNA from these clones was further tested by hybridisation to the two probes, it was found that only six of these clones showed no detectable hybridisation to the coleoptile probe indicating that they were pollen specific. Four of the clones were completely sequenced [8]
Correspondence:D.M. Lonsdale,IPSR CambridgeLaboratory,John lnnes Centre for Plant Science Research, Colney Lane, Norwich, Norfolk, NR4 7UJ, U.IC
and also used to probe Northern blots of poly(A) + RNA isolated from various tissues. The Northern probed with 3C12 (Fig. 1) is representative of all of the clones. The mRNAs corresponding to these clones were only detected in mature pollen and developing pollen/tassel tissue. The estimated size of the mRNAs was larger than the cDNA inserts (Table I) and sequencing showed that the open reading frames were incomplete at their 5' ends. The EMBL (version 25.0) and Genbank (version 65.0) databases have been searched using these sequences and except for 3C12 no significant homologies 123456789
1.5kb
Fig. 1. Northern of 3C12. The tracks contain 5 izg poly(A)+ RNA from !, mature pollen: 2, mature non-dehiscenttassels; 3, in.mature tassels; 4, very immaturetassels; 5, coleoptyle; 6, leaf; 7, root; 8, cob; 9, s.ks.
412 1
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GCGACCACGG&CCT~GCCAGTAC~GG~CCATGGT~TTGGATCGAGATTCT~CGTGTGGAT~CCTGGTCATCA~CGGC~GGG~AC
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1171
GT~CGC&C~CAGT,~k,~kCGAC&G~CATCCG&C~GCTATATT&TGTTCG~CGGTGT~CACCCTG~TTTGAGG
1245
Fig. 2. The ~quence and translation of clone 3CI2 and its comparison to the r e , t i e d c ~ o n sequences of the Oeno~era and tomato ~lygalacturona~ (Genbank accession number: X05656) cDNAs. *, identical amino acids between all three sequences. :, ~sition where a conse~ative amino acid replacement has occurred in one of the three sequences. The alignment, ~tween the three ~quences (not s h ~ n ) , was per~rmed using the Wi~onsin program GAP [12].
to known proteins have been detected to date. 3C12 shows significant homology to the polygalacturonase associated with fruit-ripening in tomato [9] (amino acid identity 40%, similarity 61%) and to the reported pollen-specific polygalacturonase of Oenothera [10] TABLE I Clone
eDNA size (bp)
mRNA (size kb)
EMBL accession No.
3A6 3C12 4H3 4H7
661 1245 793 837
! .2 1.5 1.8 0.9
X57272 X57274 X57273 X57275
(amino acid identity 44%, similarity 62%) as shown in Fig. 2. Polygalacturonase has been found in the pollen of a number of plant species [10,11] and it is thought that it may have a role in the expansion of the pollen tube and in dissolution of a pectin-rich stratum within the stigma. As more precise data on the timing and Iocalisation of the eDNA clones becomes available it is hoped that the role of the remaining eDNA clones may be elucidated. This work was supported by the Agricultural Genetics Company, to whom we are grateful for the technical assistance of J.C. Timans and D.A. Harrison, and latterly by Pioneer Hi-Bred International Inc. We
413
would also like to thank S.M. Brown and M.L. Crouch, University of Bloomington, IN, U.S.A. for providing the sequence of the Oenothera P2 cDNA [10]. References 1 Mascarenhas, J.P. (1975) Bot. Rev. 41,259-314. 2 Mascarenhas, N.T., Bashe, D., Eisenberg, A., Willing, R.P., Xiao, C.M. and Mascarenhas, J.P. (1984} Theor. Appl. Genet. 68, 323-326. 3 Tanksley, S.D., Zamir, D. and Rick, C.M. (1981) Science 213, 453-455. 4 Sari Gorla, M., Frova, C., Binelli, G. and Ottaviano, E. (1986) Theor. Appl. Genet. 72, 42-47.
5 Pedersen, S., Simonsen, V. and Loeschcke, V. (1987) Theor. Appl. Genet. 75, 200-206. 6 Willing, R.P., Bashe, D. and Mascarenhas, J.P. (1988) Theor. Appl. Genet. 75, 751-753. 7 St. John, T.P. and Davis, R.W. (1979) Cell 16, 443. 8 Murphy, G. and Kavanagh, T.A. (1988) Nucleic Acids Res. 16, 5198. 9 Grierson, D., Tucker, G.A., Keen, J., Ray, J., Bird, C.R. and Schuch, W. (1986) Nucleic Acids Rcs, 14, 8595. 10 Brown, S.M. and Crouch, M.L (1990)The Plant Cell 2, 263-274. I1 Pressey, R. and Reger, B.J. (1989) Plant Science 59, 57-62. 12 I2¢vereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395.
411
© 1991 ElsevierSciencePublishersB.V. 0167-4781/91/$03.50 ADONIS 0167478191001864 BBAEXP 90250
Short Sequence-Paper
Pollen specific c D N A clones from Zea mays H.J. Rogers i, R.L. Allen 2, W.D.O. Hamilton 3 and D.M. Lonsdale 2 7 Royal Holloway and Bedford New College, Egham Hill, Egham (U.K.), 2 IPSR Cambridge Laboratory, lohn lnnes Centre for Plant Science Research, Norwich (U.K~) and 3 Applied Plant Technology Laboratory, Agricultural Genetics Company Ltd., Babraham, Camlm'dge (U.K.)
(Received5 February 1991) (Revisedmanuscript7 May 1991) Key words: Pollen; cDNA: Polygalacturonase;(Maize) We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-r/pening polygalacturonase and to a pollen-specific polygalacturonase from Oenothem. The other three clones have no significant homologies to any reported sequence.
The male gametophyte (pollen) is a terminally specialised ,issue and its development requires a unique pattern of gene expression involving both sporophytic and gametophytic genes. The cytological differentiation occurring during this process is accompanied by biochemical changes which involve synthesis of complex polysaccharides, pigments and storage products [1]. It has also been shown that stored messages are present in the pollen grain which encode proteins important for pollen maturation, pollen germination and early pollen tube growth [2]. Both biochemical evidence relating to isozyme studies [3-5], and reassociation kinetics experiments [6] indicate that there is a class of genes which are uniquely expressed in pollen. It therefore seemed possible to isolate pollen-specific messages by differential screening. A library of 1000 cDNA clones was constructed from maize pollen using dG tailed pUC9 vector (Pharmacia). Colony lifts from the library were screened using cDNA probes made from poly(A) ÷ RNA isolated from pollen and a vegetative tissue: the coleoptile [7]. 40 cDNA clones were found by this method to be differentially expressed. However, when the DNA from these clones was further tested by hybridisation to the two probes, it was found that only six of these clones showed no detectable hybridisation to the coleoptile probe indicating that they were pollen specific. Four of the clones were completely sequenced [8]
Correspondence:D.M. Lonsdale,IPSR CambridgeLaboratory,John lnnes Centre for Plant Science Research, Colney Lane, Norwich, Norfolk, NR4 7UJ, U.IC
and also used to probe Northern blots of poly(A) + RNA isolated from various tissues. The Northern probed with 3C12 (Fig. 1) is representative of all of the clones. The mRNAs corresponding to these clones were only detected in mature pollen and developing pollen/tassel tissue. The estimated size of the mRNAs was larger than the cDNA inserts (Table I) and sequencing showed that the open reading frames were incomplete at their 5' ends. The EMBL (version 25.0) and Genbank (version 65.0) databases have been searched using these sequences and except for 3C12 no significant homologies 123456789
1.5kb
Fig. 1. Northern of 3C12. The tracks contain 5 izg poly(A)+ RNA from !, mature pollen: 2, mature non-dehiscenttassels; 3, in.mature tassels; 4, very immaturetassels; 5, coleoptyle; 6, leaf; 7, root; 8, cob; 9, s.ks.
412 1
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1245
Fig. 2. The ~quence and translation of clone 3CI2 and its comparison to the r e , t i e d c ~ o n sequences of the Oeno~era and tomato ~lygalacturona~ (Genbank accession number: X05656) cDNAs. *, identical amino acids between all three sequences. :, ~sition where a conse~ative amino acid replacement has occurred in one of the three sequences. The alignment, ~tween the three ~quences (not s h ~ n ) , was per~rmed using the Wi~onsin program GAP [12].
to known proteins have been detected to date. 3C12 shows significant homology to the polygalacturonase associated with fruit-ripening in tomato [9] (amino acid identity 40%, similarity 61%) and to the reported pollen-specific polygalacturonase of Oenothera [10] TABLE I Clone
eDNA size (bp)
mRNA (size kb)
EMBL accession No.
3A6 3C12 4H3 4H7
661 1245 793 837
! .2 1.5 1.8 0.9
X57272 X57274 X57273 X57275
(amino acid identity 44%, similarity 62%) as shown in Fig. 2. Polygalacturonase has been found in the pollen of a number of plant species [10,11] and it is thought that it may have a role in the expansion of the pollen tube and in dissolution of a pectin-rich stratum within the stigma. As more precise data on the timing and Iocalisation of the eDNA clones becomes available it is hoped that the role of the remaining eDNA clones may be elucidated. This work was supported by the Agricultural Genetics Company, to whom we are grateful for the technical assistance of J.C. Timans and D.A. Harrison, and latterly by Pioneer Hi-Bred International Inc. We
413
would also like to thank S.M. Brown and M.L. Crouch, University of Bloomington, IN, U.S.A. for providing the sequence of the Oenothera P2 cDNA [10]. References 1 Mascarenhas, J.P. (1975) Bot. Rev. 41,259-314. 2 Mascarenhas, N.T., Bashe, D., Eisenberg, A., Willing, R.P., Xiao, C.M. and Mascarenhas, J.P. (1984} Theor. Appl. Genet. 68, 323-326. 3 Tanksley, S.D., Zamir, D. and Rick, C.M. (1981) Science 213, 453-455. 4 Sari Gorla, M., Frova, C., Binelli, G. and Ottaviano, E. (1986) Theor. Appl. Genet. 72, 42-47.
5 Pedersen, S., Simonsen, V. and Loeschcke, V. (1987) Theor. Appl. Genet. 75, 200-206. 6 Willing, R.P., Bashe, D. and Mascarenhas, J.P. (1988) Theor. Appl. Genet. 75, 751-753. 7 St. John, T.P. and Davis, R.W. (1979) Cell 16, 443. 8 Murphy, G. and Kavanagh, T.A. (1988) Nucleic Acids Res. 16, 5198. 9 Grierson, D., Tucker, G.A., Keen, J., Ray, J., Bird, C.R. and Schuch, W. (1986) Nucleic Acids Rcs, 14, 8595. 10 Brown, S.M. and Crouch, M.L (1990)The Plant Cell 2, 263-274. I1 Pressey, R. and Reger, B.J. (1989) Plant Science 59, 57-62. 12 I2¢vereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395.
