Brassica napus pollen oleosins possess a characteristic C-terminal domain

Planta (1995) 195:469-470

Pl~lJ.~l__t3~ 9 Springer-Verlag 1995

Rapid communication Brassica napus pollen oleosins possess a characteristic C-terminal domain Michael R. Roberts, Rachel Hodge, Rod Scott Department of Botany, University of Leicester, University Road, Leicester LE1 7RH, UK Received: 25 August 1994 / Accepted: 6 October 1994

Abstract. The sequences of Brassica napus L. pollen oleosins have been determined and examined. Contrary to a recent report, inferred primary sequences of pollen oleosins do include a unique C-terminal domain characterised by the presence of a repetitive motif of three alanine and one proline residue (AAAP). This motif appears to be present in all oleosins expressed in pollen, but not in oleosins from other tissues.

reading frame, lacking the original characteristic C-terminus, and proposed that the second pollen oleosin, C98, must by inference also have been incorrectly sequenced. Here we report the sequence of a third antherspecific oleosin, homologous to I3, and a revised I3 sequence, which together with information from other sequences, confirm the presence of the characteristic AAAP motif in the C-terminal domains of all three pollen oleosins.

Key words: Brassica (oleosin, pollen) - Oleosin - Pollen Sequencing. The cDNAs and subclones were sequenced as single-

Oleosins are the major class of proteins found in association with storage lipid bodies, and have been identified in a range of oil-storing seeds (Huang 1992). More recently, oleosins have also been found in pollen (Roberts et al. 1993), the female gametophyte (Lee et al. 1994) and the anther tapetum (Robert et al. 1994; Ruiter et al. 1994). Seed oleosins possess three distinct domains, the most significant of which is the conserved central hydrophobic region, which is believed to interact directly with the internal lipid domain of the oil body (Huang 1992; Murphy 1993). We recently identified a previously described anther-specific cDNA clone (Roberts et al. 1991) as an oleosin on the basis of similarity to this domain and confirmed this identification by purification and microsequencing of B. napus pollen oleosin protein (Roberts et al. 1993). This clone, termed I3, and a second pollen oleosin identified in our laboratory, termed C98 (Roberts et al. 1993), contain an unusual C-terminal domain including a repeated AAAP motif. Subsequently, Lee et al. (1994) have highlighted an error in the original sequence of I3 following PCR amplification of a region of the I3 gene. These authors presented a new deduced Accession numbers: The nucleotide sequence data reported in this

paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X82019 (I3 cDNA) and X82020 (po13 cDNA). Correspondence to: R. Scott; Fax: 44 (116) 2522791; Tel.: 44

(116) 25233 87

stranded DNA templates rescued from pBluescript SK- or SK+ (Stratagene, Cambridge, UK) either manually using the Sequenase version 2.0 (USB, Cleveland, Ohio, USA) kit, or automatically using cycle sequencing and an Applied Biosystems (Warrington, Lancs., UK) model 373A sequencer. Both strands of the DNA were sequenced twice. Following the paper of Lee et al. (1994) which pointed out an error in the published I3 sequence (Roberts et al. 1991), we re-sequenced this region of the original I3 cDNA clone. In order to examine the possibility of errors introduced during cDNA cloning, we also fully sequenced a different cDNA previously isolated using I3 as a probe. This new clone was not in fact an independent isolate of I3, but represented a different but homologous anther oleosin with 9 1 % nucleotide sequence identity with I3. We termed this new clone po13, and its sequence and the deduced amino acid sequence are shown in Fig. 1. The figure shows that the clone encodes a C-terminal domain comprising a repeated motif of alanine and proline residues, as was originally reported for I3 and C98. By thoroughly re-sequencing the I3 cDNA across the central domain, we confirmed the presence of the extra GC pair between bases 255,256 in the original I3 sequence, as reported by Lee et al. (1994). These bases were not visible in sequencing gels from all templates sequenced, due to compressions caused by G/C richness in this area. However, our re-sequencing also uncovered a second error in the published sequence, this being the presence of an additional adenosine base at position 333,

470

M.R. Roberts et al.: Pollen oleosin sequences

GAATTCC ATTCAAACTAGTAAACAATGGGGATACTCAGGAAGAAAAAACACGAGCGAAAG M G I L R K K K H E R C CGTCGTTTAAGAGTGTTTTAACCGCAATATTAGCTACACATGC P S P K S V L T A I L A T H

K

CGCAACATTC CTCTTG A T F L L

A

TTGATCGCCGGTGTATCCCTCGCCGGCACAGCCGCCGCATTTATCGCTACCATGC L I A G V S I, A G T A A A F I A T

M

P

CACTA L

TTCGTAGTATTCAGTCCGATTCTCGTACCAGC F V V F S P I L V P

A

GGTTTAGCAGCCGCCGGTC-GCGCCGGCGCG G L A A A G G A G A

AC T G C T G T C A C C A T C A T C C T G T G G C T C T A C T A V T I I L W L Y

AAGCGAGCAACGGGCAAGGCGC K R A T G K A

P

TGGTATTACCACTGGTTTACTGACTACG G I T T G L L T T

CGCC AAAAGTCCTAGAAAAAGTCTTGAAAAAGATAATA P K V L N K V L K K I I

CCAGGTGCTGCAGCTGCACCAGCAGCCGCTCCAGGAGCCGCTCCAGCAGCGGCGCCAGCA P G A A A A P A A A P G A A P A A A GCCGCACCAGCTGTGGCGCCAGCAGCCGCACCAGC A A P A V A P A A A P

P

A

TGCTGCGCCAGCACCTAAGCCAGCA A A P A P K P A

A

GCCCCACCAGCACC TAAGC CAGCAGCCGCACCGAGTATATGAAAAGAAGTGGTGGGCATG A P P A P K P A A A P S I AGTAAAGGTTGATATGGAAAACTGGATACATAGAAAAAAGAGTAATCCAACTTTTAAAAA ATAAATAACAACTTCACGTGGGGATAGAAAAATTTTCAAATATTATTTTACTAATGGATG TCGCGGTACAAAATAATAAC GCTATGTAGCGTTGACA

AAATGTAAGCCTTTTTATTGTATAGTATTTTAAGAACGAA (n )

Fig. 1. Nucleotide and deduced amino acid sequence of the po13 cDNA from Brassica napus pollen. The poly(A) tail is represented A(n). The amino acid sequence is shown beneath the nucteotide sequence with the stop codon indicated by an asterisk (*) po13 13 GRP8 C98

po13 13 GRP8 C98

i MGILRKKKHE MGILRKKKHE MFS ....... MFS

RKPSFKSVLT RNASFKSVLT FLIFLLEAFQ FIFPFVNVIK

AILATHAATF SILATQAATF VVIATVVSIV LIIASVTSLV

LLLIAGVSLA LLLISGVSLA FLVFAGLTLV CLAFSCVALG

51 PLFVVFSPIL PLFVVFSPIL PLFIIFSPIL PLFIMFSPIL *** *****

VPAGITTGLL VPAGITTGLL VPATIATAVI VPATIATTLL *** * *

TTGLAAAGGA TTGLAAAGGA TTGFTTGGAL ASGLMAGTTL *

GATAVTIIL. GATAVTIIL. GAMAVALIRR GLTGIGLI.~ * *

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i00

i01 po13

13

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GRP8 C98

TESAQPLLK VSLLQSPLR

po13 I3 GRP8 C98

151 T~3KAPP . . . . . . . K V L E K V L TGKEPP ....... AVLSKVL AGGGAPGGLG GGGNPFGNIS GG~SRLA RLKKILGLLN *

po13 I3 GRP8 C98

201 PAAAPAAAPA PAPKPAAAPA PAAGAAPAAG PAAAPAAAPA ** * *

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.

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L P\rYGGYGGFW .

50 GTAAAFIATM GTAAAFIATM GSATALTITT GSAVALIVST

similar in computer comparisons. We also re-sequenced the C98 cDNA, which had been presumed to be incorrect by Lee et al. (1994) and found no errors in the originally reported sequence which includes the AAAP motif. In addition to these three B. napus pollen cDNA sequences, a B. napus genomic clone for a different pollen oleosin homologous to C98 has been isolated and sequenced (J.H.E. Ross, Cambridge Laboratory, John Innes Centre, Norwich; personal communication) and this clone also encodes a C-terminal domain including A A A P repeats. In Arabidopsis too, a family of flowerspecific cDNAs and corresponding genomic clones encoding an oleosin-like domain has been reported (de O1iveira et al. 1993). The sequence of one of these genes in particular, atGRP-8, is significantly similar to our B. napus pollen oleosins and includes the A A A P repeated motif. An alignment of the three B. napus pollen oleosins and the a t G R P - 8 amino acid sequence is presented in Fig. 2. The a t G R P - 8 sequence was not considered by Lee et al. (1994). Studies in our laboratory have demonstrated that the a t G R P - 8 promoter is active predominantly in developing microspores and pollen, and also in the carpels of transgenic plants (data not shown), an expression pattern consistent with the proposition that a t G R P - 8 may encode an oleosin. We thus propose that pollen oleosins from the Brassicaceae do indeed possess a characteristic C-terminal domain, dissimilar to those found in seeds or other tissues, the defining feature of which is the repeated amino acid motif A A A R The function of this domain and in particular the role of the repeated motif in pollen remains unknown.

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KKIIPGAAAA KKIIPGAAAA KWFGPGAAGG KLRGMGAGGA * **

.

150 WLYKRA . . . . . . . . . . . . WLYKQA NKPGGGNPFG DISKWLGPGA K I IVNRIKARL .

GGKKFSGTFG .

References KMGAKPTAEG 9M G L V R T A G G

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PAAAPGAAPA PRAAPAAAPA DASAAGGAPA AAPAAEPAPA * ***

200 AAPAAAPAVA AAPAAAPAAA AEAAPAAGAA AEAAPAAEAA * * * *

230 PKPAAPPAPK PAAAPSI.. PKPAAPPAL .......... AAPAAGAAPA AGGSTPpTW AAP ................ *** *

Fig. 2. Multiple sequence alignment of pollen oleosin proteins. po13, I3 and C98 are the B. napus pollen oleosins, and GRP8 the putative Arabidopsis pollen oleosin (de Oliveira et al. 1993). Residues present in all sequences are indicated by an asterisk (*) below the alignment 77 bases 3' of the first difference. The inclusion of these three additional bases in the I3 sequence restores the original reading frame including the repeated A A A P motif. The deduced amino acid sequences of the revised I3 cDNA and the po13 cDNA are 90% identical and 92%

de Oliveira, D.E., Franco, L.O,, Simoens, C., Seurinck, J., Coppietters, J., Botterman, J., van Montagu, M. (1993) Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins. Plant J. 3,495-507 Huang, A.H.C. (1992) Oil bodies and oleosins in seeds. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, t77-200 Lee, K., Bih, F.Y., Learn, G.H., Ting, J.T.L., Sellers, C., Huang, A.H.C. (1994) Oteosins in the gametophytes of Pinus and Brassica and their phylogenetic relationship with those in the sporophytes of various species. Planta 193, 461-469 Murphy, D.J. (1993) Structure, function and biogenesis of storage lipid bodies and oleosins in plants. Prog. Lipid Res. 31, 247-280 Ruiter, R.K., van Eldik, G J., van Herpen, M.M.A., Schrauwen, J.A.M., Wullems, G.J. (1994) Molecular analysis of pollen coat proteins of Brassica oleracea in relation to anther development. Proceedings 4th Intl. Congress Plant Mol. Biol., Amsterdam Robert, L.S., Gerster, J.L., Allard, S., Cass, L., Simmonds, J. (1994) Molecular characterization of two Brassica napus genes related to oleosins which are highly expressed in the tapetum. GenBank Nucleotide Sequence Database Roberts, M.R., Robson, F., Foster, G.D., Draper, J., Scott, RJ. (199t) A Brassica napus mRNA expressed specifically in developing microspores. Plant Mol. Biol. 17, 295-299 Roberts, M.R., Hodge, R., Ross, J.H.E., Sorensen, A., Murphy, D.J., Draper, J., Scott, R. (1993) Characterization of a new class of oleosins indicates a male gametophyte-specific lipid storage pathway. Plant J. 3, 629--636