Article Figures & Data
Figures
- Fig. 1.
A, SCR sequences are extremely polymorphic except for a putative highly conserved secretion signal. The amino acid sequences of the SCR alleles were deduced from cDNA sequences with the exception of SCR910. Based on homology to SCR6, SCR8, and SCR13 (Schopfer et al., 1999), we identified exons of SCR910 in the genomic sequence of the S910 haplotype (submitted to GenBank by Cui et al., 1999; accession number AJ245479.1). In the absence of an unambiguous splice site in the SCR910 gene, we tentatively joined the conserved Gln at position 20 in the first exon with Val encoded by the second exon. The SCR allele of theS9 haplotype was reported as SP11 without assignment of function (Suzuki et al., 1999). The SCR alleles of the haplotypes S12 andS52 were reported as SP11-12 and SP11-52, respectively (Takayama et al., 2000). Gaps were introduced to force the alignment of the conserved Cys residues C1 to C8. Residues conserved in at least four of the seven sequences are marked in bold. Classification as secretory proteins and signal peptide prediction was done using the TargetP and SignalP web servers (O. Emanuelsson, H. Nielsen, and G. von Heijne, unpublished data; Nielsen at al., 1997). Residues representing the potential N terminus residues of the mature polypeptide are underlined. The exact signal peptidase cleavage site will have to be determined experimentally. Bc, Brassica campestris; Bo, Brassica oleracea. B, The predicted secreted SCR polypeptides have a similar surface probability profile. The surface probability of hexapeptide regions of the amino acid sequences was calculated according to Emini et al. (1985) using LASERGENE software (version 4.03, 1999, DNASTAR, Inc., Madison, WI). Values above the default surface decision threshold of 1.0 indicate a likely localization at the surface of the SCR polypeptide. Dotted lines connect the positions of corresponding Cys, which are indicated by black circles. The asterisk marks the C terminus of the SCR polypeptide. C, Cys patterns in other small Cys-rich polypeptides in plants. Pollen-borne polypeptides are represented by PCP-A1 (Doughty et al., 1998) and plant defensins are represented by γ1-P (Colilla et al., 1990). Secondary structure elements of PCP-A1, outlined with dotted boxes (β-strands) and black boxes (α-helix), were predicted based on homology modeling (Doughty et al., 1998) onto the solution structure of γ1-P (Bruix et al., 1993). The brackets indicate disulfide bridges determined for γ1-P. Similar to γ1-P, PCP-A1 is predicted to adopt a triple-stranded β-sheet oriented parallel to the α-helix, with the Cys in the α-helix (motif C3xxxC4) connecting to the Cys in the third β-strand (motif C6xC7).
- Fig. 2.
The SCR promoter is active both gametophytically in microspores and sporophytically in the tapetum. Anthers were stained overnight at 37°C (1 mg/mL 5-bromo-4-chloro-3-indolylglucuronide in 0.1 m sodium phosphate buffer [pH 7.0], 0.5% [v/v] Triton X-100, 10% [v/v] methanol, and 0.5 mmferrocyanide) and embedded in paraffin before sectioning (A and B). Pollen grains isolated from dehisced anthers were stained for 20 min at 37°C (C and D). β-Glucuronidase (GUS) staining of five independentB. oleracea transformants carrying the SCR8:: uidA reporter construct showed the patterns of GUS activity illustrated in A and C. In contrast, negative control anthers and microspores showed no GUS staining as shown in B and D. T, Tapetum; M, microspores.
