Genes Expressed in Pinus radiata Male Cones Include Homologs to Anther-Specific and Pathogenesis Response Genes

Temporal expression analysis. Northern blots of total RNA extracted from male cones during two flowering seasons. Abbreviations for male cones are given in Table I. Needle, root, and shoot data (where presented) are as indicated, and the abbreviations for female cones are as follows: Fc8, female cones 8 months before meiosis; Fc7, female cones 7 months before meiosis; Fc4, female cones 4 months before meiosis. Expression data and transcript size are summarized in Figure 3 and Table II. Loading differences were assessed by probing blots with a 26S rRNA probe as indicated. Note that there is wide variation in the loading of samples between lanes, with some samples underloaded (e.g. T1, Misi); this is the likely cause of the apparent transient down-regulation of genes such as PrLTP1 in T1-stage cones. Faint smears immediately adjacent to lanes containing strongly hybridizing bands (e.g. PrMC104, T1 sample) have been interpreted as noise in the summary in Figure 3.

Most cDNAs are expressed from before to just after meiosis. This figure illustrates the expression of the 13 cDNAs at different stages of pollen development (top). Development progresses from left to right. The cDNAs are labeled on the left side of the figure. An oval spot represents expression. Abbreviations for male cones are given in Table I. Bars indicating the size of developing pollen are 9 μm. Me-iep- to Me-at-stage meiocytes are enlarged by a factor of two.

Spatial expression of five genes in P. radiata male cones. Me-tIpII-stage- and T2-stage male cones (top and bottom, respectively) were sectioned and probed with sense (control) and antisense transcripts of five cDNAs. The results for PrChS1, PrLTP1, PrMC1, and PrMC2 were similar and are represented above by the results of PrChS1 and PrLTP1. The result for PrLTP2 is illustrated separately above. The bar beneath each image represents 250 μm; rectangles indicate the origin of the enlarged images. Detection of transcript is indicated by the blue/black tetrazolium blue signal (arrow) and in the control sections arrows indicate corresponding tissue.

Southern-blot analysis. Blots of genomic DNA (10 μg per lane, digested as indicated) were probed with the cDNA clones indicated. Clones with an average of less than three signals per digest were scored as low (e.g. PrChS1 and PrMC3), three to seven bands were scored as intermediate (e.g. PrMC187), and eight or more bands were scored as high copy (e.g. PrLTP1, data summarized Table II).

Phylogenetic analysis of ChS and StS-like sequences. The figure illustrates the tree generated when ChS and StS amino acid sequences corresponding to bases 8 to 386 of PrChS1 were aligned and subjected to a phylogenetic analysis. Five of the six sequences (indicated by asterisks) in the PrChS1 clade are specific to male reproductive tissues. ChS sequences from plant species corresponding to those represented in the PrChS1 clade are shaded gray. The distribution of StS sequences (boxed) throughout the phylogram suggests that StS evolved from ChS several times independently during evolution (also see Tropf et al., 1994). ChS sequences from plant species corresponding to the StS sequences are underlined. Abbreviations are as follows: Ph, Petunia hybrida; Ah,Arachis hypogaea; Pl, Pueraria lobata;Gm, Glycine max; Ms, Medicago sativa; Ps,Pisum sativum; Ts, Trifolium subterraneum; Psy, Pinus sylvestris; Pst,Pinus strobus; Cs, Camellia sinensis; Vv,Vitis vinifera; Bn, Brassica napus; At, Arabidopsis; Br, Brassica rapa; Ns, Nicotiana sylvestris; Os, Oryza sativa; Pr, P. radiata; Hv, Hordum vulgare; Sc, S. cereale; Zm, Z. mays; Mm, Matthiola incana; Pc, Petroselinum crispum; Am,Antirrhinum majus; Le, Lycopersicon esculentum.

Comparison of deduced LTP and A9 anther-expressed proteins. Deduced sequences of nsLTPs (PrLTP1, PrLTP2, E2, OsLTP, and bLTP) and A9 homologs (PrMC1, PrMC2, Men-8, Lhm7, and A9) were aligned using the GCG program PileUp. The eight conserved Cys residues are shown beneath the alignment. The four helical domains of the barley LTP are labeled A, B, C, and D as indicated. The cleavage site for removal of the secretory sequence predicted by the GCG program SPSCAN is indicated by asterisk. The eight conserved Cys residues are shaded. Accession numbers are Men-8, y08780; LHm7, x80719; A9, q05772; barley LTP, p07597; OsLTP, u29176; and E2, x60318.