Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS) gene expression patterns

Abstract

Background: A number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan) walls and septae (callose plugs) of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS). Of 12 CalS gene family members in Arabidopsis, only one (CalS5) has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been.

Results: We investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms) and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5) (Nymphaeales). Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda.

Conclusion: The finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression and pollen-specific functions in early seed plants and was then recruited to novel expression patterns and functions within pollen tube walls in an ancestor of extant angiosperms.

Figure 1

Putative CalS5 orthologue (CcCalS5) topology and expression in Cabomba caroliniana and Nymphaea odorata. A, Predicted topology for CcCalS5 isolated from Cabomba pollen. Numbers indicate predicted amino acids in each domain. B, Semi-quantitative RT-PCR. Gels on left are from Cabomba cDNA derived from different tissues: S-stem tissue, L-leaf tissue, M-meristem tissue, A-pre-dehiscent anther and pollen, P-mature pollen grains. Gels on right are from Nymphaea cDNA: G-genomic DNA control, 0-mature pollen stage, 1, 3 and 6-pollen tubes at 1, 3 and 6 h post-inoculation.

Figure 2

Phylogenetic tree for full length CDSs of Arabidopsis, Physcomitrella CalS gene families and CalS5 orthologues. Phylogenetic tree based upon alignment of predicted polypeptides for full length CDSs of Arabidopsis, Physcomitrella CalS genes, and putative CalS orthologues identified in this study. Sequences for Amborella trichopoda CalS5 and Pinus taeda CalS13 are from partial cDNA fragments containing 196 and 471 amino acids, respectively.

Figure 3

Cell wall staining in Ginkgo biloba and Gnetum gnemon. A, Ginkgo pollen grain showing aniline blue staining of internal male gametophyte walls and intine (arrows; one day of in vitro growth). B, Aniline blue stain localized to inner wall of Gnetum pollen (arrow) before exine shedding (one day of in vitro growth). C, D, Gnetum pollen grain after exine shedding (eight days of in vitro growth). C, Lack of aniline blue staining of intine (compare with DIC view in D). Note pollen is larger than first-day pollen in B and now contains abundant starch grains. Scale bars, 10 μm.

Figure 4

Cell wall staining and CalS RT-PCR in Pinus taeda pollen tubes grown in vitro. A, Semi-quantitative RT-PCR of PtCalS13 from 0 to 72 h on growth medium. B, Ungerminated P. taeda pollen after 12 h on growth medium. Note the weak band stained by aniline blue in distal aperture area (at bottom) and strong staining of prothallial cell walls on proximal side of pollen grain (white arrows). C-E, Pollen tube after 48 h on growth medium (24 h after germination). In D, note strong aniline blue staining at the distal aperture area, and lack of stain in tube walls (compare with C). Calcofluor localizes to both intine and tube wall (E). Scale bars, 20 μm.

Figure 5

Hypothesized evolution of CalS5 gene expression in seed plant pollen. CalS gene relationships from Figure 2 and Additional file 3 are superimposed on a Moss (Physcomitrella) + Seed plant species tree [71]. Filled boxes indicate aniline blue staining of the inner wall of mature pollen (lower) or pollen tube walls/callose plugs (upper) and have been associated with CalS5 or CalS13 expression in all but Amborella (see text). Unfilled boxes represent lack of staining for callose. Branches are coloured to reflect inferred changes in CalS5 gene expression patterns, based on data from this study. Nymphaeaceae includes Nymphaea and Nuphar.