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Abercrombie et al. EvoDevo 2011, 2:14
http://www.evodevojournal.com/content/2/1/14

RESEARCH

Open Access

Developmental evolution of flowering plant
pollen tube cell walls: callose synthase (CalS)
gene expression patterns
Jason M Abercrombie, Brian C O’Meara, Andrew R Moffatt and Joseph H Williams*

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-b-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.

Background
The pollen tube is a unique feature of male gametophytes
of seed plants. In cycads and Ginkgo, pollen tubes are
long-lived and function solely as haustorial, highly
branched structures that grow invasively into female tissues [1-3]. In conifers and Gnetales pollen tubes function
in a new way to deliver non-motile sperm to the egg
(siphonogamy), while generally retaining a haustorial
growth pattern [2,3]. Flowering plant (angiosperm) pollen
tubes have lost most features of haustorial growth - their
* Correspondence: joewill@utk.edu
Department of Ecology and Evolutionary Biology, University of Tennessee,
Knoxville, TN, USA

pollen tubes are typically short-lived and seem to function
exclusively to deliver sperm to the egg [4,5]. The origin of
siphonogamy has been held up as a classic example of
exaptation [6], because the plesiomorphic function of the
pollen tube - nutritional support for the male gametophyte
- was subsequently co-opted for a novel role in sperm
delivery [3]. Yet siphonogamy is clearly a complex process,
and it is not at all obvious which aspects have common
origins, which represent modifications of an ancestral pattern, and which have arisen independently in separate
lineages [1,3,4,7]. Understanding the homologies of pollen
tube structure and growth pattern may provide deeper
insights into the origin(s) of this remarkable innovation.

© 2011 Abercrombie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Abercrombie et al. EvoDevo 2011, 2:14
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Angiosperm pollen tubes have a unique wall structure.
Their thin growing tip is comprised almost entirely of
pectins. Just behind the pectic tip, cellulose synthases
operate to form a very thin, pecto-cellulosic primary
wall. Then, still in the subapical region, (1,3)-b-glucan
(callose) is synthesized beneath the thin primary wall to
form a thick layer [8]. The mature pollen tube wall of
most angiosperms is primarily made of callose (81% by
weight in Nicotiana; ref. 9). As an amorphous polysaccharide, callose can be synthesized more rapidly than an
equivalent weight of fibrous cellulosic cell wall [9] and it
provides resistance to tensile and compression stress
[10]. Callose also severely reduces wall permeability and
since angiosperm pollen tube walls are also prone to
forming septae ("callose plugs”) [11], the plesiomorphic
haustorial function of tubes is largely precluded. These
patterns are general features of all angiosperms, from
Amborella and water lilies to Arabidopsis and maize
[4,12]. Yet, despite their ubiquity, the ancestral function
of callose walls and plugs is not obvious. Tubes that
lack callose in their walls retain their function in some
derived eudicot lineages, such as Lamiales [13] and in
an Arabidopsis mutant line [14,15], though they have
reduced competitive ability in the latter [14].
Pollen tubes in ovules of gymnosperms rarely contain
callose in lateral walls, and callose plugs have never
been reported [16]. Callose is found in the tip wall of
growing pollen tubes of some conifers [17], a pattern
never seen in angiosperms. Studies of in vitro-grown
gymnosperm pollen tubes do sometimes find callose (or
mixed-glucans) in lateral tube walls [17,18]. Importantly,
the deposition of callose is generally transient in gymnosperm male gametophytes and its extent and location
varies even among closely related species [17-20]. Such
transient and variable phenotypes contrast with the relatively invariant and persistent expression pattern seen in
angiosperm pollen tubes.
Callose synthesis is mediated by the enzyme, callose
synthase, encoded by the callose synthase gene, and originally described as a glucan synthase-like gene (GSL) in
Nicotiana alata [21]. Hong et al. (2001) named the callose
synthase gene family CalS after identifying 12 gene family
members in Arabidopsis thaliana [22]. AtCalS5 and its
characterized orthologues (NaGSL1 from N. alata) have
been directly linked to pollen tube wall formation and callose plug deposition, as well as to pollen exine development [14,15,23,24].
CalS5 appears to have an ancient origin by duplication.
A comparative phylogenetic analysis of all CalS paralogs
from the genomes of the moss, Physcomitrella patens
[25] and Arabidopsis found that AtCalS5 was more closely related to a Physcomitrella CalS gene copy (PpCalS5)
than to any other Arabidopsis paralog. Because callose
was observed in the moss spore aperture region and

Page 2 of 13

PpCalS5 was identified as a putative orthologue to
AtCalS5, PpCalS5 was hypothesized to play a role in
moss spore germination [25]. If so, then CalS5 involvement in pollen tube growth may ultimately derive from a
more ancient function involving the germination process.
As such, changes in gene regulation were likely prerequisites for the acquisition of novel callose deposition patterns in angiosperm pollen tube walls. Alternatively, the
patterns arose via duplication and functional divergence
of a CalS gene within seed plants, or perhaps within the
stem lineage leading to angiosperms.
In this paper we present molecular evidence that CalS5
orthologues are expressed in mature pollen and pollen
tubes of several extant early-diverging angiosperms in
Nymphaeales and Austrobaileyales and likely also in
Amborella trichopoda. CalS5 orthologues are also
expressed in mature gymnosperm pollen, including one
siphonagam (Gnetum) and one non-siphonogam (Ginkgo).
In the siphonogamous conifer, Pinus, we report a potentially unique CalS gene expressed in germinated pollen.
We discuss the implications of these findings for the evolution of the angiosperm pollen tube wall and suggest new
avenues of research to clarify the functional roles of CalS
in the seed plant male gametophyte.

Results
Putative orthologues of CalS5 are expressed in pollen and
pollen tubes of early-diverging angiosperms

A nearly full length coding sequence was obtained from
Cabomba caroliniana (Cabombaceae; Nymphaeales)
(CcCalS5) comprising 5,562 bp which translated into a
predicted 1854 amino acid polypeptide with 78% identity
to Arabidopsis thaliana CalS5 (AtCalS5) and 66% identity
to the moss orthologue (PpCalS5) from the Physcomitrella
patens genome [25]. The deduced polypeptide has a predicted topology containing between 13 and 17 transmembrane helices (as predicted by TMHMM v. 2.0; CBS,
Lyngby, Denmark and SOSUI engine ver. 1.11; Nagoya
University, Nagoya, Japan), a cytoplasmic N-terminal loop
domain containing > 423 amino acids, and a large hydrophilic loop domain consisting of 758 amino acids, with
loops between the helices ranging from 4 to 106 amino
acids in length (Figure 1A).
RT-PCR was used to assess the presence of CcCalS5
transcript in various tissues of C. caroliniana, as well as in
a pollen tube time-course experiment in Nymphaea odorata (Figure 1B) using intron-spanning primers. A 1,497 bp
nucleotide sequence that shared 100% nucleotide identity
with the CcCalS5 was amplified from N. odorata pollen
and was used to designate the Nymphaea CalS5 orthologue (NoCalS5) in our RT-PCR experiments (Figure 1B).
CcCalS5 expression was clearly observed in pre-dehiscent
anthers and mature pollen of Cabomba (Figure 1B; left
panel gel), but was also detected in low abundance in stem

Abercrombie et al. EvoDevo 2011, 2:14
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A

Page 3 of 13

40
35

28

106

25
34

6

4

14
14
40

41

423

B

758

S

L M A P

G

0 1 3

6

CalS5
Actin

Fig 1.

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.

and leaf tissues during our RT-PCR optimization experiments (See Additional file 1). Expression of CcCalS5 in
both Cabomba stem and leaf tissues was confirmed by
sequencing clones of PCR products. NoCalS5 was consistently expressed during a time-course of in vitro-grown
pollen tubes harvested at 0, 1, 3 and 6 hrs after inoculation
in liquid medium (Figure 1B; right panel gel).
In the 18-taxon phylogenetic tree constructed to infer
orthologous relationships, all putative angiosperm CalS5
orthologues plus CcCalS5, formed a clade with 99%
bootstrap support (Figure 2). A partial sequence from

the putative CalS5 orthologue in Amborella trichopoda
[26] also falls within the CalS5 clade (Figure 2). The
putative CalS5 orthologues of Physcomitrella [25] and
Selaginella (this study) are strongly supported as falling
in an angiosperm clade of CalS paralogs, but not necessarily as sister to the CalS5 clade.
RT-PCR also recovered CalS transcripts from the
mature pollen of the early-diverging angiosperms, Austrobaileya scandens (Austrobaileyaceae; Austrobaileyales),
Nuphar advena (Nymphaeaceae; Nymphaeales), and
Trithuria austinensis (Hydatellaceae; Nymphaeales). These

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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.

Abercrombie et al. EvoDevo 2011, 2:14
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partial sequences from the hydrophilic loop domain align
with the partial NoCalS5 sequence and are orthologous to
CalS5, based on phylogenetic analysis (Additional files 2
and 3).
Putative orthologues of CalS5 are expressed in pollen of
Gnetum gnemon and Ginkgo biloba

At 24 h after incubation, Ginkgo pollen stained for callose
in the aperture area, intine, and also in the walls that separated prothallial, generative, and tube cells (Figure 3A).
Gnetum pollen sheds its exine before tube growth, and
prior to exine shedding, aniline blue staining was observed
in the inner pollen wall (Figure 3B). After exine shedding,
callose was not observed in the intine (Figure 3C, D).
Partial cDNA fragments from putative CalS5 orthologues were amplified from mature pollen of Gnetum gnemon (GgCalS5) and Ginkgo biloba (GbCalS5). Their
predicted amino acid sequences aligned with the central
loop domains of other CalS proteins, including those
known to function during Arabidopsis pollen development (see Additional file 2). Phylogenetic analysis placed
the Ginkgo and Gnetum sequences within a strongly
supported clade of angiosperm CalS5 sequences (Additional file 3).
Interestingly, the aligned predicted protein sequences
identified a short NASQ motif that Ginkgo shares with all

Page 5 of 13

members of Nymphaeales but that is absent from all
other CalS5 sequences (see Additional file 2). Prosite
scans of the aligned sequences identified the shared motif
as a putative N-glycosylation site (Prosite scan data not
shown). Other putative functional motifs common within
this alignment are CK2 and PKC phosphorylation sites
that are highly conserved within all other Arabidopsis
CalS sequences, however one predicted cAMP-dependent
phosphorylation site, K(R/K)ES, was unique to most taxa
within the CalS5 clade (see Additional file 2).
A unique CalS orthologue is expressed in Pinus taeda pollen

A 1,413 bp CalS transcript was strongly expressed in
mature and germinated Pinus taeda pollen (Figure 4A).
Phylogenetic analysis of the 471 amino acid predicted
polypeptide indicates 99% bootstrap support for its
inclusion within a clade that does not include CalS5
(Figure 2). Because it is distantly related to any of the
known Arabidopsis paralogs in that clade, we named the
gene PtCalS13. PtCalS13 transcripts were abundant over
a 72-hour time period of in vitro growth (Figure 4A),
however repeated attempts to amplify CalS5-like transcripts over the same developmental stages failed. All
combinations of primers that amplified CalS5-like fragments from Ginkgo, Gnetum, and early-divergent angiosperm pollen cDNA were attempted.

A

B

C

D

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.

Abercrombie et al. EvoDevo 2011, 2:14
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A

0

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12 24 36 48 72

B

PtCalS13
Actin

C

D

E

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.

Aniline blue staining was localized to the inner walls of
the mature pollen grain, but not the tube wall (Figure 4BD). In pollen, strong staining was observed within the
third intine layer at the proximal face of the microgametophyte, whereas weaker staining was apparent in the aperture region, the leptoma (Figure 4B). Upon germination,
the aperture area became strongly stained, but the thick
lateral walls of the pollen tube did not (Figure 4C, D). Calcofluor staining for cellulose was strong throughout all
time periods observed during in vitro pollen tube growth
(Figure 4E).
Comparative analysis of predicted functional motifs
among callose synthases

Protein motif searches [27,28] were performed on the
sequences of CalS5 orthologues, as well as on all other

callose synthases in Physcomitrella and Arabidopsis to
evaluate conserved patterns of short linear motifs that
may have functional significance. Comparison of the callose synthase protein sequences revealed conservation of
seven different site patterns in all callose synthases: Nglycosylation, cAMP- and cGMP-dependent protein
kinase phosphorylation, protein kinase C phosphorylation (PKC), casein kinase II phosphorylation (CK2), tyrosine kinase phosphorylation, N-myristoylation, and
amidation (data not shown). The majority of putative
functional motifs were found within the N-terminal
loop and the large central loop domain. The central
loop domains of all callose synthases were consistently
enriched with predicted PKC and CK2 phosphorylation
sites. There were no clear distinguishing features of
CalS5 orthologues with respect to selected patterns of

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Page 7 of 13

predicted linear motifs when compared to the other callose synthases. Zea mays, Sorghum bicolor, and Selaginella moellendorfii all appear to have truncated CalS5
proteins, with the entire N-terminal domains completely
absent. This may reflect incorrect annotation, given the
conserved nature of this large functional domain.

also identified a putative CalS5 N-glycosylation site unique
to seed plants - present in Ginkgo and all members of the
Nymphaeales clade (see Additional file 2) but absent from
Austrobaileya and all monocots and eudicots. It is not
known whether these variants cause functional differences
in CalS5 expression.

In silico identification and phylogenetic analysis of
putative CalS5 orthologues

CalS genes involved in male gametophyte development

BLAST searches of NCBI [29] and Phytozome [30] databases revealed putative CalS5 orthologues from a phylogenetically diverse group of plant species which ranged
from the unicellular green algae, Chlamydomonas reinhardtii to most completely sequenced angiosperms. Amino
acid sequences from these putative orthologues were
aligned with all known callose synthase gene family members from Physcomitrella, Arabidopsis, as well as other
putative CalS5 orthologues for other angiosperms and the
spikemoss, Selaginella moellendorfii. Also included in the
alignment were two sequences of particular interest, the
471 amino acid PtCalS13 sequence obtained from Pinus
taeda pollen cDNA, and a putative CalS5 orthologue identified in silico from the early-diverging angiosperm, Amborella trichopoda. The Amborella translated uniscript was
obtained from the 454-EST build from the Ancestral
Angiosperm Genome Project website [26] and comprised
196 amino acids. Several other translated uniscripts from
Amborella displayed homology to CalS5, but because contig assembly was not feasible, they were not included in
this analysis (data not shown).

Discussion
CcCalS5 contains both general CalS functional motifs and
CalS5-specific motifs

All CalS proteins studied to date share a common topology with a large N-terminal hydrophilic domain followed
by two clusters of transmembrane (TM) domains that
flank a large central hydrophilic domain [15,22,23,31,32].
The large hydrophilic loop is thought to accommodate
interactions with other proteins, such as Rop1, UDP-glucose transferase (UGT1), sucrose synthase, and annexin,
which enable the formation of a CalS enzyme complex
[31]. Scans for functional motifs in CcCalS5 support the
model of Verma and Hong [31], in which most known callose synthase proteins exhibit similarities in putative glycosylation and phosphorylation sites, particularly within the
N-terminal domain and the large central loop.
With respect to pollen tube growth, cAMP is a known
signalling molecule for pollen tube guidance and growth
[33]. In our alignment of the loop domains, we identified
one putative cAMP and cGMP-dependent phosphorylation site common to all seed plant members of the CalS5
clade, but absent from PpCalS5 of Physcomitrella and
from all other CalS paralogs (see Additional file 2). We

Five of 12 callose synthase genes (CalS5, CalS9, CalS10,
CalS11, and CalS12) have been shown to function during
microsporogenesis and microgametogenesis in Arabidopsis [15,34-37]. In contrast to other CalS genes, CalS11 and
CalS12 contain only two or three exons [22] and are Ca2
+
-dependent [38]. CalS11 and CalS12 are genetically
linked and perform partially redundant roles in the formation of walls separating microspores of the tetrad and in
late maturation of the male gametophyte [34]. CalS12 is
also activated during wound response and on stigmatic
papillae [39].
The other 10 CalS genes contain between 39 and 50
exons and are Ca2+-independent [40,41]. CalS5 is sporophytically expressed to form the callose wall of pollen
mother cells and microspore tetrads and is also the predominant gametophytically-expressed transcript in germinating pollen and growing pollen tubes [14, 15, this study].
CalS9 and CalS10 function early in microgametogenesis
[35,36] since mutant lines independently exhibited functional aberrations during the entry of microspores into
mitosis. Mutants of CalS9 caused failure of mitosis II as
well as abnormal positioning of nuclei in mature pollen
and precocious germination, inside the anther [37].
CalS10 is known to be involved in cell plate formation
[22]. Silencing of CalS9 and CalS10 using gene-specific
dsRNAi constructs also resulted in a dwarfed growth
habit, suggesting that both also function in the sporophytic
phase [35]. These studies show that male gametophyte
development in Arabidopsis is mediated by transient
expression of a number of CalS genes, whereas in all
angiosperms studied to date CalS5 is abundant and predominant during pollen germination and in all stages of pollen tube growth.
The Pinus taeda male gametophyte expresses a unique
CalS gene in pollen

We found strong expression of a novel CalS gene in
mature and in germinated pollen of P. taeda. PtCalS13
was strongly supported as falling within a clade of AtCalS
paralogs that does not include any of the known gametophytically-expressed paralogs (AtCalS5 and AtCalS9-12).
PtCalS13 cannot be a deeply divergent copy of CalS5 or
CalS9-12, but is most likely a novel copy of CalS (alternatively, it may be a deeply divergent orthologue of
CalS1-4 or CalS6-8; Figure 2). CalS5 transcripts were not
found at the same stages of development, suggesting

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PtCalS13 functions in place of CalS5. However, more
work is needed to test this hypothesis.
Pinus species are quite variable in the location and
extent of pollen callose deposition [42,43]. In P. taeda, callose was present in the intine and male gametophyte walls
before pollen germination. Upon germination, it became
strongly expressed in the aperture region surrounding the
exiting pollen tube, but did not extend into the tube wall,
as it does in angiosperms [41,44,45]. Thus, the callose distribution pattern associated with PtCalS13 expression is
quite different from the CalS5 pattern in germinating
angiosperm pollen.
Ginkgo and Gnetum express an orthologue of CalS5 in
pollen

There is strong evidence that the CalS genes expressed in
mature pollen of Gnetum and Ginkgo are orthologous to
angiosperm CalS5 since their protein sequences were
nested within the angiosperm CalS5 clade. Callose deposition is apparently restricted to pollen in these species. In
Gnetum it was present in the inner wall of pollen before
exine shedding, but after shedding it was absent from the
intine which is continuous with the emerging pollen tube
wall. In Ginkgo, callose was abundant in the intine and in
the walls of the male gametophyte. A study of in vitro pollen tube growth in Ginkgo found that their tube walls
stained weakly for aniline blue but reacted strongly to calcofluor white and b-(1,3)(1,4) antibodies, suggesting the
presence of b-(1,3)(1,4) mixed glucans [18].
The evolutionary developmental origins of callose
synthase expression in angiosperm pollen tubes

Fast growing pollen tubes are arbiters of the rapid reproductive cycles of angiosperms and their unique wall structure may have been a trigger for extensive pollen tube
growth rate evolution in the group [1,4,46]. Gene family
expansions are thought to have been important for trait
diversification early in angiosperm history or pre-history
[47,48]. Six CalS paralogs from three ancient land plant
lineages - five from Arabidopsis [22] and one from Pinus
(this study) - are now known to be expressed in male
gametophytes of seed plants (Figure 5). To date, only
CalS5 has been shown to be expressed in pollen tubes,
and only in a few model system eudicots [15,23,24]. The
finding that pollen tubes of a broad set of extant earlydiverging angiosperms also utilize CalS5 in their pollen
tubes supports the homology of callose walls and plugs in
flowering plants [4,5].
CalS5 transcripts were also found in mature and/or germinating pollen, of two distantly-related gymnosperms,
Ginkgo and Gnetum. The predominant anatomical location of callose in Ginkgo was in the intine and internal
gametophytic cell walls, whereas the pollen tube-forming
intine of germinated Gnetum pollen did not contain

Page 8 of 13

callose. In all early-divergent angiosperms used in this
study, callose was present within the intine of germinating
pollen and continuous with the callose inner wall of the
pollen tube [4,43,49]. Gametophytic expression of CalS5
has a similar pattern in Arabidopsis and tobacco
[14,15,23,24,32,37,50]. Thus, it seems likely that CalS5 had
an ancestral expression pattern within the inner pollen
wall that later became modified via the evolution of gene
regulation to function in growing pollen tubes of an ancestor of extant angiosperms (Figure 5).
A number of hypotheses have been proposed as to what
the ancestral function of callose in germinating pollen or
spores might have been. In the moss, Physcomitrella, callose was deposited in the inner exine layer (not the intine)
near the aperture at the proximal pole of the spore just
before germination [25]. Callose was inferred to function
in spore germination and it was suggested that a CalS5
orthologue was involved [25]. Expression data are needed
to determine if these results reflect an ancient aspect of
land plant spore germination or an apomorphic feature of
moss spores (Figure 5). We also found callose thickenings
in the aperture area of germinating Pinus pollen, but these
were associated with PtCalS13, not a CalS5 orthologue
(Figure 5). Callose is localized to the inner intine of mature
pollen of some Pinus species [51-53], the outer layer
in P. sylvestris [17,54], and is absent from the intine of
P. wilsonii [55]. Pacini et al. (1999) concluded that such
variable and transient expression of callose in Pinus pollen
indicates that it functions as a reserve polysaccharide,
rather than serving a structural or prophylactic function
[45]. Górska-Brylass (1970) argued its presence in the
proximal outer intine was due to non-retrieval of callose
plates from the prior divisions of prothallial cells [56].
Alternatively, its co-localization with degenerate prothallial
cells may indicate the prior involvement of callose as a
wall sealant that initiates cell death, a pattern also seen
during megasporogenesis in seed plants [57].
To date no study has convincingly shown callose to be
the predominant and permanent constituent of any gymnosperm pollen tube wall, nor is there any finding of a
callose plug in a gymnosperm pollen tube. For example,
callose is reported from young but not old tubes in Pinus
and Cycas [17,20]; it is a transient feature of long-lived
Pinus tubes during winter dormancy [17]; and it can
appear at the growing tube tip but does not persist in the
lateral walls as the tip continues its growth [16,17]. From
a functional standpoint, one should note that the origin
of persistent callose walls and plugs in angiosperm pollen
tubes must have been contingent on a shift from a primarily haustorial function, such as occurs in root hairs,
rhizoids and pollen tubes of Cycads and Ginkgo, to a new
one in which a short-lived and fast-growing tip functions
primarily to carry sperm to egg. Pollen tubes of gymnosperms are long-lived and extend from a multicellular

Abercrombie et al. EvoDevo 2011, 2:14
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Page 9 of 13

Physcomitrella

Arabidopsis

?

*

moss-angiosperm
divergence

CalS5 expressed in mature pollen
only

1

2

3

CalS5 expressed in mature pollen
and growing pollen tubes

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.

male gametophyte, which typically lives within a pollen
grain attached to female tissues at the pollination site [3].
In most conifers, sperm are formed late in male gametophyte ontogeny and must travel from the pollen grain to
the tip late in life. Thus, the biology of the fertilization
process in most gymnosperms prevents their pollen
tubes from utilizing callose as a semi-permanent structural feature of their walls.
At some point(s) along the lineage leading to angiosperms, two shifts in CalS5 localization occurred - callose
deposition became restricted to a short subapical region
of the growing pollen tube tip and to a small distal region
of the tube where a callose plug forms. Importantly, both
of these changes in localization must also have involved
changes in callose retrieval, giving rise to persistent callose walls and plugs. One model for the origin of angiosperm pollen tube morphology is that an ancestral
conifer-like pollen tube became transformed by the

evolution of faster growth rates [46], causing the callose
synthesis and retrieval machinery to be displaced from
the newly-forming tube tip to a subapical position [1,17].
Comparative analyses support the notion that early
angiosperm pollen tubes grew faster than those of their
gymnosperm-like ancestor (extant gymnosperm pollen
tubes are characterized by exceptionally slow pollen tube
growth rates) [5]. What is not clear is whether nonretrieval of callose is a cause or a consequence of the origin of faster growth rates.
Given the increased tendency of molecular phylogenetic analyses to place conifers/Gnetales in an isolated
position relative to angiosperms [58], it is worth considering that angiosperm pollen tube morphology may have
evolved independently from an ancestral Ginkgo/Cycadlike haustorial tube [20], rather than from a transitional
siphonogamous, conifer-like predecessor. Resolving the
question of pollen tube origins will require careful

Abercrombie et al. EvoDevo 2011, 2:14
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developmental analyses of pollen tube growth and the
many genes that mediate differences among extant seed
plant groups. It would be especially interesting to look at
the evolution of the callase (b-1,3-glucanase) gene family,
which catalyzes the retrieval of callose [59].

Conclusion
This study supports the homology of callose pollen tube
walls and plugs across flowering plants at the level of
CalS5 gene expression. Since CalS5 was also found to be
actively transcribed in mature pollen of Ginkgo and Gnetum, we suggest that CalS5 localization to the inner intine
of mature germinating pollen was present in a distant
angiosperm ancestor, perhaps extending into the walls of
young pollen tubes, and was later co-opted as a non-transient feature of angiosperm pollen tubes (Figure 5). CalS5
is a structural gene that originated by duplication long
before the origin of extant angiosperms. Thus, the novel
callose deposition patterns of angiosperm pollen tubes
must be a consequence of the evolution of novel regulation of an ancient gene. It remains to be seen to what
extent this involved duplication and divergence of other
genes involved in the callose synthesis or retrieval pathways, and to what degree it was or was not a developmental outcome of the evolution of faster growth rates.
Methods
Plant material

Whole flowers from Austrobaileya scandens White were
collected near Millaa Millaa, Queensland, Australia (17°
31’ 15” S, 145° 33’ 53” E). A. scandens pollen tubes were
grown in hanging drops of BK media [60] containing 2.5%
sucrose inside closed petri plates for 5 to 12 hours. Pollen
from Trithuria austinensis Sokoloff was collected in Branchinella Lake, shire of Manjimup, Western Australia (34°
16’ S, 116° 42’ E). Pollen or pollen tubes of these two species were centrifuged briefly and resuspended in RN Later
(Ambion, Austin, TX, USA) and RNA was isolated within
several weeks. RNA was isolated from fresh pollen or pollen tubes for the remaining species below. Pollen from
Ginkgo biloba L. was collected from trees growing on the
University of Tennessee campus in Knoxville, TN, USA.
Nuphar advena Aiton. flowers were collected near Sparta,
TN, USA (35° 55’ 11” N, 85° 20’ 41” W) and pollen tubes
were grown in BK media with 5% sucrose for two hours
before RNA isolation. Nymphaea odorata Aiton. flowers
were collected from a pond in Knoxville, TN, USA (35° 53’
51” N, 84° 10’ 23” W). Cabomba caroliniana A. Gray
plants were grown in greenhouse water tanks and were
originally collected from Racoon Creek, Jackson County,
AL, USA (34° 46’ N, 85° 50’ W) or purchased from Carolina Biological Supply (Burlington, NC, USA). Gnetum
gnemon L. flowers with dehiscent anthers were collected
from greenhouse-grown plants in DEPC-treated water,

Page 10 of 13

vortexed to separate pollen from all other flower parts,
and briefly centrifuged prior to RNA isolation. Pollen from
Pinus taeda L. and P. strobus L. was collected from trees
on the University of Tennessee campus. Pinus pollen was
grown in a liquid medium containing 10% sucrose,
15 mM MES, 1 mM H3B, 1 mM CaCl2, pH 4.0 in petri
plates sealed with parafilm.
In vitro pollen tube experiments with Nymphaea and
Cabomba

Due to the poor germination observed for both Nymphaea
and Cabomba pollen in standard BK media, stigmatic fluid
from first day Nymphaea flowers was collected on site
with a disposable pipette and used as the pollen tube
growth medium for these species. Both species exhibited
70 to 90% germination success when grown in the fresh
stigmatic fluid, and thus fresh stigmatic fluid was used for
all pollen tube experiments described here. Stigmatic fluid
was collected shortly after flower opening (9 to 10 am),
and centrifuged for three minutes at 13,000 rpm to
remove any contaminating debris and/or pollen grains.
Anthers from Cabomba and Nymphaea were removed
with forceps and placed into 1.7 ml tubes that contained
1 ml of stigmatic fluid. Anther number was used to standardize samples for pollen density during tube growth.
Tubes were vortexed briefly to separate pollen grains from
anthers, anthers were then removed, and contents were
transferred to a small petri plate for pollen tube growth at
room temperature. Nymphaea pollen tubes were grown
for various lengths of time (one, three, and six hours postinoculation) for gene expression experiments. For RNA
extractions, pooled pollen tube samples were harvested at
each time point, centrifuged at a 2,000 rpm for 30 seconds
to maintain pollen tube integrity, and immediately frozen
in liquid nitrogen after growth medium was removed.
Bioinformatics and primer design

In order to search for orthologous CalS5 gene sequence
in the early-diverging angiosperms, the local BLAST tool
on the ancestral angiosperm genome project website [26]
was used to blast the Arabidopsis CalS5 protein sequence
(tBlastn) against all available 454-Sanger hybrid databases. After performing non-redundant nucleotide NCBI
BLAST searches of individual uniscript hits, primers
were designed to amplify a Nuphar advena uniscript
sequence (c78546) with the lowest E-value corresponding
to AtCalS5. The primers designed towards Nuphar
advena sequence (Sec16F; Sec17R) amplified a 600 bp
highly conserved region of sequence within the predicted
hydrophilic domain of the putative CalS5 orthologue.
These primers were also used for amplifying putative
CalS5 orthologous pollen-derived cDNA from Ginkgo
biloba, Austrobaileya scandens, and the more closely
related water lily species, Cabomba caroliniana,

Abercrombie et al. EvoDevo 2011, 2:14
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Nymphaea odorata, and Trithuria austinensis. Another
forward primer (Sec17F) nested within this sequence and
the reverse primer (Sec17R) enabled PCR amplification
of the 250 bp Gnetum sequence. Various multiple
sequence alignments were carried out on all callose
synthase DNA and protein sequences in Physcomitrella
patens and Arabidopsis to aid in primer selection. To
amplify the Pinus taeda CalS cDNA, two EST sequences
showing the highest homology to AtCalS5 and PpCalS5
sequence (AI812992 and FJ114840) obtained from nonredundant NCBI BLAST of Pinus were aligned with
PpCalS5 and AtCalS5. Forward FJ114840-F and reverse
primer AI812992-R amplified a product that reflected the
alignment. To prevent amplification of other pollenexpressed CalS genes (CalS9, CalS10, CalS11, CalS12),
particular attention was given to these sequences during
primer design for PCR applications. All primer design
and sequence alignments were performed using Vector
NTI software (Invitrogen, Carlsbad, CA, USA). A complete set of primers used in this study is listed in Additional file 4.
Molecular analyses

Total RNA from pollen and pollen tubes was isolated
using Tri Reagent J (Ambion, Austin, TX, USA) and a
modified CTAB protocol was required for Cabomba vegetative tissues [61]. For cDNA synthesis, 1.5 μg of total
RNA was used, according to the manufacturer’s protocol
(ArrayScript; Ambion, Austin, TX, USA). Prior to cDNA
synthesis, all samples were subjected to DNase I (TurboFree DNase kit; Ambion). RNA was assessed for quality
with agarose gel electrophoresis and quantified with a
NanoDrop (Thermo Scientific, Waltham, MA, USA) spectrophotometer. A 3’ RACE procedure was used according
to the First Choice RLM RACE kit (Ambion, Austin, TX,
USA) to acquire the 3’ end of the Cabomba caroliniana
CalS5 gene. However, due to difficulties with the 5’ RACE
protocol 5’ products were amplified using a forward primer designed towards highly conserved sequence within a
multiple sequence alignment of CalS5 orthologues
(CalS515F). Inverse PCR was performed to obtain the 5’
end of the full length cDNA using a digest/re-ligation/
digest strategy with HindIII/PstI, respectively [62].
Although attempts to obtain the 5’ end of the cDNA were
incomplete, the inverse PCR procedure did enable the
sequencing of introns that flanked the 5’-most exon. Identification of these intron-exon boundaries enabled the
design of intron-spanning primers for semi-quantitative
RT-PCR. All PCR reactions that required cloning were
performed with Herculase II DNA fusion polymerase
(Agilent, Santa Clara, CA, USA). Products were gel purified using a Qiaex II gel purification kit (Qiagen, Valencia,
CA), cloned into pcr8-GW-TOPO (Invitrogen, Carlsbad,
CA, USA), and sequenced on an ABI 3100 capillary

Page 11 of 13

sequencer at the University of Tennessee Molecular Biology Resource Facility. cDNA tissue sources are listed in
Additional file 5. All sequences were deposited in Genbank [http://www.ncbi.nlm.nih.gov/genbank/index.html].
Semi-quantitative RT-PCR

To prevent amplification of possible orthologous pollenexpressed CalS genes (CalS9, CalS10, CalS11, CalS12),
both protein and nucleotide alignments were used to
design primers to amplify a 631 bp intron-flanking
sequence that shared a predicted low homology to nontarget CalS cDNA. Primers were designed to amplify an
Actin gene isolated from Nuphar advena pollen. Actin
was also used as a reference gene to confirm equal template loading in Pinus taeda RT-PCR. A master mix of
PCR reagents (described above) was used to amplify the
CalS5 fragment and Actin control gene in separate reactions at equal template concentrations and cycling parameters. Threshold cycle optimization was determined by
performing PCR amplifications over a range of cycles.
The number of PCR cycles selected corresponded to
where the trend line exhibited the highest correlation to
exponential amplification. Bands representing each tissue type were purified, cloned, and the sequence was
verified to confirm single product amplification.
Phylogenetic analysis

Alignments of predicted amino acid sequences were performed using MAFFT (Cambridge, Engand) [63]. Mesquite (Vancouver, BC, Canada) [64] was used to
truncate taxon names. Gblocks software (Barcelona,
Spain) [65] with gap mode ALL was used to exclude
poorly aligned regions. Model testing was performed
using ProtTest 2.4 (Vigo, Spain) [66] using only those
substitution models present in RAxML (San Diego, CA,
USA) [67,68]. RAxML was used on the CIPRES web server [68,69] to infer phylogenetic trees and do fast bootstrapping (maximum likelihood method). Trees were
visualized using FigTree (Edinburgh, Scotland, UK) [70].

Additional material
Additional fie 1: N-terminal alignment of CcCalS5 with AtCalS5 and
PpCalS5, and CcCalS5 expression in various tissues of Cabomba
caroliniana. A) Amino acid alignment showing the expected missing
sequence of the N-terminal end of the CcCalS5 cDNA. B) Agarose gel
showing amplified PCR products that were cloned and sequenced to
confirm the presence of CcCalS5 transcript in vegetative and
reproductive tissues of Cabomba. S, stem tissue, L, leaf tissue, M,
meristem tissue, A, pre-dehiscent anther, P, pollen from dehiscent anther.
Additional file 2: Amino acid alignment of selected sequence within
CalS central hydrophilic loop domain. Amino acid alignment of
putative CalS5 orthologues with other selected CalS genes. Red boxes
indicate putative functional motifs. Note that a smaller fragment was
amplified from Gnetum. *N-glycosylation site, **Casein kinase 2
phosphorylation site, ***Protein kinase 2 phosphorylation site, ****cAMPand cGMP-dependent phosphorylation site.

Abercrombie et al. EvoDevo 2011, 2:14
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Additional file 3: Phylogenetic tree from central loop domains of
Arabidopsis and Physcomitrella CalS genes and putative CalS5
orthologues. Phylogenetic tree based on alignment of predicted
polypeptides for central loop domains of known Arabidopsis,
Physcomitrella CalS genes and putative CalS orthologues identified in this
study.
Additional file 4: Primers used in this study. Primers used to amplify
CalS orthologues and conduct semi-quantitative RT-PCR.
Additional file 5: Sources for putative CalS orthologues amplified in
this study. Tissue sources and cDNA fragment sizes for putative CalS
orthologues amplified from taxa in this study.

Abbreviations
AsCalS5: Austrobaileya scandens CalS5; CalS: callose synthase; CK2: casein
kinase II; PKC: protein kinase C; CcCalS5: Cabomba caroliniana CalS5;
GbCalS5: Ginkgo biloba CalS5; GgCalS5 Gnetum gnemon CalS5; GSL: glucan
synthase-like; NoCalS5: Nymphaea odorata CalS5; PtCalS13: Pinus taeda
CalS13; TaCalS5: Trithuria austinensis CalS5; UGT1: UDP-glucose transferase I.
Acknowledgements
The authors wish to thank Richard Moore, Barry Bruce and three anonymous
reviewers for their insightful comments. For technical assistance we thank
Mark Lazzaro (gymnosperm pollen tube growth), Ken McFarland
(greenhouse), Joe May (sequencing of clones), Nick Buckley (lab assistance)
and Karen Hughes (use of lab equipment). We also thank Mackenzie Taylor for
Trithuria austinensis samples and David McFarland for access to the Nymphaea
odorata population. The work of all authors was supported by US National
Science Foundation awards DEB 0640792 and IOS 1052291 to J. H. W.
Authors’ contributions
JA conceived of the study, carried out the experiments, and drafted the
manuscript. AM performed troubleshooting of RNA isolation protocols and
assisted in experiments. BO performed phylogenetic analyses and tree
construction. JW also conceived of the study, provided experimental
guidance, and shared in writing the manuscript. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 1 February 2011 Accepted: 1 July 2011 Published: 1 July 2011
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doi:10.1186/2041-9139-2-14
Cite this article as: Abercrombie et al.: Developmental evolution of
flowering plant pollen tube cell walls: callose synthase (CalS) gene
expression patterns. EvoDevo 2011 2:14.

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