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This light and electron microscope study reveals considerable uniformity in hornwort stomata morphology
and density in contrast to common spatial and developmental abnormalities in tracheophytes and mosses.
Stomata arise from a median longitudinal division of sporophyte epidermal cells morphologically
indistinguishable from their neighbours apart from the retention of a single chloroplast whilst those in the
other epidermal cells fragment. Chloroplast division and side-by-side repositioning of the two daughter
chloroplasts determines the division plane in the stomatal mother cell. The nascent guard cells contain
giant, starch-filled chloroplasts which subsequently divide and, post aperture opening, regain their
spherical shape. Accumulation of wall material over the guard cells and of wax rodlets lining the pores
follows opening. While the majority of stomata are bilaterally symmetrical those lining the dehiscence
furrows display dextral and sinistral asymmetry due to differential expansion of the adjacent epidermal
cells.
The ubiquity of open stomata suggests that these never close with the maturational wall changes rendering
movement extremely unlikely. These structural limitations, a liquid-filled stage in the ontogeny of the
intercellular spaces, and spores already at the tetrad stage when stomata open, suggest that their primary
role is facilitating sporophyte desiccation leading to dehiscence and spore dispersal rather than gaseous
exchange. Stomata ontogeny and very low densities, like those in Devonian fossils, suggest either ancient
origins at a time when atmospheric carbon dioxide levels were much greater than today or a function other
than gaseous exchange regulation. We found no evidence for stomatal homology between hornworts,
mosses and tracheophytes.
Keywords: Anthocerotophyta, Capsule dehiscence, Desiccation biology, Guard cells, Stomatal evolution
Introduction
Genetic and hormonal manipulation of stomatal
density in flowering plants (Tal et al., 1970; Martin
& Stimart, 2005; Doheny-Adams et al., 2012; Zhang
et al., 2012), together with investigations into the
effects of carbon dioxide concentrations (McElwain &
Chaloner, 1995; Woodward & Kelly, 1995; Beerling,
2007; Konrad et al., 2008), are at the forefront of
current research on yield improvement and climate
change. Alongside these experimental studies a wide
variety of stomatal defects, affecting both guard cell
and subsidiary cell morphology and vitality, have been
reported from numerous vascular plants (Dehne, 1961;
Takahashi, 1962; Inamdar & Patel, 1969; Inamdar
et al., 1969; Gopal & Shah, 1970; Inamdar et al., 1970;
Inamdar & Patel, 1976).
In contrast, there has never been a thorough
investigation of stomatal abnormalities in bryophytes.
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Confocal microscopy
For confocal imaging, fresh samples of the four hornwort
species (sporophytes), the moss species Bryum radiculosum Brid. (sporophyte) and Polytrichastrum formosum
(Hedw.) G.L.Sm. (stem) and of Prunus (stem) were cut
into thin sections and placed onto a microscope slide in a
droplet of water and sealed with a coverslip and nail
varnish to prevent dehydration. Confocal images were
acquired with a Nikon A1-Si laser-scanning confocal
microscope using either a 10x objective (NA50.3) or
60x oil-immersion objective (NA51.4). Images were
recorded with pixel dimensions of 1.24 mm (for 10x
objective) and 0.21 mm (for 60x objective). To investigate the presence and possible distribution of lignin the
phloroglucinol/HCl histochemical test (which selectively
quenches lignin autofluorescence; Biggs, 1985) was used
in conjunction with confocal microscopy. Stems of the
moss P. formosum (known to contain lignin; Ligrone
et al., 2008) and of Prunus were used as controls. Lignin
fluorescence was excited with a 405-nm line of 100mW
cube laser (Coherent Inc., USA, http://www.coherent.com) and a 488-nm line of 50mW sapphire laser
(Coherent Inc., USA, http://www.coherent.com) was
used for exciting chlorophyll fluorescence. The fluorescence signal was collected with a 32-channel spectral
detector at 10nm resolution on a diffraction grating
covering a range of wavelengths between 410 nm
730 nm. Stomata were visualised using a 34.5 mm (1.2
airy units) confocal pinhole and a number of z-stacks
(typically between 60 and 130) with optical thickness of
125 nm each were acquired. The fluorescence signal on
collected raw images was spectrally unmixed with a
Nikon NIS-Elements software (http://www.nis-elements.
Pressel et al.
Observations
Light microscopy
The early ontogeny of the stomata in Phaeoceros is
illustrated in Figure 1. Nascent stomata mother cells,
only 13 mm above the base of the sporophytes, can
be identified by the presence of a single small
spherical to ovoid chloroplast, less than 5 mm in
diameter lying usually along a side wall (Figure 1a),
in contrast to early fragmentation of the chloroplasts
in all the other epidermal cells which occurs immediately above the foot. At this stage in sporophyte
ontogeny the epidermal cells have reached only O of
their final lengths, but are already the same width as
those in the mature sporophytes. The lengths of the
stomata mother cells are not appreciably different
from the other epidermal cells but these tend to be
slightly wider than the other epidermal cells. The
mother cell chloroplast then divides and initially the
two daughter chloroplasts, still less than 5 mm in
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Figure 1 Early ontogeny of the stomata of Phaeoceros carolinianus. The apex of the sporophytes is beyond the top of each
image. Note the absence of an intact single chloroplast in all the epidermal cells other than those forming the stoma.
a) Stomatal mother cell containing a single spherical chloroplast on the lateral wall (arrowed); b-d) dividing chloroplasts
(arrowed) in various positions in stomatal mother cells; e) chloroplasts becoming aligned side-by-side (arrowed) immediately
prior to the division of the stomatal mother cell; f) nascent stoma with spherical centrally located chloroplasts (arrowed). Scale
bars: 50 mm.
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Figure 3 Phaeoceros laevis. Abnormal stomata lining the sporophyte grooves (G). The tip of the sporophytes is towards the
top of the page in every image. aj) Stomata on the left hand-side of grooves; (kt) stomata on the right hand-side of grooves.
These are mirror images of those on the left. Positions where abnormal walls separating the guard cells meet adjacent cells are
arrowed; a, k) wall between the guard cells meeting the cell above normally but with the lower end abutting the cells away from
the grooves; b, c and l, m) wall between the guard cells meeting the cell below normally but with the upper end abutting the cell
lying towards the grooves; d, n) stomata with displaced guard cells, the upper with its outer wall lying away from the groove;
e-h and o-r) sigmoid walls between guard cells: in every case the outer wall of the guard cell displaced upwards lies away from
the grooves; i, j and s, t) near 45 degree divisions have produced short fat guard cells. Scale bars: 50 mm.
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Discussion
Electron microscopy
SEM clearly shows the dehiscence groove and the
stomata widely scattered over the sporophyte surface
(Figure 6a). The guard cells of young stomata have
completely smooth external walls (Figure 6b, c, f)
Developmental considerations
Development from epidermal cells, otherwise morphologically indistinguishable, apart for their chloroplasts, from their neighbours up until the division
producing the two guard cells, is very different from
all the various patterns of cell division leading to
stomatal formation in vascular plants (Evert, 2006).
Even in the simplest case of anomocytic stomata,
where the epidermal cells around the guard cells are
not distinguishable from their neighbours and subsidiary cells are absent, the stomatal mother cells are
much smaller than the other epidermal cells (Evert,
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Figure 5 Confocal images showing major differences between the chloroplasts of the guard cells and those of other epidermal
cells. All images were spectrally unmixed (as described in methods) into separate blue and red autofluorescence components that
match the pure fluorescence emission spectra of spruce lignin and chlorophyll a, respectively.) ae) chlorophyll autofluorescence
component shown in red; fi, k, m) lignin or spectrally close to lignin autofluorescence component shown in blue and overlaid with
the red chlorophyll autofluorescence component; j, l, n) lignin or spectrally close to lignin autofluorescence component (blue) after
treatment with phloroglucinol overlaid with the red chlorophyll autofluorescence component; a) large pleiomorphic plastids
immediately below the outer guard cell wall in a young stoma; b) plastids in epidermal cells after fragmentation, those in the
subepidermal cells and in the guard cells are rounded; c, d) epidermal plastids in the estomate Megaceros flagellaris (d) also
fragment, whilst (c) the subepidermal ones remain intact; e) plastids in the epidermal and guard cells of the moss Bryum
radiculosum; f, g) young, unopened stomata of Anthoceros punctatus (f) and Phaeoceros laevis (g) the blue autofluorescence
component is restricted to the lining of the nascent pores; h, i) mature stomata of the same two species - the blue autofluorescence
component becomes widespread in the guard cell walls; j) sporophyte of Anthoceros punctatus after treatment with phloroglucinol
the blue autofluorescence component remains unchanged; k, l) stems of Polytrichastrum formosum, untreated (k) and treated (l)
with phoroglucinol the blue autofluorescence component is quenched by the treatment; m, n) stems of Prunus, untreated (m) and
treated with phloroglucinol (n) the blue autofluorescence component is quenched by the treatment. Scale bars: (m, n) 300 mm; (al)
50 mm.
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Figure 6 Scanning electron micrographs, all cryo except g and h (critical point dried). Anthoceros agrestis (a, c, g, h);
Phaeoceros laevis (b, e, f); Phaeoceros carolinianus (d, i, j). a) Fractured sporophyte showing a dehiscence groove (arrowed)
and widely scattered stomata (arrowed); b, c) young unopened normal (b) and abnormal stomata (c); d, e) abnormal stomata;
f) newly opened stoma; note the smooth surface of the guard cells; gi) older stomata with irregular deposits of wall material
over the guard cells; j) a coating of wax rodlets lining an open pore. Scale bars: (a) 200 mm; (bi) 30 mm; (j) 5 mm.
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Figure 7 Cryo-scanning electron micrographs. Anthoceros agrestis. a) Fractured sporophyte, lower portion of within
involucre: all the intercellular spaces are liquid-filled. Stomata are unopened (arrowed) and spore mother cells (arrowed) (b)
have yet to undergo meiotic division; c) young, open stoma; the subtending intercellular spaces are all filled with liquid (*). It is
only after stomatal opening that the liquid in the ISs begins gradually to dry out (df) (arrows in d and f indicate gas-filled
spaces). Scale bars: (a) 100 mm; (bf) 50 mm.
systems are predicated by plastid division and positioning. As in other mitotic cells in hornworts, migration of
the two daughter chloroplasts to a central position in
the stomatal mother cells determines the future plane of
division. Thus the central side-by-side location of the
two chloroplasts in the stomatal mother cells ensures
their longitudinal division and the distribution of a
single chloroplast to each guard cell. This key role for
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Figure 8 Cryo-scanning electron micrographs. Anthoceros agrestis. a) Fractured sporophyte, just above involucre. Liquid
gradually drying out from ISs. Spores are at the young tetrad stage (enlarged and arrowed in (b)); c) fractured sporophyte, 1cm
above involucre: most of the ISs are now gas-filled, stomata have irregular deposits of wall material over the guard cells
(arrowed) and spores are almost mature (enlarged and arrowed in (d)). Scale bars: (a, c) 100 mm; (b, d) 50 mm.
Functional considerations
The presence of waxes lining the pore chambers
(Figure 6j), a feature not previously noted in hornworts,
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Figure 9 Schematic representation of stomatal ontogeny in hornworts; tangential views (top) and transverse sections (bottom).
a) Nascent mother cells with a single spherical chloroplast (Fig. 1a); b) division of the mother cell chloroplast (Fig. 1 bd); c)
immediately before the longitudinal division of the mother cell the chloroplasts increase in size and migrate to lie side-by-side
centrally (Fig. 1e); d) nascent stoma with each guard cell containing a single spherical chloroplast (Fig. 1f); the initial stage in
aperture formation (Fig. 2ac) coincides with division of the subepidermal chloroplasts and the almost complete disappearance
of the fragmented epidermal cell chloroplasts. Huge pleiomorphic, starch-filled chloroplasts in the guard cells. Fluorescent
additional wall material above the aperture (Fig. 5f, g). Mucilage-filled intercellular spaces (Fig. 7a, c); f) pore formation coinciding
with division of the guard cell chloroplasts (Fig. 2df) and spread of fluorescent material over the external surface (Fig. 5h, i).
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Figure 10 Schematic representation of stomatal ontogeny in hornworts; cont. from Fig. 9. a) Two spherical chloroplasts near
the surface of each of the guard cells (Fig. 2g). The mucilage in the intercellular spaces below the epidermis has dried down to
the cell corners but remains between the cells further inside the sporophytes (Fig. 7d, f); b) mature stoma with irregular
deposits of fluorescent wall material covering the external surface (Figs. 5h, i; 6h, i); wax rodlets lining the pore (Fig. 6j) and
chloroplasts lying along the inner walls of the guard cells (Fig. 2hj). All the intercellular spaces are now gas-filled with drieddown mucilage along the cell junctions (Fig. 8a, c).
the fact that they often completely cover the pore lumina
seriously calls into question the ability of hornwort
stomata to open and close and thus to respond to
external stimuli as reported by other authors (Hartung
et al., 1987, 1994; Bopp & Werner, 1993; Christianson,
2000). Our confocal data (Figure 5) further suggest
substantial developmental changes in the guard cells,
with blue fluorescing compounds associated exclusively
with the developing pores in young stomata but
becoming widespread in the outer walls of the guard
cells of mature ones. Our preliminary histochemical tests
indicate that these are not lignin-like compounds whose
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synthesis of both the organic osmoticum that generates opening (Lucas & Renzaglia, 2002) and to the
synthesis of the additional wall materials rather than
providing the energy source for active opening and
closing via a potassium pump. Whilst the present study
confirms Cavers (1911) original observation that the
mature guard cells contain 2 chloroplasts typical of
sporophytic cells, the fragmentation of the other
epidermal chloroplasts in Anthoceros, Phaeoceros
and Megaceros, which lacks stomata, appears to be
unique to these cells in the hornwort life cycle (Duckett
& Renzaglia, 1988).
The contrast between the ontogeny of the guard
cell and epidermal cell chloroplasts in hornworts
from that in mosses is perhaps suggestive of possible
functional differences between the two groups. The
presence of similar chloroplasts throughout the
epidermis of moss sporophytes (Sack & Paolillo,
1983) may perhaps relate to active opening and
closing recently reported by Chater et al. (2011) in
Physcomitrella and Funaria in response to CO2
concentrations, ABA, darkness and fusicoccin (an
activator of stomatal opening) and using mutants in
the former, in contrast to earlier experimental studies
that showed either ambiguous (Garner & Paolillo,
1973) or no responses (Paton & Pearce, 1957)
though here the negative results are now attributed to
inappropriate techniques. From their results Chater
et al. (2011) argue that core regulatory components
involved in guard cell signalling are common to both
angiosperms and mosses and originated in their
common ancestor over 400MYA. Presenting similar
data on Selaginella, Ruszala et al. (2011) come to the
same conclusion. In striking contrast Brodribb and
McAdam (Brodribb & McAdam, 2011; McAdam &
Brodribb, 2012a,b) found that lycopods and ferns
lack the key responses to ABA and epidermal cell
turgor and present the counter argument that active
control of water balance by stomata evolved after the
divergence of the ferns 360MYA. Our developmental
data showing that hornwort stomata never close, not
to mention the very different origin of the intercellular spaces from those in vascular plants, are
more closely in line with the views of Brodribb and
McAdam and are further underlined by our ongoing
experiments where we have failed to detect significant
stomatal responses in hornworts to ABA, desiccation
and darkness, not to mention the absence, as in
Sphagnum (Duckett et al., 2009), of potassium fluxes
between the guard cells and adjacent epidermal cells
(Pressel & Duckett, unpublished data). To add fuel to
this ongoing and unresolved debate it should be
noted that most of experimental treatments in Chater
et al. (2011) induced only small changes in stomatal
aperture areas (which translate into only very slight
changes in aperture widths) and none induced
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Evolutionary considerations
In a very recent review of functional relationships
between the two generations in mosses Haig (2013)
further discusses the possibility that the original
function of stomata might have been sporophyte
desiccation in preparation for spore dispersal prior to
their becoming regulators of gaseous exchange.
Concentrations of stomata around the bases of the
sporangia in several early tracheophytes (Edwards
et al., 1996) are in line with this reasoning. Haig sets
out different competing schemes for the evolutionary
origin of stomata: 1) a single origin in a common
ancestor of all bryophytes and tracheophytes as
previously proposed by Raven (2002) and Ligrone
et al. (2012). This scheme is supported by the
supposed shared mechanisms of stomatal control
between mosses and tracheophytes (Chater et al.,
2011; Ruszala et al., 2011) but presents a problem
of explaining secondary losses in the basal moss
clades Sphagnum, Takakia and Andreaea; 2) stomata
evolved twice, once in the ancestor of peristomate
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Acknowledgements
Zophia Ludlinska (Nanovision Centre, Queen Mary
University of London) for skilled technical assistance
using the cryo-SEM. Travel funds to SP from the
Natural History Museum and a Leverhulme Emeritus
Fellowship to JGD enabled the collection of the
materials used in this study.
Taxonomic Additions and Changes: Nil.
References
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