Symplasmic and apoplasmic transport inside feather moss stems of Pleurozium schreberi and Hylocomium splendens

Abstract

Background and Aims

The ubiquitous feather mosses Pleurozium schreberi and Hylocomium splendens form a thick, continuous boundary layer between the soil and the atmosphere, and play important roles in hydrology and nutrient cycling in tundra and boreal ecosystems. The water fluxes among these mosses and environmental factors controlling them are poorly understood. The aim of this study was to investigate whether feather mosses are capable of internal transport and to provide a better understanding of species-specific morphological traits underlying this function. The impacts of environmental conditions on their internal transport rates were also investigated

Methods

Cells involved in water and food conduction in P. schreberi and H. splendens were identified by transmission electron microscopy. Symplasmic and apoplasmic fluorescent tracers were applied to the moss stems to determine the routes of internal short- and long-distance transport and the impact of air humidity on the transport rates.

Key Results

Symplasmic transport over short distances occurs via food-conducting cells in both mosses. Pleurozium schreberi is also capable of apoplasmic internal long-distance transport via a central strand of hydroids. These are absent in H. splendens. Reduced air humidity significantly increased the internal transport of both species, and the increase was significantly faster for P. schreberi than for H. splendens.

Conclusions

Pleurozium schreberi and Hylocomium splendens are capable of internal transport but the pathway and conductivity differ due to differences in stem anatomy. These results help explain their varying desiccation tolerance and possibly their differing physiology and autecology and, ultimately, their impact on ecosystem functioning.

INTRODUCTION

Pleurocarpous feather mosses (e.g. Pleurozium schreberi and Hylocomium splendens) are major components of the ground layer of boreal, sub-alpine and arctic ecosystems, and play a crucial role in the maintenance and regulation of many ecosystem processes (Longton, 1992; Nilsson and Wardle, 2005; Lindo and Gonzalez, 2010). Specifically, they form a thick, continuous boundary layer between the soil and the atmosphere which strongly impacts on soil temperature (Gornall et al., 2007), biogeochemical cycling (Brown and Bates, 1990; Turetsky, 2003; Gundale et al., 2011), nitrogen input via association with cyanobacteria (DeLuca et al., 2002; Gundale et al.; 2010) and ecosystem evapotranspiration rates (Lafleur and Rouse, 1988; Heijmans et al., 2004; Shimoyama et al., 2004). While the influence of feather mosses on hydrological processes in boreal and arctic ecosystems is largely overlooked (but see Beringer et al., 2001; Bond-Lamberty et al., 2010), it is known that many mosses (including feather mosses) can retain large quantities of moisture and help maintain high humidity within the soil and understorey vegetation for extended periods (Gornall et al., 2007; Lakatos, 2011). High moisture retention by feather mosses has been attributed to overlapping concave leaves on the moss stem and the presence of paraphyllia (leaf-like scales) which serve as water reservoirs (Hébant, 1977; Proctor, 2009). These structures also promote a continuous ectohydric loss of water driven by capillary forces and evaporation (Green et al., 2011). As such, the main regulator of water flow in most boreal and arctic moss colonies is thought to result from an ectohydric transport of water (Raven, 2003; Elumeeva et al., 2011; Green et al., 2011) despite the general lack of studies investigating endohydric (internal) pathways of water movement inside the stems.

While the water-holding ability of feather mosses is regarded as important for controlling key ecological processes of northern ecosystems (Beringer et al., 2001; Cornelissen et al., 2007; Lindo and Gonzales, 2010), our understanding of the mechanisms of water uptake and transport is fragmentary and incoherent. It is generally assumed that feather mosses, which lack a well-developed rhizoid system, are entirely dependent on water from the atmosphere (cf. Brown, 1982; Glime, 2007; Lüttge et al., 2011). However, this assumption that the atmosphere is their single source of water and, implicitly, of water-soluble mineral nutrients has been challenged by studies showing that feather mosses may obtain nutrients from the soil and then recycle these inside their stems (e.g. Binkley and Graham, 1981; Van Tooren et al., 1990; Eckstein and Karlsson, 1999; Okland et al., 1999). While experimental work on solute movements in H. splendens (Brümelis and Brown, 1997; Eckstein and Karlsson, 1999) and Sphagnum sp. (Rydin and Clymo, 1989) suggested the possibility of long-distance internal transport, the possible cellular pathways of solute movement were not examined. Although anatomical studies have shown that P. schreberi has conducting elements in the form of a central strand (Bowen, 1933), and that stems of both P. schreberi and H. splendens contain elongate parenchyma with perforations in lateral and terminal walls (Finocchio, 1967; Noailles, 1974; Hébant, 1977), their actual role in internal solute transport has yet to be determined.

Solute transport in mosses over short and long distances can proceed via both the symplasm system and the apoplasm system (sensuErickson, 1986; see also Raven, 2003; Evert, 2006). In the symplasm system, solutes are transported between living food-conducting cells interconnected by numerous plasmodesmata, while in the apoplasm system, the transport occurs inside the cell walls and intercellular spaces (Evert, 2006; Sowiński, 2013). The most highly differentiated food-conducting cells in mosses are the leptoids found in Polytrichales. They are characterized by highly distinctive cytology including longitudinal arrays of endoplasmic microtubules and strong cytoplasmic polarity (Ligrone and Duckett, 1994; Ligrone et al., 2000; Pressel et al., 2006). However, other less specialized food-conducting cells with lower degrees of cytological specialization are widespread among non-polytrichaceous mosses including Sphagnum (Ligrone and Duckett, 1994, 1996, 1998). The apoplasmic transport in most mosses is via water-conducting cells known as hydroids. These cells have unevenly thickened walls in the Polytrichopsida, while those present in the Bryopsida are uniformly thin walled (Hébant, 1977; Ligrone et al., 2000; Ligrone and Duckett, 2011). As such, food- and water-conducting cells in bryophytes are regarded as important conducting elements that serve as the main pathway for solute transport from the soil to the apex (Thomas et al., 1988; Ligrone et al., 2000; Pressel et al., 2006). However, the extent of internal transport between these cells remains poorly known and this is particularly true for the pleurocarpous mosses. Pleurozium schreberi supposedly lacks leptoids, whereas in H. splendens both leptoids and hydroids are reported to be absent (Hébant, 1977; Glime, 2007). Thus, we simply do not know how feather mosses might transport solutes internally over short and long distances via the symplasmic route, apoplasmic route or both.

The rates of internal and external solute fluxes are mainly determined by evaporation which, in turn, is driven by temperature, wind speed and the humidity of the surrounding environment (Proctor, 2009). Rates of solute fluxes may also be determined by species- specific morphological traits and growth form characteristics (Léon, 2006; Proctor, 2009). In a controlled greenhouse study, Elumeeva et al. (2011) showed that H. splendens and P. schreberi had the highest desiccation rates among 22 boreal and arctic moss species but were unable to correlate this process to the leaf cell wall thickness. However, if these mosses are capable of transporting solutes internally, it appears reasonable to suggest that the nature of internal water transport, and not just water-holding leaf traits, may in part be responsible for the rate of water loss. Given the overlooked possible presence of internal conducting elements and our incomplete understanding of how long-distance transport of solutes may occur within these species, we aimed to provide understanding about how feather mosses transport solutes internally and the significance of this under different environmental conditions.

Thus, we examined the anatomy of possible conducting structures in two feather mosses, P. schreberi and H. splendens, and assessed how these might impact on their internal transport routes and efficiency under two different levels of air humidity. Based on previous studies, we hypothesized that the stems of both P. schreberi and H. splendens would absorb solutes and transport them internally but that the transport pathways might differ between the species. We expected short-distance transport to be similar for both mosses and to occur mainly via elongate parenchyma cells (Finocchio, 1967; Noailles, 1974; Hébant, 1977), while P. schreberi might also be capable of long-distance transport due to the presence of more specialized tissue within its central strand. In addition, we expected that both species would transport solutes via plasmodesmata but that apoplasmic transport would not occur in H. splendens as this would require the presence of hydroids, which these species is reported to lack (Hébant, 1977; Glime, 2007). Secondly, given that P. schreberi has a central strand (Bowen, 1933) we predicted that P. schreberi would be capable of transporting solutes at a much faster rate than H. splendens. Finally, we hypothesized that the transport rates of P. schreberi but not that of H. splendens would increase at reduced air humidity because the central strand would facilitate transport during drier conditions similar to that shown for Polytrichum commune (Blaikley, 1932; Bayfield, 1973). As such, we expected the transport rates of P. schreberi to be more responsive to changes in air humidity than those of H. splendens due to the presence of the central strand.

MATERIALS AND METHODS

Specimens and growth conditions

Intact gametophytes of Pleurozium schreberi and Hylocomium splendens were randomly sampled from a natural, moss-dominated, late fire successional boreal forest stand near the town of Arvidsjaur, northern Sweden (65°80’N; 19°10’E) in June 2012. We collected approximately ten gametophytes of each species from each of 50 randomly chosen spots in 10 × 10 m openings in the forest. The mosses were brought to the laboratory and stored at 13–16 °C in semi-open transparent plastic boxes (70 × 70 × 100 mm), under a long photoperiod (12 h day:12 h night) with a photon flux density of 120 mmol s^–1 m^–2 and a wavelength of 400–700 nm. The gametophytes were sprayed three times a week with deionized water until further used.

Anatomical studies

We sampled 5 mm long pieces from stems (about 3–5 cm from the apex) from each of 20 gametophytes per species. These pieces were either hand-sectioned with a razor blade or sectioned with a rotary microtome (Leica RM 2135), both transversely and longitudinally. Hand sections were either transferred directly to a drop of water on microscopic slides or first stained with Alcian Blue–Safranin O solution (Davis, 1997), to increase contrast and visualize potential differences between cell types. All hand sections, except those prepared for callose immunolocalization (see below), were obtained and analysed as living specimens. The microtome sections were 7 and 10 μm thick and prepared from stem pieces that had been fixed in 4 % paraformaldehyde (PFA) in 10 mm phosphate-buffered saline (PBS) solution, pH 7.4, and further embedded in paraffin. The sections were stained with Safranin O–Fast Green and subsequently mounted in Euparal according to Johansen (1940).

Callose localization

We used the presence of callose as an indicator of plasmodesmata (Overall et al., 2013) and the likelihood of symplasmic transport (Roberts and Oparka, 2003; Sowiński, 2013). Transverse and longitudinal hand-cut stem sections were stained with a 0.1 % aqueous solution of aniline blue (Currier and Strugger, 1955) for 30 min and then analysed for blue–white fluorescence of callose under an epi-fluorescence microscope (see ‘Microscopy’ section). Control sections were immersed in water instead of aniline blue. To confirm further the presence of callose, immunolocalization of callose was performed by applying 1,3-β-d-glucan-directed mouse monoclonal primary antibody (Biosupplies Australia, Melbourne, Australia) and the Alexa Fluor 488 rabbit anti-mouse secondary antibody (Invitrogen, Carlsbad CA, USA) to each of the 20 longitudinal stem sections following the protocols of Ferguson et al. (1998), Hofmann et al. (2010) and Sokołowska et al. (2013). The antibodies were diluted 100× and 200× respectively. A more detailed protocol of the 1,3-β-d-glucan immunolocalization is given in Supplementary Data Appendix 1.

Electron microscopy

Approximately 10 mm long pieces of stems of Pleurozium with the leaves removed with a razor blade were fixed in a mixture of 3 % glutaraldehyde, 1 % formaldehyde (freshly prepared from PFA) and 0.5 % tannic acid in 0.05 m phosphate buffer at pH 6.8, for 1 h at room temperature and under low vacuum, following the protocols of Ligrone and Duckett (1994) and Pressel et al. (2006). The pre-fixed stem fragments were then cut into smaller, 2–3 mm long, segments and left in the fixative for 2 h at room temperature and then overnight at 4 °C before being thoroughly rinsed in 0.1 m phosphate buffer, pH 6.8. Samples were then post-fixed in a mixture of 1 % osmium tetroxide and 0.8 % potassium ferrocyanide (McDonald, 1984) overnight at 4 °C, dehydrated in an acetone series and embedded in Epon 812 (Serva, Heidelberg, Germany). The stem fragments were gradually infiltrated using the acetone–epon mixtures in ratios of 3:1, 2:1 and 1:1. Semi-thin sections (0.8 μm thick) were stained with 1 % methylene blue in 1 % borax and examined under a light microscope. Ultrathin sections were contrasted with uranyl acetate and lead citrate (Reynolds, 1963) and examined by transmission electron microscopy (TEM; see ‘Microscopy’ section).

Tracer application studies

We set up two tracer application studies in which stems of both species were loaded with symplasmic or apoplasmic fluorescent tracers (Table 1). First, we applied fluorescent tracers both to the stem surface (external loading) and at the point of excision (internal loading), to assess how solutes may be absorbed and transported internally to the apex of the stems. We then compared the relative rate of internal movement of each tracer type under two different levels of air humidity to determine which transport pathway (whether apoplasmic or symplasmic) is more efficient at low air humidity.

Table 1.

Summary of the fluorescent tracers used to study the internal transport in feather mosses.

Transport type	Tracer type	Concentration	References
Symplasmic	CFDA	30 μg mL–1 in 10 mM PBS at pH 7.4 prepared from a 1 % stock solution in acetone	Sokołowska and Zagórska-Marek (2012)
	HPTS	2.5 mg of HPTS in 1 mL of 10 mM PBS with pH 7.4	Gisel et al. (1999).
Apoplasmic	SRB	0.02 mm aqueous solution derived from a 40 mm stock solution in dimethyl sulphoxide	Botha et al. (2008)
	TR	1 mg of TR in 1 mL of distilled water	Roberts et al. (1997)

HPTS, 8-hydroxypyrene-1,3,6-trisulphonic acid (Sigma-Aldrich, Co.); CFDA, carboxyfluorescein diacetate (Sigma-Aldrich, Co.); TR, 3 kDa Texas Red dextran (Invitrogen); SRB, sulphorhodamine B acid chloride (Sigma-Aldrich, Co.).

External loading

A mixed solution of the HPTS (8-hydroxypyrene-1,3,6-trisulphonic acid) and TR (3 kDa Texas Red dextran) tracers (1:1 ratio; Table 1) was applied on the stem surface of five gametophytes of each moss species to visualize simultaneously the movement of the two tracers and to provide a comparative analysis of their pathways (cf. Botha et al., 2008). Before loading, the upper and lower 3 cm of each stem were covered with liquid Steedman’s wax, leaving a central area of approx. 10 mm wax free (Supplementary Data Fig. S1A). A 20 μL aliquot of the mixed tracer solution was applied to this area every 30 min for 4 h, to ensure that a sufficient amount of the tracer solution had been absorbed. Longitudinal hand sections were cut from the dye application site and from stem regions above and below it. The presence of a fluorescent signal was verified under confocal laser scanning microscopy (CLSM; see ‘Microscopy’ section).

Internal loading.

 The stems of 20 gametophytes (50 mm long) of P. schreberi and H. splendens were excised under water with a razor blade. Leaves and lateral branches from the lower part of the stem (approx. 10 mm from the cut end) were gently removed with tweezers to minimize transport of tracers on the outer stem surface. The gametophytes were lightly blotted with paper towel to remove the extra water and each transferred to open Eppendorf tubes (Supplementary Data Fig. S1B) filled with 5 μL of either symplasmic tracers [HPTS or carboxyfluorescein diacetate (CFDA)], apoplasmic tracers [TR or sulphorhodamine B acid chloride (SRB)] or water (control). After 2 h at room temperature, gametophytes were removed from the tubes and hand-sectioned both transversely and longitudinally in a successive manner from the lower part of the stem towards the apex and immediately analysed under an epi-fluorescence microscope (see ‘Microscopy’ section).

Impact of air humidity on internal transport rates

Preliminary studies showed that HPTS and TR provided the most intensive fluorescence and minimal leakage from the cells and were, therefore, selected as the symplasmic and apoplasmic tracers for this experiment. Forty gametophytes of H. splendens and 80 gametophytes of P. schreberi were misted with water to reduce variation in water content among individuals and were then exposed to TR or HPTS as described before. Half of the gametophytes for each species and tracer type were covered with a transparent plastic lid to increase air humidity (to about 75 %) and the other half were left at ambient conditions (air humidity approx. 30 %).

After 30 min, gametophytes were successively removed one at a time and at regular intervals from the tracer solutions, with the last gametophytes removed after 300 min. The lower 2 mm portion of each gametophyte stem was discarded and stems were cut into 5 mm segments which were then longitudinally hand-sectioned. The 5 mm stem sections were observed under an epi-fluorescence microscope, and the distance travelled by the tracer from the base of the stems towards the apex was recorded. Transport rates (mm h^–1) were calculated by dividing the distance the tracer had travelled by tracer exposure time.

Microscopy

All the semi-thin sections prepared within this work were analysed using either an epi-fluorescence microscope with bright-field optics (Olympus BX50, Olympus Optical), with blue (470–490 nm), green (530–550 nm) and UV (360–370 nm) excitation filters and with a digital camera (DP71, Olympus Optical) equipped with CellˆB software (Olympus Optical) or using CLSM (Fluo View1000, Olympus Optical) with blue (488 nm) and green (536 nm) lasers. The ultrathin sections were examined by TEM (Zeiss EM 900 at 80 kV and Tesla BS 300). Stem sections prepared for the anatomical studies were analysed using bright-field optics. Callose after aniline blue staining was detected under UV light in the epi-fluorescence microscope and after immunolocalization using CLSM with blue (for Alexa Fluor 488) and green (for propidium iodide) lasers. The HPTS and TR tracers applied externally were examined under CLSM using blue and green lasers, respectively. All tracers loaded internally to the stems were detected by epi-fluorescence microscopy using green light (TR and SRB tracers) and blue light (HPTS and CFDA tracers).

Statistical analyses

To assess differences in internal transport rates, we first ran a three-way analysis of variance (ANOVA) with moss species, tracer type and air humidity as main factors. When this ANOVA revealed significant main effects of moss species – including several interactive effects (Supplementary Data Table S1) – we used two-way ANOVA testing for each species, the effects of tracer type/air humidity and tracer type and air humidity interactions. When the effects of ANOVAs were significant at P < 0.05, Tukey’s HSD post-hoc test was used to compare means of transport rates between tracer types and air humidity for each species. Data were spread-level plot transformed to meet the assumptions of homoscedasticity of error variance and normality. All analyses were performed in R (R Core Team, 2015).

RESULTS

Stem anatomy

Anatomically the main stems of P. schreberi consist of an epidermis, inner and outer cortical regions and a small central strand (Fig. 1A). The epidermis forms a multistratose external structure composed of thick-walled cells with a small lumen (Fig. 1B). The central strand is composed of elongated and thin-walled hydroids (Fig. 1C) lacking cytoplasmic contents (Fig. 1D, F) apart from small cytoplasmic remnants (Fig. 1D, F). Hydroids in P. schreberi are imperforate and possess uniformly thin cell walls (Fig. 1D, F), and lack plasmodesmata. The lateral branches lack a central strand (data not shown). The inner and outer cortical cells in the stems of P. schreberi are filled with vertically orientated conducting parenchyma cells with abundant, oval or circular regions of thinner walls (Fig. 1G). These regions are larger in the terminal walls and smaller, but more frequent, in the lateral walls (Fig. 1G, H) and are filled with callose (Fig. 2A–D). This was inferred from the blue–white fluorescence after aniline blue staining as well as from the immunolocalization assay against 1,3-β-d-glucan. Numerous plasmodesmata also span these thinner cell wall areas of the conducting parenchyma (Fig. 2E–J). The plasmodesmata have a constricted neck region (Fig. 2G, H) and are sometimes slightly enlarged in the middle lamella region of the wall (Fig. 2H). The neck region of plasmodesmata is sometimes occluded with plugs of electron-opaque material (Fig. 2H). A desmotubule is sometimes visible throughout the neck region (Fig. 2J), but is not visible in the median enlarged region.

Fig. 1.

Light and TEM microphotographs showing anatomical characteristics of the Pleurozium schreberi (A–H) and Hylocomium splendens (I–N) gametophyte stems. (A) Transverse section of the main stem of P. schreberi. (B) Cell to cell contacts (arrowheads) in the outer cortical region. (C) Thin-walled hydroids forming the central strand. (D) The hydroids lack cytoplasmic contents in contrast to the neighbouring cortical cells. Cytoplasmic remnants in hydroids are marked with arrowheads. (E) Longitudinal section of the main stem of P. schreberi. (F) Details of the central strand and the inner cortical region; note the lack of contents in the hydroids with only small cytoplasmic remnants (arrowhead). (G) Conducting parenchyma cells with numerous thin-walled regions (arrowheads). (H) Thin-walled areas on lateral and terminal walls (arrowheads). (I) Transverse section of the main stem of H. splendens. (J) A central strand is absent. (K) Cell to cell contacts (arrowheads) in the outer cortical region. (L) Longitudinal section of the main stem of H. splendens. (M) Conducting parenchyma cells with numerous thin-walled regions (arrowheads). (N) The thin-walled areas (arrowheads) on the terminal and lateral walls. E, epidermis; Ocr, outer cortical region; Icr, inner cortical region; Cs, central strand; Cpc, conducting parenchyma cells; H, hydroids; P, paraphyllia. Microphotographs were made from cross hand sections (A–C and I–K) and from median longitudinal hand sections (E, G, H, L–N), stained with Alcian Blue–Safranin O solution (G, H, M, N) and observed with the use of light microscopy. Microphotographs in (D) and (F) are made with the use of TEM. Scale bars are 3 μm for (D, F), 20 μm for (B, C, G, H, J, K, M, N) and 100 μm for (A, E, I, L).

Fig. 2.

Characteristics of thin-walled regions in the conducting parenchyma cells of P. schreberi. Callose deposits (A–D) and abundant plasmodesmata (E–J) localized in the thin-walled regions between the conducting parenchyma cells. (A) Accumulation of callose (arrowheads) in the cortical region detected by aniline blue staining. (B) Control section without secondary antibody. (C and D) Immunolocalization of 1,3-β-d-glucan (arrowheads) in the walls of the conducting parenchyma in the outer and inner cortical regions. (D) At higher magnification, the fluorescent signal is clearly located in the thin-walled regions. (E–J) Longitudinal (E, G, H) and transverse (F, I, J) sections of plasmodesmata in the thin-walled regions. (E) Numerous plasmodesmata (arrows) between two conducting parenchyma cells. (F) Lateral cell wall of a cortical cell. The cell wall region close to the plasmodesmata is clearly thinner. (G) Terminal cell wall of a cortical cell with abundant plasmodesmata (arrows). The polarity of the cytoplasm between the neighbouring cells is clearly visible. (H–J) Details of ultrastructure of the plasmodesmata in the longitudinal (H) and transverse (I and J) view. The neck region is marked by empty arrowheads. White arrowheads mark desmotubules. Ocr, outer cortical region; Icr, inner cortical region; Cpc, conducting parenchyma cells; Pd, plasmodesmata. Longitudinal hand sections were excited with a UV filter in an epi-fluorescence microscope (A), with a blue laser (B–D), and with a green laser in CLSM (C and D). Microphotographs in (E–J) were made with the use of TEM. Scale bars are 0.5 μm for (H–J), 1 μm for (E–G), 20 μm (for A, B, D) and 100 μm (for C).

Conducting parenchyma cells in both inner and outer cortical regions display polar organization of the cytoplasm with an asymmetric localization of the nucleous and an aggregation of elongated plastids and mitochondria to one end of the cell (Fig. 3A–E, G, H). However, this asymmetric organization is not evident for all cells in these regions, as some lack cytoplasmic polarity under the light microscope (Fig. 3C). The cytology of the cells from the inner cortical region is similar to that of the cells of the outer cortical region; both are clearly food-conducting cells (Fig. 3G–L). The elongate plastids in food-conducting cells have central grana and peripheral vesiculate thylakoids (Fig. 3I, J), the elongate mitochondria are filled with swollen vesiculate cristae (Fig. 3K) and the nuclei are elliptical (Fig. 3L). The transparent cytoplasm of the food-conducting cells contains numerous vesicles (Fig. 2E–G) and possible refractive spherules (Fig. 2E, F), and their walls are thicker than those of the hydroids (Fig. 3F).

Fig. 3.

Cytology of the conducting parenchyma cells in P. schreberi stems. Longitudinal (A–E) and transverse (F) semi-thin sections of the conducting parenchyma cells from outer and inner cortical regions; and longitudinal ultrathin sections of food-conducting cells from the inner cortical region (G–L). (A–D) Elongated cells in the cortical regions have elliptical (A, B, D) or round (C) nuclei (marked with arrows). Elliptical nuclei are present at one end of the cell. (E) Higher magnification of (D) showing elongate plastids (arrowheads) in close proximity to the nucleus. (F) Conducting parenchyma cells around the central hydroids with many small vacuoles. Note their thicker walls compared with those of the hydroids. (G–L) Ultrastructure of the cortical food-conducting cells. (G) Cytoplasmic polarity between two neighbouring cells. (H) Polarized distribution of elongated plastids and mitochondria in a food-conducting cell. (I) Distinct thylakoid system of a food-conducting cell plastid. (J) Elongated plastids located near the lateral wall. (K) Elongated mitochondria. (L) Eliptical nucleous. The nucleolus is clearly visible. Ocr, ourter cortical region; Icr, inner cortical region; H, hydroids; Fcc, food-conducting cells, P, plastid; M, mitochondrium; N, nucleous. Scale bar: 1 μm (H– L), 2 μm (G), 10 μm (F) and 20 μm (A–E).

The stems of H. splendens comprise an epidermis, an inner and an outer cortical region (Fig. 1I), but differ from those of P. schreberi in the absence of a central strand (Fig. 1J). As in P. schreberi, the epidermis is formed by thick-walled cells with small lumina (Fig. 1K) and the cortical regions are composed of elongated conducting parenchyma cells (Fig. 1L). These cells have numerous thin-walled regions (Fig. 1M, N) containing callose (Supplementary Data Fig. S2), suggesting the presence of plasmodesmata.

Uptake of tracers and their short- and long-distance internal transport

Gametophore stems of P. schreberi are able to absorb and transport both symplasmic and apoplasmic tracers when applied to the stem surfaces (Fig. 4). The symplasmic tracers were mostly visible in the cytoplasm and in the plastids of the cortical regions, and very faintly in the lumina of the central strand cells (Fig. 4A), while the apoplasmic dyes were mainly observed in the central strand (Fig. 4B). This observation provides evidence of solute uptake by the main stem and possible solute transport over long distances via the hydroids in the central strand inside the gametophore stems. Hylocomium splendens is, like P. schreberi, able to absorb and transport the symplasmic HPTS dye (Fig. 4C). The signal of the HPTS tracer was visible in the cortical regions of H. splendens; however, in contrast to P. schreberi, in H. splendens the TR dye was only present on the stem surface (Fig. 4D).

Fig. 4.

Fluorescent microphotographs of longitudinal hand sections of P. schreberi (A and B) and H. splendens (C and D) stems after external application of symplasmic (green) and apoplasmic (red) tracers. (A) Distribution of HPTS tracer in the symplasm of the inner cortical and central strand cells (arrowheads). (B) Localization of TR tracer in the cells of the central strand (arrow). (C) The presence of the HPTS tracer in the cortical cells (arrowheads). (D) Localization of the TR tracer on the stem surface (arrows). Please note that this dye is absent from the cortical region. Microphotographs were taken above (A, D), below (B) and at the tracer application site (C). Cr, cortical region; Cs, central strand. Scale bars are 100 μm.

When the tracers were applied at the base of the cut stem, P. schreberi transported both symplasmic (CFDA and HPTS) and apoplasmic (TR, SRB) tracers internally over short and long distances (Fig. 5). The HPTS and CFDA symplasmic tracers and the TR apoplasmic dye were transported from cell to cell over short distances at the lower end of the P. schreberi stem (Fig. 5A, B). The symplasmic tracers were localized in the cytoplasm of the outer cortical cells, whereas the apoplasmic dye was only found in the cell walls. Furthermore, both tracer types were loaded into the hydroids of the central strand via the cortical cells and transported over long distances (Fig. 5C–F). Similar results were obtained for the SRB tracer (data not shown). It is therefore evident that the long-distance transport within the central strand occurs via the hydroids regardless of the symplasmic or apoplasmic nature of the applied tracer. In addition, the symplasmic tracers were localized in the lumen of the hydroids of the central strand (Fig. 5C), whereas the apoplasmic tracers moved into their walls (Fig. 5D). In the upper part of the stem, both the symplasmic and the apoplasmic tracers were frequently localized in the cytoplasm of cortical cells in the unloading region beneath the shoot apex (Fig. 5E, F).

Fig. 5.

Distribution of symplasmic and apoplasmic fluorescent tracers in P. schreberi stems after internal loading. The localization of the tracers is from (A and B) the lower (loading), (C and D) the middle and (E and F) the upper (close to the shoot apex – unloading) regions of the stem. The symplasmic tracers CFDA (A) and HTPS (C, E) are visible in green and the apoplasmic tracer TR (B, D, F) in red. Sub-panels in (C) and (D) show transverse sections of the stem and the presence of the symplasmic and apoplasmic tracers in the central strand, respectively. Note that the HPTS tracer is present in the cytoplasm (arrowheads) whereas the TR tracer is only visible in the cell wall. Sub-panels in (B) and (F) show localization of the TR tracer in the apoplast and in the cytoplasm of cortical cells, respectively. Cr, cortical region; Cs, central strand. Scale bars are 100 μm (for A–F and for sub-panels in B and F) and 5 μm (for sub-panels in C and D).

In contrast to P. schreberi, internal transport in H. splendens stems occurred only from cell to cell and exclusively via the symplasmic route (Fig. 6). This result is supported by the presence of the CFDA and HTPS symplasmic tracers in the cortical cells along the stem (Fig. 6A–C) and the absence of apoplasmic tracers inside the stem (Fig. 6D, E). However, while the apoplasmic tracers (TR and SRB) were visible at the application site (Fig. 6F; data not shown for SRB), they were not transported upwards inside the stem (Fig. 6E). For the short-distance transport, the symplasmic tracers were absorbed by the epidermis and cortical cells, and moved further upwards from cell to cell along the stem (Fig. 6A–C). The transport of these tracers was slow and the dyes did not reach the main apical shoot during 2 h of observation.

Fig. 6.

Epi-fluorescent microphotographs showing the distribution of symplasmic (A–C) and apoplasmic (D–F) tracers in H. splendens stems after internal loading. (A) Longitudinal stem section showing localization of the CFDA tracer in the symplasm (arrows) of cortical cells. (B and C) Transverse sections illustrating the presence of the HPTS tracer in the cytoplasm (arrowheads) of cortical cells; at both 0.5 mm (C) and 5.0 mm (B) above the application site. (D) The TR tracer is present on the outer stem surface (arrowheads) and absent from the cortical region (except at the loading site). (E and F) Transverse sections showing localization of the TR tracer in the upper part of the stem (E) and at the site of tracer application (F). Open yellow arrowheads indicate the site of tracer application. Cr, cortical region; Cs, central strand. Scale bars are 100 μm.

Impact of air humidity on internal transport rates

Internal relative transport rates of P. schreberi were significantly higher than those of H. splendens. For both mosses, transport rates were nearly three times higher at low air humidity compared with high air humidity (Fig. 7; Supplementary Data Table S1). For P. schreberi, the relative transport rates of the symplasmic and the apoplasmic tracers within each humidity condition did not differ from each other (Fig. 7A). There was, however, a significant interactive effect of tracer type and air humidity for H. splendens (Fig. 7B) since apoplasmic tracer transport was close to zero in H. splendens.

Fig. 7.

Transport rates of symplasmic and apoplasmic tracers (mean ± s.e.) in gametophyte main stems of P. schreberi and H. splendens at low and high air humidity. Data were analysed using two-way ANOVA for each species. Different lower case letters indicate where α < 0.05 between symplasmic and apoplasmic tracers, upper case letters indicate where α < 0.05 between low and high moisture conditions.

DISCUSSION

The main finding of this work was the discovery of food-conducting cells that have a major role in determining internal conduction in the pleurocarpous mosses P. schreberi and H. splendens. We also found that the central strand in the stems of P. schreberi consists of hydroids which contribute to long-distance transport in this species. Both P. schreberi and H. splendens are able to absorb solutes from the stem surface and transport them horizontally towards the stem interior and vertically towards the apex. These findings are the first direct evidence of internal (endohydric) transport in feather mosses and contradict the general assumption that these mosses are exclusively ectohydric. In P. schreberi, solutes are quickly transported via the apoplasmic route from the stem cortical region via the central strand to the apex and, from there, transported back to the cortical region. These results are indicative of loading and unloading regions in the main stem, and further suggest that the central strand in P. schreberi assists in the internal transport and solute movement over long distances (cf. Sowiński, 2013). Thus the central strand does not mainly serve as a strengthening structure as previously suggested (Bowen, 1933). In both P. schreberi and H. splendens, the intercellular, short-distance transport inside the stem cortical region mainly occurs via the symplasmic route through abundant plasmodesmata. The presence of food-conducting cells in P. schreberi was confirmed by our TEM analyses. Given that the morphology of the stem cortical region of H. splendens resembles that of P. schreberi, we assume that the cortical cells of H. splendens are also likely to be food-conducting cells.

Our data also showed that the food-conducting cells in P. schreberi have a transparent cytoplasm with vesicles and possible refractive spherules plus cytoplasmic polarity, as has been observed in other mosses (Ligrone and Duckett, 1994; Pressel et al., 2008). We also found that these cells contained elongated plastids and mitochondria, but no endoplasmic microtubules. This suggests that feather mosses may have less highly differentiated food-conducting cells than those in many other mosses such as Mnium and Plagiomnium, and most closely resemble those in Neckera which, like Hylocomium, also lacks hydroids (compare Ligrone and Duckett, 1994; Ligrone et al., 2000). Interestingly, the rate by which the applied fluorescent tracers spread inside the moss stems is similar to that reported for molecule transport through plasmodesmata in higher plants (Goodwin et al., 1990; Rutschow et al., 2011). Our data also confirm that plasmodesmata serve as efficient structures for cell to cell transport of symplasmic tracers in feather mosses and raise further questions about the role of plasmodesmata in the developmental and physiological processes in mosses. Plasmodesmata have, for example, a key role in regulating cell to cell communication not only of small molecules, but also of macromolecules such as transcription factors (Roberts and Oparka, 2003; Sowiński, 2013). Therefore, the presence of abundant plasmodesmata between the food-conducting cells and the ubiquitous occurrence of these cells (Ligrone and Duckett, 1994, 1998) highlights the general importance of symplasmic transport in mosses.

The different pathways for internal transport in the two mosses are clearly related to anatomical differences. While the stem interior of both species consists of food-conducting cells responsible for short-distance transport, fast transport over long distances from the base of the stem to the apex in P. schreberi is linked to the presence of dead hydroids in the central strand, which facilitate the long-distance transport of solutes in this species via the apoplasmic route (c.f. Raven, 2003). Surprisingly, the hydroids transported the symplasmic HPTS tracer within their lumen despite the absence of cytoplasm (Ligrone et al., 2000; Raven, 2003). The mechanism underlying the HPTS tracer transport in hydroids is unknown, but is most likely to be due to the membrane-impermeable property of the dye and its inability to diffuse freely through plasma membranes.

In agreement with our second hypothesis, we found that the rates of internal solute transport differed between the two species. Pleurozium schreberi transported the dyes about 3.5 times faster than H. splendens. We attributed this to the presence of hydroids in P. schreberi. Interestingly, solute movement in the central strand occurred equally as fast via the apoplasmic route, with similar relative rates for both tracer types (e.g. the HPTS and TR tracer). This suggests that similar mechanisms are responsible for the long-distance transport of both types of tracers, and is most probably akin to mass flow found in the tracheary elements of vascular plants. In comparison with species from the Polytrichaceae and the Mniaceae, the transport rate of P. schreberi appears relatively low. These species may transport solutes 16-fold (Eschrich and Steiner, 1967) or even 50- to 100-fold (Zacherl, 1956 after Hébant, 1977) faster than P. schreberi. The slower rate is most probably due to the smaller size of the central strand of P. schreberi.

Overall our finding of faster internal transport in both P. schreberi and H. splendens at the lower air humidity is in line with previous work showing that mosses have a general capacity to increase internal solute movement due to the lack of active control over water status when environmental humidity decreases (Bayfield, 1973; Longton, 1992). However, contrary to expectation, the relative increase was slightly greater for H. splendens (2.9 times higher compared with 2.6 times higher for P. schreberi). The underlying mechanism for this is unknown, but our results are supported by studies showing that H. splendens is likely to dry out faster than P. schreberi under drier conditions (Heijmans et al., 2004; Elumeeva et al., 2011). This result may also explain why H. splendens preferentially occupies wetter habitats (Busby et al., 1978). The faster internal transport of P. schreberi under low air humidity suggests that the central strand may help to maintain stable tissue water content/metabolic activity as well as heat tolerance and nitrogen/carbon economy (Kappen and Valladare, 2007; Gundale et al., 2009) and therefore make P. schreberi better adapted to drier conditions. These traits may also enhance buffering against extreme changes in environmental conditions.

In summary, this study contributes to an improved understanding of feather moss water transport by demonstrating that these mosses are not exclusively ectohydric. We have identified, for the first time, the presence of hydroids and food-conducting cells in feather mosses and showed that their capacity for internal transport is strongly dependent on species-specific tissue characteristics. We have demonstrated that both apoplasmic and symplasmic pathways can be involved in solute transport inside the gametophyte stems. We also showed that internal transport of P. schreberi increased significantly under drier conditions due to the presence of hydroids in the central strand.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Appendix 1: callose immunolocalization using 1,3-β-d-glucan-directed mouse monoclonal primary antibody and the Alexa Fluor 488 rabbit anti-mouse secondary antibody. Figure S1: illustration of fluorescent tracer loading methodology of moss gametophytes. Figure S2: localization of callose deposition in the stem cortical region of H. splendens following aniline blue staining and 1,3-β-d-glucan immunolocalization. Table S1: results from a three-way ANOVA testing for the effects of moss species (P. schreberi and H. splendens), tracer type (apoplasmic and symplasmic), air humidity (low and high) and all possible two- and three-way interactions among these factors, on the relative internal transport rate of moss gametophytes.