Skip to main content
NCBI home page
Annals of Botany logoLink to Annals of Botany
. 2016 Apr 23;117(6):985–994. doi: 10.1093/aob/mcw029

Patterning of stomata in the moss Funaria: a simple way to space guard cells

Amelia Merced 1,✉, Karen S Renzaglia 1
PMCID: PMC4866314  PMID: 27107413

Abstract

Background and Aims Studies on stomatal development and the molecular mechanisms controlling patterning have provided new insights into cell signalling, cell fate determination and the evolution of these processes in plants. To fill a major gap in knowledge of stomatal patterning, this study describes the pattern of cell divisions that give rise to stomata and the underlying anatomical changes that occur during sporophyte development in the moss Funaria.

Methods Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold labelling was used to investigate the presence of pectin in the newly formed cavities.

Key Results Substomatal cavities are liquid-filled when formed and drying of spaces is synchronous with pore opening and capsule expansion. Stomata in mosses do not develop from a self-generating meristemoid as in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e. there are no stomatal lineage ground cells.

Conclusions Development of stomata in moss occurs by differentiation of guard mother cells arranged in files and spaced away from each other, and epidermal cells that continue to divide after stomata are formed. This research provides evidence for a less elaborated but effective mechanism for stomata spacing in plants, and we hypothesize that this operates by using some of the same core molecular signalling mechanism as angiosperms.

Keywords: Funaria, GMC, guard cell, LM19, mosses, immunolocalization, stomata, stomatal development, stomatal patterning, ultrastructure

INTRODUCTION

The elucidation of the unique cell division patterning during the development of guard cells in Arabidopsis and other flowering plants established the foundation for understanding the genes involved in stomatal distribution and spacing (Geisler et al., 2000; Liu et al., 2009; Rudall et al., 2013; Rudall and Knowles, 2013). Stomatal patterning and cell lineages that lead to guard cell formation are poorly known in seedless plant groups. As one of two bryophyte groups with stomata, mosses are crucial in ascertaining the function and evolution of stomatal development genes, some of which have orthologues present in Physcomitrella (Peterson et al., 2010; MacAlister and Bergmann, 2011).

Sack and Paolillo (1983a, b, 1985) describe the differentiation of the peculiar binucleate single-cell stoma in Funaria through incomplete cytokinesis of guard mother cells (GMCs) in the subapical region of the capsule. Garner and Paolillo (1973a, b) reported that stomata differentiate before capsule expansion and opening of the pore occurs during the first few days of capsule expansion. This is similar to other mosses where stomata are fully formed before full expansion and maturation of capsules (Paton and Pearce, 1957). These studies on Funaria focused on the ultrastructural development of individual guard cells, the timing of stomatal development in the sporophyte and stomatal function. However, the patterning, distribution and spacing of stomata, relationship within the developing epidermis and associated anatomical changes during capsule expansion remain unknown.

In Arabidopsis, stomata develop from a protodermal cell through a series of asymmetrical and symmetrical divisions. Protodermal cells produce pavement cells and self-renewing meristemoids that can divide asymmetrically several times, generating meristemoids and pavement cells known as stomatal lineage ground cells (Rudall et al., 2013). Meristemoids can eventually differentiate into GMCs and a final symmetrical division of a GMC produces two guard cells (Geisler et al., 2000). Spacing between stomata is the result of meristemoid divisions (Pillitteri and Dong, 2013). The development of stomata requires cell-to-cell communication and many of the genes involved in the process of spacing and differentiation have been identified (reviewed in Casson and Gray, 2008; Vaten and Bergmann, 2012; Pillitteri and Dong, 2013).

To fill a significant gap in knowledge that is essential for evo-devo studies of stomata, the present study details the pattern of cell division that leads to guard cell formation in the moss Funaria. Funaria serves as an excellent proxy for the model moss Physcomitrella for this work because of the close phylogenetic relationship between these two taxa and because, unlike the tiny capsules with 12–14 stomata in Physcomitrella, Funaria capsules are large, with up to 200 stomata (Paton and Pearce, 1957; Field et al., 2015). Changes in sporophyte anatomy, especially the formation of substomatal cavities and internal air spaces, are followed throughout the developmental process. Recently formed substomatal cavities were probed for pectin to investigate whether the liquid filling the cavities consists of pectin residues from the separation of the middle lamella (Sack, 1987; Sack and Paolillo, 1983a). The stomatal development pattern and capsule anatomy are presented from the spear stage to mature capsule, with special attention to early capsule expansion. The three major questions addressed in the study are: (1) is the development of guard cells perigineous, specifically do GMC and epidermal cells develop from different protodermal cells? (2) what is the developmental pattern for stomata distribution in Funaria and how does it compare to other plants? and (3) when and how do substomatal cavities and internal spaces form in Funaria capsules? Due to the manner in which moss capsules develop, we hypothesize that stomata develop from GMCs that are delineated after seta elongation but before capsule expansion, and not by a self-generated meristemoid, as in Arabidopsis. In addition to following guard cell lineages, we detail internal anatomical changes that are coordinated with and integral to guard cell development during capsule expansion.

MATERIALS AND METHODS

Plants of Funaria flavicans and Funaria hygrometrica with developing sporophytes at different stages were collected between the spring seasons of 2012–2014. Voucher specimens have been deposited at SIU (K. S. Renzaglia, 3441, 3444; A. Merced, 113).

Transmission electron microscopy

Spear and expanding sporophytes were fixed in 2 % glutaraldehyde in 0·05 M NaPO4 buffer for 1 h at room temperature, then overnight at 20 °C. The next day specimens were washed three times in 0·05 M NaPO4 buffer, 30 min each time, and postfixed for 20 min in 1 % OsO4 in 0·05 M NaPO4 buffer, then rinsed three times in distilled water (total rinsing time 30 min) and dehydrated in a graded ethanol series ending with three changes of 100 % ethanol. Gradual infiltration with LR White resin (London Resin Company, Berkshire, UK) was done over 4 d using a graded ethanol series by increasing the ratio of resin to ethanol. Specimens were placed in moulds with fresh resin and cured at 65 °C for 2 d. Semithin sections (250–750 nm) for light microscopy were mounted on glass slides and stained with 1·5 % toluidine blue in distilled water. Thin sections (60–90 nm) were collected on nickel grids and allowed to dry for 1–3 h at room temperature.

Immunogold localization

Protocols are described in Merced and Renzaglia (2013, 2014) and are briefly summarized here. Grids were incubated overnight at 4 °C in a humid chamber with 2 % bovine serum albumin (BSA) in 0·02 M phosphate-buffered saline solution, pH 7·2 (PBS). Two or three grids were transferred to the LM19 primary antibody (diluted 1 : 20 in 2 % BSA/PBS) for 3 h and controls (one grid) were left in buffer during that time. All grids were rinsed in 2 % BSA/PBS four times for 3 min each, then incubated in goat anti-rat IgG secondary antibody (Sigma-Aldrich, St Louis, Missouri, MO, USA) diluted 1 : 20 in 2 % BSA/PBS for 30 min. Grids were rinsed four times with PBS for 3–5 min each, followed by distilled/deionized autoclaved filtered water, and dried at room temperature. Grids were observed unstained with a Hitachi H7650 transmission electron microscope at 60 kV.

Scanning electron microscopy

Specimens were fixed in 2 % glutaraldehyde in 0·05 M NaPO4 buffer for 1 h at room temperature, then overnight at 20 °C. Sporophytes were postfixed in 1 % OsO4 in 0·05 M NaPO4 buffer for 2 h then rinsed in dH2O three times for 30 min each followed by dehydration in a graded ethanol series and ending with three changes of 100 % ethanol, 25 min each. Specimens were critical-point dried using CO2 as the transitional fluid, mounted on stubs, sputter-coated for 230 s with palladium–gold and viewed using a FEI 450 scanning electron microscope.

Light microscopy

Living Funaria sporophytes were studied under a light microscope to determine critical stages in stomata development. Clear silicone (Loctite, Henkel Corporation, Rocky Hill, USA) was used to make impressions of spear and expanding sporophytes (n =9) and expanded capsules (n= 7). Silicone moulds were filled with clear fingernail polish and left to dry at room temperature. Epidermal peels of spear and early expanding capsules (n=5) were prepared using clear tape and scraping the tissue under the epidermis. Mature expanded capsules (n=7–11) were examined for stomata number per capsule, percentage of stomata next to each other and number of epidermal cells surrounding each stoma. Measurements were made using ImageJ (http://rsb.info.nih.gov/ij/).

Fresh capsules of spear and expanding sporophytes (n=8) were stained with 1 % aniline blue in 0·067 M sodium phosphate buffer, pH 8·5, for 1 h in the dark at room temperature. Controls were prepared using buffer only (n=2). Aniline blue binds to callose and emits yellow–green fluorescence under UV light. Callose fluorescence was observed using an excitation filter at 350 nm and an emission filter with a transmission cut-off at 460 nm.

Fresh material, semithick sections, epidermal peels and impressions were observed using light, differential interference contrast and fluorescence microscopy with a Leica DM 5000B compound microscope equipped with UV fluorescence and a Q-Imaging Retiga 2000R digital camera.

RESULTS

Mature capsules of Funaria have ∼150 stomata located in the apophysis, the basal section of the capsule that connects to the seta (Fig. 1A). Stomata are arranged in files of five to seven rows around the apophysis and spaced by at least one cell, with the exception of juxtaposed stomata, occurring 1–4 % of the time (Table 1). The ventral guard cell walls, which separate to form the pore, do not reach the entire length of the cell and the two guard cells are connected at the polar ends, forming a continuous cytoplasm. Mature stomata are elliptical and have a long pore that usually aligns with the length of the sporophyte (Fig. 1B). Each stoma is surrounded by 10–16 epidermal cells that are radially arranged around the guard cells (average 13, n=55 guard cells, 11 sporophytes).

Fig. 1.

Fig. 1.

Open in a new tab

Mature capsules of Funaria. (A) Scanning electron micrograph of expanded capsule with stomata in irregular rows and files on apophysis (arrowheads). (B) Drawing of stomata distribution in the apophysis of mature capsule. (C, D) Scanning electron micrographs of spongy tissue inside the capsule. (E) Scanning electron micrograph of apophysis showing slightly raised stomata covered by smooth cuticle that is thickened around the pore (arrow). Scale bars: (A, C) = 500 µm; (B) = 35 µm; (D) = 100 µm; (E) = 10 µm.

TABLE 1. Number of stomata per capsule and percentage of stomata next to each other

Sporophyte specimen Number of stomata Number of stomata without space % of contiguous stomata
1 177 7 4·0
2 131 4 3·1
3 157 2 1·3
4 137 6 4·4
5 145 2 1·4
6 132 2 1·5
7 170 2 1·2
Average 149·9
Open in a new tab

Inside the capsule the spongy photosynthetic tissue extends above the apophysis (Fig. 1C). The spongy tissue consists of elongated aerenchyma cells forming an interconnected network of intercellular spaces. The innermost layer of the spongy tissue has larger intercellular spaces and consequently fewer aerenchyma cells than those towards the outside of the capsule (Fig. 1D). The sporophyte is covered by a smooth, thin cuticle that is thicker along the outer ledges of the guard cells (Fig. 1E).

Stomata develop during the spear stage, which occurs after seta elongation and before capsule expansion (Fig. 2A–E). The calyptra covers the apex and distal part of the sporophyte until expansion of the capsule. The twisted apex of the sporophyte at the spear stage develops into the operculum and peristome, while the subapical region of the capsule becomes the urn and apophysis (Fig. 2A). At this stage, the seta can be distinguished from the capsule in having highly elongated cells (Fig. 2A). Capsule expansion occurs over several days and stomata differentiate early in the process (Fig. 2B–E). At the spear stage, cells are arranged in longitudinal files and stomatal initials are indistinguishable from other epidermal cells. Before capsule expansion, most epidermal cells of the capsule are square to rectangular, and in the subapical region, about 40 cells from the tip, putative GMCs are visible as round and larger than other cells (Fig. 2B, F, G). There is no evidence of meristemoids or asymmetrical divisions that lead to the formation of GMCs; instead, differentiation of putative GMCs from protodermal cells follows the file arrangement of the developing sporophyte, with at least one cell between neighbouring GMCs (Fig. 2F, G). Epidermal cells above and below putative GMCs divide first, in a plane more or less parallel to the length of the sporophyte (Fig. 2F). Multiple chloroplasts are arranged around the periphery of each paradermal GMC, affirming their identification as GMCs (Fig. 2G).

Fig. 2.

Fig. 2.

Open in a new tab

Development of Funaria sporophyte. (A) Epoxy impression of early spear stage after seta elongation. Cells are arranged in files and three different areas can be distinguished based on cell shape: cells of the seta (arrowhead) are rectangular and elongated; cells of the urn are small and square; and cells of the operculum (o) are small and slightly twisted. (B–E) Light micrographs of capsule expansion. (B) Spear stage. Box delimits the area drawn in (F) that will become the apophysis. (C) Beginning of capsule expansion, which starts at the apophysis. (D) Stomata are visible but do not have pores or subtending air spaces. (E) Later stage when the pore is formed and air spaces are formed. (F) Line drawing of cells in the area of the spear stage that will become the apophysis (box in B). Large round cells are putative GMCs (in grey). Epidermal cells divide parallel to the longitudinal axis of the capsule (red dotted lines). Arrow points towards the sporophyte apex. (G) Light micrograph of the apophysis region with slightly raised, rounded, putative GMCs with multiple chloroplasts (arrow) in the same area of the spear sporophyte as in (F). Asterisks in F and G identify the same GMC. Scale bars: (A) = 35 µm; (B–E) = 75 µm; (F, G) = 20 µm.

The apophysis is the first part of the capsule to expand and GMCs differentiate into stomata in this area before expansion is completed (Fig. 2D, E). Based on aniline blue staining of callose, a polysaccharide present in newly formed cell walls (Ivakov and Persson, 2012), epidermal cell divisions are mostly parallel to the sporophyte axis at the onset of capsule expansion (Fig. 3A). Almost all stomata are differentiated at this point, but the orderly arrangement in rows and files is distorted by divisions and expansion of the epidermal cells (Fig. 3B). Ventral walls are formed in stomata but do not separate to open the pore and no gas is visible inside the capsule at this stage (Fig. 3A–C). Distal to stomata, epidermal cells are small and continue to divide in several planes. Several cells that resemble GMCs in this area appear to be undifferentiated stomata arrested in development (Fig. 3C). Epidermal cell divisions continue mostly parallel to the longitudinal axis of the sporophyte (Fig. 3D). With further expansion, epidermal cells undergo less orderly divisions in varying planes, thus contributing to a dramatic increase in width of the sporophyte. Multiple round chloroplasts extend around the periphery of the entire stoma, and guard cells are slightly elevated above epidermal cells (Fig. 3E). At a later stage of capsule expansion, similar to the stage shown in Fig. 2E, epidermal cell divisions radiate in different directions from the stomata (Fig. 3F). These epidermal cell divisions separate the stomata from each other and produce the radial arrangement of cells around the guard cells (Fig. 1B). Thus, spacing of stomata occurs during capsule expansion as a result of divisions of epidermal cells but only well after the stomata have differentiated.

Fig. 3.

Fig. 3.

Open in a new tab

Early capsule expansion at the same stage as in Fig. 2D. (A) Predominantly longitudinal cell divisions of epidermal cells in the expanding apophysis as visualized by bright turquoise fluorescence of callose in new cell walls, detected by aniline blue. (B) Differential interference contrast image of capsule during expansion. Most stomata have differentiated and have ventral walls but no pore. Line drawing overlay of part of the capsule shows the arrangement of stomata. (C) Guard cells have abundant chloroplasts and are bigger than epidermal cells; stomata are arranged in files and rows. Distal round cells in the same files as stomata appear to be arrested stomata (arrowheads). (D) Aniline blue fluorescence of the same area as (C), identifying callose (bright turquoise) in newly formed walls of epidermal cells (arrows); divisions are mostly parallel to the sporophyte axis and are consistent with expansion in width of the capsule. Asterisks in C and D indicate the same stoma. (E) Light micrograph of capsule expansion with fully formed stomata with prominent peripheral chloroplasts. (F) Aniline blue fluorescence of the same area as in (E), showing callose (bright turquoise) in newly formed walls of epidermal cells in various planes around stomata (arrows). No callose is found in guard cell walls. Asterisks in (E) and (F) indicate the same stoma. Scale bars: (A) = 75 µm; (B, E, F) = 35µm; (C, D) = 10 µm.

Stomatal development is synchronized with internal changes in sporophyte anatomy. During the spear stage, cells of the sporophyte are tightly compacted without intercellular spaces (Fig. 4A–C). At the apical region of the sporophyte where the peristome develops, cells follow the fundamental square formation (Fig. 4A); this arrangement continues further down the capsule but is less obvious at the apophysis (Fig. 4B). The endothecium, which gives rise to the columella and archesporium, is visible in the spear stage at the centre of the sporophyte. Surrounding the endothecium is the amphithecium, which will give rise to the peristome, outer spore sac, spongy tissue and epidermis (Fig. 4A). Lower on the capsule, in the area of the future apophysis, cells are rounded and less compacted (Fig. 4B). Epidermal cells of the capsule are undifferentiated and without cuticle (Fig. 4A, B); along the seta, however, epidermal cells are smaller, with thickened walls and cuticle (Fig. 4C). Water-conducting tissue consists of a cylinder of thin-walled cells devoid of protoplasm at the centre of the seta (Fig. 4C). At the spear stage the calyptra adheres tightly to the capsule but is separate around the seta. As the capsule expands the calyptra is gradually pushed upwards.

Fig. 4.

Fig. 4.

Open in a new tab

Anatomy of spear stage capsules of Funaria. (A–C) Cross-section of spear sporophyte from apex to seta entirely covered by the calyptra. (A) Apical area of capsule. The columella (c) and archesporium (a) are located at the centre of the sporophyte, surrounded by the amphithecium (am), which contains the peristomial layers and epidermis. (B) Developing apophysis. (C) Upper seta with central water-conducting cells (arrowhead), loosely covered by the calyptra. (D–G) Transmission electron micrograph of the junction between calyptra and sporophyte. (D) Cells of the calyptra have thickened walls and cellular content. (E) Calyptra cells with nucleus (n), round chloroplasts (ch) and multiple small vacuoles (v); a thick cuticle (*) covers the exterior walls. (F) Large round chloroplast with folded thylakoids in the calyptra. (G) Space (arrowhead) between the calyptra and the spear sporophyte appears to be filled with fluid. Scale bars: (A–C) = 35 µm; (D) = 10 µm; (E–G) = 500 nm.

The calyptra consists of several layers of cells, with one or two compacted layers near the apex (Fig. 4A) and three or four loose layers at the base (Fig. 4C). Cells of the external layer of the calyptra have thickened walls and dense cytoplasm with round chloroplasts and multiple small vacuoles (Fig. 4D, E). A thick cuticle covers the calyptra (Fig. 4E). The rounded chloroplasts have narrow elongated sparse grana without starch grains (Fig. 4F). The space between the calyptra and the developing sporophyte is filled with an electron-transparent liquid devoid of substructure (Fig. 4G).

The anatomy of the capsule at the onset of expansion is similar to that of the epidermis, with cells more or less arranged in files (Fig. 5A). In the early stage of capsule expansion, internal spaces form by separation of cells in the amphithecium (Fig. 5A), and liquid-filled substomatal cavities form under developing stomata (Fig. 5B) (Field et al., 2015). At this stage some putative GMCs remain single celled while others have ventral walls but pores are not yet open (Fig. 5C). Most stomata differentiate and remain arranged in files as expansion begins (Fig. 5D). Although internal spaces at the centre of the sporophyte seem empty or gas-filled (Fig. 5A), the small substomatal cavities are liquid-filled (Fig. 5B, E). At this stage ventral walls have not separated to open the pore, but substomatal cavities are already forming. Substomatal cavities are filled with an electron-light material of blotchy appearance (Fig. 5F). Unlike the liquid between the calyptra and the sporophyte (Fig. 4G), the liquid filling the substomatal cavities has substructure that suggests a different composition (Fig. 5F). Abundant pectin, as localized by LM19, is found in all cell walls, but the liquid in the substomatal cavity does not react with this antibody (Fig. 5G).

Fig. 5.

Fig. 5.

Open in a new tab

Anatomy of the expanding capsule of Funaria and immunolocalization of pectin. Round black dots in (F) and (G) are colloidal gold labels attached to the LM19 antibody. (A–C) Longitudinal sections of expanding sporophytes. (A) Light micrograph of stomata (arrowheads) in the apophysis and intercellular spaces that result from separation of cortical cells (arrows). (B) Light micrograph of substomatal cavities forming under stomata (arrowheads). (C) Tangential light micrograph section of apophysis with guard mother cells (*) and guard cells with ventral walls but no pore. (D) Superficial light micrograph of section of apophysis with stomata. (E) Transmission electron micrograph of longitudinal section of stoma (arrowhead) with substomatal cavity (*). (F) Transmission electron micrograph of substomatal cavity filled with electron-lucent liquid with blotchy substructure. (G) The liquid filling the space does not localize for LM19, but this pectin is present in cell walls (arrow). (H, I) Light micrograph of cross-section of a later stage in capsule expansion with dried air spaces. (H) Apical region of the sporophyte and formation of internal spaces; the columella (c) at the centre is surrounded by the archesporium (a). (I) Apophysis with stomata (arrowheads) over substomatal cavities that connect to prominent air spaces. Scale bars: (A, H) = 75 µm; (B) = 20 µm; (C, D, I) = 35 µm; (E) = 10 µm; (F, G) = 500 nm.

Further into capsule expansion, the anatomy of the capsule changes considerably. Towards the apex, cells of the amphithecium separate while the columella and archesporium, where spores develop, remain intact (Fig. 5H). After stomata have formed the pore, prominent air spaces and large substomatal cavities occupy a large volume of the apophysis (Fig. 5I). Funaria capsules expand almost 10-fold from spear stage to mature capsules (Garner and Paolillo, 1973b). Cell arrangement in the apophysis is less regular than in the apex of the capsule, and separation of cells to form the internal air spaces leads to a more random distribution of spaces in the apophysis (Figs 1C and 5I).

DISCUSSION

It is evident from this study that stomatal development and distribution in the moss Funaria is different from that in Arabidopsis. Based on exhaustive morphological observations of developing sporophytes, it can be concluded that differentiation of GMCs in Funaria occurs directly from protodermal cells without amplifying divisions. There are no stomatal lineage ground cells because pavement cells develop directly from protodermal cells, not from meristemoids, and moss stomata are perigineous (sensu Rudall et al., 2013). Putative GMCs are visible before capsules expand and are arranged in cell files. The ‘one-cell spacing rule’ is typical in Funaria, with <4 % of stomata touching each other. Positioning stomata away from others with at least one surrounding epidermal cell is critical for stomatal function in tracheophytes, and may be necessary for pore and substomatal cavity formation in mosses (Peterson et al., 2010; Rudall et al., 2013). A peculiarity of Funaria epidermal development is that, after stomata differentiate, surrounding epidermal cells divide perpendicularly to guard cells, creating a radial arrangement of epidermal cells. These cell divisions are integral to the expansion of the capsule and obscure the original files of stomata, leading to a more random distribution in the fully expanded apophysis. Thus, asymmetrical cell divisions are common during capsule expansion, resulting in increased numbers of epidermal cells, but there are none of the meristemoid-like cells typical of stomatal development in Arabidopsis.

In Funaria, as in most mosses, capsule expansion is preceded by seta elongation, which is brought about by an intercalary meristem (Campbell, 1895; French and Paolillo, 1975c). Maturation of epidermal cells of the capsule is acropetal in Funaria, occurring upwards from the apophysis. Development of stomata is synchronized with changes in the anatomy of the capsule, beginning with separation of cells from the amphithecium to create the spongy tissue in concert with separation of the inner walls of guard cells from cortical cells to form substomatal cavities. During early stages of capsule expansion there is no evidence of air inside the capsule; air spaces are visible later after stomatal pores open. This supports the hypothesis proposed for hornwort stomata that air enters liquid-filled spaces after pores open, drying out the liquid and forming air spaces (Pressel et al., 2014).

Formation of substomatal cavities presumably involves dissolution of the middle lamella (Sack, 1987), but the blotchy material filling the cavities in Funaria does not label for LM19. The LM19 antibody recognizes unesterified homogalacturonans, the most abundant pectins in cell walls and the only ones that localize in the middle lamella (Merced and Renzaglia, 2013, 2014). This epitope is present in the spaces where walls start to separate to form cavities in Dicranum (A. Merced, unpubl. res.), suggesting that pectins may be present at the beginning of cell separation (Sack and Paolillo, 1983a), but are absent later in the liquid that fills the spaces. This liquid is considered to be mucilage, but it has a different substructure from that of pectin (Lofgren et al., 2002).

In mosses, the calyptra is necessary for proper stomatal development and pore orientation, but its role is mechanical rather than chemical (French and Paolillo, 1975a, b). The calyptra is of gametophytic origin and remains alive after detachment from the rest of the plant, as is evident by the intact cellular content described here. The calyptra provides the main protection for the developing sporophyte against desiccation (Budke et al., 2011, 2012). Although stomata develop in the apophysis while it is covered by the calyptra, capsule expansion begins with displacement of the calyptra. Opening of the pore coincides with displacement and splitting of the calyptra, which no longer adheres to the sporophyte as it did during the spear stage (Garner and Paolillo, 1973b).

The origin of moss stomata in files is reminiscent of that in Selaginella (Brown and Lemmon, 1985), hornworts (Pressel et al., 2014), corn and rice (Liu et al., 2009; Luo et al., 2012), which suggests that the developmental pattern of stomatal distribution is related to the elongated organ shape (Croxdale, 2000; Rudall et al., 2013; Villarreal and Renzaglia, 2015). In the first divergent angiosperms (ANITA grade), the development and distribution of stomata is more heterogeneous and protodermal cells divide in a squared pattern (Rudall and Knowles, 2013). In Amborellales and Austrobaileyales, protodermal cells can directly become GMCs or divide asymmetrically to produce a GMC and stomatal lineage ground cells, while in Nymphaeales GCMs develop solely from protodermal cells without a preceding asymmetrical division (Rudall and Knowles, 2013). In the fern Asplenium, GMCs develop from a polarized protodermal cell after one or two asymmetrical divisions (Apostolakos et al., 1997). Radial arrangement of epidermal cells surrounding guard cells occurs in other mosses, but moss stomata are not always regularly spaced and there is considerable variation in the number and distribution of stomata in the capsule (Paton and Pearce, 1957). Differentiation of GMCs in hornworts occurs in the youngest section of the sporophyte and includes changes in chloroplasts (Pressel et al., 2014). Asymmetrical divisions to produce adjacent guard cells are associated with the sporophyte suture and dehiscence, and not with GMC development (Villarreal and Renzaglia, 2015). These studies reveal the diversity in stomatal development and the importance of understanding stomatal patterning in an evolutionary context.

Three closely related transcription factors of the basic helix–loop helix (bHLH) family act sequentially to regulate stomatal development in Arabidopsis; the transition from protodermal cell to meristemoid is regulated by SPEECHLESS (SPCH), MUTE controls the transition from meristemoid to GMC, and finally FAMA is responsible for guard cell differentiation from GMCs (Pilliteri and Dong, 2013). In grasses, stomata are arranged in files, and although spacing and developmental patterning are modified, the function of these key regulatory elements, i.e. SPCH, MUTE and FAMA, is highly conserved (Liu et al., 2009; Luo et al., 2012). An evolutionary study of the SMF (SPCH, MUTE, FAMA) domain found orthologues of MUTE and FAMA in Selaginella and FAMA in Physcomitrella (MacAlister and Bergmann, 2011). Considering the developmental pattern described here for Funaria stomata, it is not surprising that SPCH and MUTE do not exist in the moss Physcomitrella, a close relative of Funaria. Since stomata in mosses do not develop from a self-generating meristemoid as in Arabidopsis, but instead from protodermal cells that differentiate directly into GMCs, it follows that SPCH would not be present because there are no amplifying divisions in protodermal cells. Regulation of stomatal development in mosses requires only differentiation of a protodermal cell into a GMC and the symmetrical partial division that generates the two connected guard cells. According to a cross-complementation assay that partially recovers function in Arabidopsis mutants, the functions attributed to MUTE and FAMA can be executed by the moss SMF1 (MacAlister and Bergmann, 2011). Consistent with this finding is that the SMF1 transcription factor is significantly upregulated in early sporophytes of Physcomitrella compared with mature sporophytes (O’Donoghue et al., 2013).

It is plausible that, through gene duplications, an ancestral multifunctional SMF that regulated the transition from protodermal cell to GMC and guard cell gave rise to functional specialization in newly evolved genes, leading to the to diversification of stomatal patterns in tracheophytes (MacAlister and Bergmann, 2011; Ran et al., 2013). More studies are necessary to assess whether the genes involved in stomatal development in angiosperms have the same role in mosses and other seedless plants. Although a complementary approach using genetic markers would be optimal to confirm the cellular identity of GMCs and other stomatal lineage cells, this study shows that the developmental pattern found in mosses that distributes stomata away from each other is quite simple. This foundation is necessary to understand and pursue the common molecular underpinnings with stomata of hornworts and the more derived tracheophytes.

ACKNOWLEDGEMENTS

We are very grateful to Handling Editor S. Pressel and two anonymous reviewers for comments and suggestions about an earlier version of the manuscript. This work was supported by the National Science Foundation (DEB 0638722 to K.R.).

LITERATURE CITED

  1. Apostolakos P, Panteris E, Galatis B. 1997. Microtubule and actin filament organization during stomatal morphogenesis in the fern Asplenium nidus. Protoplasma 198: 93–106. [DOI] [PubMed] [Google Scholar]
  2. Brown RC, Lemmon BE. 1985. Development of stomata in Selaginella: division polarity and plastid movements. American Journal of Botany 72: 1914–1925. [Google Scholar]
  3. Budke JM, Goffinet B, Jones CS. 2011. A hundred-year-old question: is the moss calyptra covered by a cuticle? A case study of Funaria hygrometrica. Annals of Botany 107: 1279–1286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Budke JM, Goffinet B, Jones CS. 2012. The cuticle on the gametophyte calyptra matures before the sporophyte cuticle in the moss Funaria hygrometrica (Funariaceae). American Journal of Botany 99: 14–22. [DOI] [PubMed] [Google Scholar]
  5. Campbell DH. 1895. The structure and development of the mosses and ferns (Archegoniatae). London: MacMillan. [Google Scholar]
  6. Casson S, Gray JE. 2008. Influence of environmental factors on stomatal development. New Phytologist 178: 9–23. [DOI] [PubMed] [Google Scholar]
  7. Croxdale JL. 2000. Stomatal patterning in angiosperms. American Journal of Botany 87: 1069–1080. [PubMed] [Google Scholar]
  8. Field KJ, Duckett JG, Cameron DD, Pressel S. 2015. Stomatal density and aperture in non-vascular land plants are non-responsive to atmospheric CO2 concentrations. Annals of Botany 115: 915–922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. French J, Paolillo D. 1975a. The effect of the calyptra on the plane of guard cell mother cell division in Funaria and Physcomitrium capsules. Annals of Botany 39: 233–236. [Google Scholar]
  10. French J, Paolillo D. 1975b. On the role of the calyptra in permitting expansion of capsules in the moss Funaria. Bryologist 78: 438–446. [Google Scholar]
  11. French JC, Paolillo DJ. 1975c. Intercalary meristematic activity in the sporophyte of Funaria (Musci). American Journal of Botany 62: 86–96. [DOI] [PubMed] [Google Scholar]
  12. Garner DLB, Paolillo DJ. 1973a. On the functioning of stomates in Funaria. Bryologist 76: 423–427. [Google Scholar]
  13. Garner DLB, Paolillo DJ. 1973b. A time-course of sporophyte development in Funaria hygrometrica. Bryologist 76: 356–360. [Google Scholar]
  14. Geisler M, Nadeau J, Sack FD. 2000. Oriented asymmetric divisions that generate the stomatal spacing pattern in Arabidopsis are disrupted by the too many mouths mutation. The Plant Cell 12: 2075–2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ivakov A, Persson S. 2012. Plant cell walls. eLS. doi:10.1002/9780470015902.a0001682.pub2. [Google Scholar]
  16. Liu T, Ohashi-Ito K, Bergmann DC. 2009. Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses. Development 136: 2265–2276. [DOI] [PubMed] [Google Scholar]
  17. Lofgren C, Walkenstrom P, Hermansson AM. 2002. Microstructure and rheological behavior of pure and mixed pectin gels. Biomacromolecules 3: 1144–1153. [DOI] [PubMed] [Google Scholar]
  18. Luo L, Zhou W-Q, Liu P, Li C-X, Hou S-W. 2012. The development of stomata and other epidermal cells on the rice leaves. Biologia Plantarum 56: 521–527. [Google Scholar]
  19. MacAlister CA, Bergmann DC. 2011. Sequence and function of basic helix–loop–helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants. Evolution and Development 13: 182–192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Merced A, Renzaglia KS. 2013. Moss stomata in highly elaborated Oedipodium (Oedipodiaceae) and highly reduced Ephemerum (Pottiaceae) sporophytes are remarkably similar. American Journal of Botany 100: 2318–2327. [DOI] [PubMed] [Google Scholar]
  21. Merced A, Renzaglia K. 2014. Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata. Annals of Botany 114: 1001–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O’Donoghue MT, Chater C, Wallace S, Gray JE, Beerling DJ, Fleming AJ. 2013. Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens. Journal of Experimental Botany 64: 3567–3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paton JA, Pearce JV. 1957. The occurrence, structure and functions of the stomata in British bryophytes. Transactions of the British Bryological Society 3: 228–259. [Google Scholar]
  24. Peterson KM, Rychel AL, Torii KU. 2010. Out of the mouths of plants: the molecular basis of the evolution and diversity of stomatal development. The Plant Cell 22: 296–306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pillitteri LJ, Dong J. 2013. Stomatal development in Arabidopsis. Arabidopsis Book. doi:10.1199/tab.0162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pressel S, Goral T, Duckett JG. 2014. Stomatal differentiation and abnormal stomata in hornworts. Journal of Bryology 36: 87–103. [Google Scholar]
  27. Ran JH, Shen TT, Liu WJ, Wang XQ. 2013. Evolution of the bHLH genes involved in stomatal development: implications for the expansion of developmental complexity of stomata in land plants. PloS One 8:e78997. doi: 10.1371/journal.pone.0078997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rudall PJ, Knowles EV. 2013. Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning. Annals of Botany 112: 1031–1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rudall PJ, Hilton J, Bateman RM. 2013. Several developmental and morphogenetic factors govern the evolution of stomatal patterning in land plants. New Phytologist 200: 598–614. [DOI] [PubMed] [Google Scholar]
  30. Sack FD. 1987. The development and structure of stomata. In: Zeiger E, Farquhar GD, Cowan IR, eds Stomatal function. Stanford: Stanford University Press, 59–89. [Google Scholar]
  31. Sack FD, Paolillo DJ. 1983a. Structure and development of walls in Funaria stomata. American Journal of Botany 70: 1019–1030. [Google Scholar]
  32. Sack FD, Paolillo DJ. 1983b. Stomatal pore and cuticle formation in Funaria. Protoplasma 116: 1–13. [Google Scholar]
  33. Sack FD, Paolillo DJ. 1985. Incomplete cytokinesis in Funaria stomata. American Journal of Botany 72: 1325–1333. [Google Scholar]
  34. Vaten A, Bergmann DC. 2012. Mechanisms of stomatal development: an evolutionary view. EvoDevo 3: 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Villarreal JC, Renzaglia KS. 2015. The hornworts: important advancements in early land plant evolution. Journal of Bryology 37: 157–170. [Google Scholar]

Articles from Annals of Botany are provided here courtesy of Oxford University Press

RESOURCES