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. 2013 Aug 21;112(6):1031–1043. doi: 10.1093/aob/mct169

Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning

Paula J Rudall 1,*, Emma V W Knowles 1
PMCID: PMC3783234  PMID: 23969762

Abstract

Background and Aims

Angiosperm stomata consistently possess a pair of guard cells, but differ between taxa in the patterning and developmental origin of neighbour cells. Developmental studies of phylogenetically pivotal taxa are essential as comparative yardsticks for understanding the evolution of stomatal development.

Methods

We present a novel ultrastructural study of developing stomata in leaves of Amborella (Amborellales), Nymphaea and Cabomba (Nymphaeales), and Austrobaileya and Schisandra (Austrobaileyales), representing the three earliest-divergent lineages of extant angiosperms (the ANITA-grade).

Key Results

Alternative developmental pathways occur in early-divergent angiosperms, resulting partly from differences in pre-patterning and partly from the presence or absence of highly polarized (asymmetric) mitoses in the stomatal cell lineage. Amplifying divisions are absent from ANITA-grade taxa, indicating that ostensible similarities with the stomatal patterning of Arabidopsis are superficial. In Amborella, ‘squared’ pre-patterning occurs in intercostal regions, with groups of four protodermal cells typically arranged in a rectangle; most guard-mother cells are formed by asymmetric division of a precursor cell (the mesoperigenous condition) and are typically triangular or trapezoidal. In contrast, water-lily stomata are always perigenous (lacking asymmetric divisions). Austrobaileya has occasional ‘giant’ stomata.

Conclusions

Similar mature stomatal phenotypes can result from contrasting morphogenetic factors, although the results suggest that paracytic stomata are invariably the product of at least one asymmetric division. Loss of asymmetric divisions in stomatal development could be a significant factor in land plant evolution, with implications for the diversity of key structural and physiological pathways.

Keywords: Epidermal pre-patterning, meristemoids, mesogenous stomata, perigenous stomata, stomatal development, ANITA, early-divergent angiosperms

INTRODUCTION

Stomatal structure is highly conserved across land plants, in which a symmetric pair of specialized guard cells delimits a central pore (Sack, 1987; Ziegler, 1987). However, when viewed from a developmental perspective, the patterning of the stomatal complex (i.e. the stoma and neighbouring cells) differs among taxa, depending on asymmetric divisions of one or more specialized precursor cells and lateral divisions of neighbouring cells. Most hypotheses of stomatal evolution in angiosperms are based on comparative studies of mature stomata of both extant and fossil taxa, with a primary focus on three widely recognized stomatal types – anomocytic, paracytic and stephanocytic – which differ in the patterning of their neighbour cells (e.g. Wilkinson, 1979; Doyle and Endress, 2000; Carpenter, 2005; Rudall et al., 2012; for definitions see Table 1). In anomocytic stomata, the neighbour cells (located immediately adjacent to the guard cells) resemble other pavement epidermal cells. Paracytic stomata possess one or more pairs of modified lateral neighbour cells (termed subsidiary cells), whereas stephanocytic stomata possess a rosette of several distinct subsidiary cells surrounding the guard cells. However, although such descriptive terms are useful for classification, they frequently combine non-homologous patterns that have been achieved via contrasting developmental pathways (e.g. Fryns-Claessens and Van Cotthem, 1973; Payne, 1979).

Table 1.

Glossary of stomatal terms (see also Bünning, 1952; Payne, 1979; Carpenter, 2005; Rudall et al., 2013)

Term Definition
 Guard cell One of a pair of specialized epidermal cells that together delimit the stomatal pore
 Guard-mother cell (GMC) Final stomatal precursor cell that divides symmetrically to form a pair of guard cells
 Meristemoid Specialized precursor cell
 Meristemoid-mother cell (MMC) Initial precursor cell that divides asymmetrically
 Stoma (plural stomata) Pair of guard cells plus the central pore that they delimit
 Stomatal complex Stoma plus all adjacent modified epidermal cells (subsidiary cells)
 Stomatal-lineage ground-cell (SLGC), also termed a mesogenous subsidiary cell Larger daughter cell resulting from an asymmetric division of a meristemoid; can differentiate into a pavement cell or can divide again asymmetrically
Mature stomata
 Anomocytic Lacking modified subsidiary cells (term applied to mature stomata)
 Paracytic Possessing one or more pairs of lateral subsidiary cells (applied to mature stomata; includes laterocytic types)
 Stephanocytic Possessing a distinct rosette of subsidiary cells (applied to mature stomata; includes actinocytic and tetracytic stomata)
Developing stomata
 Mesogenous Stomata with subsidiary cells formed from the same cell lineage as the guard cells, following one or more asymmetric divisions
 Mesoperigenous Stomata with a combination of both mesogenous and perigenous subsidiary cells
 Perigenous Stomata formed entirely by symmetric divisions; subsidiary cells not resulting from the same immediate cell lineage as the guard cells
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In all angiosperm species that have been studied in detail to date, stomatal development typically commences with the asymmetric division of a meristemoid mother cell (MMC) to form a smaller meristemoid and a larger stomatal-lineage ground cell (SLGC) (Payne, 1979; Nadeau and Sack, 2003; Robinson et al., 2011; Pillitteri and Torii, 2012). In many species, only one asymmetric division occurs, and the resulting meristemoid forms a guard-mother cell (GMC), which divides symmetrically to form a pair of guard cells (e.g. in many monocots, including rice and Tradescantia: Croxdale, 1998). This type of development, resulting in a single SLGC, is termed mesoperigenous (e.g. Payne, 1979). By contrast, in some eudicots (e.g. Arabidopsis), two or more successive asymmetric divisions (termed amplifying divisions) can occur in the same cell lineage. In Arabidopsis, a series of asymmetric amplifying divisions occurs, each producing an SLGC and a meristemoid (Zhao and Sack, 1999; Robinson et al., 2011; Pillitteri and Torii, 2012). After two or three amplifying divisions, the final meristemoid becomes a GMC and divides symmetrically to form a pair of guard cells. The asymmetric divisions are orientated away from each other, so that the SLGCs form a spiral surrounding the guard cells. This type of development, resulting in an encircling ring of SLGCs, is termed mesogenous (e.g. Payne, 1979). Molecular developmental studies, especially on Arabidopsis, have identified numerous genes that together contribute to the regulation of stomatal development (e.g. Peterson et al., 2010; Serna, 2011).

Understanding evolutionary pathways requires a more explicit phylogenetic context than over-simplistic comparisons between dicotyledons (a non-monophyletic group) and monocotyledons. Such comparisons are most commonly exemplified by the model organisms Arabidopsis and rice, respectively (e.g. Facette and Smith, 2012). Modern classifications of extant angiosperms, based primarily on molecular phylogenetic data (e.g. APG III, 2009), recognize two major species-rich clades – monocots and eudicots – plus about five relatively species-poor relictual lineages (Fig. 1). Three of these relictual lineages (Amborellales, Nymphaeales, Austrobaileyales) form a stepwise series of early-divergent angiosperms (sometimes termed the ANITA-grade or ANA-grade) that is placed immediately above the root node of the angiosperms in most analyses (e.g. Graham and Iles, 2009; summarized by Rudall et al., 2013).

Fig. 1.

Fig. 1.

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Relationships of angiosperms, based on the Angiosperm Phylogeny Group classification (APG III, 2009).

ANITA-grade angiosperms are disproportionately significant as potential yardsticks for morphological evolution in more derived angiosperms. Yet, although a few studies have examined mature stomatal patterns in these taxa, remarkably little is known about their development. Carpenter (2005) reported that Amborella, Austrobaileya and Schisandra possess both paracytic and stephanocytic stomata, but anomocytic stomata are rare. In contrast, Nymphaeales possess anomocytic and more-or-less stephanocytic stomata but entirely lack paracytic stomata. However, a comparative study of mature stomata cannot readily determine whether a neighbour cell is mesogenous or perigenous (Table 1).

Developmental studies of these phylogenetically pivotal taxa are essential to understand both the homologies of stomatal types and the evolution of stomatal development in angiosperms. Here, we present an ultrastructural study of developing stomata in leaves of Amborella, Nymphaea, Cabomba, Austrobaileya and Schisandra, together representing the three ANITA-grade lineages (Fig. 1).

MATERIALS AND METHODS

Leaves of five species representative of early-divergent angiosperms were examined for this study, all sampled from specimens growing at RBG Kew (listed here as HK followed by the Kew accession number or sine numero). For each species, a selection of foliage leaves at different stages of development was removed from each plant. No cotyledons were examined in this study.

  1. Amborella trichopoda Baill., a shrub endemic to New Caledonia, is the sole extant species of the family Amborellaceae and order Amborellales; this species is usually placed molecularly as sister to all other extant angiosperms, or sometimes as sister to Nymphaeales. Leaves were collected from a specimen that was kindly supplied by the Bonn Botanic Garden.

  2. Austrobaileyales includes three families of woody plants: Austrobaileyaceae, Schisandraceae and Trimeniaceae, together encompassing approx. 70 species in five genera. Material examined consisted of specimens grown at RBG Kew (HK): Austrobaileya scandens C.T.White (HK 2012–64) and Schisandra rubriflora Rehder and E.H.Wilson (HK 1969–19803).

  3. The order Nymphaeales consists of approx. 90 aquatic or semi-aquatic species assigned to eight or nine genera. The order includes the water-lily families Nymphaeaceae and Cabombaceae. Two specimens grown at RBG Kew were examined: Nymphaea violacea Lehm. (HK 2008–566) and Cabomba aquatica Aubl. (HK s.n.). Leaves of N. violacea examined ranged from a full-sized leaf with mature stomata to submerged leaves at different sizes. C. aquatica has two leaf types: finely divided submerged leaves that lack stomata, and floating peltate leaves that have stomata on the upper surface only. A range of developmental stages of floating leaves were examined.

For light microscopy (LM) and transmission electron microscopy (TEM) of all species except C. aquatica, samples from the mid-regions of leaves were cut into small squares and fixed in 3 % phosphate-buffered glutaraldehyde followed by 1 % osmium tetroxide. Samples were taken through a graded ethanol and ethanol/LR-White resin series before embedding and sectioning. Ultrathin sections (50–100 nm thick) were collected on Formvar-coated copper slot grids and imaged in a Hitachi H-7650 TEM with integral AMT XR41 digital camera. Semithin sections (0·5 µm thick) were mounted onto microscope slides, stained with toluidine blue, mounted using DPX mountant (a mixture of distyrene, a plasticizer and xylene), and examined using a Leitz Diaplan photomicroscope. Composite images were merged using Adobe Photoshop. Measurements were taken from scanning electron microscopy (SEM) and LM images using the image processing program ImageJ 1·46r (http://imagej.nih.gov/ij/docs/guide).

For SEM, leaves were fixed in 70 % ethanol. Dissected leaves were critical-point dried using an Autosamdri 815B CPD, mounted onto SEM stubs, coated with platinum using an Emitech K-550 sputter coater, and examined at 2 kV using a Hitachi cold-field emission SEM S-4700.

In C. aquatica, leaves at a range of developmental stages were cleared using a modified version of Herr's clearing fluid (lactic acid/chloral hydrate/phenol/clove oil/Histoclear, in proportions 2 : 2 : 2 : 2 : 1, by weight) and examined using differential interference contrast optics on a Leitz Diaplan photomicroscope fitted with a Leica DC500 digital camera.

RESULTS

Stomatal distribution

In all four species examined, most stomata are placed at least one cell apart, a pattern that is often said to follow the one-cell-spacing rule (e.g. Hara et al., 2007). However, occasional immediately contiguous stomata are not uncommon in Amborella (Fig. 2I), Austrobaileya and Schisandra, although they were not observed here in Nymphaea.

Fig. 2.

Fig. 2.

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Amborella trichopoda, abaxial surface of mature leaf. (A) SEM showing stomatal distribution, primarily intercostal, but with some stomata present over veins. (B, C) Details of intercostal stomata (SEM). (D) Paradermal view of a single stoma (TEM). (E) Transverse section of a single stoma showing differential wall thickenings, prominent outer cuticular ridges and less conspicuous ridging inside stomatal chamber (TEM). (F) Transverse section (LM) of a single stoma showing differential wall thickenings, prominent outer cuticular ridges and inconspicuous ridging inside stomatal chamber. (G) Transverse section of leaf (LM). (H) Paradermal section of stoma (LM). Abbreviations: ir = inner ridging, n = nucleus, ocr = outer cuticular ridge, s = stoma, st = starch, vb = vascular bundle, wt = wall thickening. Scale bars: (A) = 1 mm, (B) = 100 µm, (C, G) = 50 µm, (D) = 2 µm, (E, F, H) = 10 µm.

Amborella trichopoda (Figs 2–5)

Fig. 3.

Fig. 3.

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Amborella trichopoda: light micrographs and diagrams illustrating patterns of cell divisions during early development of the abaxial leaf epidermis. (A) Young developing leaf, with red lines outlining the likely boundaries of the original longitudinal cell files that existed prior to squared divisions. (B) Region with squared groups of cells. (C, D) Two versions of the same micrograph, with a group of cells outlined in red in (D) to illustrate division pattern. More recent walls are drawn as not interrupting older ones, to indicate their sequence of formation. (E) Group of cells highlighted in dark green box in (C), showing the squared pattern of division (diagram, Fig. 7A), and illustrating how sister cells divide at different rates. Cell A was sister to cell B; they each divided into A1 and A2 and B1 and B2, respectively. B2 has already divided again, forming B2a and B2b; A2 is in the process of dividing. (F) Diagram showing the series of divisions highlighted in (D). The cells would initially have been smaller and more regularly shaped than they appear at this stage. (G, H) stomata formed directly by symmetric division of protodermal cells, without asymmetric division (diagram, Fig. 5B). (I) Stoma and meristemoid both formed by asymmetric divisions (diagram, Fig. 5C). (J) Meristemoid formed by asymmetric division. (L) Stoma formed by asymmetric division. (L) Older stoma; developmental origin not clear. Abbreviations: g = guard cell, gmc = guard mother cell (GMC), m = meristemoid, sd = symmetric division, slgc = stomatal lineage ground cell (SLGC). Scale bars: (A, B) = 20 µm; (C–E, G–K) = 10 µm.

Fig. 4.

Fig. 4.

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Amborella trichopoda: TEM micrographs illustrating patterns of cell divisions during stomatal development on the abaxial leaf epidermis. (A) Group of protodermal cells showing squared arrangement. (B, C) Meristemoids formed by asymmetric division (diagram, Fig. 5C). (D) Pair of guard cells formed by symmetric division of protodermal cells, without asymmetric division (diagram, Fig. 5B). (E) Pair of guard cells showing starch. (F) Pair of guard cells and SLGC. Abbreviations: dp = division plate, g = guard cell, m = meristemoid, sd = symmetric division, slgc = stomatal lineage ground cell (SLGC).

Fig. 5.

Fig. 5.

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Amborella trichopoda: diagrams to illustrate different orientations of cell division. (A) Protodermal ‘squared’ division. Each cell divides symmetrically across its narrowest width, so that each division is usually perpendicular to the previous one. (B, C) Two contrasting trajectories of stomatal formation from squared pattern: (B) perigenous stoma formed by symmetric division of protodermal cells; (C) mesoperigenous stoma formed by asymmetric division of protodermal cells to form a guard-mother cell (GMC: red) and a stomatal-lineage ground cell (SLGC: yellow). Stomata are coloured green, GMC red and SLGC yellow. Other cells are not coloured.

Mature stomata

The leaf is relatively thick (Fig. 2A) and bears stomata only on the abaxial surface. Mature stomata are fairly regular in both shape and size, ranging from approx. 30 µm long in intercostal areas to approx. 40–45 µm long in costal areas; they are orientated apparently randomly with respect to each other (Fig. 2B, C). The guard cells have dense, prominent thickenings of the inner and outer periclinal walls that extend across the top and bottom of the cell in transverse section (Fig. 2E, F) and across the cell in paradermal section (Fig. 2H–K). A thick cuticle is present, exhibiting prominent outer cuticular ridges around the pore opening (Fig. 2E, F). Inside the substomatal cavity, very small ridges are present on the neighbouring pavement cells that partially underlie the guard cells (Fig. 2E, F). Starch granules are present in the guard cells (Fig. 2D), but are relatively infrequent in neighbouring epidermal cells. Some stomata have an irregular pattern of neighbouring cells, but the majority have a narrow subsidiary cell (or sometimes two smaller subsidiary cells) on each side.

Leaf abaxial epidermal development

In very young leaves, the protodermal cells on the midrib and leaf margins are arranged in linear cell files, so that each cell division is parallel with the previous division (Fig. 3A). Stomata located on the midrib are initiated before the intercostal stomata. In intercostal regions, prior to GMC formation, protodermal cells show a ‘squared’ arrangement, consisting of groups of four cells roughly arranged in a rectangle or less often a T-shape (Fig. 3B). (A squared pattern is also present on the adaxial surface, where stomata are absent.) To form this squared pattern during early leaf development, each (approximately rectangular) epidermal cell divides symmetrically across its narrowest width, usually perpendicular to the previous division (see Fig. 5A). Occasional groups of only three cells resulted from failure of one of the cell divisions. The daughter cells then each iteratively expand and divide again symmetrically across their narrowest width, resulting in a squared pattern that is only slightly disordered by different rates of expansion and division. The pavement cells expand as the leaf matures.

We observed GMCs, GCs and meristemoids in the same small intercostal region, demonstrating that stomata develop sequentially. The stomata are all either perigenous or mesoperigenous; none is entirely mesogenous (Glossary: Table 1). The perigenous stomata develop by symmetric division of cells that have already divided symmetrically (Figs 3G, H, 4D and 5B); no asymmetric divisions have occurred in their developmental pathway. Perigenous stomata are apparently among the earliest to develop and are possibly relatively few in number, although more data are required to confirm this hypothesis. In mesoperigenous stomata, one of the protodermal cells divides asymmetrically to form a smaller GMC and a larger SLGC; subsequently, the GMC divides symmetrically to form a pair of guard cells (Figs 3I–K, 4B, C, F and 5C). GMCs are identifiable by their relatively small size, small or absent vacuole, and darker appearance, but especially by their angular shape, which is triangular or trapezoidal, the longest wall being adjacent to its sister cell (the SLGC). Intercostal GMCs appear randomly distributed; distances between them differ, at least partly due to the variable orientation of cell division. Asymmetric divisions of cells adjacent to stomata are uncommon, and are typically orientated with the smaller cell (the GMC) furthest from the older stoma, thus maintaining one-cell spacing (e.g. Fig. 3I).

Austrobaileya scandens (Figs 6 and 7)

Fig. 6.

Fig. 6.

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Austrobaileya scandens: SEM micrographs of abaxial leaf surfaces. (A–C) Mature leaves, showing giant stoma with radiating striations in (B) and broken stoma with encircling striations in (C). (D–G) Developing leaves. (D) Stoma with encircling striations starting to develop. (E, F) Young surfaces with pre-patterning still visible, linear patterning outlined in (E) and squared patterning outlined in (F). (G) Young surface with a single pair of guard cells. Scale bars: (A, B, E, F) = 100 µm, (C) = 10 µm, (D, G) = 50 µm.

Fig. 7.

Fig. 7.

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Austrobaileya scandens: abaxial leaf surfaces, all LM, except (C), TEM. (A) Mature stomata with wall thickenings. (B, C) Stomata before wall thickenings developed, with starch plastids. (D–I) Developing surfaces with a range of stomatal stages. Scale bars: (A–C, E–I) = 10 µm, (D) = 20 µm.

Mature stomata

Mature intercostal leaf stomata are approx. 45 µm long, with occasional ‘giant’ stomata up to 60 µm long (Fig. 6B). Each stoma is surrounded by a ring of 5–7 neighbour cells, partly resulting from lateral divisions of neighbour cells (Fig. 7A). Most stomata are bordered by a ring of concentric cuticular striations, although giant stomata reliably have radiating striations (Fig. 6A–D). The cuticular ridges that extend over the pore are not striated. The guard cells have thickened anticlinal walls opposite the pore, and relatively thin walls bordering the pore. Two prominent cuticular ridges (inner and outer) are present around the pore opening. Stomata in older leaves have prominent wall thickenings in the guard cells (Fig. 7A). In younger stomata, before the walls become thickened, numerous chloroplasts are present in the guard cells (Fig. 7B, C, E, F, I).

Leaf epidermal development

Formation of stomata starts earliest on the midrib, then begins over veins and near the midrib, and finally in intercostal regions. Protodermal cells occur in linear files over the midrib, the leaf margin and some veins (Figs 6E and 7D). Orientation of older stomata suggests that midrib GMCs are formed by a division parallel with the other divisions within the cell file. Developing stomata over veins are slightly longer than intercostal stomata on the same leaf. In intercostal regions, protodermal cell division follows the ‘squared’ pattern already documented in Amborella (Fig. 6F). As in Amborella, cellular arrangement also indicates an underlying intercostal pre-patterning that is linear (Fig. 6E). On both the midrib and the intercostal regions, GMCs, GCs and meristemoids are all present in the same small sample.

In intercostal regions, GMC orientation is often difficult to determine because of the circular cell shape (Fig. 7E, F). Intercostal GMCs and new guard cell pairs appear circular or pentagonal in paradermal section, probably depending on the angle and/or level of sectioning (Fig. 7E–I). Guard cells are initially thin walled and circular (Fig. 7B, C), but become more elongated and their walls thicken (Fig. 7A).

Schisandra rubriflora (Fig. 8)

Fig. 8.

Fig. 8.

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Schisandra rubriflora: abaxial leaf surfaces. (A, B) Developing leaves with patterning distrupted by the presence of large oil cells (LM). (D) Mature surface with stomata randomly orientated (SEM). Abbrevaitions: g = guard cell, oc = oil cell, wt = wall thickening of mature guard cell. Scale bars: (A, B, D) = 10 µm, (C) = 100 µm.

Mature stomata

As in Austrobaileya, the mature guard cells are very thick-walled, leaving relatively little space for cytoplasm (Fig. 8D). Two small cuticular ridges (inner and outer) are present around the pore opening. Mature intercostal leaf stomata are approx. 50 µm long, with occasional ‘giant’ stomata up to 75 µm long; they appear randomly orientated in intercostal regions (Fig. 8C).

Leaf epidermal development

In the developing leaf, cells divide in linear files along the midrib, but it was difficult to distinguish cell-division patterning in intercostal regions, as the presence of large oil cells disrupts the underlying pattern (Fig. 8A, B).

Nymphaea violacea (Figs 9 and 10)

Fig. 9.

Fig. 9.

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Nymphaea violacea. (A) Adaxial surface of floating leaf (SEM). (B) Transverse section of leaf. (C, D, F) Transverse sections of stomata. (E) Paradermal section of adaxial epidermis with mature stomata. Abbreviations: ch = cuticular chamber, g = guard cell, sc = sclereid, ss = substomatal chamber. Scale bars: (A, C, E, F) = 10 µm, (B) = 100 µm, (D) = 5 µm.

Fig. 10.

Fig. 10.

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Nymphaea violacea. Adaxial leaf surfaces: (A–E) LM, and (F–I) TEM. (A, B) Two versions of the same micrograph, with a group of cells outlined in (B) to illustrate squared patterning, prior to GMC fomation. (C) Stage with GMCs. (D) Stage with GMCs just divided. (E) Stage with maturing stomata. (F–I) Successive stages of developing stomata. Scale bars: (A) = 5 µm, (C–E) = 10 µm, (F–I) = 2 µm.

Mature stomata

Stomata are present on the adaxial (upper) surface of the floating leaf, which is very thick, including a palisade adaxial mesophyll and spongy abaxial mesophyll containing astrosclereids (Fig. 9B). Mature stomata are relatively small (approx. 20 µm long) and are regular in size, shape and orientation (Fig. 9A, E). Each stoma is surrounded by 4–8 neighbour cells, which have undulating anticlinal walls. Each neighbour cell is often shared by two stomata. The guard-cell walls are generally thin, but slightly thickened bordering the pore. A single small cuticular ridge surrounds the narrow pore. The pore opens into a substomatal cavity (Fig. 9C, D, F). Ziegler (1987) suggested that stomata in Nymphaea alba and Nuphar lutea are non-functional because they lack substomatal cavities, but our results clearly show substomatal cavities in the palisade mesophyll of Nymphaea violacea. In paradermal view, each guard cell contains a row of starch plastids along the outer anticlinal edge, and a large, elongated central nucleus (Fig. 10I).

The cuticle is apparently chambered and irregularly thickened (Fig. 9C, D, F). A cuticular thickening above the wall that separates the guard cells eventually splits when the guard cells pull apart to generate the stomatal pore, thus forming a cuticular ridge. Although a narrow pore forms between the guard cells when the leaf is still submerged, until it reaches the surface the cuticular ridges keep the pore sealed to prevent water from entering.

Leaf epidermal development

Prior to GMC formation, adaxial cell division follows a squared pattern (Fig. 10A, B), except over the main veins, where epidermal cells occur in approximately linear files. We found no evidence of asymmetric divisions during stomatal development in Nymphaea violacea, indicating that protodermal cells give rise directly to GMCs. GMCs are formed more-or-less simultaneously and are regularly spaced in a grid pattern (Fig. 10C). Younger GMCs are usually pentagonal or hexagonal, depending on the number of neighbour cells (typically 4–7). Their walls are straight or slightly convex in paradermal section. GMCs each have a large, round central nucleus; they lack the large vacuoles visible in developing pavement cells and appear darker than pavement cells.

With a few exceptions, most neighbouring GMC divisions are orientated in the same direction (Figs 9E and 10D, E). After guard-cell formation, both the guard cells and the pavement cells enlarge. The pavement cells probably do not divide further, as the spacing between stomata appears to be the same in developing and mature leaves. Their anticlinal walls become sinuous. The guard cells elongate and differentiate; starch granules form soon after guard-cell formation, and the vacuole enlarges as the leaf extends upwards towards the water surface.

Cabomba aquatica (Fig. 11)

Fig. 11.

Fig. 11.

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Cabomba aquatica: adaxial leaf surfaces (DIC). (A) Very young leaf (located above water surface, but with margins still tightly rolled) showing squared protodermal patterning prior to GMC differentiation. (B, C) Slightly older (still rolled) leaf in which some prodermal cells have directly differentiated into GMCs; some have already divided symmetrically to form a pair of GCs. (D–F) Older (unfurled, floating) leaf with stomata present, mostly orientated in the same direction, with a few exceptions. (G–I) Fully enlarged floating leaf with surface crystals. Images (H, I) show optical sections of a single stoma (H) and substomatal cavity (I). Scale bars are all 10 µm, except (D, G) = 20 µm.

Cabomba aquatica has two leaf types: finely divided submerged leaves that lack stomata, and floating peltate leaves that have stomata on the upper surface. A range of developmental stages of floating leaves were examined using LM of cleared leaves. Very young leaves (Fig. 11A) show the squared patterning that is also typical of N. violacea. At this stage, it is impossible to determine which cells will form stomata. In slightly older leaves (Fig. 11B, C), some of the protodermal cells have become rounded and slightly domed; some of these cells have already divided symmetrically to form a pair of guard cells. Subsequently, all the cells enlarge, although pavement cells enlarge more than the guard cells. Most stomata are orientated on the same direction (Fig. 11D, E), with the division plane parallel to the veins that radiate from the centre of the peltate leaf. However, occasional stomata are orientated differently, often at right angles to the other stomata (Fig. 11F). The mature surface is covered in tiny crystals (Fig. 11G, H). Each mature stoma has a substomatal cavity (Fig. 11H, I).

DISCUSSION

Mature stomata: wall thickenings and ‘giant’ stomata

In fully expanded leaves, the guard cells of Amborella, Austrobaileya and Schisandra possess similar strong thickenings of their inner and outer periclinal walls. In paradermal view, these characteristic wall thickenings traverse the cell entirely (Figs 2H–K, 7A and 8D). The cell lumen is deeper at the poles and hence almost dumbbell-shaped in profile, as Sack (1987) noted for some other angiosperms with reniform (kidney-shaped) guard cells such as Quercus ilex. In contrast, such pronounced differential wall thickenings are entirely lacking in Nymphaea, in which the wall is only slightly thickened on the side bordering the pore (Fig. 9E). It is tempting to speculate that such contrasting wall thickenings reflect differences in stomatal mechanics in different taxa, although Sack (1987) was sceptical of their significance in this respect.

So-called ‘giant’ stomata are unusually large stomata that are interspersed among more normal-sized stomata on a leaf surface; recorded examples are present on leaves of the eudicot genera Mangifera and Limoma (Sitholey and Pandey, 1971). We observed giant stomata dispersed among smaller stomata across the leaf blade in Austrobaileya scandens (Fig. 6B); indeed, the giant stomata are distinguishable by their radiating cuticular striations (see also Wilkinson, 1979). The difference in cuticular patterning between giant and regular stomata in this species indicates a difference in developmental timing, as they are initiated at different times as the leaf expands. We hypothesize that the giant stomata are formed before smaller stomata. In Austrobaileya the midrib stomata are generally larger than those on the lamina, and are probably formed earlier. In some other angiosperms, specialized enlarged stomata occur over hydathodes on marginal leaf teeth. Such specialized stomata usually develop relatively early (e.g. Payne, 1979) and represent water pores with a different function to typical photosynthetic stomata, although studies of Arabidopsis have shown that the same genes control their early development (Pillitteri et al., 2008). Thus, it seems likely that differences in stomatal size across a single leaf are influenced by differences in developmental timing rather than by contrasting spatial constraints.

Stomatal development and epidermal pre-patterning

Stomatal diversity is governed by several primary morphogenetic factors, which are regulated by a complex signalling cascade of genes from several families (e.g. Peterson et al., 2010; Rychel et al., 2010; Serna, 2011; Facette and Smith, 2012). These morphogenetic factors include: (1) the presence of an asymmetric division in the stomatal cell lineage preceding GMC formation; (2) a subsequent series of asymmetric cell divisions (termed amplifying divisions: Nadeau, 2009) in the GMC lineage; and (3) the developmental origin of neighbouring cells by lateral divisions in adjacent cell lineages. Stomata of Arabidopsis are characterized by both asymmetric and amplifying divisions, whereas asymmetric and lateral divisions characterize many monocots, most notably maize.

Our investigation necessarily incorporates epidermal pre-patterning, which occurs prior to GMC initiation and has rarely been described in detail in studies of stomatal development. In Amborella, abaxial epidermal cells divide in linear cell files during the initial elongation phase of the leaf, prior to leaf expansion (Fig. 5A). This linear pattern resembles the linear cell files found in most monocot leaves, which are typically relatively narrow. Thus, we speculate that the persistence of linear cell files in monocots represents a neotenous condition, resembling early development of laminar leaves. This hypothesis will be tested in a future investigation. Marx and Sachs (1977) also noted that in the eudicot Anagallis arvensis, epidermal cells are arranged in files prior to stomatal formation. We also observed linear pre-patterning in Austrobaileya, although the early initiation of numerous large oil cells distorts protodermal patterning in Schisandra.

In Amborella, following the onset of lateral expansion of the leaf blade, cell divisions remain in linear files on the midrib and margins but adopt a squared (or sometimes irregular) pattern in intercostal regions. The squared groups of cells are formed by perpendicular (rather than parallel) protodermal divisions, illustrated diagrammatically in Fig. 5A. Each cell divides symmetrically across its narrowest width, so that each division usually occurs perpendicular to the previous division. Sometimes a daughter cell is square or unusually wide or short, so that its division is approximately parallel to the previous one. A similar squared pre-patterning is also evident in Austrobaileya (Fig. 6F), Nymphaea (Fig. 10A, B) and Cabomba (Fig. 11A), thus characterizing all three ANITA-grade lineages and indicating that the squared condition could be ancestral (plesiomorphic) in angiosperm leaves.

No obvious pre-patterning has been reported in Arabidopsis, except in seedling hypocotyls, where stomata are formed in files of cells overlying the junctions between the mesophyll cells (Berger et al., 1998). The apparent lack of an obvious squared pattern in Arabidopsis could be related to the presence of amplifying divisions during stomatal development (see below), perhaps due to differences in timing of leaf expansion. Studies of pre-patterning in a taxonomically broad range of angiosperms will help to determine the evolutionary significance of this feature, which is clearly related to broader – and inevitably complex – issues of leaf development.

Lateral divisions in neighbouring cells

The presence of lateral divisions in the cells neighbouring the stomata can increase the complexity of the epidermis and often makes interpretation of stomatal patterns difficult. For example, lateral divisions of neighbour cells are frequent, although not ubiquitous, during stomatal development in leaves of the ‘living fossil’ gymnosperm species Ginkgo biloba (Rudall et al., 2012). Highly consistent lateral divisions of neighbour cells occur in commelinid monocots such as maize and Tradescantia (e.g. Tomlinson, 1974; Croxdale, 1998; Cartwright et al., 2009). At least in maize, asymmetric division of neighbouring cells is promoted by the PANGLOSS1 (PAN1) gene (e.g. Facette and Smith, 2012). The function of narrow lateral subsidiary cells is not clear; they could have a physiological role, or (perhaps more likely) they could help to compensate for the contrasts in growth rate between stomata and their neighbours (see also Payne, 1979).

In the present study, Amborella proved an unexpectedly useful subject for studying stomatal development because it apparently lacks lateral divisions of neighbour cells. We also found no evidence for lateral divisions of neighbour cells during stomatal development in Nymphaea violacea. In contrast, lateral divisions are common in Austrobaileya and Schisandra, in which the lateral neighbour cells divide parallel to the guard cells and often asymmetrically, thereby generating narrow neighbour cells. This finding supports Carpenter's (2005) observation that stomata encircled by two concentric rings of neighbours are common in Austrobaileyales but not in Amborella or water-lilies. Studies of Austrobaileya and Schisandra are also complicated by the presence of oil cells in the epidermis that are relatively large at early developmental stages, although less obvious in the mature leaf.

Presence of an asymmetric division preceding GMC formation

Our study shows two clear pathways to stomatal formation in Amborella. Either a protodermal cell directly forms a GMC and divides symmetrically (Fig. 5B), or a protodermal cell divides asymmetrically to give rise to a GMC and an SLGC (Fig. 5C). Similar pathways probably exist in both Austrobaileya and Schisandra, although the presence of lateral divisions and oil cells both obscure the fundamental stomatal patterning in these taxa. Carpenter (2005) also noted some evidence for asymmetric divisions in Illicium, another genus of the order Austrobaileyales.

In contrast, we found no evidence of asymmetric divisions during stomatal development in either species of Nymphaeales examined (Cabomba aquatica and Nymphaea violacea), indicating that protodermal cells always give rise directly to GMCs in this species. In these water-lily species, following squared pre-patterning of protodermal cells, there emerges a highly regular arrangement of pavement cells in mature leaves, with stomata of a relatively consistent size, orientation and spacing. The relatively regular spacing of stomata could be due to lateral inhibition of meristemoids or GMCs, and is a common feature of angiosperm leaves. However, consistent alignment of stomata is highly unusual in a species with non-linear leaves. In species with protodermal cells in linear files (such as monocots), stomata are regularly aligned simply by orientating the division of the GMC perpendicular to previous divisions. Similarly, in Amborella, persistent linear patterning over the midrib results in regular stomatal orientation in this region, as GMC divisions are orientated perpendicular to the GMC-forming division.

However, this linear patterning does not explain stomatal alignment in Nymphaea and Cabomba, in which protodermal pattering is squared rather than linear. In Amborella, asymmetric divisions that follow squared pre-patterning result in apparently random orientation of stomata in intercostal regions. The reason that stomatal orientation is so regular in water-lilies is that they apparently lack asymmetric divisions in the stomatal cell lineage, so divisions are always aligned with other cells. Water-lilies are also apparently unusual in that the stomata are initiated almost simultaneously and mature synchronously. Epidermal cell division ceases soon after stomatal formation; thereafter, the leaf enlarges primarily by cell expansion.

Conclusions: amplifying divisions and asymmetric divisions

With respect to mature stomatal types, Carpenter (2005) reported that Amborella, Austrobaileya and Schisandra produce mostly paracytic and stephanocytic and rarely anomocytic stomata, whereas Nymphaeales have anomocytic and stephanocytic stomata but lack paracytic stomata entirely. We found only anomocytic stomata in mature leaves of Nymphaeales, and it appears that paracytic stomata are absent from water-lilies. Is there a developmental basis for this difference? Our developmental study shows that stomata of water-lilies (at least in Nymphaea violacea and Cabomba aquatica) entirely lack asymmetric divisions in their developmental pathway, and are therefore always perigenous. In contrast, stomatal development in Amborella and Austrobaileya (and probably Schisandra) is often mesoperigenous (i.e. with a single asymmetric division in the stomatal cell lineage: Table 1). Thus, in water-lilies there is a clear correlation between anomocytic stomata and perigenous development. However, this correlation fails for most other taxa. In linear-leaved species, such as many monocots and conifers, anomocytic stomata invariably result from asymmetric divisions (e.g. Tomlinson, 1974; Johnson and Riding, 1981). These results demonstrate that anomocytic and stephanocytic stomata can result from contrasting morphogenetic factors, thus confirming earlier assumptions that mature stomatal types rarely reflect developmental patterns (e.g. Payne, 1979).

On the other hand, our results suggest that paracytic stomata are invariably the product of at least one asymmetric division, at least in early-divergent angiosperms. This finding is potentially significant in assessing stomatal evolution in taxa known only from fossils (Rudall et al., 2013). Paracytic stomata are highly characteristic of angiosperms (e.g. Doyle and Endress, 2000; Carpenter, 2005). If the topology of relationships of extant angiosperms shown in Fig. 1 is correct, then our observations suggest that asymmetric divisions were lost in Nymphaeales, in which the aquatic environment could promote a neotenous habit. This hypothesis contrasts with Carpenter's (2005) suggestion of recurrent formation of paracytic types in early angiosperms, but these alternative hypotheses both appear plausible, and more data are needed to test them. Evolutionary loss of highly polarized asymmetric divisions that form meristemoids has also been reported in other angiosperms, albeit in non-stomatal cell lineages; this phenomenon is most obvious when it results in a loss of epidermal long–short cell alternation, as in the silica cells and root epidermis of rice and its close allies (Kim and Dolan, 2011; Rudall et al., in press). It remains unclear whether this evolutionary switch has adaptive significance, although clearly the diverse range of stomatal patterning could imply functional and mechanical diversity (Franks and Farquhar, 2007).

We found no evidence for amplifying divisions in either Amborella or any of the other ANITA-grade angiosperms examined here, indicating that ostensible similarities with the stomatal patterning of Arabidopsis are superficial. Among other angiosperms that are putatively early-divergent, Peterson et al. (2010) hypothesized that the extended spiral of cells around each stoma in Houttuynia, a magnoliid, develops by amplifying divisions, as in Arabidopsis. To test more broadly the evolutionary origin and phylogenetic distribution of both asymmetric and amplifying divisions in angiosperms, our future studies will examine stomatal development in magnoliids and net-veined monocots, as well as in well-preserved fossils of putative angiosperm relatives.

ACKNOWLEDGEMENTS

We thank Carlos Magdalena and Sara Redstone for growing the plants examined, and Richard Bateman for critically reading the manuscript.

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