Bars = 5 μm except for F, where bar = 2 μm. The general structure and development of an idealized hornwort sporophyte is presented in Figure 1. Grids were observed unstained with a Hitachi H7650 transmission electron microscope at 60 kV. The stomatal aperture is lined by adjacent ventral guard cell walls composed of radial fibrils that often buckle along their length (Figs. (2015) that CO2 levels are inconsequential to guard cell development. Sporogenous tissue in the spore sac is surrounded by mucilage that dries progressively as spores differentiate (Fig. Guard cells collapse inwardly, increase in surface area, and remain perched over a substomatal cavity and network of intercellular spaces that is initially fluid filled. Following stomatal collapse, continued drying of the sporophyte results in the death of epidermal and assimilative cells and browning of the sporophyte (Fig. Without a rapid osmotic control of pore opening and closing, the constraints of guard cell size that suggest that small is faster do not exist (Raven, 2014). It is possible that the well-developed chloroplast in guard cells may play a role in the perception of environmental cues and perhaps signals the onset of senescence. These are released from the capsule when it splits lengthwise from the tip. The largest guard cells among the 16 hornwort species are those in Phymatoceros bulbiculosus (Brot.) The common name refers to the elongated horn-like structure, which is the sporophyte. A, Outer ledge with thickened cuticle (arrow). Examination of more early fossil stomata on or near sporangia is necessary to test these inferences. 4, B and E). 1B and 2D). All three taxa are tropical and produce highly elongated involucres and spiraled pseudoelaters. Adjacent epidermal cells have thickened radial walls and are beginning to collapse in the opposite direction from the guard cells. It also shows that the three groups of bryophytes share a common ancestor that branched off from the other landplants early in evolution, and that liverworts and mosses are more closely related to each other than with hornworts. Meristem cells at the base of the plant keep dividing and adding to its height. Stomata on the earliest fossil land plants share remarkable similarities with hornwort stomata and sporophyte surfaces (Fig. Directly above the involucre where stomata collapse, young spores in tetrads develop spore walls but remain surrounded by the spore mother cell wall along most of the length of the sporophyte (Fig. A, P. carolinianus. A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species, Cell wall arabinan is essential for guard cell function, ABA signal transduction at the crossroad of biotic and abiotic stress responses, Major transitions in the evolution of early land plants: a bryological perspective, The origin of the sporophyte shoot in land plants: a bryological perspective, Reconstructing relative genome size of vascular plants through geological time, An experimental evaluation of the use of C3 δ13C plant tissue as a proxy for the paleoatmospheric δ13CO2 signature of air, Genome size as a predictor of guard cell length in, Structure and function of hornwort stomata, Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants, In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage, Ancestral stomatal control results in a canalization of fern and lycophyte adaptation to drought, Abscisic acid controlled sex before transpiration in vascular plants, Novel insights on the structure and composition of pseudostomata of, Developmental changes in guard cell wall structure and pectin composition in the moss, Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation, Nuclear DNA C-values in 30 species double the familial representation in pteridophytes, The occurrence, structure and functions of the stomata in British bryophytes, Stomatal differentiation and abnormal stomata in hornworts, A comparative morphology and developmental anatomy of the Anthocerotophyta, Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny, Estimates of nuclear DNA content in bryophyte sperm cells: phylogenetic considerations, Bryophyte phylogeny: advancing the molecular and morphological frontiers, Land plants acquired active stomatal control early in their evolutionary history, Development of multicellular spores in the hornwort genus, Changing the light environment: chloroplast signaling and response mechanisms. Chloroplasts are large and prominent in assimilative cells throughout sporophyte maturation and until cells dry and die. Guard cell walls, especially outer walls, in tracheophytes and true mosses are dense, thickened, and do not normally collapse (Sack, 1987; Ziegler, 1987; Everet, 2006; Merced and Renzaglia, 2013). Each guard cell contains a thin outer wall (ow), an outer ledge (ol), dorsal (dw) and ventral (vw) walls, and a thickened inner wall (iw). The pyrenoid is comprised predominantly of RuBisCO, the key enzyme in carbon fixation. The gametophyte stage is the dominant stage in both liverworts and hornworts; however, liverwort sporophytes do not contain stomata, while hornwort sporophytes do. It allows them to concentrate carbon dioxide inside their chloroplasts, making the production of sugar more efficient.. from Puerto Rico and Makanda, Illinois, and Anthoceros agrestis Paton from Makanda, Illinois. This gradualistic model proposes that stomata evolved the ability to close to ABA in seed plants by coopting the ABA mechanism that was already in place in early land plants (Sussmilch et al., 2017). In contrast to bryophyte stomata, vascular plant stomata are essential … 3I) and LM13 (Fig. 6, D and E). Following maturation, stomata die and collapse, while the surrounding cells remain alive. Phaeoceros carolinianus. Stomata appear in the hornworts and are abundant on the sporophyte. Out of the 24 hornwort species with available genome sizes (Bainard and Villarreal, 2013), eight lack stomata; thus, only 16 taxa were used in our correlation. Finally, through measurements of 16 hornwort species from all seven genera with stomata, we assessed the existence of an evolutionary correlation between guard cell size and genome size in hornworts as occurs in angiosperms (Beaulieu et al., 2008; Hodgson et al., 2010). In fully developed stomata, the guard cells are turgid with large vacuoles, and an open aperture connects the outside environment to the schizogenous substomatal cavity (Figs. Meristem cells at the base of the plant keep dividing and adding to the height of the sporophyte. DUE 1136414) and National Institutes of Health (grant no. S2). Epidermal cells are identical to dried hornwort epidermal cells. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. The gametophyte stage is the dominant stage in both liverworts and hornworts; however, liverwort sporophytes do not contain stomata, while hornwort sporophytes do. 4, F–I). (Fig. D, A. adscendens. As in Physcomitrella and the hornworts, the pseudostomata of Sphagnum also are implicated in sporangial drying (Duckett et al., 2009). Stotler et al. Grids were placed in 2% BSA in 0.02 mol L−1 PBS solution, pH 7.2 (PBS), overnight at 4°C in a humid chamber. , Recent studies of molecular, ultrastructural, and morphological data have yielded a new classification of hornworts. The outer aperture remains open and increases in diameter during the drying process. To our knowledge, there are no other stomata in extant plants that have the structure and developmental fate of those in hornworts. The thin ventral walls of guard cells form a folded convoluted inner pore that reduces the passage way for pathogens to enter (Lee and Luan, 2012) as the sporophyte differentiates. In half of the roughly 200 hornwort species, this chloroplast is fused with other organelles to form a large pyrenoid that both enables more efficient photosynthesis and stores food. In the middle of the sporophyte (just above the foot), is a meristem that will continue to divide and produce new cells for the third region. C, Thin fibrillar ventral wall with scattered cuticle/waxes (arrow). Copyright © 2021 by The American Society of Plant Biologists. Guard cells of hornworts are similar in length to those of Psilotum (72.7 μm) and Ophioglossum (65.6 μm; Obermayer et al., 2002), both of which have genome sizes 300 times that of the largest hornwort genome. Guard cells and epidermal cells of hornworts show striking similarities with the earliest plant fossils. SEM shows the epidermis in desiccated and dehisced sporophyte with ridges of collapsed epidermal cell surrounding an enlarged stoma that has a broadened outer aperture. 1.  Such colonies of bacteria growing inside the thallus give the hornwort a distinctive blue-green color. One control that excluded incubation of the primary antibody and two treatments were made for each antibody on three to five individual stomata. Dehiscence in the epiphytic Dendroceros is irregular and appears to be influenced by the continued growth and expansion of the precocious, multicellular spores (Renzaglia, 1978; Schuette and Renzaglia, 2010). A thin cuticle and cuticular region overlie the outer and ventral guard cell walls (Fig. Stomatal guard cell length from selected early Devonian fossils of rhyniophytes, zosterophllyloids, aglaophytes, and lycophytes taken from Lomax et al. It is difficult to test the impact of character loss on organisms, but hornworts do present a clear case of the loss of stomata in well-defined genera with specific life history strategies. Among land plants, hornworts are one of the earliest-diverging lineages of the early land plant ancestors; cladistic analysis implies that the group originated prior to the Devonian, around the same time as the mosses and liverworts. Outer walls on adjacent epidermal cells are thick, lack radial fibrils, and are covered by a jagged cuticular layer of alternating cutin and branched fibrils (Fig. The total number of species is still uncertain. Sporophytes of L. dussii were examined using immunogold labeling to identify pectin epitopes in guard cell walls and intercellular spaces. Stomata (air pores that can be opened and closed) appear in the hornworts and are abundant on the sporophyte. C, Differential interference contrast image of older stoma. Cross section light micrograph shows dead guard cells with degenerated protoplasm at the onset of collapse of the outer cell wall and while fluid is still within the substomatal cavity (asterisk) and intercellular spaces (double asterisks). I, P. carolinianus. The following supplemental materials are available. SEM images of hornwort stomata compared with fossil stomata. SEM images of hornwort sporophytes. Black dots in images are secondary gold labels attached to specific antibodies. Hornwort spores are relatively large for bryophytes, measuring between 30 and 80 µm in diameter or more. C, P. carolinianus. 1C and 4, C and D). In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. The stomata in Figure 6 from a Silurian fossil are similar in size to those of hornworts. 3G), and inner (Fig. collapsed guard cells and the scattered distribution along the sporangium) leave open the possibility of a common origin, as suggested by Merced (2015a). The loss of stomata in Notothylas, the sister taxon to Phaeoceros, can be explained by their highly reduced sporophytes that are often cleistocarpic and remain within the involucre throughout development (Renzaglia, 1978). Due to differential wall thickenings on epidermal and guard cell walls, guard cells remain perched in position over the substomatal cavity, expanding the surface area in contact with the environment, including the width of the outer aperture. , When the sporophyte is mature, it has a multicellular outer layer, a central rod-like columella running up the center, and a layer of tissue in between that produces spores and pseudo-elaters. Average guard cell lengths, measured in green stomata, across all seven hornwort genera with stomata range from 51 to 81 μm, and genome sizes of these same species vary from 159 to 269 Mb (Supplemental Table S1). 4). Liverworts are autotrophs that require light to make food by photosynthesis. It is estimated that there are about 9000 species of liverworts. Following senescence, guard cells collapse inwardly until the outer walls rest against the inner walls (Fig. 1D and 4, H and I). However, arabinan-rich pectins that are essential for the opening and closing of guard cells and the resilience of walls in tracheophytes (Jones et al., 2003, 2005; Moore et al., 2013) are not wall constituents of hornwort stomata. 4, D and G). We present data from development and immunocytochemistry that identify a role for hornwort stomata that is correlated with sporangial and spore maturation. In A. Jonathan Shaw & Bernard Goffinet (Eds. These earliest plants produced terminal sporangia and lacked leaves, as do hornworts, suggesting that the collapsed condition originated in the colonizing stages of plant evolution in the Upper Silurian and was conserved over hundreds of millions of years. E, L. dussii. S1; Macquet et al., 2007). Briefly, sporophytes were processed as for TEM up to 3× 100% ethanol. Cell walls are thicker than those in A, and epidermal cells contain large amyloplasts (black arrow). In most species, there is a single cell inside the spore, and a slender extension of this cell called the germ tube germinates from the proximal side of the spore. 5E). - Australian National Botanic Gardens, Hornwort pyrenoids, carbon-concentrating structures, evolved and were lost at least five times during the last 100 million years - PNAS, BTI researchers unlocking hornworts' secrets | EurekAlert! Mosses and hornworts are the earliest among extant land plants to have stomata, but unlike those in all other plants, bryophyte stomata are located exclusively on the sporangium of the sporophyte. At this time, the inner walls separate from cortical cells to form substomatal cavities (Fig. Stomata on hornworts are larger in width and depth than the surrounding epidermal cells, which is an unusual character in plants. Particularly notable are the guard cells of fossils from the Early Devonian, which are over 80 μm in length in Horneophyton lignieri and Aglaophyton major. Stomata appear in the hornworts and are abundant on the sporophyte. As in mosses and liverworts, the flattened, green plant body of a hornwort is the gametophyte plant. This stage usually grows as a thin rosette or ribbon-like thallus between one and five centimeters in diameter. We examined the composition of guard cell walls for the occurrence of arabinan-containing polysaccharides that allow for flexibility and resilience in actively moving stomata (Jones et al., 2003, 2005; Merced and Renzaglia, 2014). When developed, guard cell walls are rich in unesterified homogalacturonans similar to mosses (Merced and Renzaglia, 2013, 2014; Merced, 2015a) and Arabidopsis (Arabidopsis thaliana (L.) Heynh. A. agrestis and L. dussii have the smallest genome sizes among hornworts (0.085 and 0.16 pg, respectively [1 pg = 0.978 × 109 bp; Dolezel et al., 2003]), while the largest genome size is found in P. bulbiculosus (0.28 pg). Sporophyte structure in the neotropical hornwort, The hornworts: important advancements in early land plant evolution, RBR-Type E3 Ligases and the Ubiquitin-Conjugating Enzyme UBC26 Regulate Abscisic Acid Receptor Levels and Signaling, CLE40 Signaling Regulates Root Stem Cell Fate, Design Principle for Decoding Calcium Signals to Generate Specific Gene Expression Via Transcription, by The American Society of Plant Biologists. TEM images showing wall ultrastructure in guard cell walls of Leiosporoceros dussii. A large vacuole occupies most of the guard cells with nucleus (n) and plastids (p), with pyrenoids (arrow) toward the inside of the stoma. Assimilative cells begin to die around the substomatal cavity (asterisk) and intercellular space (double asterisks). When present, LM6 (Fig. These structures are morphologically similar to stomata in the rest of land plants, consisting of a pair of guard cells surrounding a pore. (2017) identified othologs of SPCH/MUTE/FAMA (SMF), ICE/SCREAM (SCRM), and EPIDERMAL PATTERNING FACTOR (EPF), genes required for stomatal development, in the draft genome of the hornwort Anthoceros punctatus. Do bryophytes have stomata? Indeed, the thin outer walls and collapse of ventral walls in hornwort guard cells are strikingly similar to those of Sphagnum pseudostomata (Merced, 2015a). Open pores of stomata provide a larger area for gas exchange and allow the assimilative tissue to be thicker, consequently increasing the self-sufficiency of sporophytes while developing spores. Liverworts have colonized every terrestrial habitat on Earth and diversified to more than 7000 existing species (Figure 1). Dehiscence occurs along two clearly defined sutures that separate the sporophyte into two valves and expose the dried spore mass. and A.M. wrote the article with contributions of all the authors; A.M. conducted the immunogold labeling experiments; K.S.R., A.M., B.T.P., and J.C.V. Large tropical and sub-tropical species of Dendroceros may be found growing on the bark of trees. SEM preparation followed that described by Merced and Renzaglia (2013). We thank Nicholas Flowers for technical assistance. 3F), outer (Fig. The same goes for hornworts, but not for liverworts, which completely lack stomata. As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Copyright © 2021 by the disappearance of liquid in the substomatal cavity asterisk... ) leads to internal air spaces are dry within the spore sac is surrounded by hydrated epidermal cells of show! Microscopic studies focused on four hornwort genera with stomata to assess developmental changes in size and to analyze correlation! Plant Biology, Southern Illinois University, Carbondale, Illinois, and open stoma pseudoperine ( Fig of. 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