All tracheophytes (blue tags) have green, living stomata. As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. 2D and 3, A–D). The following supplemental materials are available. SEM shows completely collapsed guard cells surrounded by hydrated epidermal cells. The pyrenoid is comprised predominantly of RuBisCO, the key enzyme in carbon fixation. conceived and designed the project; K.S.R. There is no correlation between the mean guard cell length and genome size in hornworts (multiple r2 = 0.03607, adjusted r2 = −0.02819; F statistic = 0.5613, P = 0.4653). Microscopic studies focused on four hornwort genera with stomata. For example, moss grows into a dense covering like a mat. D, Thin fibrillar outer wall with a thin layer of cuticle. A large substomatal cavity (asterisk) leads to internal air spaces. Stomata of bryophytes are only present in the sporophyte generation of mosses and hornworts; liverworts do not have stomata. 4, F and H). Mature stomata were examined using three primary monoclonal antibodies: LM19 (unesterified homogalacturonan), LM6 (arabinan rhamnogalacturonan I), and LM13 (linear arabinan rhamnogalacturonan I; Plant Probes, University of Leeds). The biflagellate sperm must swim from the antheridia, or else be splashed to the archegonia. Specimens were infiltrated in LR White resin (London Resin) by increasing the percentage of resin to ethanol over 4 d. After two changes in 100% resin, the material was place in molds with fresh resin and cured for 2 d at 65°C. C, Tetrad with spore mother cell wall drying down on the papillate distal wall ornamentation. (Fig. 6). The sporophyte of a hornwort is unusual in that it grows from a meristem near its base, instead of from its tip the way other plants do. Spores develop their thickened walls while still enclosed in the spore mother cell wall, remaining in tetrads until spores separate where they are dispersed at the sporophyte tip. D to F, Fossil stomata reproduced with permission from Edwards et al. 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. These include the sporangia of some hornworts and mosses, as well as in fossils of the earliest known vascular plants, such as Cooksonia and Zosterophyllum from around 400 Myr ago (Edwards, 1993). Openings that allow the movement of gases may be observed in liverworts. They argued that high levels of atmospheric CO2 as demonstrated in vitro for angiosperms (Edwards, 2003; Franks et al., 2012; Lomax et al., 2012) and paleopolyploidy may have resulted in exceptionally long guard cells. Unlike liverworts, most hornworts have true stomata on their sporophyte as mosses do . Stomata are expendable in hornworts, as they have been lost twice in derived taxa. Liverworts also have rhizoids (hair-like filaments) that function similarly to roots in that they hold the plant in place. Among bryophytes, only the sporophytes of mosses and hornworts have stomata, whereas stomata are missing from liverwort sporophytes. Hornworts, liverworts, and mosses are all examples of bryophytes. Stomata are expendable in hornworts, as they have been lost twice in derived taxa. Stomata (air pores that can be opened and closed) appear in the hornworts and are abundant on the sporophyte. At this time, the inner walls separate from cortical cells to form substomatal cavities (Fig. Our findings identify an architecture and fate of stomata in hornworts that is ancient and common to plants without sporophytic leaves. However, these are not sto… Oil bodies in their leaves*****, 3. By contrast, species of the family Dendrocerotaceae may begin dividing within the spore, becoming multicellular and even photosynthetic before the spore germinates. Based on the evidence presented here, we hypothesize that hornworts have retained ancestral features of stomata that occurred on axes with solitary terminal sporangia in the earliest land plants. 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.  Such colonies of bacteria growing inside the thallus give the hornwort a distinctive blue-green color. The female organs are known as archegonia (singular archegonium) and the male organs are known as antheridia (singular antheridium). However, recent studies contradict that hypothesis and show that stomatal responses to leaf water status are controlled passively in ferns and lycophytes, with ABA signaling associated with drought stress and sex determination, not stomatal closure (Brodribb and McAdam, 2011; McAdam and Brodribb, 2013; McAdam et al., 2016). Note the liquid-filled subtending intercellular spaces (*) in (E). Following senescence, guard cells collapse inwardly until the outer walls rest against the inner walls (Fig. Supplemental Table S2. 3, B and D). Newly opened stomata are raised slightly above the epidermal surface (Fig. Stomata ontogeny and very low densities, like those in Devonian fossils, suggest either ancient origins at a time when atmospheric carbon dioxide levels were much greater than today or a function other than gaseous exchange regulation. First, stomata on sporangia, as occur in hornworts and mosses, are expendable. Average genome sizes (Bainard and Villarreal, 2013) and stomatal guard cell length from mature guard cells in sixteen hornwort species. 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. Each guard cell contains two large amyloplasts, and the aperture (white arrow) is fully developed. 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). The fluid in the sporogenous region is presumed to be mucilage because it labels with pectin epitopes (Supplemental Fig. 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). The sporophyte is green in this region above the involucre due to chloroplasts in the assimilative region, and the intercellular spaces may have some fluid (Fig. 3I) and LM13 (Fig. More recently, a second class Leiosporocertotopsida has been segregated for the singularly unusual species Leiosporoceros dussii. 5; Supplemental Fig. Scarce labeling is shown for LM6 (I) and LM13 (J), both localized toward the inside of the wall at the plasmalemma. Prosk. The CO2 sensitivity of stomata evolved by the time modern tracheophytes radiated, as this physiological response is found in ferns (Franks and Britton-Harper, 2016). Sporogenous tissue is continually produced, meiosis is always occurring in a progressive and spatial (but not temporal) fashion, and all stages of spore differentiation are visible along the length of the sporophyte. The outer ledge (black arrow) and substomatal cavity (white arrow) are beginning to form. Both the large stomatal size and pectin composition are counterindicators of active opening and closing of hornwort stomata, even in green portions of the sporophyte. Hornwort genomes are among the smallest of all land plants (Renzaglia et al., 1995). The sporophyte of most hornworts are also photosynthetic, which is not the case with liverworts. A and B, L. dussii. Further divisions produce three basic regions of the sporophyte. However, it now appears that this former division is paraphyletic, so the hornworts are now given their own division, Anthocerotophyta (sometimes misspelled Anthocerophyta). Hornworts are considered to be ecological pioneers as they often grow in areas where there is little to no competition, such as on mineral soils. This third region is the capsule. 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. The complete absence of stomata in liverworts may be interpreted either as a loss or a pleisiomorphy, depending on whether hornworts or liverworts are sister to land plants (Villarreal and Renzaglia, 2015). Here, we examined stomatal development and fate vis-a-vis spore differentiation and sporophyte maturation. It is estimated that there are about 9000 species of liverworts. 2k,n). Stotler et al. Structural features that have been used in the classification of hornworts include: the anatomy of chloroplasts and their numbers within cells, the presence of a pyrenoid, the numbers of antheridia within androecia, and the arrangement of jacket cells of the antheridia. 1. 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. B, Juncture of inner and ventral guard cell walls with wax deposits on cell walls in the substomatal cavity (arrows). S1; Macquet et al., 2007). B, Mature, living, and open stoma. 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). Cross section light micrograph of a newly opened stoma shows large starch-filled plastids in guard cells and differentially thickened epidermal and guard cell walls. By using inorganic c… 2D and 3B). 3F), outer (Fig. Following pore formation, the sporophyte dries from the outside inwardly and continues to do so after guard cells die and collapse. Stomata on hornworts are larger in width and depth than the surrounding epidermal cells, which is an unusual character in plants. It follows that stomata are intricately involved in these processes. , Hornworts are unique in having a gene called LCIB, which is not found in any other known land plants but occurs in some species of algae. 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. Stomata on the earliest fossil land plants share remarkable similarities with hornwort stomata and sporophyte surfaces (Fig. Bars = 0.5 μm except for E, where bar = 2 μm. Esau’s Plant Anatomy: Meristems, Cells and Tissues of the plant Body—Their Structure, Function and Development. The loss of stomata in the hornwort crown group that includes Nothoceros, Megaceros, and Dendroceros may be a function of their life history traits. SEM cross section shows the epidermis and a stoma with dead collapsing guard cells that contain degenerated protoplasm (arrow). Do Hornworts have stomata? SEM images of hornwort stomata compared with fossil stomata. C, A adscendens Lehm. Phaeoceros carolinianus. The inner wall typically lacks a ledge and has an inner layer of radial fibrils and a thickened outer fibrillar network (Figs. Loss of stomata in moss species is much more complicated and remains to be analyzed (Paton and Pearce, 1957; Merced, 2015b). In contrast, Arabidopsis has a comparable genome size (0.16 pg) to Leiosporoceros, P. carolinianus, and Anthoceros punctatus L. but produces much smaller guard cells that are approximately 25 × 7.5 μm (Lomax et al., 2009). Stomatal guard cell length from selected early Devonian fossils of rhyniophytes, zosterophllyloids, aglaophytes, and lycophytes taken from Lomax et al. Additionally, this structure can be found in the sporophyte phase of pants which is a stage in the life of some plants including hornworts that have long horn-like structures that contain the stomate. Stomata are expendable in hornworts, as they have been lost twice in derived taxa. A thin cuticle and cuticular region overlie the outer and ventral guard cell walls (Fig. The smallest guard cells are those in Anthoceros fusiformis Aust. These findings come together with a paucity of arabinans in the cell walls and no correlation between guard cell and genome sizes to challenge the possibility of diurnally active stomata in hornworts. There are about 200 species known, but new species are still being discovered. 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). from Puerto Rico and Makanda, Illinois, and Anthoceros agrestis Paton from Makanda, Illinois. What are Liverworts – Definition, Characteristics, Reproduction 4). SEM images of hornwort sporophytes. Most plants are monoecious, with both sex organs on the same plant, but some plants (even within the same species) are dioecious, with separate male and female gametophytes. The adjacent epidermal cell contains degenerated cytoplasm and has begun to collapse like an accordion in the opposite direction from the guard cells. Stomata occur in all major groups of extant land plants except liverworts, but they are found on sporangia (capsules) only in hornworts and mosses. All three taxa are tropical and produce highly elongated involucres and spiraled pseudoelaters. Supplemental Figure S1. Key Areas Covered. A to D, P. carolinianus. 3J) labeling is restricted to the inside of the walls at the plasmalemma. Stomata appear in the hornworts and are abundant on the sporophyte. Once open, stomata never close, but the outer aperture increases slightly in width after guard cell collapse. DUE 1136414) and National Institutes of Health (grant no. 4H). During the drying process, the spore mother cell wall adheres to individual spores, forming a pseudoperine (Fig. The earliest fossil stomata from the Silurian (yellow tag) exhibit the collapsed condition. Cell walls are thicker than those in A, and epidermal cells contain large amyloplasts (black arrow). Spores form walls early in development but remain bathed in mucilage as the sporophyte dries until dehiscence. , One of the three Divisions of bryophytic plants, The name Hornwort also refers to aquatic plants of the genus, What is a hornwort? Do Pteridophytes have stomata? Guard cell lengths of 16 hornwort species, representing approximately 9% of all hornwort species and 16% of those with stomata, were measured, and their means were compared with published genome size data (Bainard and Villarreal, 2013) using a correlation implemented in the R package. E, Early Devonian fossil stoma at the base of terminal sporangium of Sporogonites NMW96.5G.3. Large tropical and sub-tropical species of Dendroceros may be found growing on the bark of trees. 1C), which remain larger than newly formed stomata (green zone in Fig. 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. Our findings identify an architecture and fate of stomata in hornworts that is ancient and common to plants without sporophytic leaves.