Plant Physiology Preview. Published on March 28, 2017, as DOI:10.1104/pp.17.00120
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Short title: Growth conditions affect fern stomatal responses
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Corresponding Author:
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Name: Ebe Merilo
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Affiliation: Plant Signal Research Group, University of Tartu, Institute of Technology,
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Nooruse 1, Tartu, 50411, Estonia
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Address: Nooruse 1, Tartu, 50411, Estonia
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E-mail: [email protected]
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Article Type: Research Report
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Research Area: stomatal responses, ferns, physiology, evolution
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Fern stomatal responses to ABA and CO2 depend on species and growth conditions
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Hanna Hõrak1, Hannes Kollist1, Ebe Merilo1*
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Plant Signal Research Group, University of Tartu, Institute of Technology, Nooruse 1, Tartu,
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50411, Estonia
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One sentence summary: Growth conditions and species affect fern stomatal responsiveness to
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ABA and CO2.
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Funding information: This work was supported by Estonian Ministry of Science and
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Education (PUT1133, E.M. and IUT2-21, H.K.) and by European Regional Development
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Fund (Center of Excellence in Molecular Cell Engineering CEMCE).
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*
Address correspondence to [email protected]
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Author contributions: E.M. and H.H. conceived and designed the study, E.M. performed most
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of the experiments, E.M. analyzed the data, H.H. wrote the article with E.M. and H.K.
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Keywords: stomata, guard cell, fern, ABA signaling, CO2 signaling
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Copyright 2017 by the American Society of Plant Biologists
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ABSTRACT
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Changing atmospheric CO2 levels, climate and air humidity affect plant gas exchange that is
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controlled by stomata, small pores on plant leaves and stems formed by guard cells. Evolution
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has shaped the morphology and regulatory mechanisms governing stomatal movements to
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correspond to the needs of various land plant groups over the past 400 million years. Stomata
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close in response to the plant hormone abscisic acid (ABA), elevated CO2 concentration and
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reduced air humidity. Whether the active regulatory mechanisms that control stomatal closure
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in response to these stimuli are present already in mosses, the oldest plant group with stomata,
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or were acquired more recently in angiosperms, remains controversial. It has been suggested
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that the stomata of the basal vascular plants, such as ferns and lycophytes, close solely
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hydropassively. On the other hand, active stomatal closure in response to ABA and CO2 was
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found in several moss, lycophyte and fern species. Here we show that the stomata of two
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temperate fern species respond to ABA and CO2 and an active mechanism of stomatal
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regulation in response to reduced air humidity is present in some ferns. Importantly, fern
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stomatal responses depend on growth conditions. The data indicate that the stomatal behavior
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of ferns is more complex than anticipated before, and active stomatal regulation is present in
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some ferns and has possibly been lost in others. Further analysis that takes into account fern
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species, life history, evolutionary age and growth conditions is required to gain insight into
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the evolution of land plant stomatal responses.
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INTRODUCTION
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Stomatal pores, formed by guard cells on plant leaves and stems, mediate CO2 uptake for
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photosynthesis and water loss via transpiration. Adequate regulation of stomatal aperture in
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response to changing environmental conditions is essential for thriving plant growth. In
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angiosperms, whose stomatal regulation has been studied the most, stomata close in response
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to abscisic acid (ABA), elevated CO2 concentration, reduced air humidity, darkness and air
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pollutants, whereas they open in response to light, increased air humidity and decrease in CO2
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concentration.
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Over the recent years, the evolution of plant stomatal signaling pathways has become a
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subject of intensive studies and passionate debate. The central role of ABA in angiosperm
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stomatal responses has been known for a long time (Cutler et al., 2010). More recently,
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experiments assessing stomatal responses to ABA have been conducted with mosses and
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basal vascular plants such as ferns and lycophytes. Lack of stomatal closure in response to
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ABA treatment in several fern and lycophyte species led to the hypothesis that these plant
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groups use only hydropassive mechanisms for stomatal regulation (Brodribb and McAdam,
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2011). Moreover, high ABA levels induced in response to drought did not inhibit the opening
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of fern and lycophyte stomata upon rehydration, suggesting that endogenous ABA was not
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controlling stomatal responses in these plant species (McAdam and Brodribb, 2012).
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On the other hand, stomata in the epidermal strips of the lycophyte Selaginella uncinata and
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in the sporophytes of the mosses Physcomitrella patens and Funaria hygrometrica closed in
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response to ABA in a concentration-dependent manner (Chater et al., 2011; Ruszala et al.,
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2011), indicating a conserved role for ABA in the stomatal responses of plants. Recently,
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dose-dependent ABA-induced stomatal closure was also shown to be present in the ferns
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Polystichum proliferum and Nephrolepis exaltata (Cai et al., 2017). Several genes encoding
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proteins involved in ABA signal transduction are expressed in the stomata-bearing sporophyte
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of the moss P. patens (O’Donoghue et al., 2013) and in the epidermal fraction of the fern P.
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proliferum (Cai et al., 2017). Furthermore, the P. patens and Selaginella moellendorffii
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homologues of OPEN STOMATA 1 (OST1), a SnRK-type kinase that participates in ABA-
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induced stomatal closure via phosphorylation of the central guard cell anion channel SLOW
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ANION CHANNEL 1 (SLAC1) in Arabidopsis thaliana (Geiger et al., 2009; Lee et al., 2009;
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Vahisalu et al., 2010), complemented the ABA-insensitivity of stomatal closure in A. thaliana
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ost1 mutant (Chater et al., 2011; Ruszala et al., 2011). The P. patens deletion mutant that
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lacked OST1 had impaired ABA-induced stomatal closure (Chater et al., 2011) and the P.
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patens OST1 and several other OST1-like SnRKs from different non-vascular plants could
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activate A. thaliana SLAC1 in oocytes (Lind et al., 2015). These data indicate that the core
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stomatal ABA-signaling pathway is conserved in plants. Recently, a homologue of OST1 was
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shown to regulate sex determination in the fern Ceratopteris richardii (McAdam et al., 2016),
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suggesting that at least some of the components of the ABA-signaling pathway that control
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stomatal responses in angiosperms could have different or additional functions in ferns.
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Moreover, the ABA signal transduction pathways of ferns and angiosperms may be at least
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partly different and thus the analysis of fern homologues of components involved in ABA-
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response pathways of angiosperms is not sufficient to fully understand the mechanisms of
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ABA-responsiveness in ferns.
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Plant stomata open or close in response to sub-ambient or above-ambient CO2 concentration,
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respectively. This is the basic mechanism through which the supply of photosynthetic CO2 is
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regulated. The question of stomatal responsiveness to CO2 in plant groups older than
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angiosperms remains controversial. Gas-exchange analysis of several fern and lycophyte
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species indicated that the stomata of these plants open in response to sub-ambient CO2
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concentrations but do not close at above-ambient CO2 levels (Brodribb et al., 2009; Brodribb
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and McAdam, 2013). Similarly, stomata in the mosses P. patens and F. hygrometrica had
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strong CO2-response in the range of 0 to 400 ppm CO2, but did not close markedly at above-
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ambient CO2-levels (Chater et al., 2011). However, stomata of the lycophyte S. uncinata
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responded to both sub-ambient and above-ambient CO2-concentration (Ruszala et al., 2011),
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as did the stomata of the fern Phyllitis scolopendrium (Mansfield and Willmer, 1969).
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Recently, several fern species were shown to close their stomata in response to CO2 elevation
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from the ambient 400 ppm to 800 ppm, similar to the angiosperm A. thaliana (Franks and
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Britton-Harper, 2016). It was hypothesized that different growth conditions and species-
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specific behavior may account for the varied results obtained in the analysis of stomatal
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responses of mosses, ferns and lycophytes (Roelfsema and Hedrich, 2016). For example, in
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some fern species stomata closed in response to CO2 elevation from 0 to 400 ppm, in others
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they did not respond or even opened, whereas in some species the response depended on
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growth light intensity (Creese et al., 2014). Thus, a consensus on whether there is a universal
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mechanism that governs stomatal responses to CO2 and ABA in all land plants, has not been
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reached to date and further experiments assessing the effect of growth conditions and species
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are required. Importantly, above-mentioned studies paid little attention to the fact that fern
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stomata are morphologically different from those of angiosperms – they lack subsidiary cells
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and there is little mechanical interaction between guard cells and epidermal cells during guard
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cell swelling i.e. diminished mechanical advantage of epidermal cells over guard cells (Franks
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and Farquhar, 2007). Large kidney-shaped stomata of ferns that grow in the forest understory
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characterized by deep shade and more humid air, have been suggested to have slow dynamics
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of stomatal movements (Hetherington and Woodward, 2003). These differences in guard cell
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morphological and mechanical properties undoubtedly translate into differences in stomatal
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functioning.
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Here, we provide evidence of growth condition- and species-dependent stomatal
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responsiveness to ABA and CO2 in three temperate fern species. The results indicate that
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while the stomata of some ferns can respond to ABA and CO2, the kinetics and thus
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underlying mechanisms of stomatal regulation differ, at least partly, between ferns and
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angiosperms. Furthermore, the stomatal response to decreased air humidity is slowed down at
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low CO2 concentration in Athyrium filix-femina, indicating that fern stomatal responses are
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not purely hydropassive. Thus, species and growth conditions affect fern stomatal
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responsiveness and should be taken into account when drawing major conclusions about the
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evolution of the mechanisms that control stomatal regulation in ferns.
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RESULTS AND DISCUSSION
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Stomatal response to ABA is species-specific and depends on growth conditions in ferns
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As the presence of active stomatal responses in ferns has remained controversial to date, we
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studied whether their stomatal responses to ABA and CO2 could be influenced by growth
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conditions. Three temperate fern species (Athyrium filix-femina, Dryopteris carthusiana and
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Dryopteris filix-mas) were collected from nature and transferred to the laboratory where they
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were grown in two different growth conditions: in a growth cabinet characterized by constant
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relative air humidity of 70% both day and night (from here on referred to as “the growth
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cabinet”), or in a growth room with lower and variable relative air humidity (RH ~30-40%
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during day and 40-65% at night, from here on referred to as “the growth room”). The light
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regime was 16/8 and 12/12 hours light/dark in the growth room and the growth cabinet,
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respectively. After the ferns had developed new leaves indoors, gas exchange analysis was
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carried out with a custom-built device that enabled parallel recording of stomatal conductance
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in four plants (see methods; one measurement cuvette is shown in Fig. 1A). Similar
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experiments were carried out with an angiosperm Arabidopsis thaliana (Col-0) grown in
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identical conditions and using another custom-built device that enabled parallel recording of
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stomatal conductance in eight plants (see methods). Stomatal responses of the ferns and A.
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thaliana to 10 μM ABA sprayed on leaves or changes in CO2 concentration were measured.
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The stomatal conductance of the angiosperm A. thaliana strongly decreased in response to 10
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μM ABA application both in plants grown in the growth cabinet as well as in the growth room
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(Fig. 1B), pointing at prominent ABA-responsiveness of Arabidopsis stomata irrespective of
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growth conditions. However, the growth conditions affected stomatal responsiveness in ferns.
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When grown in the growth cabinet at 70% RH, the stomatal conductance of A. filix-femina
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and D. filix-mas decreased in response to spraying with ABA, whereas D. carthusiana did not
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respond to ABA-treatment (Fig. 1C). None of the tested fern species responded to ABA when
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grown in the growth room at lower RH (Fig. 1C). Thus, fern species differ in their ability to
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close stomata in response to ABA and stomatal responsiveness is affected by growth
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conditions.
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Air humidity has been shown to affect stomatal morphology and responsiveness in various
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angiosperm species. High relative air humidity during growth has been associated with longer
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and more open stomata (Torre et al., 2003; Nejad and van Meeteren, 2005; Fanourakis et al.,
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2011; Aliniaeifard et al., 2014), lower levels of ABA (Zeevaart, 1974; Nejad and van
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Meeteren, 2007; Okamoto et al., 2009; Aliniaeifard et al., 2014) and decreased stomatal
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responsiveness to ABA (Pospíšilová, 1996; Fanourakis et al., 2013; Pantin et al., 2013b; Arve
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et al., 2014). Exposure to low RH can enhance stomatal ABA-responsiveness in the young
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leaves that are constantly in a microenvironment with high RH (Pantin et al., 2013b). This
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indicates that reduced RH is required to prime stomatal ABA responses, potentially via
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increased ABA levels, as ABA application can restore stomatal ABA-responsiveness (Pantin
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et al., 2013b) and results in normal stomatal development (Fanourakis et al., 2011) in the
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plants grown at high RH. However, in these reports, the RH that led to reduced ABA-
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sensitivity was very high: 90% or more. In our experiment, lower growth RH (30-65%) led to
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the loss of stomatal ABA-responsiveness in two ferns as compared to growing them at 70%
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RH (Fig. 1C). This could be explained by increased ABA levels induced by low air humidity
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as observed in other species (Bauer et al., 2013; McAdam and Brodribb, 2015), that could
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potentially lead to partial ABA-induced closure of the stomata of ferns grown in the growth
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room and no further effect of ABA-treatment on stomatal conductance. If ABA-response is
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already activated in ferns grown at lower RH, these plants would be expected to have lower
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stomatal conductance. However, as the magnitude of ABA-induced stomatal closure was
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modest even in the ferns grown at 70% RH, such a difference in stomatal conductance could
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remain undetected. It has been shown that the ABA levels of ferns do not increase in response
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to short-term low humidity treatment (McAdam and Brodribb, 2015), but ABA levels in ferns
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subject to long-term growth at low RH remain to be characterized.
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Guard cell ABA response appears to be present in some fern species and not in others (Fig.
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1C, Brodribb and McAdam, 2011; Cai et al., 2017), suggesting that the ABA responsiveness
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may have been lost in some lineages of ferns. In addition to the direct effect on stomata, ABA
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reduces leaf hydraulic conductance in angiosperms (Shatil-Cohen et al., 2011; Pantin et al.,
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2013a). Thus, the ABA-induced decrease in stomatal conductance in A. filix-femina and D.
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filix-mas grown at 70% RH could have occurred due to changes in leaf hydraulics and not due
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to the direct effect of ABA on guard cells. Different procedures for ABA application could
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explain the varying ABA-responsiveness of ferns and lycophytes found in experiments. It is
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possible that ABA applied via the transpiration stream is incapable of inducing stomatal
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closure in ferns and lycophytes, whereas ABA applied to epidermal peels or by leaf spraying
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enters guard cells directly and thus is more efficient in promoting stomatal closure (compare
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Fig.1 C, Brodribb and McAdam, 2011; Ruszala et al., 2011; Cai et al., 2017).
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Although the stomata of two fern species closed in response to ABA, the extent of the closure
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was small compared to the angiosperm A. thaliana (Fig. 1, B and C). This indicates that while
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several ferns are capable of ABA-perception and response, the kinetics and underlying
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molecular mechanisms of this response could be different in ferns and in angiosperms. The
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components of core stomatal ABA signaling pathway have been shown to be present in
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mosses, lycophytes, ferns and angiosperms (Hanada et al., 2011; Cai et al., 2017), indicating
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an early origin of ABA signal transduction. Nevertheless, it is possible that not all of these
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proteins have strictly stomata-related functions in ferns as was shown recently for a
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homologue of the OST1 kinase that participates in sex determination in a fern (McAdam et
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al., 2016). However, other kinases besides OST1 can activate anion channels towards
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stomatal closure and although OST1 is the most important among them in Arabidopsis
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(Kollist et al., 2014), ABA-induced signaling pathways do not have to be identical in ferns
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and angiosperms. Thus, it is possible that the classical stomatal ABA-response pathway
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functions only partially in ferns, and is not capable of inducing stomatal closure in response to
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ABA treatment in all fern species. A wider study of stomatal ABA-responsiveness across the
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evolutionary tree of ferns would be required to understand how guard cell ABADownloaded from on June 15, 20177- Published by www.plantphysiol.org
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responsiveness has evolved. For example, an analysis of different fern classes showed that
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whereas the stomatal opening in response to blue light is present in lycophytes and most
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classes of ferns, it has been lost in the fern group Polypodiopsida (Doi et al., 2015). Thus, it is
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conceivable that the stomatal ABA-responsiveness has been lost in some branches of the
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evolution tree of ferns and systematic analysis of stomatal responses in different taxa is
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required to bring further insight into this question.
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Stomatal responses to sub-ambient and above-ambient CO2 concentrations differ and depend
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on growth conditions in ferns
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Next we studied stomatal responses to sub- and above-ambient CO2 concentrations in
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Arabidopsis and the three fern species. In A. thaliana, strong and steady stomatal opening
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occurred in response to the reduction of CO2 concentration from 400 to 100 ppm and stomata
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closed quickly, when CO2 was returned to 400 ppm (Fig. 2, A and B, Supplemental Table 1).
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Fast stomatal closure occurred also in response to further elevation of CO2 concentration from
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400 ppm to 800 ppm (Fig. 2, A and B, Supplemental Table 1). Stomatal responsiveness to
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changes in CO2 levels did not markedly depend on growth conditions in Arabidopsis.
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Fast stomatal opening in response to a reduction of CO2 concentration from 400 ppm to 100
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ppm occurred in all ferns irrespective of growth conditions (Fig. 2, C and D, Supplemental
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Tables 2 and 3). Elevation of CO2 from 100 ppm back to 400 ppm and then further to 800
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ppm caused stomatal closure in nearly all tested fern species and conditions (Fig. 2, C and D,
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Supplemental Tables 2 and 3). There was one exception: D. filix-mas grown in the growth
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room did not respond significantly to above-ambient levels of CO2, whereas its stomatal
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opening in response to a change from 400 ppm to 100 ppm and closure induced by CO2
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elevation from 100 ppm to 400 ppm was one of the largest (Fig. 2, C and D, Supplemental
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Tables 2 and 3). Previously, high RH has been shown to enhance the response to CO2
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elevation in Vicia faba (Talbott et al., 2003). In accordance with this, stomatal responses to
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CO2 elevation were at least partially reduced in ferns grown at lower RH. In addition to D.
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filix-mas, the low RH of the growth room also affected CO2 responsiveness in A. filix-femina,
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whose final stomatal conductance achieved after transition from 100 to 400 ppm was
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significantly higher than the pre-treatment value at 400 ppm, indicating a slowed-down
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closure response (Fig. 2D, Supplemental Table 2). Thus, as in experiments with ABA, growth
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in the growth room with lower RH tended to reduce the stomatal sensitivity in D. filix-mas
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and A. filix-femina. As stomatal responses to CO2 are tightly coupled with ABA-responses
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(Xue et al., 2011; Merilo et al., 2013; Chater et al., 2015), the analogous dampening of
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responses to ABA and elevated CO2 found in A. filix-femina and D. filix-mas when grown at
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low RH suggests that the underlying mechanisms responsible for the effect of growth
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conditions could be similar and potentially related with ABA levels.
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All the fern species studied here had the ability to open stomata due to CO2 withdrawal and
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close stomata due to CO2 enrichment, both in the sub-ambient and above-ambient range of
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CO2 levels. However, their stomatal opening in response to low CO2 was markedly faster than
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stomatal closure in response to elevated CO2 (Fig. 2C). Thus, the previously documented
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presence and absence of stomatal responses to sub-ambient and above-ambient CO2
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concentration, respectively (Brodribb et al., 2009; Brodribb and McAdam, 2013), could be
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explained by the relatively slow response to elevated CO2 in ferns. The closure kinetics of
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elevated CO2 response was clearly different between the studied ferns and the angiosperm A.
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thaliana; the latter showed a fast drop in stomatal conductance in response to transition from
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400 ppm to 800 ppm compared to the steady decrease in ferns, whereas in ferns the opening
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under sub-ambient CO2 levels was faster than in Arabidopsis (Fig. 2, A and C). A similar
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difference in the kinetics of high CO2-induced stomatal closure in ferns and A. thaliana has
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been found before (Franks and Britton-Harper, 2016). These differences in the kinetics of CO2
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responses between ferns and angiosperms may result from their different stomatal
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morphology and related mechanical properties. Ferns have lower stomatal conductance and
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diminished mechanical advantage of epidermal cells over guard cells (Franks and Farquhar,
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2007) that could result in faster opening of their stomata. However, ferns also show reduced
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rate of changes in stomatal aperture compared to angiosperms due to less lateral movement of
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guard cells and reduced shuttling of osmotica between guard and epidermal cells that could
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explain their slower closure responses (Franks and Farquhar, 2007).
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Interestingly, the pre-treatment stomatal conductance of Arabidopsis plants grown at lower
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humidity in the growth room was higher than in the plants grown in the growth cabinet both
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in experiments with ABA and CO2 (Fig. 1B, 2A and 2B, Students t-test, p < 0.05). This seems
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counterintuitive, as previous studies have shown higher stomatal conductance or wider
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stomatal aperture at higher RH in several angiosperm species (Torre et al., 2003; Nejad and
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van Meeteren, 2005; Fanourakis et al., 2011; Aliniaeifard et al., 2014). However, here the
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measurements of stomatal behavior were carried out at ~70% RH, which was higher than the
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growth room RH of ~40%. Thus, it is possible that in Arabidopsis, stomatal opening occurred
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when plants were transferred from the growth room to the measurement chamber. This fast
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opening response was not present in any of the tested fern species, where stomatal
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conductance of plants grown in the growth room was similar to that in the growth cabinet
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(Fig. 1B, 2C and 2D, two-way ANOVA with Tukey post hoc test where appropriate,
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Supplemental Tables 4 and 5). These data suggest a greater flexibility and faster stomatal
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responsiveness to changes in vapor pressure deficit (VPD) in angiosperms. This could be
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expected as the responses to changes in VPD are linked with the biosynthesis and presence of
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ABA (Bauer et al., 2013; McAdam and Brodribb, 2015) as well as ABA signaling (Merilo et
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al., 2013), and stomatal ABA-responses in angiosperms are faster and stronger than in ferns
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(Brodribb and McAdam, 2011; Fig. 1B and C).
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Dampening of the response to high VPD at low CO2 concentration suggests the presence of
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active stomatal regulation in ferns
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Stomatal responses of ferns have been proposed to be regulated solely hydropassively by leaf
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water status (Brodribb and McAdam, 2011). If fern stomata indeed act as purely passive
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valves, it would be expected that the stomatal response to increased VPD would be faster in
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plants with higher stomatal conductance, as this would result in higher transpiration, greater
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loss of water and more negative leaf water potential. To test this, we analyzed stomatal
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responsiveness to reduction in RH corresponding to higher VPD in either low (50 ppm) or
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ambient (400 ppm) CO2 concentration in ferns grown in the growth room and the growth
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cabinet. As the stomata of A. filix-femina responded to ABA and those of D. carthusiana did
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not, we chose these two species for the experiments. As expected, the stomatal conductance
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was higher in low CO2 concentration in both species albeit the difference was greater for A.
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filix-femina (Fig. 3A, Supplemental Fig. 1A).
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Stomata of A. filix-femina closed in response to a rapid increase in VPD from 0.7-1 kPa to 2-
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2.2 kPa both in low and ambient CO2 concentration (Fig. 3A, 3B and 3C). However, the
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response to high VPD was significantly slower in low than in ambient CO2 (Fig. 3C). This
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was true for both ferns grown in the growth room as well as in the growth cabinet, indicating
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the robustness of the response. This finding is not in accordance with the model of passive
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VPD-dependent stomatal regulation in ferns since stomatal conductance was roughly two
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times higher at low CO2 and this should have caused a faster hydropassive response to VPD.
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This experiment indicates that there is more than just a passive mechanism that regulates the
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stomatal response to a change in VPD in this fern species. As ABA and the components of its
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signaling pathway mediate stomatal responses to changes in VPD in angiosperms (Bauer et
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al., 2013; Merilo et al., 2013; McAdam and Brodribb, 2015) and A. filix-femina is a fern with
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ABA-responsive stomata (Fig. 1C), ABA is an obvious candidate as a mediator of stomatal
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response to high VPD in A. filix-femina. Stomatal closure in response to increased VPD
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appeared to be delayed in low CO2 concentration also in D. carthusiana (Supplemental Fig. 1)
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grown both in the growth room and the growth cabinet, but there was no statistically
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significant difference between stomatal half-response times to increased VPD at low and
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ambient CO2 concentrations. Thus, it is possible that in D. carthusiana, hydropassive stomatal
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control is more important in VPD-responses.
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Interestingly, despite the low stomatal conductance of ferns, their net CO2 assimilation rate
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ranged between 4.1 to 4.5 μmol m-2 s-1 compared to 5.5 in Arabidopsis. This resulted in twice
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as high instantaneous water use efficiency of ferns in comparison with Arabidopsis. As ferns
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naturally grow in the shaded conditions of forest understory, where CO2 concentration is
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higher than in upper layers (Bazzaz and Williams, 1991), it is possible that the low stomatal
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317
conductance of ferns is an adaptive trait associated with growth habitat. Adaptation to growth
318
in shade and increased CO2 concentration could also explain the strong stomatal sensitivity to
319
CO2 withdrawal in ferns.
320
CONCLUSIONS
321
The results presented here indicate that while the stomatal responses of some ferns may be
322
hydropassive as described before (Brodribb and McAdam, 2011), other fern species have
323
active mechanisms for stomatal regulation as well. Large-scale studies of stomatal
324
responsiveness in ferns from different families and evolutionary age would be necessary to
325
gain further insight into the evolution of passive and active stomatal control mechanisms in
326
ferns. Moreover, as shown here, growth conditions can have strong effects on stomatal
327
responsiveness in ferns – for example, responses to ABA and CO2 were weakened in ferns
328
grown at relative air humidity of 30-40% compared to 70% RH, while fast stomatal responses
329
were maintained in the angiosperm A. thaliana grown in both conditions. Whether contrasting
330
patterns of stomatal ABA-sensitivity detected for ferns in this study are accompanied with
331
differences in ABA synthesis in variable conditions remains to be studied in the future.
332
MATERIALS AND METHODS
333
Plant material and growth conditions
334
The ferns Athyrium filix-femina, Dryopteris carthusiana and Dryopteris filix-mas were used
335
in experiments. Specimens of Dryopteris filix-mas were collected from wild gardens in
336
Kooraste and Näräpää in Põlvamaa, Southern Estonia in August 2016. Specimens of
337
Dryopteris carthusiana were collected from a clear cut forest area in Kooraste and specimens
338
of Athyrium filix-femina from forest in Näräpää between August and October 2016. All the
339
ferns were transported to the laboratory, transplanted into 4:2:3 peat:vermiculite:water
340
mixture and grown either in a growth room at 16/8h light/dark regime, low relative air
341
humidity (30-40% during the day and 40-65% at night) and 100-150 µmol m-2 s-1 light, or in a
342
growth cabinet (MCA1600, Snijders Scientific, Drogenbos, Belgia) at 12/12h light/dark
343
regime, 70% relative air humidity and 100 µmol m-2 s-1 light. These growth conditions are
344
hereafter referred to as the growth room and the growth cabinet, respectively. Only leaves
345
developed indoors in the respective conditions were used for gas exchange measurements.
346
Arabidopsis thaliana (Col-0) plants used for comparison were grown in identical conditions,
347
and were 23-27 days old during experiments.
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348
Gas-exchange experiments
349
Stomatal conductance of ferns was recorded with a thermostated 4-chamber custom-built
350
flow-through gas exchange device. The main body of the system consists of four thermostated
351
gas exchange cuvettes formed by two glass cylinders (inner diameter 10.6 cm, height 15.6
352
cm) and thermostated water jacket between them. The cuvettes are placed on a stand
353
composed of two well-fitted glass plates that form a bottom of the gas exchange cuvette. One
354
of these plates contains perforations for plant stem and is removable. The other glass plate
355
contains gas input and output ports, a temperature sensor, and a fan to guarantee high
356
turbulence and uniform gas mixing. Fan ensured that boundary layer conductance was high
357
(11-12 cm s-1) and, further, that heat exchange between leaves and chamber air was very
358
good, reducing the effect of transpiration on leaf temperature. Modeling gum was used to
359
ensure an airtight separation of plant shoots within the cuvette from roots in the soil.
360
Chambers are hermetically sealed and operate under slight overpressure of a few millibars to
361
avoid uncontrolled intake of ambient air. Air flow rate through the chamber was 2.5 l min-1.
362
Ambient air passing through a large buffer volume of 25 l was used. The air temperature
363
inside the chambers was continuously measured with negative temperature coefficient
364
thermistors (model -001; RTI Electronics, Anaheim, CA, USA) and was between 24-25°C.
365
Leaf temperature was determined from leaf energy balance based on absorbed light and
366
transpiration. All tubing and connections were made of Teflon and stainless steel. For
367
illumination, four 50W halogen lamps provided PPFD of 500 μmol m−2 s−1 to each cuvette.
368
Concentrations of CO2 and water vapor in the reference channel (i.e air entering the
369
measuring cuvette) and measurement channel (air coming out from the cuvette) were
370
measured with an infrared gas analyzer (Li-7000, Li-Cor, Nebraska, USA) and stomatal
371
conductance and net assimilation rate were calculated with a custom-written program as
372
described in Kollist et al., 2007.
373
Stomatal conductance of the plants was measured at ~70% RH, 500 µmol m-2 s-1 light and 24-
374
25°C air temperature. When stomatal conductance had stabilized, plants were treated with
375
ABA, change in CO2 concentration or change in VPD. For ABA treatment, cuvette covers
376
were removed, 10 μM ABA with 0.012% Silwet L-77 (Duchefa) and 0.05% ethanol was
377
sprayed on leaves, cuvette covers were returned and measurement of stomatal conductance
378
continued. In experiments with CO2, CO2 concentration in the chamber was reduced from 400
379
ppm to 100 ppm for 2 hours, thereafter returned to 400 ppm for two hours and then elevated
380
to 800 ppm for two hours. In experiments with high VPD, stomatal conductance of the plants
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381
was allowed to stabilize in the measurement cuvette with either 50 ppm or 400 ppm CO2.
382
Thereafter the air humidity in the chamber was lowered from ~70% to ~30-40%, yielding a
383
change in VPD from 0.7-1 kPa to 2-2.2 kPa.
384
Stomatal conductance of A. thaliana was recorded with an 8-chamber custom-built
385
temperature-controlled gas-exchange device analogous to the one described before (Kollist et
386
al., 2007). Stomatal conductance of the plants was measured at ~70% RH, 150 µmol m-2 s-1
387
light and 24-25°C air temperature. When stomatal conductance had stabilized, plants were
388
treated similar to ferns with either ABA spraying or changes in CO2 concentration.
389
Data analysis and statistics
390
In ABA and CO2 experiments, stomatal conductance values before and after application of
391
these stimuli were compared by repeated measures ANOVA with Tukey post hoc test. In
392
experiments with reduced air humidity, stomatal half-response times were calculated by
393
scaling the whole 42-min stomatal response to a range from 0 to 100% and by calculating the
394
time when 50% of stomatal closure was achieved. Half-response times were compared by
395
two-way ANOVA with Tukey post hoc test. All statistical analyses were carried out with
396
Statistica (StatSoft Inc., Tulsa, OK, USA). All effects were considered significant at p < 0.05.
397
398
Supplemental data
399
The following material is available in the online version of this report.
400
Supplemental Figure S1. Stomatal response of Dryopteris carthusiana to reduced air humidity
401
at different CO2 concentrations.
402
Supplemental Table 1. Repeated measures ANOVA with Turkey HSD post hoc test for Fig
403
2B.
404
Supplemental Table 2. Repeated measures ANOVA with Turkey HSD post hoc test for Fig
405
2D, plants grown in the growth room.
406
Supplemental Table 3. Repeated measures ANOVA with Turkey HSD post hoc test for Fig
407
2D, plants grown in the growth cabinet.
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408
Supplemental Table 4. Two-way ANOVA for pre-treatment stomatal conductances in Fig.
409
1C.
410
Supplemental Table 5. Two-way ANOVA with Turkey HSD post-hoc test for pre-treatment
411
stomatal conductances in Fig. 2D.
412
413
ACKNOWLEDGMENTS
414
We are grateful to Toomas Kukk for help with fern species determination.
415
FIGURE LEGENDS
416
Figure 1. Stomatal responses of ferns and A. thaliana grown in two different conditions to
417
foliar ABA spraying (10 µM). A, D. filix-mas in the gas-exchange measurement chamber. B,
418
Mean ± SEM stomatal conductance before and 47 min after treatment with ABA in A.
419
thaliana grown in the growth room and in the growth cabinet (n=7). C, Mean ± SEM stomatal
420
conductance before and 47 min after treatment with ABA in ferns A. filix-femina, D. filix-mas
421
and D. carthusiana grown at different conditions, n=7 and n=9 for A. filix-femina, n=4 and
422
n=7 for D. filix-mas, n=5 and n=4 for D. carthusiana, grown in the growth room or the
423
growth cabinet, respectively. Statistically significant differences are denoted with asterisks
424
(Repeated measures ANOVA with Tukey post hoc test).
425
Figure 2. Stomatal responses of ferns and A. thaliana grown in different conditions to
426
changes in CO2 concentration. A, Time-resolved stomatal conductance of the angiosperm A.
427
thaliana grown at different conditions. At time point 0, CO2 was decreased from 400 to 100
428
ppm, at time point 120 it was elevated back to 400 ppm and at time point 240 increased
429
further to 800 ppm. Mean ± SEM is shown, n=8. B, Last stomatal conductance values from
430
panel A at each CO2 concentration, mean ± SEM is shown. C, Time-resolved stomatal
431
conductance of the ferns A. filix-femina, D. filix-mas and D. carthusiana grown at different
432
conditions. CO2 treatments were applied as described above for A. thaliana. Mean ± SEM is
433
shown, n=7 and n=6 for A. filix-femina, n=4 and n=8 for D. filix-mas, n=6 and n=6 for D.
434
carthusiana grown in the growth room and the growth cabinet, respectively. D, Last stomatal
435
conductance values at each CO2 concentration from panel C.
436
Figure 3. Stomatal response of Athyrium filix-femina to reduced air humidity at different CO2
437
concentrations. A, Time-resolved stomatal conductance of A. filix-femina. At time point 0,
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438
VPD was increased from 0.7-1 kPa to 2-2.2 kPa. Mean ± SEM is shown, n=7 and n=8 for the
439
growth room or the growth cabinet, respectively. B, Stomatal response in relative units,
440
calculated based on the data presented in A. C, Stomatal half-response times, calculated based
441
on data presented in A. Different letters denote statistically significant differences (Two-way
442
ANOVA with Tukey post hoc test, the effect of CO2 concentration was statistically
443
significant).
444
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