Abscisic acid controlled sex before transpiration in vascular plants

Significance

Since the dawn of land plants, the phytohormone abscisic acid (ABA) has played a critical role in regulating plant responses to water availability. Here we seek to explain the origins of the core ABA signaling pathway found in modern seed plants. Using the characterization of mutants and gene silencing in a fern species, we find that the same hormone signaling components are used in sex determination of ferns as are used for the control of seed dormancy and transpiration in seed plants. Ferns are shown to lack downstream functionality of stomatal components, suggesting that the origins of the core ABA signaling pathway in seed plants may lie in the sexual differentiation of ferns.

Abstract

Sexual reproduction in animals and plants shares common elements, including sperm and egg production, but unlike animals, little is known about the regulatory pathways that determine the sex of plants. Here we use mutants and gene silencing in a fern species to identify a core regulatory mechanism in plant sexual differentiation. A key player in fern sex differentiation is the phytohormone abscisic acid (ABA), which regulates the sex ratio of male to hermaphrodite tissues during the reproductive cycle. Our analysis shows that in the fern Ceratopteris richardii, a gene homologous to core ABA transduction genes in flowering plants [SNF1-related kinase2s (SnRK2s)] is primarily responsible for the hormonal control of sex determination. Furthermore, we provide evidence that this ABA–SnRK2 signaling pathway has transitioned from determining the sex of ferns to controlling seed dormancy in the earliest seed plants before being co-opted to control transpiration and CO₂ exchange in derived seed plants. By tracing the evolutionary history of this ABA signaling pathway from plant reproduction through to its role in the global regulation of plant–atmosphere gas exchange during the last 450 million years, we highlight the extraordinary effect of the ABA–SnRK2 signaling pathway in plant evolution and vegetation function.

The phytohormone abscisic acid (ABA) plays a critical role in everyday plant function by translating the hydration status of plant tissue into a chemical signal that can activate metabolic responses (1). Two prominent processes regulated by ABA signaling in seed plants are highly distinct in terms of target tissue and physiology: one involving seed dormancy (2) and the other the regulation of leaf transpiration (3). In seeds, the ability to delay germination until conditions are suitable for growth is controlled by an antagonism between ABA (which promotes dormancy) and gibberellin (GA; which breaks dormancy and promotes germination) (2). In leaves, ABA regulates transpiration by activating anion channels in the guard cells on either side of tiny pores on the leaf surface (stomata), causing cell turgor loss and pore closure (4, 5). Despite involving distinct target organs, both processes share components of a common ABA-signaling cascade, including the ABA receptor complex, comprised of PYRABACTIN RESISTANCE1 (PYR1), PYR1-like (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) proteins, TYPE 2 PROTEIN PHOSPHATASES (PP2Cs), and members of the OPEN STOMATA1 (OST1) subclade of SNF1-related kinase2 (SnRK2) kinases (4, 6–9). Characterizations of ABA-deficient and ABA-insensitive mutants across a diversity of angiosperm species have confirmed the universal involvement of this ABA-signaling cascade for stomatal control and seed dormancy within the angiosperm clade (7, 9, 10). However, an absence of available mutants in nonangiosperm vascular plants has led to uncertainty about the evolution of this critical signaling pathway and the genes involved (cf refs. 11 and 12).

To provide insight into the question of how ABA signaling evolved in seed plants, we used the model fern species, Ceratopteris richardii, as a representative member of a plant lineage basal to the seed plants (13). Our specific aim was to determine the role of the ABA–SnRK2 signaling pathway in plants that evolved before seeds as a means of understanding the evolution of key SnRK2-related ABA signaling processes in vascular plants. It is known that ABA and SnRK2s are both present in early land plants, yet the only function clearly associated with ABA action in ferns involves sex determination of the free-living, haploid gametophyte generation of C. richardii (14). In many fern species, antheridiogen [a modified form of GA that is converted to bioactive GA₄ once imported into fern cells (15)] is secreted by hermaphroditic gametophytes, causing their immature neighbors to develop as males (14, 15). Experiments with C. richardii demonstrate that exogenous ABA completely blocks the sex-determining effect of antheridiogen, such that no male gametophytes develop when grown in the presence of ABA and the antheridiogen of C. richardii (A_CE) (16). The strong similarity between the antagonism of ABA and A_CE observed in C. richardii (16), and the ABA-GA antagonism known to regulate seed dormancy in angiosperms (2), raises an interesting possibility that the ABA-GA signaling system in ferns may have been co-opted to control seed dormancy in the earliest seed plants.

We sought to investigate the genetic ancestry of ABA signaling in the vascular plant lineage, using both forward and reverse genetic approaches to target core ABA-signaling genes in the fern C. richardii. We use ABA sensitivity mutants (17) to demonstrate how evolution has altered the downstream targets of ABA signaling while preserving the core components of the transduction pathway virtually unchanged.

Results and Discussion

We identified five ABA-insensitive mutant alleles in a mutagenesis screen of C. richardii (SI Appendix, Table S1). Unlike wild-type plants, gametophytes of these mutants are unable to perceive ABA, and thus do not develop as hermaphrodites when grown on media containing both A_CE and ABA (SI Appendix, Fig. S1). Further analysis revealed that these mutant alleles represent four independent loci, which we named GAMETOPHYTES ABA INSENSITIVE ON A_CE1 (GAIA1; three alleles), GAIA2 (two alleles), and GAIA3 and GAIA4 (one allele each), accordingly (SI Appendix, Table S1). The identity of these loci was further investigated, using our Trinity RNA-Seq de novo transcriptome assembly for C. richardii to screen homologs of ABA-signaling genes characterized in angiosperms as possible candidates.

SnRK2s and Sex Determination.

Through our analysis, we identified a C. richardii homolog within the OST1 subclade of SnRK2s (SI Appendix, Fig. S2) that was significantly altered in each of the three gaia1 mutant alleles relative to wild-type (Fig. 1A and SI Appendix, Fig. S3). In the gaia1-1 mutant, an A217G transition affects an invariant residue within a region of the kinase domain that is important for protein conformation and kinase activity (17). In the gaia1-2 mutant, a duplication of 134 bp affects the terminal portion of the ABA box, a domain that is conserved among members of the strongly ABA-responsive OST1 subclade and is necessary and sufficient for target protein binding (18). No portion of this gene could be isolated from gaia1-3, using PCR-based techniques (SI Appendix, Fig. S3), suggesting a significant deletion or rearrangement. On the basis of these distinct, functionally significant mutations, we concluded that this gene corresponds to the GAIA1 locus and named the gene GAIA1, accordingly. To confirm that these mutations were the cause of the ABA-insensitive phenotype, we silenced the expression of GAIA1 in wild-type gametophytes by RNA interference (RNAi) (19). Unlike wild-type gametophytes expressing GAIA1, gametophytes in which GAIA1 expression was silenced were insensitive to ABA, not switching to hermaphrodites (small meristics) when grown on media containing both ABA and A_CE, but developing only as males (Fig. 1C and SI Appendix, Fig. S4). ABA insensitivity in the gaia1 mutants or wild-type plants in which GAIA1 expression is silenced is not surprising, given the well-documented ABA-insensitive phenotypes described in all angiosperm mutants of OST1 subclade genes characterized so far (6, 7). By characterizing the GAIA1 gene, we provide very strong evidence that the ABA–SnRK2 signaling pathway in C. richardii is engaged in the regulation of sex determination of the fern gametophytes.

Fig. 1.

GAIA1, a C. richardii homolog of OST1, regulates ABA signaling for gametophyte sex determination. (A) C. richardii sporophyte. (B) Gametophyte phenotype for wild-type (Hn-n) and gaia1 mutant alleles grown on media containing A_CE and 10 µM ABA, with diagrams showing GAIA1 gene and predicted protein structure for each allele, indicating the nature of mutations. No portion of GAIA1 could be isolated from gaia1-3 (SI Appendix, Fig. S3). (Scale bars, 200 µm, 100 bp, or 10 aa; gene/protein diagrams share the same scale.) (C) Phenotypes of three representative gametophytes bombarded with only 35S::DsRed2 acting as controls and three GAIA1 RNAi gametophytes cobombarded with both 35S::DsRed2 and 35S::hpGAIA1. Knocking down the expression of GAIA1 prevents the development of a new hermaphroditic prothallus (indicated by arrows in the controls) when plants are transferred from media containing only A_CE to media containing both A_CE and 5 µM ABA. (Scale bars, 200 µm.) (D) Phylogram of C. richardii (Cr), Oryza sativa (Os), and A. thaliana (At) SnRK2-type protein kinases, with Vicia faba (Vf) AAPK also included. The OST1-type subgroup is shaded in blue with the names of ABA-signaling proteins associated with stomatal control in blue text, proteins acting in seed dormancy in green, and proteins linked to both processes in blue and underlined in green. CrGAIA1 is shown in red. Branches with bootstrap values <50% obtained from 1,000 trees have been collapsed. Representative Arabidopsis SnRK1 and SnRK3 proteins are included as outgroups. Phylogenetic analysis is based on the sequence alignment in SI Appendix, Dataset. For sequence details, see SI Appendix, Dataset. (E) Alignments showing residues in the αC-helix of the kinase domain and ABA box conserved among OST1 homologs and between SnRK2 proteins. Shading indicates degree of conservation (black = 60%, dark gray = 50%, and light gray = 40%, yellow = residues affected in gaia1 mutants). Protein names are colored as in D.

SnRK2s and Spore Germination.

The phylogenetic position of GAIA1 within the ABA-signaling SnRK2s and the strong A_CE–ABA interaction associated with this C. richardii signaling pathway suggests a common ancestry with the ABA-signaling pathway that was used, possibly as early as 300 million years ago, to regulate seed dormancy in early seed plants (8, 20). This hypothesis is supported by the fact that gaia1 mutants showed reduced sensitivity to ABA in terms of spore germination relative to wild-type plants (Fig. 2A). The high levels of ABA that we found are required to inhibit spore germination in C. richardii correspond to very high endogenous levels of ABA present in fresh spores, levels that decline rapidly upon germination (SI Appendix, Fig. S5). Interaction between ABA and A_CE to control spore germination was also observed, with the double-mutant of gaia1-3 and the A_CE-insensitive her3 mutant (21) found to restore a wild-type germination sensitivity to ABA (Fig. 2A). The recent discovery of the modulatory effect of ABA and diterpenes (GA precursors) on spore germination in the moss Physcomitrella patens (22) is intriguing, because if this process in mosses is also regulated by an SnRK2, then propagule dormancy may be the ancestral function of this core ABA signaling pathway in land plants.

Fig. 2.

The germination of ABA-insensitive gaia1 mutant spores is less inhibited by ABA compared with wild-type, whereas stomatal responses to changes in leaf water status are normal. (A) The percentage of spores germinating when sown on media containing increasing concentrations of ABA relative to the percentage of spores germinating without ABA, including the double-mutant gaia1-3 and A_CE-insensitive her3 mutant (n = 3). (B) Mean response of stomatal conductance (n = 3) to a reversible step change in VPD. Change in VPD is denoted by vertical lines; data during equilibration of the gas exchange chamber after the VPD change have been removed. (C) Mean response of stomatal conductance (n = 3, ±SE) to leaf excision. The vertical line denotes leaf excision.

SnRK2s and Stomatal Control.

To gain further insight into the evolution of SnRK2-related ABA signaling, we examined the possibility that stomatal closure, another major ABA-regulated process in seed plants that operates via SnRK2s, might also be dysfunctional in the ABA-insensitive gaia1 mutant plants. Declining leaf water content in seed plants is known to result in rapid ABA synthesis, triggering stomatal closure via an SnRK2-mediated activation of S-type anion channels (SLACs) (4). Examination of stomatal responses to humidity and desiccation in C. richardii wild-type and gaia1 mutant plants revealed identical behavior in all genotypes, whereby stomata closed relatively quickly in response to either a reduction in atmospheric water vapor content or leaf drying (Fig. 2 B and C). Thus, we can conclude that the OST1 subclade gene GAIA1 is not involved in ABA signaling for stomatal closure in C. richardii.

Angiosperm SnRK2s that function in ABA-dependent stomatal control require guard cell-specific expression patterns in leaves (6, 8). We examined the localization of expression of GAIA1 and the other C. richardii SnRK2 gene that fell within the OST1 subclade associated with ABA signaling in seed plants (SI Appendix, Fig. S2), PARALOG OF GAIA1 (PGAI). Unlike the expression of stomatal-associated OST1 subclade genes in angiosperms, neither PGAI nor GAIA1 show guard cell specificity in expression but, rather, these genes are expressed in all tissues (SI Appendix, Fig. S6).

A second, more specific test of the functionality of the SnRK2 pathway for stomatal ABA signaling in C. richardii was conducted to confirm an apparent lack of functionality suggested by nonspecific gene expression. We tested the ability of the fern SnRK2s to activate the SLACs responsible for anion efflux during ABA-driven stomatal closure (4). We examined the ability of GAIA1 and PGAI kinases to activate SLACs from both the fern and Arabidopsis (SLAC1; Fig. 3 and SI Appendix, Fig. S9). Using a Xenopus oocyte expression system, together with the two-electrode voltage-clamp technique and bimolecular fluorescence complementation (BiFC), we found that both of the native C. richardii OST1 subclade SnRK2s were able to physically interact with and activate Arabidopsis SLAC1 (Fig. 3 and SI Appendix, Fig. S8). These findings are similar to SnRK2s from other basal nonvascular species, such as algae, liverworts, and mosses (23). A different result, however, was observed when the action of C. richardii OST1 subclade SnRK2s was tested using native C. richardii SLACs. Although both PGAI and GAIA1 were able to interact with native C. richardii SLACs (SI Appendix, Fig. S8), coexpression of these native SnRK2-SLAC pairs yielded weak or immeasurable macroscopic anion channel currents (Fig. 3 and SI Appendix, Fig. S9). Our data from C. richardii suggest that unlike all angiosperm species examined thus far, the ABA-signaling pathway for stomatal closure through SnRK2-mediated SLAC activation does not appear to be operating in the fern C. richardii.

Fig. 3.

Fern and lycophyte SnRK2s are unable to activate native S-type anion channels. Mean whole-oocyte current measurements at −100 mV in chloride-based standard medium of S. moellendorffii, C. richardii, and A. thaliana SLACs coexpressed with OST1 subclade SnRK2s in Xenopus oocytes (n ≥ 4, ±SE). Shown below are example whole-oocyte currents of representative SLACs either expressed with a native OST1 subclade SnRK2 or, in the case of S. moellendorffii, SLAC1b alone. For data in nitrate-based standard medium (SI Appendix, Fig. S9), all SnRK2 proteins physically interacted with SmSLACs, except SmSLAC1d (SI Appendix, Fig. S8). SmSLAC1a was represented by two allelic variants: SLAC1a.1 and SLAC1b.1.

A recent report of activation of a moss (P. patens) SLAC protein by a native SnRK2, albeit weakly, could suggest that the evolution of ABA-SnRK2-SLAC signaling for stomatal closure predates the divergence of ferns (23), and that the absence of native anion channel activation by SnRK2s in C. richardii represents a unique loss, similar to the loss of a stomatal response to blue light in this lineage (24). To address this possibility, we examined the functionality of SnRK2–SLAC combinations in a representative of the lycophyte clade, the earliest diverging clade of vascular plants. Thus, all native SnRK2-SLAC combinations in a sequenced representative of the lycophyte clade (Selaginella moellendorffii) were identified. The S. moellendorffii genome has three OST1 subclade SnRK2s and four SLAC1 homologs. We found that the native S. moellendorffii OST1 subclade SnRK2s were able to interact with all but one of the SLACs from S. moellendorffii (SI Appendix, Fig. S8), as well as Arabidopsis SLAC1. However, similar to the situation in C. richardii, none of the S. moellendorffii OST1 subclade SnRK2s were found to activate native S. moellendorffii SLACs (Fig. 3). Only one S. moellendorffii SLAC protein was capable of conducting anions, but this activity was not further induced by coexpression with SnRK2s (SI Appendix, Fig. S10). Thus, we conclude that the elements necessary for SnRK2 activation of anion channels are not present in the most basal vascular plant lineage. That the two most basal vascular land plant clades lack an SnRK2 activation of endogenous SLACs provides strong evidence for the conclusion that this ABA signaling pathway for stomatal closure during water stress was absent in the common ancestor of all vascular land plants (Fig. 3) and remains absent in extant ferns and lycophytes.

It is possible that some form of ancestral ABA signaling via SnRK2s may have evolved in mosses, as proposed by Lind et al. (23), before being subsequently lost in the earliest vascular land plants and reemerging in seed plants, but this seems an improbable scenario. Several pieces of evidence suggest that ABA-mediated stomatal closure reported in the moss species P. patens is not analogous to that observed in seed plants. The first is that P. patens does not exhibit the guard cell-specific expression of an SnRK2 (22, 25), which is required for a stomatal response to ABA (SI Appendix, Fig. S6) (26). Questions about a lack of functional homology between the stomata of bryophytes and vascular plants (27, 28) arise; given the recent revelation that mosses are sister to liverworts (29). The high number of losses, or possible gains, of stomata across bryophyte lineages (27) make any interpretation about the function of the first land plant stomata from species in a single moss family difficult.

Conclusion

The linkage established here between ABA perception and SnRK2s in ferns provides direct evidence that this pathway, which is critical for the regulation of seed dormancy, stomatal aperture, and other dehydration responsive processes (e.g., dehydrin induction) (30) in seed plants, extends back at least 360 million years to the emergence of ferns. We propose that the origins of the ABA signaling pathway critical for regulating seed dormancy are present in the fern lineage that evolved 60 million years before the first fossil evidence of seed dormancy (20). This signaling system in ferns primarily provides a means to regulate the degree of outbreeding in populations by modifying the sex ratio of the gametophyte reproductive stage (31). The connection between ABA and stomatal control via the specific activation of transmembrane anions channels (4) appears to be a more recent innovation that did not evolve until after the divergence of ferns and seed plants (Fig. 4). This latest engagement of the ABA signaling pathway for stomatal closure occurs in the earliest seed plants and, through their success, has become one of the most influential signaling pathways on Earth.

Fig. 4.

Reconstructed evolution of the interactions between A_CE/GA (yellow), ABA (red), SnRK2s (green), and S-type anion channels (blue). Precursors of GA and ABA interact to modulate spore dormancy in mosses (22). It is not yet known whether an SnRK2 is involved in this process (dotted green line), or indeed whether these hormones and signaling pathways influence phenotypes in the most basal extant vascular plant lineage, the lycophytes (dotted lines). SnRK2s signal the A_CE–ABA regulatory antagonism of both gametophyte sex determination, as well as spore dormancy in ferns; this signaling interaction was later adopted to regulate seed dormancy in the earliest seed plants; however, it may persist in regulating sex determination in angiosperms (43). Seed plants were the first group of land plants to evolve ABA signaling through SnRK2s and S-type anion channels to regulate stomatal behavior.

Methods

Mutant Isolation.

Hn-n, the wild-type strain of C. richardii used in this study, is described by Hickok et al. (32). The antheridiogen (A_CE)-insensitive hermaphroditic (her) mutants are described by Banks (21), and the two ABA-insensitive mutants HαA48 and HαA104 (referred to here as gaia1-3 and gaia4, respectively) are described by Hickok (33). Gametophyte and sporophyte growth conditions, ethyl methyl sulfate mutagenesis, and crosses were performed according to the methods of Banks (21). To identify new gaia mutants that are insensitive to ABA, 10⁶ her13 or wild-type spores were mutagenized with ethyl methyl sulfate and plated on medium containing A_CE and 10 µM ABA. gaia mutants were selected as large hermaphrodites or male gametophytes from the ethyl methyl sulfate mutagenized populations after 21 d of growth. When grown on the same media, wild-type and her13 spores develop as small gametophytes with a multicellular meristem and no antheridia, and are referred to as small meristics (SI Appendix, Fig. S1). Of the 23 putative gaia her13 hermaphrodites selected and crossed by wild-type sperm, three (gaia1-1, gaia1-2, and gaia2-1) produced sporophytes with a gametophyte progeny that segregated large hermaphrodites (her13 gaia), small meristics (HER13 GAIA), and males (HER13 gaia) in a 1:2:1 ratio on medium containing A_CE and 10 µM ABA, indicating segregation of the mutant phenotype as a single Mendelian trait, independent of her13 (SI Appendix, Table S1). When two males (gaia2-2 and gaia4) selected from the mutagenized wild-type population of spores were switched to hermaphrodites and crossed by wild-type sperm, these produced sporophytes with a gametophyte progeny that segregated small meristics (GAIA) and males (gaia) in a 1:1 ratio on a medium containing A_CE and 10 µM ABA, also indicating that these gaia phenotypes segregate as single Mendelian traits (SI Appendix, Table S1). All seven gaia mutants develop as normal hermaphrodites in the absence of A_CE.

To determine the linkage relationships among the gaia mutants, each mutant was crossed to gametophytes of all other gaia mutants. When grown in the presence of A_CE and 10 µM ABA, segregation of the gametophyte progeny derived from these crosses as 3:1 males:small meristics indicated that the two gaia mutations were not linked, whereas all male segregants indicated loci were tightly linked and likely allelic. In some crosses, a hermaphroditic her gaia double mutant was used as the female donor, and in these instances, the expected ratio of progeny grown in the presence of A_CE and 10 µM ABA was 3:3:2 normal hermaphrodites:males:small meristics if the gaia mutations were not linked; or 1:1 male:hermaphrodite if the gaia mutations were completely linked. Linkage analysis revealed that the seven gaia mutants fall into four linkage groups: gaia1 (gaia1-1, gaia1-2, and gaia1-3), gaia2 (gaia2-1 and gaia2-2), gaia3, and gaia4 (SI Appendix, Table S1).

Gene Silencing by RNA Interference.

Eight-day-old Hn-n (wild-type) gametophytes grown on media containing A_CE were bombarded using a PDS 100 Helium System (BioRad), as described by Rutherford et al. (19), with one exception: 1.3 mM tungsten M-20 microcarriers (BioRad) were coated with plasmids purified using a NucleoBond Xtra Midi Plus kit (Macherey-Nagel). Gametophytes were bombarded with a 35S::DsRed2 plasmid (described in ref. 34) or cobombarded with the 35S::DsRed2 plasmid plus the hairpin-forming GAIA1 mRNA plasmid (called 35S::hpGAIA1). 35S::hpGAIA1 was made by amplifying two 300-bp PCR GAIA1 fragments from cDNA, using primer pairs listed in SI Appendix, Dataset, and by amplifying the Ricinus communis Catalase Intron 1 from the 35S:irint (19). These three fragments were then cloned into pFF19 (35) between the 35S promoter and poly(A)⁺ addition site, using the In-Fusion HD Cloning Plus CE kit (Clontech). To test the efficiency of cobombardment and RNAi, gametophytes were cobombarded with 35S::DsRed2 plus 35S::irintCrChl (19), which targets the C. richardii Protoporphyrin IX magnesium chelatase gene required for chlorophyll biosynthesis (SI Appendix, Fig. S4). Targeting this gene by RNAi results in colorless gametophytes (19). One day after bombardment, gametophytes with a DsRed2 fluorescent cell were transferred to A_CE-containing plates. One day later, one-half of the gametophytes were transferred to A_CE (no ABA) and the other half to A_CE and 5 µM ABA plates that were then scored and photographed 11 d after bombardment.

Phylogenetic Analysis.

Full-length coding sequences for C. richardii SnRK2 and SLAC homologs were identified by tBLASTn search, using Arabidopsis thaliana OST1 and SLAC1 and SLAH1-4 protein sequences, respectively, as queries against an unpublished Trinity RNA-Seq de novo transcriptome assembly (36) generated from gametophyte tissue. Gene identity was confirmed by reciprocal BLAST searches against A. thaliana at TAIR (www.arabidopsis.org). Genes from other species were identified from publicly available databases, as outlined in SI Appendix, Dataset. For full sequence details, see SI Appendix, Dataset. For each alignment (SI Appendix, Dataset), full-length amino acid sequence was aligned using ClustalX Version 2.7.000; distance-based methods were used for phylogenetic analyses in PAUP* 4.0b10 (paup.csit.fsu.edu/).

qRT-PCR.

To measure the expression of genes of interest in wild-type C. richardii plants across life-history stages and sporophyte tissues, spores were harvested after 24 h of imbibing in water, gametophytes after 14 d of growth in liquid medium, crozier tissue was taken from the youngest still unfurled primordial leaf, vegetative leaf tissue was taken from the newest most fully expanded leaves without sporangia, reproductive leaf tissue was taken from the newest most fully expanded leaves with sporangia, and roots were harvested from aerial asexual propagules. Total RNA was extracted using the Agilent Plant RNA Isolation Mini Kit (Agilent Technologies), and RNA concentrations determined using a NanoDrop 8000 (Thermo Scientific). Reverse transcription was conducted in 20 µL with 1 µg total RNA, using the Tetro cDNA synthesis kit (Bioline) according to the manufacturer’s instructions. RT-negative (no enzyme) controls were performed to monitor for contamination. First-strand cDNA was diluted five times, and 2 µL was used in each real-time PCR. Reactions using SYBR green chemistry (SensiFAST; Bioline) were set up with a CAS-1200N robotic liquid handling system (Corbett Research) and run for 50 cycles in a Rotor-Gene Q (Qiagen). Two technical replicates and at least two biological replicates were performed for each tissue type. All primer details are given in SI Appendix, Dataset. Expression of each gene of interest was examined relative to the BestKeeper index calculated from three reference genes evaluated using Bestkeeper and geNorm, and found to be stably expressed in these samples (CrAPT, M = 0.657; CrUBC9, M = 0.661; CrTBP, M = 0.601), using previously outlined methods (37–39).

Leaf Gas Exchange.

Stomatal responses to a reversible, mild transition in vapor pressure deficit (VPD) and after leaf excision were assessed in the most recent fully expanded nonreproductive leaves of wild-type and gaia1 mutant plants, using a portable infrared gas analyzer (Li-6400; LI-COR Biosciences). Measurements were conducted on one leaf each from three potted plants of WT and gaia1-1, gaia1-2, and gaia1-3 individuals grown under controlled glasshouse conditions and shaded natural light, supplemented and extended to a 16-h photoperiod by sodium vapor lamps, ensuring a minimum 150 µmol quanta m⁻²⋅s⁻¹ at the pot surface and 23 °C/16 °C day/night temperatures. Environmental conditions in the cuvette of the gas analyzer were controlled for the duration of the experiment at an air temperature of 23 °C, light intensity of 500 µmol quanta m⁻²⋅s⁻¹, and VPD regulated initially at 1 kPa, using a portable dewpoint generator (Li-610; LI-COR Biosciences). Leaf gas exchange and cuvette conditions were logged every 30 s. Leaves were allowed to equilibrate to the conditions inside the cuvette until leaf gas exchange had reached a maximum and stabilized. After stabilization, VPD was increased to 1.5 kPa and maintained for 20 min, after which it was returned to 1 kPa for a further 20 min. Once the VPD transition was complete and leaf gas exchange had again stabilized for at least 5 min, leaf tissue inside the cuvette was excised, and leaf gas exchange was monitored for a further 20 min.

Inhibition of Spore Germination by ABA.

Approximately 2,000 spores of wild-type, gaia1 and gaia1-3 her3 double mutants were plated on standard medium containing both A_CE and 0, 10 20, 50, 100, and 200 µM ABA and grown for 14 d, according to the methods of Banks (21). After 14 d, the numbers of germinated and nongerminated spores per plate were scored. For each genotype, the percentage of spores that germinated at each increasing concentration of ABA was calculated relative to the percentage of spores that germinated on media containing 0 µM ABA.

CRNA Generation.

Full-length coding sequence from OST1 and SLAC homologs of S. moellendorffii, C. richardii, and A. thaliana were isolated, using primers outlined in SI Appendix, Table S3, and cloned into pNB1uYN and pNB1uYC expression vectors by uracil excision–based cloning (40). For functional analysis in oocytes, cRNA was prepared with the mMESSAGE mMACHINE T7 transcription kit (Ambion). Oocyte preparation and cRNA injection were performed as described by Becker et al. (41). For oocyte BiFC and electrophysiological experiments, 10 ng SLAC:YFP^CT (vector pNB1uYC) and 10 ng OST1-homolog:YFP^NT (vector pNB1uYN) cRNA were injected.

Oocyte Recordings.

In two-electrode voltage-clamp studies, oocytes were perfused with Tris/Mes buffers. The standard solution contained 10 mM Tris/Mes (pH 5.6), 1 mM Ca (gluconate)₂, 1 mM Mg (gluconate)₂, 50 mM NaCl or NaNO₃⁻, and 1 mM LaCl₃. Osmolality was adjusted to 220 mosmol kg⁻¹ with d-sorbitol. The standard voltage protocol was as follows: starting from a holding potential (V_H) of 0 mV, 50 ms single-voltage pulses were applied in 20 mV decrements from +70 to −150 mV. Instantaneous currents were extracted right after the voltage jump from the holding potential of 0 mV to the test pulses.

BiFC Experiments.

Expression of BiFC constructs in oocytes was performed as described by Geiger et al. (4). For documentation of the oocyte BiFC results, images were taken with a confocal laser scanning microscope (Leica DM6000 CS; Leica Microsystems CMS GmbH) equipped with a Leica HCX IRAPO L25×/0.95W objective. Images were processed (low-pass filtered and sharpened) identically with the image acquisition software LAS AF (Leica Microsystems CMS GmbH).

Quantification of ABA Levels in Spores and Leaves.

Germinating spores or gametophytes were filtered from liquid culture media, or leaf material from unstressed plants was harvested and immediately weighed (±0.001 g), covered in cold (−20 °C) 80% methanol in water (vol⋅vol⁻¹) with 250 g⋅L⁻¹ (m⋅v⁻¹) of added butylated hydroxytoluene and transferred to −20 °C. ABA then was extracted from tissue at room temperature, and 15 ng [²H₆]ABA (National Research Council of Canada) was added to each sample, as an internal standard. ABA was purified by ether partitioning and quantified by ultraperformance liquid chromatography tandem mass spectrometry, according to the methods of ref. 42.