Adaptation to dehydration stress requires plants to coordinate environmental and endogenous signals to inhibit stomatal proliferation and modulate their patterning. The stress hormone abscisic acid (ABA) induces stomatal closure and restricts stomatal lineage to promote stress tolerance. Here, we report that mutants with reduced ABA levels, xer-1, xer-2 and aba2-2, developed stomatal clusters. Similarly, the ABA signaling mutant snrk2.2/2.3/2.6, which lacks core ABA signaling kinases, also displayed stomatal clusters. Exposure to ABA or inhibition of ABA catabolism rescued the increased stomatal density and spacing defects observed in xer and aba2-2, suggesting that basal ABA is required for correct stomatal density and spacing. xer-1 and aba2-2 displayed reduced expression of EPF1 and EPF2, and enhanced expression of SPCH and MUTE. Furthermore, ABA suppressed elevated SPCH and MUTE expression in epf2-1 and epf1-1, and partially rescued epf2-1 stomatal index and epf1-1 clustering defects. Genetic analysis demonstrated that XER acts upstream of the EPF2-SPCH pathway to suppress stomatal proliferation, and in parallel with EPF1 to ensure correct stomatal spacing. These results show that basal ABA and functional ABA signaling are required to fine-tune stomatal density and patterning.

Stomata represent a specialized epidermal lineage within land plants that requires a suite of regulators to adequately arise, proliferate and enforce pattern. In Arabidopsis, the stomatal lineage initiates from an undifferentiated protodermal cell called a meristemoid mother cell (MMC), which undergoes an entry asymmetric cell division (ACD) to give rise to a small stomatal precursor cell (meristemoid) and a large stomatal lineage ground cell (SLGC). Cell identity is maintained and directed through the sequential expression of three basic helix loop helix (bHLH) transcription factors, SPEECHLESS (SPCH), MUTE and FAMA (Ohashi-Ito and Bergmann, 2006; MacAlister et al., 2007; Pillitteri et al., 2007). As SPCH-expressing meristemoids can maintain an undifferentiated status, they continue to divide asymmetrically to properly pattern and distribute along the epidermis by undergoing successive amplifying and spacing divisions (Lau et al., 2014). Additionally, SLGCs may also undergo asymmetric divisions to produce satellite meristemoids, and thereby further increase stomatal proliferation. Divisions of meristemoids and SLGCs are coordinated through positional cues to orient meristemoids away from existing precursors and stomata (Rowe and Bergmann, 2010). This process of oriented asymmetric divisions ensures that stomata are separated by at least one non-stomatal lineage cell, preventing the formation of stomatal clusters that can affect regulation of pore closure (Geisler et al., 2000; Robinson et al., 2011).

To enforce spatial patterning, a ligand–receptor signaling pathway inhibits entry into the stomatal lineage and orients new asymmetric divisions. The signaling cascade is initiated by the secretion of EPIDERMAL PATTERNING FACTORs (EPFs) from stomatal lineage cells, which are perceived by the cell-surface receptor-like kinase family ERECTAs (ERs) and the co-receptor-like protein TOO MANY MOUTHs (TMM) (Nadeau and Sack, 2002; Shpak et al., 2005; Hara et al., 2007, 2009; Hunt and Gray, 2009). This binding facilitates intracellular activation of a mitogen-activated protein kinase (MAPK) cascade to repress stomatal fate via phosphorylation of SPCH, which is required to initiate stomatal lineage (Bergmann et al., 2004; Wang et al., 2007). Within the EPF family, two stomatal lineage-specific ligands, EPF1 and EPF2, coordinate cell–cell signaling in their respective ligand–receptor pairs to maintain adequate stomatal number and spacing by blocking entry into the stomatal fate (Lee et al., 2012, 2015). Various upstream environmental signals converge onto EPF1/2 to further optimize stomatal density and patterning (Engineer et al., 2014; Hronková et al., 2015). Enhanced EPF1/2 expression in Arabidopsis, poplar and barley cultivars results in decreased stomatal density that improves plants tolerance to drought, thereby establishing potential mechanisms by which extrinsic cues may guide stomatal development for environmental adaption (Hepworth et al., 2015; Wang et al., 2016; Hughes et al., 2017).

Hormones modulate stomatal density and distribution by integrating environmental cues into the stomatal signaling module (Qi and Torii, 2018; Lee and Bergmann, 2019; Wei et al., 2021). Particularly, the phytohormone abscisic acid (ABA) broadly regulates a variety of stomatal functions, including stomatal physiology and development (Brookbank et al., 2021). Genetic analysis in Arabidopsis has shown that ABA-deficient mutants (aba, bg, nced, xer) and insensitive mutants (abi1, abi2 and snrk2) display increased stomatal index (Tanaka et al., 2013; Allen et al., 2019; Vonapartis et al., 2022; Yang et al., 2022). In contrast, ABA over-accumulating mutants (cyp707a), show reduced stomatal index (Tanaka et al., 2013). Thus, adequate ABA levels and a functional ABA signaling pathway are necessary for the inhibition of stomatal development. Genetic interaction studies have revealed that ABA restricts divisions of stomatal precursor cells upstream of SPCH and MUTE during epidermal development (Tanaka et al., 2013). ABA decreases stomatal density via transcriptional repression of SPCH and MUTE (Tanaka et al., 2013). Recently, SnRK2.2/2.3/2.6 kinases, which are positive regulators of the core ABA signaling pathway, were shown to inhibit initiation and proliferation of stomatal precursors by directly phosphorylating SPCH (Yang et al., 2022). Additionally, overexpression of the ABA-responsive transcription factor HOMEODOMAIN GLABROUS11 (HDG11) promotes ABA accumulation, and is also an activator of ERECTA, indicating multiple mechanisms by which ABA affects stomatal development (Yu et al., 2013; Guo et al., 2019).

Although some environmental factors capable of influencing stomatal development have been identified, the genetic and molecular mechanisms by which they affect progression through stomatal lineage is not well characterized. The stress-responsive gene XERICO (XER) encodes for a RING E3 ubiquitin ligase that is conserved across monocot and dicot plants (Ko et al., 2006). Analysis of XER overexpression lines (35S:XER) revealed that XER increases ABA levels by reducing ABA catabolism through destabilization of the ABA catabolic enzyme ABA 8′-hydroxylase, encoded by the CYP707A gene family, thus promoting drought stress tolerance (Ko et al., 2006; Zentella et al., 2007; Brugière et al., 2017). The enhanced drought tolerance exhibited by 35S:XER plants was the result of elevated ABA and improved stomatal closure following stress (Ko et al., 2006; Zeng et al., 2015). In contrast, xer mutants showed decreased ABA levels and increased stress sensitivity (Zentella et al., 2007; Vonapartis et al., 2022). Furthermore, xer mutants exhibited increased stomatal number in Arabidopsis, indicating that XER also inhibits stomatal development aside from promoting stomatal closure (Vonapartis et al., 2022).

Here, we further characterized the role of ABA in stomatal development. We showed that mutants with reduced ABA levels, xer and aba2-2, exhibited stomatal clusters. The stomatal density and clustering defects of aba2-2 and xer could be rescued by exogenous ABA or by inhibiting ABA catabolism. The snrk2.2/2.3/2.6 mutant also displayed stomatal clustering. These findings suggest a role for basal ABA and a functional ABA signaling pathway in regulating stomatal distribution during epidermal development, aside from controlling entry into the stomatal development pathway. Mechanistically, ABA promotes EPF1/2 expression, and represses SPCH and MUTE. Genetic interaction studies showed that XER acts upstream of EPF2-SPCH to restrict stomatal density, and works in parallel with EPF1 to ensure proper stomatal spacing. Our findings indicate that basal ABA ensures proper stomatal patterning during leaf epidermal development by acting through the canonical EPF-SPCH stomatal pathway, as well as through independent pathways.

Loss of XER results in stomatal patterning defects

Previously, xer-1 and xer-2 mutant alleles were shown to display an elevated stomatal index (SI), suggesting that XER is required to restrict stomatal development (Vonapartis et al., 2022). A careful phenotypic analysis of 20-day-old cotyledons revealed that xer mutants also exhibit defective stomatal distribution, resulting in clustering of stomata (Fig. 1A-C). We quantified the percentage of stomatal clusters (defined as at least two stomata in contact) on the abaxial surface of cotyledons. Both xer mutant alleles showed a mild increase in stomatal clusters relative to wild type (WT) (Fig. 1C). Overexpression of XER (35Sp:XER-GFP) was sufficient to rescue the increased stomatal density and patterning defects of xer-1 (Fig. 1B,C; Fig. S1). This indicates that loss of XER results in improper patterning of stomatal lineage cells, and suggests that XER is important for maintaining proper stomatal distribution during epidermal development.

Fig. 1.

XERICO controls stomatal distribution in the developing epidermis. (A) Representative images of the epidermis of wild-type (WT), xer-1, xer-2 and 35S:XER-eGFP in xer-1. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustered stomata. Images are representative of n=10 samples. Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) quantified across n=10 cotyledons per genotype. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

Fig. 1.

XERICO controls stomatal distribution in the developing epidermis. (A) Representative images of the epidermis of wild-type (WT), xer-1, xer-2 and 35S:XER-eGFP in xer-1. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustered stomata. Images are representative of n=10 samples. Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) quantified across n=10 cotyledons per genotype. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

XER is required for proper stomatal spacing

During stomatal development, aberrant amplifying divisions can result in the development of two adjacent daughter cells with meristemoid-like fate (paired meristemoids), and misoriented spacing divisions can lead to stomatal precursors developing in contact with existing stomata (Gong et al., 2021). XER is expressed in stomatal lineage cells, suggesting a role regulating stomatal development (Fig. S2; Adrian et al., 2015; Vonapartis et al., 2022).

To determine how and when XER affects entry into the stomatal lineage, we monitored the expression pattern of the stomatal lineage marker SPCHp:SPCH-YFP, introduced in xer-1 (Lopez-Anido et al., 2021). First, we observed that at 4 days post-germination (DPG) xer-1 exhibits an increased number of stomatal precursors expressing SPCH, indicating that a higher number of epidermal cells progress into the stomatal lineage (Fig. 2A). Additionally, we found that TMM expression was elevated across the epidermis of xer-1 (Fig. S3). The TMM promoter is active in early stomatal precursors, including in MMCs, meristemoids and SLGCs (Nadeau and Sack, 2002; Bhave et al., 2009). This suggests that a higher number of epidermal cells progress into the stomatal lineage when the ABA level is reduced. Using the SPCHp:SPCH-YFP reporter, we quantified the frequency of pairing defects (paired meristemoids and paired guard cell–meristemoid; Fig. 2C). We found that xer-1 exhibited higher incidences of both phenotypes compared with WT (Fig. 2D). These results show that XER and ABA are required for correct stomatal spacing.

Fig. 2.

xer mutant displays enhanced expression of SPCH and defective stomatal cell divisions. (A) Abaxial epidermis of 4 DPG WT (top) and xer-1 mutant (bottom) cotyledons expressing SPCHp:SPCH-YFP reporter (green) imaged using confocal microscopy. Cell outlines were visualized by propidium iodide (magenta). Scale bars: 20 μm. Images were taken with the same acquisition settings and are representative of n=24 samples. (B) Quantification of the percentage of cells expressing SPCHp:SPCH-YFP in WT and xer-1 mutant plants. (C) Schematic of normally distributed meristemoids and guard cells (normal), paired meristemoids (paired M) and paired guard cells-meristemoids (paired GC-M). (D) Quantification of the distribution of division patterns in 4 DPG WT and xer-1. Statistical significance was determined by unpaired Mann–Whitney test (**P<0.01). Values represent mean±s.e.m. (n=20 images per line).

Fig. 2.

xer mutant displays enhanced expression of SPCH and defective stomatal cell divisions. (A) Abaxial epidermis of 4 DPG WT (top) and xer-1 mutant (bottom) cotyledons expressing SPCHp:SPCH-YFP reporter (green) imaged using confocal microscopy. Cell outlines were visualized by propidium iodide (magenta). Scale bars: 20 μm. Images were taken with the same acquisition settings and are representative of n=24 samples. (B) Quantification of the percentage of cells expressing SPCHp:SPCH-YFP in WT and xer-1 mutant plants. (C) Schematic of normally distributed meristemoids and guard cells (normal), paired meristemoids (paired M) and paired guard cells-meristemoids (paired GC-M). (D) Quantification of the distribution of division patterns in 4 DPG WT and xer-1. Statistical significance was determined by unpaired Mann–Whitney test (**P<0.01). Values represent mean±s.e.m. (n=20 images per line).

Previous work has shown that loss of the inhibitory effect of EPF1 and EPF2 results in excessive entry of epidermal cells into the stomatal cell lineage and causes altered stomatal patterning (Lee et al., 2012; Qi et al., 2017). To determine whether EPF2 and EPF1 expression is affected in the xer-1 mutant, we tracked the percentage of cells expressing EPF2p:GFP and EPF1p:GFP reporters. EPF2p:GFP is expressed in early stomatal lineage cells (MMCs, meristemoids, early guard mother cells), whereas EPF1p:GFP is expressed in later cell types (late meristemoids, guard mother cells, guard cells; Hara et al., 2009). We found that xer-1 exhibited a reduced percentage of cells expressing both EPF1 and EPF2 reporters (Fig. 3A-C). This suggests that XER and adequate ABA levels may be necessary to maintain adequate spatiotemporal expression of these ligands to ensure correct number and spacing of stomata.

Fig. 3.

XER maintains proper EPF1 and EPF2 expression to suppress over-proliferation of stomatal progenitors. (A,B) Abaxial epidermis of 4 DPG WT and xer-1 mutant cotyledons imaged using confocal microscopy. (B) EPF2:GFP is expressed in early stomatal progenitors, including MMCs and early meristemoids. (A) EPF1:GFP is expressed in late stomatal progenitors, including late meristemoids and guard mother cells. Cell walls are stained with propidium iodide (magenta). Scale bars: 20 μm. Images were taken with the same acquisition settings and are representative of n=30 samples. (C) Quantification of the percentage of cells expressing EPF1:GFP and EPF2:GFP in WT and xer-1 mutant at 5 DPG. The xer-1 mutant exhibits reduced expression of EPF1 and EPF2. (D) Quantification of the percentage of meristemoid-like (M-like) cells in WT and xer-1. (E) Quantification of the percentage of M-like cells expressing EPF2:GFP in 5 DPG WT and xer-1. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis (C; different letters between groups indicate statistical significance) or unpaired Mann–Whitney test (D,E; **P<0.01). Values represent mean±s.e.m.

Fig. 3.

XER maintains proper EPF1 and EPF2 expression to suppress over-proliferation of stomatal progenitors. (A,B) Abaxial epidermis of 4 DPG WT and xer-1 mutant cotyledons imaged using confocal microscopy. (B) EPF2:GFP is expressed in early stomatal progenitors, including MMCs and early meristemoids. (A) EPF1:GFP is expressed in late stomatal progenitors, including late meristemoids and guard mother cells. Cell walls are stained with propidium iodide (magenta). Scale bars: 20 μm. Images were taken with the same acquisition settings and are representative of n=30 samples. (C) Quantification of the percentage of cells expressing EPF1:GFP and EPF2:GFP in WT and xer-1 mutant at 5 DPG. The xer-1 mutant exhibits reduced expression of EPF1 and EPF2. (D) Quantification of the percentage of meristemoid-like (M-like) cells in WT and xer-1. (E) Quantification of the percentage of M-like cells expressing EPF2:GFP in 5 DPG WT and xer-1. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis (C; different letters between groups indicate statistical significance) or unpaired Mann–Whitney test (D,E; **P<0.01). Values represent mean±s.e.m.

In particular, expression of the EPF2:GFP reporter was greatly reduced in xer-1, indicating that XER and ABA may have a prominent role in promoting EPF2 expression in early stomatal cell progenitors. To confirm this, the presence of meristemoid-like (M-like) cells expressing the EPF2:GFP reporter was scored between WT and xer-1 (Fig. 3D). Although xer-1 exhibited a higher abundance of M-like cells at 5 DPG, EPF2:GFP reporter activity was detected at lower frequency in xer-1 compared with WT (Fig. 3D,E). These results suggests that XER and ABA may suppress stomatal precursor proliferation by maintaining proper expression of EPF2.

XER regulates stomatal development upstream of SPCH and EPF2, and stomatal spacing independently of EPF1

To determine how XER functions within the stomatal signaling module, the genetic relationship between xer and the stomatal development mutants spch-3, epf2-1 and epf1-1 was assessed.

The bHLH transcription factor SPCH is a master regulator that initiates the stomatal lineage in Arabidopsis (MacAlister et al., 2007). Loss-of-function spch epidermis is devoid of stomata, and heterozygous spch-3/+ has a partially suppressed scrm-D phenotype, suggesting that SPCH has a dosage-dependent effect on scrm-D (MacAlister et al., 2007; Kanaoka et al., 2008). Similarly, we found that heterozygous spch-3/+ develops fewer stomata compared with WT (Fig. 4A,B). Previously, spch was shown to be epistatic to aba2-2 in stomatal development (Tanaka et al., 2013). As expected, xer-1 spch-3/+ cotyledons exhibited a decreased stomatal index and no clustering, similar to spch-3/+, indicating that spch-3 is also epistatic to xer-1 in stomatal production (Fig. 4A-C).

Fig. 4.

Genetic interactions of xer with key regulators of stomatal density and spacing. (A) Representative image of the epidermis of WT, xer-1, spch-3/+, epf1, epf2-1, xer-1 spch-3/+, xer-1 epf1 and xer-1 epf2-1. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustered stomata. Images are representative of n=4-8 samples (WT, xer-1, n=8 samples; spch-3/+, n=6; xer-1 spch-3/+, n=5; epf1-1, epf2-1, xer-1 epf1-1, xer-1 epf2-1, n=4). Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) was quantified across n=4-8 cotyledons per genotype (WT, xer-1, n=8 samples; spch-3/+, n=6; xer-1 spch-3/+, n=5; epf1-1, epf2-1, xer-1 epf1-1, xer-1 epf2-1, n=4). Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

Fig. 4.

Genetic interactions of xer with key regulators of stomatal density and spacing. (A) Representative image of the epidermis of WT, xer-1, spch-3/+, epf1, epf2-1, xer-1 spch-3/+, xer-1 epf1 and xer-1 epf2-1. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustered stomata. Images are representative of n=4-8 samples (WT, xer-1, n=8 samples; spch-3/+, n=6; xer-1 spch-3/+, n=5; epf1-1, epf2-1, xer-1 epf1-1, xer-1 epf2-1, n=4). Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) was quantified across n=4-8 cotyledons per genotype (WT, xer-1, n=8 samples; spch-3/+, n=6; xer-1 spch-3/+, n=5; epf1-1, epf2-1, xer-1 epf1-1, xer-1 epf2-1, n=4). Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

The loss-of-function epf2-1 mutant exhibits excessive entry and amplifying divisions, resulting in a significant increase in total epidermal cells. The epf2-1 mutant also developed arrested meristemoids and small cells, causing a decrease in the total number of stomata (Fig. 4A,B; Hara et al., 2009; Hunt and Gray, 2009). Consequently, the single mutant epf2-1 displayed a decreased stomatal index, in contrast to xer-1 (Fig. 4A,B). The xer-1 epf2-1 stomatal index was similar to that of epf2-1, suggesting that epf2 is epistatic to xer in the control of stomatal density (Fig. 4A,B).

epf1-1 was shown to display increased stomatal index and clustering (Fig. 4A-C; Hara et al., 2007). The xer-1 epf1-1 mutant exhibited a similar stomatal index to the epf1-1 and xer-1 single mutants, indicating that XER and EPF1 act in the same pathway controlling stomatal number (Fig. 4A,B). However, xer-1 epf1-1 showed an enhanced effect in stomatal clustering compared with the xer-1 or epf1-1 single mutants (Fig. 4A,C). We assessed EPF2 transcript levels in xer-1 epf1-1 to determine whether the enhanced stomatal clustering of xer-1 epf1-1 is due to altered EPF2 expression level (Fig. S4). However, EPF2 levels were similar in both the epf1-1 single and xer-1 epf1-1 double mutants (Fig. S4). This indicates that XER and EPF1 act in the same pathway controlling stomatal number, but XER may function in a parallel pathway and acts independently of EPF1 to regulate stomatal patterning.

These results show that XER and ABA functions upstream of EPF2 and SPCH to suppress initiation of the stomatal lineage; however, XER may affect stomatal positioning independently of EPF1.

Basal ABA and core SnRK2 kinases of ABA signaling are required for correct stomatal patterning

Previously, the ABA-deficient aba2-2 mutant was shown to exhibit increased stomatal index that could be rescued by exogenous ABA (Tanaka et al., 2013). Loss of XER produces plants with reduced ABA content (Zentella et al., 2007). In contrast, overexpression of XER increases ABA levels, and this was shown to be due to destabilization of the ABA catabolic enzyme ABA 8′-hydroxylase (Ko et al., 2006; Brugière et al., 2017). This suggests that the increased stomatal proliferation and clustering in xer mutants could be due to reduced ABA levels caused by increased ABA catabolism. Therefore, we tested whether exogenous ABA or inhibition of ABA catabolism could rescue the increased stomatal production of xer. As control, we also included aba2-2.

As expected, aba2-2 exhibited a significant decrease in stomatal index following exposure to 0.1-0.5 μM ABA (Fig. 5A,B; Tanaka et al., 2013). The aba2-2 mutant was also rescued by abscinazole-E1 (Abz-E1), an inhibitor of ABA 8′-hydroxylase (Fig. 5A,B; Okazaki et al., 2011). In contrast, xer-1 and xer-2 exhibited no significant reductions in stomatal index with 0.1 μM ABA, likely owing to the increased ABA catabolism. Indeed, xer-1 and xer-2 stomatal index was rescued by 0.5 μM ABA and Abz-E1 (Fig. 5A,B).

Fig. 5.

ABA and inhibition of ABA hydrolysis rescues xer and aba2-2 stomatal defects. (A) Representative images of the epidermis of WT, xer-1, xer-2 and aba2-2 treated with DMSO control, 0.1 μM ABA, 0.5 μM ABA and 50 μM abscinazole-E1 (Abz-E1), an inhibitor of CYP707A-mediated ABA catabolism. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustering stomata. Images are representative of n=8 samples. Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) was quantified across n=8 cotyledons per genotype/treatment. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

Fig. 5.

ABA and inhibition of ABA hydrolysis rescues xer and aba2-2 stomatal defects. (A) Representative images of the epidermis of WT, xer-1, xer-2 and aba2-2 treated with DMSO control, 0.1 μM ABA, 0.5 μM ABA and 50 μM abscinazole-E1 (Abz-E1), an inhibitor of CYP707A-mediated ABA catabolism. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Mature stomata are false-colored purple for visualization. Black brackets indicate clustering stomata. Images are representative of n=8 samples. Scale bars: 100 μm. (B,C) Stomatal index (B) and percentage of clustered stomata (C) was quantified across n=8 cotyledons per genotype/treatment. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis. Different letters between groups indicate statistical significance. Values represent mean±s.e.m.

Interestingly, stomatal clustering was also found in 20-day-old cotyledons of aba2-2 (Fig. 5A,C). Both xer and aba2-2 displayed stomatal clustering also in 10-day-old seedlings (Fig. S5). Like the stomatal index, stomatal clustering of aba2-2 was significantly rescued by ABA and Abz-E1 (Fig. 5A,C). Although stomatal clustering of xer-1 and xer-2 was reduced by 0.1 μM ABA, it was not statistically significant. However, following 0.5 μM ABA and Abz-E1 treatment, increased stomatal clustering of xer-2 was also significantly reduced (Fig. 5A,C). Finally, we quantified stomatal clustering in the ABA signaling mutant snrk2.2/2.3/2.6, which was previously shown to have an increased stomatal index, and found that it exhibits mild stomatal clustering, similar to xer and aba2 mutants (Fig. S6; Yang et al., 2022).

These results support a role for basal ABA and ABA signaling in inhibition of stomatal development and regulation of stomatal spacing in the developing cotyledon epidermis.

ABA is necessary to maintain EPF1 and EPF2 expression

EPF2 signaling inhibits SPCH to regulate entry into the stomatal lineage pathway, whereas the EPF1 pathway inhibits MUTE to control spacing divisions (Jewaria et al., 2013; Qi et al., 2017). Previously, SPCH and MUTE were shown to be upregulated in the ABA-deficient mutant aba2-2, which shows increased stomatal index similar to xer-1, suggesting that ABA works upstream of these master regulators to suppress stomatal fate (Tanaka et al., 2013). To determine whether ABA modulates the expression levels of positive and negative regulators of stomatal fate, we monitored transcript levels of EPF1, EPF2, SPCH and MUTE in the ABA-deficient mutants xer-1 and aba2-2 (Fig. 6). In agreement with Tanaka et al. (2013), SPCH and MUTE were upregulated at 3 and 5 DPG in aba2-2, and a similar trend was also observed in xer-1 (Fig. 6A,B). Conversely, EPF1 and EPF2 were downregulated in xer-1 and aba2-2 (Fig. 6C,D), further supporting the results obtained with the EPF1:GFP and EPF2:GFP reporters (Fig. 3). We also quantified the expression level of EPFL9 (also known as STOM), which promotes stomatal development (Fig. S7). However, no change in EPFL9 expression in the ABA deficient mutants was found, suggesting that ABA does not regulate EPFL9 expression.

Fig. 6.

Positive and negative regulators of stomatal development exhibit altered expression in the ABA-deficient mutants aba2-2 and xer during early cotyledon development. (A-D) Expression kinetics of SPCH, MUTE, EPF1 and EPF2 across 3-7 DPG in WT, xer-1 and aba2-2 were measured by RT-qPCR relative to PP2AA3. Values represent mean±s.e.m. of three biological replicates. Statistical significance was determined by one-way ANOVA (*P<0.05, **P<0.01). ns, not significant.

Fig. 6.

Positive and negative regulators of stomatal development exhibit altered expression in the ABA-deficient mutants aba2-2 and xer during early cotyledon development. (A-D) Expression kinetics of SPCH, MUTE, EPF1 and EPF2 across 3-7 DPG in WT, xer-1 and aba2-2 were measured by RT-qPCR relative to PP2AA3. Values represent mean±s.e.m. of three biological replicates. Statistical significance was determined by one-way ANOVA (*P<0.05, **P<0.01). ns, not significant.

Collectively, these findings suggest that ABA inhibits stomatal development and clustering by promoting EPF1 and EPF2 expression, and repressing SPCH and MUTE.

ABA partially rescues epf1 and epf2 defects in stomatal density and spacing

Next, we examined whether ABA could rescue the elevated stomatal density and altered stomatal spacing caused by the loss of EPF1 and EPF2 by treating epf1-1 and epf2-1 mutants with 0.1 μM ABA, which was sufficient to rescue aba2-2 (Fig. 5). We found that epf2-1 stomatal density defects were partially rescued by 0.1 μM ABA (Fig. 7A,B). Additionally, stomatal clustering of epf1-1 was partially rescued by ABA (Fig. 7C). We reasoned that the partial rescue of epf2-1 stomatal density and epf1-1 stomatal clustering defects by ABA may be due to ABA-mediated suppression of SPCH and MUTE, which act downstream of EPF2 and EPF1, respectively. To assess this, 5 DPG WT, epf2-1 and epf1-1 seedlings were treated with 10 μM ABA for 6 h. As a control, we included aba2-2, which shows SPCH upregulation (Fig. 5; Tanaka et al., 2013). We found that SPCH and MUTE levels were upregulated in the epf mutants (Fig. 7D,E). Furthermore, following ABA treatment, elevated SPCH and MUTE levels were strongly reduced in epf2-1 and epf1-1, respectively (Fig. 7D,E). Similarly, ABA dampened SPCH and MUTE levels in aba2-2 (Fig. S8). This suggests that ABA represses SPCH and MUTE downstream of EPFs.

Fig. 7.

ABA dampens SPCH and MUTE upregulation and partially rescues stomatal defects of epf1 and epf2 mutants. (A) Representative images of the epidermis of WT, epf1-1 and epf2-1 treated with DMSO control and 0.1 μM ABA. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Black brackets indicate clustered stomata. Mature stomata are false-colored purple for visualization. Images are representative of n=4 samples. Scale bars: 100 μm. (B,C) of stomatal index (B) and percentage of clustered stomata (C) was quantified across four cotyledons per genotype/treatment. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Values represent mean±s.e.m. (D,E) SPCH and MUTE expression was measured by RT-qPCR following a 6-h 10 μM ABA treatment on 5 DPG WT, epf2-1 and epf1-1 seedlings, relative to PP2AA3. Values represent the average of three biological replicates±s.e.m. (B-E) Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis (*P<0.05, **P<0.01). Different letters between groups indicate statistical significance.

Fig. 7.

ABA dampens SPCH and MUTE upregulation and partially rescues stomatal defects of epf1 and epf2 mutants. (A) Representative images of the epidermis of WT, epf1-1 and epf2-1 treated with DMSO control and 0.1 μM ABA. Abaxial cotyledons from 20-day-old seedings were fixed and stained using 0.5% Toluidine Blue and imaged using brightfield microscopy. Black brackets indicate clustered stomata. Mature stomata are false-colored purple for visualization. Images are representative of n=4 samples. Scale bars: 100 μm. (B,C) of stomatal index (B) and percentage of clustered stomata (C) was quantified across four cotyledons per genotype/treatment. Quantification of all clustered stomata was conducted by pooling all stomatal cluster compositions (n>2 stomata in contact) and calculating the percentage of stomata in clusters. Values represent mean±s.e.m. (D,E) SPCH and MUTE expression was measured by RT-qPCR following a 6-h 10 μM ABA treatment on 5 DPG WT, epf2-1 and epf1-1 seedlings, relative to PP2AA3. Values represent the average of three biological replicates±s.e.m. (B-E) Statistical significance was determined by one-way ANOVA at P<0.05, followed by Tukey's honestly significant difference (HSD) analysis (*P<0.05, **P<0.01). Different letters between groups indicate statistical significance.

Altogether, these data indicate that ABA regulates stomatal density and spacing by modulating stomatal signaling pathways at multiple levels.

Environmental cues and hormones modulate stomatal aperture, density and distribution (Qi and Torii, 2018). However, the molecular and genetic relationships between hormone- and stress-dependent regulation of core stomatal signaling components remain less understood. Genetic analysis of ABA biosynthesis and signaling has shown that basal ABA inhibits entry into the stomatal lineage pathway (Brookbank et al., 2021). Here, we demonstrate that ABA biosynthesis, catabolism and signaling are also required for stomatal patterning. Previously, the ABA-deficient mutants aba2-2 and xer, and the ABA signaling mutant snrk2.2/2.3/2.6, were shown to exhibit increased stomatal index (Tanaka et al., 2013; Vonapartis et al., 2022; Yang et al., 2022). Here, we show that xer, aba2-2 and snrk2.2/2.3/2.6 display stomatal clusters, in addition to increased stomatal index. Stomatal index and clustering defects of aba2-2 and xer were rescued by exogenous ABA or by inhibition of ABA catabolism, indicating a role for ABA biosynthesis, catabolism and signaling in stomatal patterning. Mechanistically, we found that ABA is necessary to maintain adequate expression of SPCH, MUTE, EPF1 and EPF2 at early stages of epidermal development, as shown by altered expression of these genes in aba2-2 and xer. We also quantified expression levels of EPFL9, which promotes stomatal development (Fig. S7). However, no change in EPFL9 expression in the ABA-deficient mutants was found, suggesting that ABA does not regulate EPFL9 expression. Previously, ABA2 was shown to control stomatal development upstream of SPCH and MUTE (Tanaka et al., 2013). Our genetic analysis indicates that XER acts upstream of the EPF2-SPCH pathway to control stomatal proliferation, while acting synergistically with EPF1 to modulate stomatal spacing (Fig. 8). We show that ABA can partially rescue epf1 and epf2 stomatal defects by downregulating MUTE and SPCH. Altogether, these results demonstrate that ABA metabolism is required for correct proliferation and spacing of stomata, by regulating stomatal signaling pathways at multiple levels. They also indicate that core ABA signaling SnRK2 kinases are required for correct stomatal distribution, possibly acting through SPCH, suggesting that stomatal patterning requires a functional core ABA signaling pathway (Fig. 8). The role of basal ABA in modulating stomatal spacing, in addition to stomatal density, is likely to be important for plant growth as well as stress response.

Fig. 8.

Model of ABA action modulating stomatal proliferation and patterning. In Arabidopsis, ABA biosynthesis (ABA2) and regulation of catabolism (XER) fine-tune basal ABA levels in the developing epidermis (Nambara et al., 1998; Zentella et al., 2007). ABA maintains adequate expression of EPF ligands and the TMM receptor, and downregulates SPCH and MUTE to limit initiation of the stomatal cell lineage and control stomatal spacing (Tanaka et al., 2013; this study). Additionally, ABA activates the core ABA signaling kinases SnRK2.2/2.3/2.6 to phosphorylate and trigger SPCH degradation (Yang et al., 2022). SnRK2 kinases inhibit initiation and proliferation of stomatal precursors (Yang et al., 2022) and ensure correct stomatal spacing (this study). Therefore, ABA influences stomatal proliferation and patterning at multiple levels to coordinate stomatal development in the developing epidermis. Schematics of cells show the progression of cell type differentiation through development.

Fig. 8.

Model of ABA action modulating stomatal proliferation and patterning. In Arabidopsis, ABA biosynthesis (ABA2) and regulation of catabolism (XER) fine-tune basal ABA levels in the developing epidermis (Nambara et al., 1998; Zentella et al., 2007). ABA maintains adequate expression of EPF ligands and the TMM receptor, and downregulates SPCH and MUTE to limit initiation of the stomatal cell lineage and control stomatal spacing (Tanaka et al., 2013; this study). Additionally, ABA activates the core ABA signaling kinases SnRK2.2/2.3/2.6 to phosphorylate and trigger SPCH degradation (Yang et al., 2022). SnRK2 kinases inhibit initiation and proliferation of stomatal precursors (Yang et al., 2022) and ensure correct stomatal spacing (this study). Therefore, ABA influences stomatal proliferation and patterning at multiple levels to coordinate stomatal development in the developing epidermis. Schematics of cells show the progression of cell type differentiation through development.

ABA metabolism impacts stomatal density and patterning

During short-term drought acclimation, plants trigger stomatal closure to maintain cell turgor and reduce water loss via transpiration (Munemasa et al., 2015; Hsu et al., 2021). Drought-induced stomatal closure requires the action of ABA to decrease guard cell turgor through activation of ion channels (Munemasa et al., 2015; Hsu et al., 2021). However, long-term adaption to stress, including drought and elevated CO2, involves decreasing stomatal density through the ABA signaling pathway (Chater et al., 2015; Yang et al., 2022). ABA has also been shown to restrict stomatal lineage under non-stressed conditions, suggesting that basal ABA is required to adjust stomatal number in the developing epidermis (Tanaka et al., 2013; Brookbank et al., 2021). Notably, correct stomatal spacing is required for plant viability as well as stress response, as shown by seedling lethality and impaired drought stress response of yda and epf1 epf2 mutants with severe stomatal defects (Bergmann et al., 2004; Hepworth et al., 2015; Wang et al., 2016). However, the role of ABA in stomatal distribution and spacing has not been previously explored.

Our data show that ABA is required for proper stomatal spacing. ABA-deficient xer and aba2-2 mutants displayed elevated stomatal clustering, in addition to increased stomatal index, in the epidermis of fully developed cotyledons. We found that low amount of exogenous ABA (0.1 μM) could rescue the increased stomatal density and clustering of aba2-2, but not xer, possibly because of enhanced ABA catabolism in xer that would inactivate exogenous ABA (Saito et al., 2004; Kushiro et al., 2004; Brugière et al., 2017). Indeed, a higher amount of ABA (0.5 μM), or the CYP707A/ABA8ox inhibitor Abz-E1 (50 μM), were able to rescue the elevated stomatal index and clustering of xer, suggesting that XER suppresses stomatal proliferation and ensures correct patterning by maintaining basal ABA levels. The lack of xer rescue by the lower concentration of exogenous ABA is consistent with previous studies showing that Arabidopsis plants overexpressing CYP707A3 display increased ABA catabolism and ABA insensitivity, resulting in increased transpiration rates and wilting leaves (Umezawa et al., 2006). In contrast, plants overexpressing ZmXER have been shown to accumulate a higher level of ABA and lower levels of the ABA catabolites diphaseic acid and phaseic acid, resulting in ABA hypersensitivity and improved water use efficiency (Brugière et al., 2017). Exogenous ABA (0.5 μM) and Abz-E1 (50 μM) had a similar effect in decreasing the stomatal index of WT and rescuing xer mutants. However, 0.5 μM ABA reduced the aba2-2 stomatal index even more than WT, possible because of a lack of feedback regulation in aba2-2, which normally would ensure ABA homeostasis (Finkelstein, 2013). Furthermore, 50 μM Abz-E1 was sufficient to rescue aba2-2, but did not reduce the stomatal index to the same level as WT. This is likely because inhibition of ABA catabolism could not compensate for the strong reduction of ABA level shown by the aba2-2 mutant (20% of WT; Nambara et al., 1998). Altered ABA levels through loss of ABA2 or XER overexpression impacts the levels of other hormones that positively regulate stomatal development, including ethylene and gibberellins (LeNoble et al., 2004; Ko et al., 2006; Seo et al., 2006). Therefore, disrupted hormone profiles due to reduced ABA content may also contribute to the phenotypes exhibited by xer and aba2 mutants.

RNA-sequencing analysis has revealed that genes involved in ABA metabolism, including XER and ABA2, and ABA signaling are expressed across the stomatal lineage (Fig. S2; Adrian et al., 2015). Previous studies demonstrated that the core ABA signaling pathway is required to modulate stomatal development. Protein phosphastase 2Cs (PP2Cs) are negative regulators of the ABA signaling pathway (Cutler et al., 2010) and dominant ABA-insensitive mutants in the PP2Cs ABI1 and ABI2 showed increased stomatal index (Tanaka et al., 2013). Additionally, the snrk2.2/2.3/2.6 mutant affected in three SnRK2 kinases, which are positive regulators of ABA signaling, also displayed elevated stomatal index (Yang et al., 2022). We found that 20-day-old snrk2.2/2.3/2.6 mutants exhibit mild stomatal clustering, similar to xer and aba2 mutants. Although Yang et al. (2022) did not observe stomatal clusters in the snrk2.2/2.3/2.6 triple mutant, quantification of stomatal clusters was not shown (Yang et al., 2022). In this study, we quantified stomatal clustering over the entire cotyledon area of 20-day-old seedlings, which may uncover subtle phenotypes. Our findings indicate SnRK2s are also necessary to promote proper stomatal distribution, possibly through SPCH, suggesting that the core ABA signaling kinases are required to ensure correct stomatal spacing. It would be important to determine whether a SnRK2-insensitive SPCH variant (SPCH-S240/S271A; Yang et al., 2022) exhibits stomatal clustering owing to SPCH hyperactivity. Currently, there is no report of other ABA signaling components, such as the PYR/PYL receptors or PP2C phosphatases, being involved in stomatal spacing. This may be due to functional redundancy or specialized functions of the different ABA signaling components. In future studies, it will be important to analyze carefully different combinations of higher order mutants in ABA signaling to identify specific ABA signaling components required for correct stomatal spacing.

ABA suppresses the stomatal lineage through the canonical EPF2-SPCH pathway

Our work shows that ABA is required early during epidermal development to promote EPF1 and EPF2 expression. Reporter and transcript analysis showed that EPF1 and EPF2 expression was reduced in stomatal precursors of xer-1. Furthermore, transcript analysis showed downregulation of EPF1 and EPF2 in xer-1 and aba2-2. Altered transcriptional regulation of EPF1/2 likely contributes to their increased stomatal density, given that EPFs communicate positional information to suppress stomatal proliferation. Previously, ABA was shown to repress stomatal development through SPCH and MUTE, as these genes were upregulated in aba2-2 and abi1 mutants (Tanaka et al., 2013). Here, we corroborated these findings by showing that SPCH and MUTE are also upregulated in xer-1. Interestingly, ABA could partially compensate for the loss of EPF1 and EPF2 by repressing elevated SPCH and MUTE levels, which partially reduced epf1 stomatal clustering and increased the epf2 stomatal index. Collectively, these results show that basal ABA is required to modulate the expression level of positive and negative regulators of the stomatal signaling pathway to ensure tight control of stomatal density (Fig. 8). Although it is unclear how ABA regulates SPCH, MUTE, EPF1 and EPF2 expression, the core ABA-responsive element (ABRE) is present in the promoter and coding regions of SPCH, MUTE, EPF1 and EPF2 (Fig. S7). Thus, ABA-regulated transcription factors may directly mediate these transcriptional changes. Furthermore, SNRK2 kinases were recently shown to inhibit stomatal development by phosphorylating and inhibiting SPCH, which positively regulates EPF2 expression (Lau et al., 2014; Yang et al., 2022). Thus, ABA regulates the canonical stomatal signaling pathway at the transcriptional and post-translational level.

ABA maintains optimal stomatal distribution through EPF1 and EPF2

Reduced EPF expression, particularly EPF1, likely impacts the patterning defects exhibited by the ABA-deficient mutants xer-1 and aba2-2. EPF1 has been shown to influence stomatal spacing by inhibiting MUTE activity in neighboring cells (Qi et al., 2017). As expected, we found that MUTE was upregulated in xer-1 and aba2-2. However, increased stomatal clustering was found in xer-1 epf1-1 compared with the single mutants, suggesting that XER may affect stomatal patterning through other pathways. Previously, the mute aba2-2 mutant was shown to have increased meristemoids compared with mute, which was attributed to the role of ABA in inhibiting cell proliferation (Tanaka et al., 2013). Given the roles of SPCH, MUTE and ABA in cell cycle regulation through induction of cyclin D (CYCD) genes, ABA and the stomatal lineage pathway may converge onto cell cycle-related genes to regulate stomatal index and spacing (Wang et al., 1998; Lau et al., 2014; Adrian et al., 2015; Han et al., 2018, 2022; Zuch et al., 2023). Therefore, ABA may work together, as well as in parallel with, the EPF1-MUTE pathway to control meristemoid proliferation and stomatal spacing.

The xer mutant displays enhanced frequency of stomatal pairing, suggesting that ABA may be required to maintain proper stomatal spacing and amplifying divisions to ensure correct stomatal patterning (Fig. 2D). Factors that guide and control stomatal lineage divisions are well understood, including position and polarity markers such as BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), POLAR LOCALIZATION DURING ASYMMETRIC DIVISION AND REDISTRIBUTION (POLAR) and BREVIS RADIX/BREVIS RADIX-like (BRX/BRXL) (Dong et al., 2009; Pillitteri et al., 2011; Rowe et al., 2019 preprint). These polarity factors work in collaboration to direct, scaffold and segregate the SPCH-repressive machinery towards the SLGC post-division, to ensure fate asymmetry of ACD daughter cells (Zhang et al., 2015). ABA may also modulate stomatal patterning by maintaining EPF2 expression. EPF2 binding to the ERECTA receptor triggers higher MAPK activation and activity; MAPK signaling is then recruited by BASL to newly divided daughter cells following BASL polarization (Lee et al., 2015; Zhang et al., 2015). Lack of adequate recruitment of repressive machinery may alter stomatal cell asymmetry and produce increased stomatal density and patterning defects (Zhang et al., 2015; Houbaert et al., 2018).

Collectively, our work indicates that basal ABA and a functional ABA signaling pathway regulate stomatal patterning, in addition to entry into the stomatal development pathway. We show that ABA functions upstream of the EPF2-SPCH pathway to inhibit stomatal proliferation (Fig. 8). ABA suppresses stomatal identity by promoting EPF1 and EPF2 expression, and represses SPCH and MUTE, to produce even stomatal distribution. Core SnRK2 kinases are also required to ensure correct stomatal patterning, possibly through SPCH phosphorylation. Regulation of stomatal patterning by ABA may impact stomatal performance, affecting plant development and stress responsiveness. Indeed, clustered stomata result in poor guard cell functioning, which then impairs stomatal conductance and overall plant performance (Dow et al., 2013, 2014; Lehmann and Or, 2015). Thus, ABA likely promotes stress resistance by maintaining proper stomatal density and patterning.

Plant materials and growth conditions

All Arabidopsis lines characterized in this study are from the Col-0 background. Following sterilization, seeds were stratified at 4°C for 2-4 days. Seeds were sown on ½ Murashige and Skoog (MS) medium (Sigma-Aldrich) for 4-20 days under long-day conditions (16 h light at 21°C/8 h dark at 18°C). Transgenic lines were screened using 30 μg/ml BASTA (glufosinate ammonium; Sigma-Aldrich). Plants were propagated in growth chambers (Enconair) under long-day conditions (16 h light at 21°C /8 h dark at 18°C).

Previously published lines used in this study were: xer-1 (Zentella et al., 2007), xer-2 (Vonapartis et al., 2022), aba2-2 (Nambara et al., 1998), epf1-1 (Hara et al., 2007), epf2-1 (Hara et al., 2009), spch-3 (MacAlister et al., 2007), snrk2.2/2.3/2.6 (srk2d/e/i; Fujita et al., 2009), SPCHp:SPCH-YFP (Lopez-Andio et al., 2021), TMMp:GFP-GUS (Nadeau and Sack, 2002), EPF1p:erGFP and EPF2p:erGFP (Hara et al., 2007, 2009). New lines generated by crossing were: TMMp:GFP-GUS xer-1, SPCHp:SPCH-YFP xer-1, EPF1p:erGFP xer-1, EPF2p:erGFP xer-1, xer-1 epf1-1, xer-1 epf2-1, xer-1 spch-3. To generate the 35Sp:XER-eGFP construct, the XER coding sequence was PCR-amplified from clone RAFL-08-12-F01 (RIKEN BRC) and cloned into the pEGAD vector (Cutler et al., 2010). The 35Sp:XER-eGFP construct was transformed into xer-1 by floral dip (Clough and Bent, 1998). At least ten primary independent transgenic lines were generated and at least two homozygous lines were chosen for further propagation and molecular characterization.

For long-term ABA rescue assays, seeds were germinated on ½ MS medium for 2 days. Seedlings were then transferred to either 0.1 μM ABA, 0.5 μM ABA or 50 μM abscinazole-E1 (Abz-E1) and grown for 20 days before epidermal sampling, microscopy and image analysis.

Epidermal sampling, microscopy and image analysis

Epidermal staining of 20-day-old cotyledons was performed as described by Vonapartis et al. (2022). Seedlings were fixed in a 9:1 ethanol:acetic acid solution overnight. Following a series of ethanol washes (from 70%, 50%, down to 20%; 20 min each), seedlings were stained with 0.5% Toluidine Blue and then washed three times in double-distilled water. Cotyledons were then mounted on their abaxial side in 15% glycerol and imaged on the brightfield setting of an Axioplan 2 microscope (Zeiss). Four to ten cotyledons were imaged per genotype.

Fluorescence imaging using SPCH:SPCH-YFP, EPF1:GFP, EPF2:GFP and TMM:GFP transcriptional reporters was performed on a LSM510 META confocal laser-scanning microscope (Zeiss) and a Leica Stellaris 5 confocal microscope. GFP/YFP was excited at 488 nm; emission was detected using a 505-530 nm BP filter or the HyD detectors. At 4-5 DPG, seedlings were stained with 10 mg/ml propidium iodide for 5 min to visualize cell boundaries. Seedlings were subsequently washed and mounted in water. Serial, maximum projection z-stacks were obtained from an area of 250×250 μm for 20-40 images captured from six cotyledons per genotype. Meristemoids were scored based on cell shape and cell size (≤100 μm).

Quantification of stomatal phenotypes

Stomatal index and stomatal clusters were measured across the whole cotyledon and quantified per mm2 using the cell counter plugin from ImageJ. Stomatal indices were calculated as a percentage using the following formula:
formula
For assessing the stomatal index of epf2-1, all non-mature stomata (including pavement cells, small cells and precursor-like cells) were scored in the total epidermal cell count. Only mature stomata were considered for the number of stomata.
The percentage of stomatal clusters was calculated using the following formula:
formula

Quantitative analysis of cell type-specific reporter expression was conducted using cell counter plugin from ImageJ. Stomatal clustering was assessed with the SPCHp:SPCHFP reporter, where the percentage of normally distributed (normal), paired meristemoids (paired M) and paired guard cells-meristemoids (paired GC-M) was scored based on the presence of SPCH-YFP in meristemoids.

Gene expression kinetics analysis

Fifty milligrams of tissue was harvested from WT, xer-1 and aba2-2 cotyledons for time-course analysis. Transient 10 μM ABA treatment was performed for 6 h on 5 DPG epf1-1 and epf2-1. RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN), followed by on-column digestion with RNase-free DNase I (Thermo Fisher Scientific). cDNA synthesis was performed on 1 μg of RNA using the SensiFAST cDNA Synthesis Kit (BioLine). RT-qPCR was conducted in triplicate with Advanced qPCR mastermix with SUPERGREEN Lo-ROX (Wisent Bioproducts) using primers specific for SPCH, MUTE, EPF1 and EPF2 (see Table S1) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Data were analyzed using the ΔΔCT method normalized to PP2AA3 as the reference gene (Schmittgen and Livak, 2008). Three biological replicates per genotype per DPG were conducted.

Statistical analyses

Statistical tests were completed using built-in packages in R studio, with default settings. One-way ANOVA at P<0.05, followed by Tukey's honestly significant different (HSD) was performed between multiple genotypes. Unpaired Mann–Whitney test or Student's t-test was performed, where stated, for comparisons between two sets of data.

We thank Jin Suk Lee (Concordia University) for providing epf1, epf2 and the TMMp:GFP-GUS reporter, Dominique Bergmann (Stanford University) for the SPCHp:SPCH-YFP reporter, Takuya Yoshida (Technische Universität München) for the srk2d/e/i (snrk2.2,2.3,2.6) seeds, Tatsuo Kakimoto (Osaka University) for the EPF1:GFP and EPF2p:GFP reporter lines, Y. Todoroki (Shizuoka University) and E. Nambara (University of Toronto) for providing abscinazole-E1 inhibitor. We also thank Kenneth Corpuz for help with the EPF expression analysis, Bruno Chue (Centre for the Neurobiology of Stress, University of Toronto Scarborough) for technical assistance with optimization of confocal microscopy imaging, and the Gazzarrini lab for helpful feedback and discussion on the manuscript.

Author contributions

Conceptualization: D.M., S.G.; Methodology: D.M., E.V., D.Y.C.; Validation: D.M.; Formal analysis: D.M.; Investigation: D.M., E.V., S.G.; Resources: S.G.; Data curation: D.M.; Writing - original draft: D.M.; Writing - review & editing: E.V., S.G.; Visualization: D.M.; Supervision: S.G.; Project administration: S.G.; Funding acquisition: S.G.

Funding

This study was funded by NSERC-DG 480529 (Natural Sciences and Engineering Research Council of Canada - Discovery Grant) to S.G.

Data availability

All relevant data can be found within the article and its supplementary information.

Adrian
,
J.
,
Chang
,
J.
,
Ballenger
,
C. E.
,
Bargmann
,
B. O.
,
Alassimone
,
J.
,
Davies
,
K. A.
,
Lau
,
O. S.
,
Matos
,
J. L.
,
Hachez
,
C.
,
Lanctot
,
A.
et al. 
(
2015
).
Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population
.
Dev. Cell
33
,
107
-
118
.
Allen
,
J.
,
Guo
,
K.
,
Zhang
,
D.
,
Ince
,
M.
and
Jammes
,
F.
(
2019
).
ABA-glucose ester hydrolyzing enzyme ATBG1 and PHYB antagonistically regulate stomatal development
.
PLoS One
14
,
e0218605
.
Bergmann
,
D. C.
,
Lukowitz
,
W.
and
Somerville
,
C. R.
(
2004
).
Stomatal development and pattern controlled by a MAPKK kinase
.
Science
304
,
1494
-
1497
.
Bhave
,
N. S.
,
Veley
,
K. M.
,
Nadeau
,
J. A.
,
Lucas
,
J. R.
,
Bhave
,
S. L.
and
Sack
,
F. D.
(
2009
).
TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems
.
Planta
229
,
357
-
367
.
Brookbank
,
B. P.
,
Patel
,
J.
,
Gazzarrini
,
S.
and
Nambara
,
E.
(
2021
).
Role of basal ABA in plant growth and development
.
Genes
12
,
1936
.
Brugière
,
N.
,
Zhang
,
W.
,
Xu
,
J.
,
Scolaro
,
E. J.
,
Lu
,
C.
,
Kahsay
,
R. Y.
,
Kise
,
R.
,
Trecker
,
L.
,
Williams
,
R. W.
,
Hakimi
,
S.
et al. 
(
2017
).
Overexpression of RING domain E3 ligase ZmXerico1 confers drought tolerance through regulation of ABA homeostasis
.
Plant Phys.
175
,
1350
-
1369
.
Chater
,
C.
,
Peng
,
K.
,
Movahedi
,
M.
,
Dunn
,
J. A.
,
Walker
,
H. J.
,
Liang
,
Y. K.
,
Mclachlan
,
D. H.
,
Casson
,
S.
,
Isner
,
J. C.
,
Wilson
,
I.
et al. 
(
2015
).
Elevated CO2-induced responses in stomata require ABA and ABA signaling
.
Curr. Biol.
25
,
2709
-
2716
.
Clough
,
S. J.
and
Bent
,
A. F.
(
1998
).
Floral dip: a simplified method for Agrobacterium–mediated transformation of Arabidopsis thaliana
.
Plant J.
16
,
735
-
743
.
Cutler
,
S. R.
,
Rodriguez
,
P. L.
,
Finkelstein
,
R. R.
and
Abrams
,
S. R.
(
2010
).
Abscisic acid: emergence of a core signaling network
.
Annu. Rev. Plant Biol.
61
,
651
-
679
. doi:10.1146/annurev-arplant-042809-112122
Dong
,
J.
,
Macalister
,
C. A.
and
Bergmann
,
D. C.
(
2009
).
BASL controls asymmetric cell division in Arabidopsis
.
Cell
137
,
1320
-
1330
.
Dow
,
G. J.
,
Berry
,
J. A.
and
Bergmann
,
D. C.
(
2013
).
The physiological importance of developmental mechanisms that enforce proper stomatal spacing in Arabidopsis thaliana
.
New Phytol.
201
,
1205
-
1217
.
Dow
,
G. J.
,
Bergmann
,
D. C.
and
Berry
,
J. A.
(
2014
).
An integrated model of stomatal development and leaf physiology
.
New Phytol.
201
,
1218
-
1226
.
Engineer
,
C. B.
,
Ghassemian
,
M.
,
Anderson
,
J. C.
,
Peck
,
S. C.
,
Hu
,
H.
and
Schroeder
,
J. I.
(
2014
).
Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development
.
Nature
513
,
246
-
250
.
Finkelstein
,
R.
(
2013
).
Abscisic acid synthesis and response
.
Arabidopsis Book
11
,
e0166
.
Fujita
,
Y.
,
Nakashima
,
K.
,
Yoshida
,
T.
,
Katagiri
,
T.
,
Kidokoro
,
S.
,
Kanamori
,
N.
,
Umezawa
,
T.
,
Fujita
,
M.
,
Maruyama
,
K.
,
Ishiyama
,
K.
et al. 
(
2009
).
Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis
.
Plant Cell Physiol.
50
,
2123
-
2132
.
Geisler
,
M.
,
Nadeau
,
J.
and
Sack
,
F. D.
(
2000
).
Oriented asymmetric divisions that generate the stomatal spacing pattern in Arabidopsis are disrupted by the too many mouths mutation
.
Plant Cell
12
,
2075
-
2086
.
Gong
,
Y.
,
Alassimone
,
J.
,
Varnau
,
R.
,
Sharma
,
N.
,
Cheung
,
L. S.
and
Bergmann
,
D. C.
(
2021
).
Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division
.
eLife
10
,
e63335
.
Guo
,
X. Y.
,
Wang
,
Y.
,
Zhao
,
P. X.
,
Xu
,
P.
,
Yu
,
G. H.
,
Zhang
,
L. Y.
,
Xiong
,
Y.
and
Xiang
,
C. B.
(
2019
).
AtEDT1/HDG11 regulates stomatal density and water–use efficiency via ERECTA and E2Fa
.
New Phytol.
223
,
1478
-
1488
.
Han
,
S. K.
,
Qi
,
X.
,
Sugihara
,
K.
,
Dang
,
J. H.
,
Endo
,
T. A.
,
Miller
,
K. L.
,
Kim
,
E. D.
,
Takashi
,
M.
and
Torii
,
K. U.
(
2018
).
MUTE directly orchestrates cell-state switch and the single symmetric division to create stomata
.
Dev. Cell
45
,
303
-
315
.
Han
,
S. K.
,
Herrmann
,
A.
,
Yang
,
J.
,
Iwasaki
,
R.
,
Sakamoto
,
T.
,
Desvoyes
,
B.
,
Seisuke
,
K.
,
Gutierrez
,
C.
,
Kim
,
E. D.
and
Torii
,
K. U.
(
2022
).
Deceleration of the cell cycle underpins a switch from proliferative to terminal divisions in plant stomatal lineage
.
Dev. Cell
57
,
569
-
582
.
Hara
,
K.
,
Kajita
,
R.
,
Torii
,
K. U.
,
Bergmann
,
D. C.
and
Kakimoto
,
T.
(
2007
).
The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule
.
Genes Dev.
21
,
1720
-
1725
.
Hara
,
K.
,
Yokoo
,
T.
,
Kajita
,
R.
,
Onishi
,
T.
,
Yahata
,
S.
,
Peterson
,
K. M.
,
Torii
,
K. U.
and
Kakimoto
,
T.
(
2009
).
Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves
.
Plant Cell Phys.
50
,
1019
-
1031
.
Hepworth
,
C.
,
Doheny-Adams
,
T.
,
Hunt
,
L.
,
Cameron
,
D. D.
and
Gray
,
J. E.
(
2015
).
Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake
.
New Phytol.
208
,
336
-
341
.
Hsu
,
P. K.
,
Dubeaux
,
G.
,
Takahashi
,
Y.
and
Schroeder
,
J. I.
(
2021
).
Signaling mechanisms in abscisic acid–mediated stomatal closure
.
Plant J.
105
,
307
-
321
.
Houbaert
,
A.
,
Zhang
,
C.
,
Tiwari
,
M.
,
Wang
,
K.
,
De Marcos Serrano
,
A.
,
Savatin
,
D. V.
,
Urs
,
M. J.
,
Zhiponova
,
M. K.
,
Gudesblat
,
G. E.
,
Vanhoutte
,
I.
et al. 
(
2018
).
POLAR-guided signalling complex assembly and localization drive asymmetric cell division
.
Nature
563
,
574
-
578
.
Hronková
,
M.
,
Wiesnerová
,
D.
,
Šimková
,
M.
,
Skůpa
,
P.
,
Dewitte
,
W.
,
Vráblová
,
M.
,
Zažímalová
,
E.
and
Šantrůček
,
J.
(
2015
).
Light-induced STOMAGEN-mediated stomatal development in Arabidopsis leaves
.
J. Exp. Bot.
66
,
4621
-
4630
. doi:10.1093/jxb/erv233
Hughes
,
J.
,
Hepworth
,
C.
,
Dutton
,
C.
,
Dunn
,
J. A.
,
Hunt
,
L.
,
Stephens
,
J.
,
Waugh
,
R.
,
Cameron
,
D. D.
and
Gray
,
J. E.
(
2017
).
Reducing stomatal density in barley improves drought tolerance without impacting on yield
.
Plant Physiol.
174
,
776
-
787
.
Hunt
,
L.
and
Gray
,
J. E.
(
2009
).
The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development
.
Curr. Biol.
19
,
864
-
869
.
Jewaria
,
P. K.
,
Hara
,
T.
,
Tanaka
,
H.
,
Kondo
,
T.
,
Betsuyaku
,
S.
,
Sawa
,
S.
,
Sakagami
,
Y.
,
Aimoto
,
S.
and
Kakimoto
,
T.
(
2013
).
Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level
.
Plant Cell Phys.
54
,
1253
-
1262
.
Kanaoka
,
M. M.
,
Pillitteri
,
L. J.
,
Fujii
,
H.
,
Yoshida
,
Y.
,
Bogenschutz
,
N. L.
,
Takabayashi
,
J.
,
Zhu
,
J. K.
and
Torii
,
K. U.
(
2008
).
SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation
.
Plant Cell
20
,
1775
-
1785
.
Ko
,
J. H.
,
Yang
,
S. H.
and
Han
,
K. H.
(
2006
).
Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis
.
Plant J.
47
,
343
-
355
.
Kushiro
,
T.
,
Okamoto
,
M.
,
Nakabayashi
,
K.
,
Yamagishi
,
K.
,
Kitamura
,
S.
,
Asami
,
T.
,
Hirai
,
N.
,
Koshiba
,
T.
,
Kamiya
,
Y.
and
Nambara
,
E.
(
2004
).
The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA catabolism
.
EMBO J.
23
,
1647
-
1656
. doi:10.1038/sj.emboj.7600121
Lau
,
O. S.
,
Davies
,
K. A.
,
Chang
,
J.
,
Adrian
,
J.
,
Rowe
,
M. H.
,
Ballenger
,
C. E.
and
Bergmann
,
D. C.
(
2014
).
Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells
.
Science
345
,
1605
-
1609
.
Lee
,
L. R.
and
Bergmann
,
D. C.
(
2019
).
The plant stomatal lineage at a glance
.
J. Cell Sci.
132
,
jcs228551
.
Lee
,
J. S.
,
Kuroha
,
T.
,
Hnilova
,
M.
,
Khatayevich
,
D.
,
Kanaoka
,
M. M.
,
Mcabee
,
J. M.
,
Sarikaya
,
M.
,
Tamerler
,
C.
and
Torii
,
K. U.
(
2012
).
Direct interaction of ligand–receptor pairs specifying stomatal patterning
.
Genes Dev.
26
,
126
-
136
.
Lee
,
J. S.
,
Hnilova
,
M.
,
Maes
,
M.
,
Lin
,
Y. C. L.
,
Putarjunan
,
A.
,
Han
,
S. K.
,
Avila
,
J.
and
Torii
,
K. U.
(
2015
).
Competitive binding of antagonistic peptides fine-tunes stomatal patterning
.
Nature
522
,
439
-
443
.
Lehmann
,
P.
and
Or
,
D.
(
2015
).
Effects of stomata clustering on leaf gas exchange
.
New Phytol.
207
,
1015
-
1025
.
Lenoble
,
M. E.
,
Spollen
,
W. G.
and
Sharp
,
R. E.
(
2004
).
Maintenance of shoot growth by endogenous ABA: genetic assessment of the involvement of ethylene suppression
.
J. Exp. Bot.
55
,
237
-
245
.
Lopez-Anido
,
C. B.
,
Vatén
,
A.
,
Smoot
,
N. K.
,
Sharma
,
N.
,
Guo
,
V.
,
Gong
,
Y.
,
Gil
,
M. X. A.
,
Weimer
,
A. K.
and
Bergmann
,
D. C.
(
2021
).
Single-cell resolution of lineage trajectories in the Arabidopsis stomatal lineage and developing leaf
.
Dev. Cell
56
,
1043
-
1055
.
Macalister
,
C. A.
,
Ohashi-Ito
,
K.
and
Bergmann
,
D. C.
(
2007
).
Transcription factor control of asymmetric cell divisions that establish the stomatal lineage
.
Nature
445
,
537
.
Munemasa
,
S.
,
Hauser
,
F.
,
Park
,
J.
,
Waadt
,
R.
,
Brandt
,
B.
and
Schroeder
,
J. I.
(
2015
).
Mechanisms of abscisic acid-mediated control of stomatal aperture
.
Curr. Opin. Plant Biol.
28
,
154
-
162
.
Nadeau
,
J. A.
and
Sack
,
F. D.
(
2002
).
Control of stomatal distribution on the Arabidopsis leaf surface
.
Science
296
,
1697
-
1700
.
Nambara
,
E.
,
Kawaide
,
H.
,
Kamiya
,
Y.
and
Naito
,
S.
(
1998
).
Characterization of an Arabidopsis thaliana mutant that has a defect in ABA accumulation: ABA-dependent and ABA-independent accumulation of free amino acids during dehydration
.
Plant Cell Phys.
39
,
853
-
858
.
Ohashi-Ito
,
K.
and
Bergmann
,
D. C.
(
2006
).
Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development
.
Plant Cell
18
,
2493
-
2505
.
Okazaki
,
M.
,
Nimitkeatkai
,
H.
,
Muramatsu
,
T.
,
Aoyama
,
H.
,
Ueno
,
K.
,
Mizutani
,
M.
,
Hirai
,
N.
,
Kondo
,
S.
,
Ohnishi
,
T.
and
Todoroki
,
Y.
(
2011
).
Abscinazole-E1, a novel chemical tool for exploring the role of ABA 8′-hydroxylase CYP707A
.
Bioorg. Med. Chem.
19
,
406
-
413
.
Pillitteri
,
L. J.
,
Sloan
,
D. B.
,
Bogenschutz
,
N. L.
and
Torii
,
K. U.
(
2007
).
Termination of asymmetric cell division and differentiation of stomata
.
Nature
445
,
501
-
505
.
Pillitteri
,
L. J.
,
Peterson
,
K. M.
,
Horst
,
R. J.
and
Torii
,
K. U.
(
2011
).
Molecular profiling of stomatal meristemoids reveals new component of asymmetric cell division and commonalities among stem cell populations in Arabidopsis
.
Plant Cell
23
,
3260
-
3275
.
Qi
,
X.
and
Torii
,
K. U.
(
2018
).
Hormonal and environmental signals guiding stomatal development
.
BMC Biol.
16
,
21
.
Qi
,
X.
,
Han
,
S. K.
,
Dang
,
J. H.
,
Garrick
,
J. M.
,
Ito
,
M.
,
Hofstetter
,
A. K.
and
Torii
,
K. U.
(
2017
).
Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling
.
eLife
6
,
e24102
.
Robinson
,
S.
,
De Reuille
,
P. B.
,
Chan
,
J.
,
Bergmann
,
D.
,
Prusinkiewicz
,
P.
and
Coen
,
E.
(
2011
).
Generation of spatial patterns through cell polarity switching
.
Science
333
,
1436
-
1440
.
Rowe
,
M. H.
and
Bergmann
,
D. C.
(
2010
).
Complex signals for simple cells: the expanding ranks of signals and receptors guiding stomatal development
.
Curr. Opin. Plant Biol.
13
,
548
-
555
.
Rowe
,
M. H.
,
Dong
,
J.
,
Weimer
,
A. K.
and
Bergmann
,
D. C.
(
2019
).
A plant-specific polarity module establishes cell fate asymmetry in the Arabidopsis stomatal lineage
.
bioRxiv, 614636
.
Saito
,
S.
,
Hirai
,
N.
,
Matsumoto
,
C.
,
Ohigashi
,
H.
,
Ohta
,
D.
,
Sakata
,
K.
and
Mizutani
,
M.
(
2004
).
Arabidopsis CYP707A s encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid
.
Plant Physiol.
134
,
1439
-
1449
.
Seo
,
M.
,
Hanada
,
A.
,
Kuwahara
,
A.
,
Endo
,
A.
,
Okamoto
,
M.
,
Yamauchi
,
Y.
,
North
,
H.
,
Marion–Poll
,
A.
,
Sun
,
T. P.
,
Koshiba
,
T.
et al. 
(
2006
).
Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism
.
Plant J.
48
,
354
-
366
.
Schmittgen
,
T. D.
and
Livak
,
K. J.
(
2008
).
Analyzing real-time PCR data by the comparative CT method
.
Nat. Protoc.
3
,
1101
-
1108
.
Shpak
,
E. D.
,
Mcabee
,
J. M.
,
Pillitteri
,
L. J.
and
Torii
,
K. U.
(
2005
).
Stomatal patterning and differentiation by synergistic interactions of receptor kinases
.
Science
309
,
290
-
293
.
Tanaka
,
Y.
,
Nose
,
T.
,
Jikumaru
,
Y.
and
Kamiya
,
Y.
(
2013
).
ABA inhibits entry into stomatal–lineage development in Arabidopsis leaves
.
Plant J..
74
,
448
-
457
.
Umezawa
,
T.
,
Okamoto
,
M.
,
Kushiro
,
T.
,
Nambara
,
E.
,
Oono
,
Y.
,
Seki
,
M.
,
Kobayashi
,
M.
,
Koshiba
,
T.
,
Kamiya
,
Y.
and
Shinozaki
,
K.
(
2006
).
CYP707A3, a major ABA 8′-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana
.
Plant J.
46
,
171
-
182
. doi:10.1111/j.1365-313X.2006.02683.x
Vonapartis
,
E.
,
Mohamed
,
D.
,
Li
,
J.
,
Pan
,
W.
,
Wu
,
J.
and
Gazzarrini
,
S.
(
2022
).
CBF4/DREB1D represses XERICO to attenuate ABA, osmotic and drought stress responses in Arabidopsis
.
Plant J.
110
,
961
-
977
.
Wang
,
H.
,
Qi
,
Q.
,
Schorr
,
P.
,
Cutler
,
A. J.
,
Crosby
,
W. L.
and
Fowke
,
L. C.
(
1998
).
ICK1, a cyclin–dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid
.
Plant J.
15
,
501
-
510
.
Wang
,
H.
,
Ngwenyama
,
N.
,
Liu
,
Y.
,
Walker
,
J. C.
and
Zhang
,
S.
(
2007
).
Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis
.
Plant Cell
19
,
63
-
73
.
Wang
,
C.
,
Liu
,
S.
,
Dong
,
Y.
,
Zhao
,
Y.
,
Geng
,
A.
,
Xia
,
X.
and
Yin
,
W.
(
2016
).
Pd EPF 1 regulates water–use efficiency and drought tolerance by modulating stomatal density in poplar
.
Plant Biotech. J.
14
,
849
-
860
.
Wei
,
H.
,
Jing
,
Y.
,
Zhang
,
L.
and
Kong
,
D.
(
2021
).
Phytohormones and their crosstalk in regulating stomatal development and patterning
.
J. Exp. Bot.
72
,
2356
-
2370
.
Yang
,
X.
,
Gavya
,
S. L.
,
Zhou
,
Z.
,
Urano
,
D.
and
Lau
,
O. S.
(
2022
).
Abscisic acid regulates stomatal production by imprinting a SnRK2 kinase-mediated phosphocode on the master regulator SPEECHLESS
.
Sci. Adv.
8
,
eadd2063
.
Yu
,
L.
,
Chen
,
X.
,
Wang
,
Z.
,
Wang
,
S.
,
Wang
,
Y.
,
Zhu
,
Q.
,
Li
,
S.
and
Xiang
,
C.
(
2013
).
Arabidopsis enhanced drought tolerance1/HOMEODOMAIN GLABROUS11 confers drought tolerance in transgenic rice without yield penalty
.
Plant Physiol.
162
,
1378
-
1391
.
Zeng
,
D. E.
,
Hou
,
P.
,
Xiao
,
F.
and
Liu
,
Y.
(
2015
).
Overexpression of Arabidopsis XERICO gene confers enhanced drought and salt stress tolerance in rice (Oryza sativa L.)
.
J. Plant Biochem. Biotech.
24
,
56
-
64
.
Zentella
,
R.
,
Zhang
,
Z. L.
,
Park
,
M.
,
Thomas
,
S. G.
,
Endo
,
A.
,
Murase
,
K.
,
Fleet
,
C. M.
,
Jikumaru
,
Y.
,
Nambara
,
E.
,
Kamiya
,
Y.
et al. 
(
2007
).
Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis
.
Plant Cell
19
,
3037
-
3057
.
Zhang
,
Y.
,
Wang
,
P.
,
Shao
,
W.
,
Zhu
,
J. K.
and
Dong
,
J.
(
2015
).
The BASL polarity protein controls a MAPK signaling feedback loop in asymmetric cell division
.
Dev. Cell
33
,
136
-
149
.
Zuch
,
D. T.
,
Herrmann
,
A.
,
Kim
,
E. D.
and
Torii
,
K. U.
(
2023
).
Cell cycle dynamics during stomatal development: window of MUTE action and ramification of its loss-of-function on an uncommitted precursor
.
Plant Cell Physiol.
64
,
325
-
335
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information