1. Plant Biology

Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division

  1. Yan Gong
  2. Julien Alassimone
  3. Rachel Varnau
  4. Nidhi Sharma
  5. Lily S Cheung
  6. Dominique C Bergmann  Is a corresponding author
  1. Department of Biology, Stanford University, United States
  2. Howard Hughes Medical Institute, Stanford University, United States
  3. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, United States
https://doi.org/10.7554/eLife.63335
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Cite this article
  1. Yan Gong
  2. Julien Alassimone
  3. Rachel Varnau
  4. Nidhi Sharma
  5. Lily S Cheung
  6. Dominique C Bergmann
(2021)
Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division
eLife 10:e63335.
https://doi.org/10.7554/eLife.63335
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Abstract

Asymmetric and self-renewing divisions build and pattern tissues. In the Arabidopsis stomatal lineage, asymmetric cell divisions, guided by polarly localized cortical proteins, generate most cells on the leaf surface. Systemic and environmental signals modify tissue development, but the mechanisms by which plants incorporate such cues to regulate asymmetric divisions are elusive. In a screen for modulators of cell polarity, we identified CONSTITUTIVE TRIPLE RESPONSE1, a negative regulator of ethylene signaling. We subsequently revealed antagonistic impacts of ethylene and glucose signaling on the self-renewing capacity of stomatal lineage stem cells. Quantitative analysis of cell polarity and fate dynamics showed that developmental information may be encoded in both the spatial and temporal asymmetries of polarity proteins. These results provide a framework for a mechanistic understanding of how nutritional status and environmental factors tune stem-cell behavior in the stomatal lineage, ultimately enabling flexibility in leaf size and cell-type composition.

Introduction

The cellular composition of a tissue defines its structure and functions. Tissue-embedded stem cells can control cell fate and distribution by modulating their division behaviors, often using symmetric cell divisions (SCDs) to renew stem-cell capacity and asymmetric cell divisions (ACDs) to diversify daughter cell fates (De Smet and Beeckman, 2011; Losick et al., 2011; Morrison and Kimble, 2006; Motohashi and Asakura, 2014). Tuning the relative proportion of ACDs to SCDs can modulate tissue size and organ composition in response to changes in the external and internal environment. For example, by shifting the ratio of ACDs to SCDs, intestinal stem cells in Drosophila melanogaster can resize the intestine in response to food availability (O'Brien et al., 2011), and in rat brains, stem cells are able to replenish differentiated neurons during stroke recovery (Zhang et al., 2004).

Cell polarity, the restricted localization of proteins, organelles, and activities to one region of the cell, is often linked to ACDs. Cell polarity can precede a division and dictate division orientation, thereby affecting daughter cell size and fate asymmetries, often through differential inheritance of specific materials (Knoblich, 2001; Muroyama and Bergmann, 2019). Although less studied, post-divisional polarity is also important, particularly in situations where cells undergo successive rounds of ACDs. Here, polarity must either be maintained or regenerated at each ACD. When the degree of polarity is not sufficient to ensure differential segregation of proteins to one daughter, it can trigger a developmental switch from ACDs to SCDs (and subsequent differentiation), as was demonstrated for PAR proteins in Caenorhabditis elegans embryo development (Hubatsch et al., 2019).

The stomatal lineage in the epidermis of leaves is an excellent model to study how cell polarity and division behaviors interface with developmental and physiological flexibility. In Arabidopsis, the stomatal lineage produces two essential cell types, stomatal guard cells and pavement cells (Figure 1A). At any given time during development, stomatal lineages at different developmental stages can be found dispersed across the surface of a leaf. These lineages are initiated by ACDs that produce meristemoids and stomatal lineage ground cells (SLGCs). Successive ACDs in either meristemoids (amplifying divisions) or SLGCs (spacing divisions) are self-renewing. Terminal differentiation coincides with the SCD, and subsequent differentiation, of a guard mother cell (GMC) into guard cells. Altering the balance of differentiation and self-renewal (approximated by the SCD/ACD ratio) in the stomatal lineage changes the size, cellular composition, and pattern of the epidermis (Bergmann and Sack, 2007; Vatén et al., 2018). Because the epidermis largely determines leaf size (Gonzalez et al., 2012; Vaseva et al., 2018), SCD/ACD ratio also influences overall leaf properties. These stomatal lineage divisions are often downstream of systemic and environmental cues (Engineer et al., 2014; Lau et al., 2018; Lee et al., 2017; Schroeder et al., 2001). For example, a recent analysis of cytokinin hormone signaling showed that regulating the ability of SLGCs to undergo spacing ACDs contributes to developmental flexibility (Vatén et al., 2018).

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Quantitative analysis of BRXL2-YFP reporter during stomatal lineage divisions reveals reduction in polar localization in the loss-of-function mutant ctr1G738R.

(A) Schematic diagram of stomatal lineage (left) and organization of leaf epidermis (right). From dispersed protodermal precursors, each asymmetric cell division (ACD) produces a small meristemoid (green) and a large stomatal lineage ground cell (SLGC, white). The meristemoid can self-renew by undergoing amplifying ACD(s) or differentiate into guard mother cells (GMCs, blue). Each GMC divides symmetrically to produce paired guard cells (purple). The SLGC can also undergo another ACD (spacing division) or differentiate into a pavement cell. Multiple stomatal lineages are initiated and undergo divisions and differentiation in a dispersed and asynchronized fashion. (B) BRXL2 localization epidermal in cells of 4 dpg Col-0 (top panels) and ctr1G738R (bottom panels) cotyledons. pBRXL2::BRXL2-YFP (left), pATML1::RCI2A-mCherry (middle), and merged (right) are shown separately. (C) Output of POME measurement of depolarized (cell A, left) and polarized BRXL2 (cell B, right). Fluorescence intensity measurements of BRXL2 at each angle are plotted in black dots, and the nonlinear regression models per each cell are plotted in red. (D) POME quantification of BRXL2 polarity index (BPI) in Col-0 (n = 30 cells). Each point represents a BPI score calculated from the BRXL2 cortical localization pattern of one cell (details in Materials and methods and Gong et al., 2021). (E) Output of POME quantification of BRXL2 polarity in 4 dpg Col-0 and ctr1G738R cotyledons (n = 30 cells/genotype, three replicates, statistical analysis reported in Figure 1—figure supplement 3). Scale bar in (B), 10 μm.

Several proteins play crucial roles in determining cell fates and division behaviors in the stomatal lineage, including transcription factors (Kanaoka et al., 2008; MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007), secreted peptide ligands, and cell surface receptors that mediate cell–cell communication (Bergmann et al., 2004; Hara et al., 2007; Hunt and Gray, 2009; Nadeau and Sack, 2002; Qi et al., 2017; Shpak et al., 2005). The transcription factor SPEECHLESS (SPCH) initiates ACDs, and its expression is maintained briefly in both daughter cells, then preferentially lost in the SLGC (MacAlister et al., 2007). Downstream targets of SPCH include ‘polarity proteins’: BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), BREVIS RADIX-LIKE 2 (BRXL2), and POLAR LOCALIZATION DURING ASYMMETRIC DIVISION AND REDISTRIBUTION (POLAR) (Dong et al., 2009; Lau et al., 2014; Pillitteri et al., 2011; Rowe et al., 2019). These polarity proteins localize to cortical crescents and are required for ensuring the size and fate asymmetries of the ACD (Dong et al., 2009; Houbaert et al., 2018; Pillitteri et al., 2011; Rowe et al., 2019; Zhang et al., 2016; Zhang et al., 2015). Each of these polarity proteins can physically interact with signaling kinases and potentially act as scaffolds to ensure the kinases are active in the appropriate cell types and subcellular locations (Houbaert et al., 2018; Marhava et al., 2018; Zhang et al., 2015). The scaffolded kinases include MITOGEN ACTIVATED PROTEIN KINASES (MAPKs), Arabidopsis SHAGGY-LIKE kinases (ATSKs), and AGC kinases—all multifunctional kinases capable of mediating both developmental and environmental signals, thus potentially linking cell polarity to flexible and tunable development.

It is largely unknown how polarity proteins and their clients polarize during stomatal ACDs, and how polarity is linked to self-renewing capacity. From a genetic screen to identify regulators of cell polarity in the stomatal lineage, we found a mutation in CONSTITUTIVE TRIPLE RESPONSE (CTR1), a core component of the ethylene signaling pathway, that resulted in overall depolarization of BRXL2. To understand the connection between CTR1 and BRXL2 polarity, and how the change of BRXL2 polarity affected leaf development, we created quantitative polarity analysis tools (Gong et al., 2021) and long-term tissue-wide lineage tracing methods. We discovered that ethylene and glucose signaling, respectively involved in environmental and nutritional pathways, antagonistically regulate the balance of asymmetric and symmetric cell divisions in the stomatal lineage. Additionally, we uncovered a new interaction between the two cells resulting from an ACD, where the temporal dynamics of BRXL2 polarity in an SLGC was linked to the self-renewing capacity of its sister meristemoid. Together, these results reveal previously underappreciated mechanisms that tune stem-cell behavior in the stomatal lineage.

Results

Loss of CTR1 reduces the fraction of stomatal lineage cells with polarized BRXL2-YFP

To identify regulators of cell polarity in Arabidopsis stomatal lineage, we performed a microscope-based screen for mutations that affected the subcellular localization of BRXL2. We screened ethylmethanesulfonate (EMS)-mutagenized seedlings expressing a native promoter-driven, yellow fluorescent protein (YFP) tagged, BRXL2 reporter (pBRXL2::BRXL2-YFP, Figure 1B, top panels). Among recovered mutants, we were particularly intrigued by a line where the BRXL2-YFP signal was mostly depolarized (Figure 1B, bottom panels). Through mapping and cloning by sequencing, we found this recessive mutant contained a G to A mutation in the coding region sequence of CTR1. CTR1 is a Raf-like kinase that couples with ethylene receptors, and its activity is required to inhibit the downstream ethylene signaling cascade (Huang et al., 2003). The mutation we found is predicted to cause a glycine to arginine substitution at position 738 in the kinase domain (Figure 1—figure supplement 1A), and thus, we will refer to this allele as ctr1G738R. Like the previously reported amorphic allele ctr1-1 (Kieber et al., 1993) and the hypomorphic allele ctr1-btk (Ikeda et al., 2009), ctr1G738R shows a strong constitutive ethylene response phenotype at the seedling level (Figure 1—figure supplement 1B). The ctr1G738R mutant also displayed a small leaf phenotype in older plants, similar to ctr1-1 and stronger than ctr1-btk. This phenotype is consistent with leaf epidermal cells undergoing fewer stem-cell-like ACDs and an overall decreased number of cells in the leaf epidermis (Figure 1—figure supplement 1C).

To confirm that the disruption of CTR1 caused the reduction in BRXL2 polarity, we introduced the pBRXL2::BRXL2-YFP reporter into the established ctr1-btk and ctr1-1 mutants. We found that ctr1-btk and ctr1-1 also caused different degrees of BRXL2 depolarization (Figure 2A–D). We also created a heteroallelic combination of ctr1G738R with ctr1-btk. The F1 progeny of a cross between ctr1G738R pBRXL2::BRXL2-YFP and ctr1-btk displayed an intermediate disruption in BRXL2 polarity, as well as an intermediate ethylene response phenotype (Figure 2E). These results confirm CTR1 as the causal locus, but because ctr1G738R (like ctr1-1) has severe developmental defects, we wanted to eliminate the possibility that the BRXL2 polarity disruption phenotype was a secondary consequence of broad and excessive ethylene signaling. We generated an artificial microRNA (amiRNA) knockdown line targeting CTR1 under a stomatal lineage-specific promoter (pTMM::amiRNA-CTR1). pTMM::amiRNA-CTR1 lines did not show a strong ethylene response at the seedling stage (Figure 1—figure supplement 1D), but did show reduced polarity of BRXL2 in the stomatal lineage (Figure 2F). Additionally, shoot epidermal-only expression of CTR1 (pATML1::CTR1) was sufficient to rescue the reduced BRXL2 polarity phenotype and the small leaf phenotype of ctr1G738R (Figure 1—figure supplement 1E–I). These rescue results support a direct role of CTR1 in the epidermis and reinforce previous work showing that the epidermis drives organ growth in leaves (Marcotrigiano, 2010; Vaseva et al., 2018).

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Active ethylene signaling decreases BRXL2 polarity.

(A–I) BRXL2 localization pattern in 4 dpg cotyledons from different ethylene mutants or treatment (pBRXL2::BRXL2-YFP in green). (A) Col-0, (B) ctr1G738R, (C) ctr1-1, (D) ctr1-btk, (E) ctr1G738R/ctr-btk F1, (F) pTMM::amiRNA-CTR1, (G) 10 μM 1-aminocyclopropane-1-carboxylic acid-treated Col-0, (H) EIN3ox, and (I) ein2-5. (J) POME quantifications of BRXL2 polarity index from (A–I) conditions (n = 30 cells/genotype). All p-values are calculated by Mann–Whitney test. Scale bars in (A–I), 10 μm.

Time-lapse imaging combined with quantitative polarity analysis reveals nuances of stomatal lineage cell polarity

Among ctr1 mutant lines, BRXL2-YFP was consistently depolarized in most, but not all, cells. This suggested that CTR1 is not absolutely essential for BRXL2 polarity establishment and emphasized that to accurately interpret the role of CTR1 we needed a full picture of BRXL2 polarity dynamics throughout the stomatal lineage. We therefore monitored the dynamics of BRXL2-YFP during development of the entire cotyledon over 2 days in 40 min intervals. Consistent with previous reports (Rowe et al., 2019), a polar crescent of BRXL2 is visible before asymmetric division (Figure 1—figure supplement 2A, top-left panels; Video 1), and after division, this crescent is inherited by the large daughter cell (SLGC). Time lapse, however, revealed two additional features of BRXL2 behavior: first, BRXL2 persists in the SLGC for more than 8 hr after division (Figure 1—figure supplement 2A, top-left panels); and second, BRXL2 is still expressed in symmetrically dividing GMCs, but is depolarized in these cells (Figure 1—figure supplement 2A, top-right panels). These results suggest a correlation between the degree of BRXL2 polarity and cell identity. Thus, we applied our recently developed polarity measurement tool (POME, Gong et al., 2021) to quantify the distribution of BRXL2-YFP signal at the periphery of each cell. With POME, the pixel intensity of the BRXL2 reporter and an evenly distributed PM reporter are captured along the entire cell circumference, and their relative distributions were used to compute a ‘polarity index’ (Figure 1C; Gong et al., 2021). The BRXL2 polarity index (BPI) is defined as the fraction of measurements above the half maximum; it represents the fraction of the plasma membrane occupied by BRXL2 and ranges between 0 and 1. A BPI close to 0 represents a cell with highly polarized BRXL2, while a BPI of 1 represents a cell with completely depolarized BRXL2. As illustrated for Col-0 in Figure 1D, it is also possible to capture the distribution of BPI measurements in the population of BRXL2-expressing cells. Because of the tight correlation we found between cell identity and BPI, this ‘snapshot’ population measure can also be used to estimate the ratio of SCDs to ACDs occurring in the leaf epidermis.

Video 1
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BRXL2 dynamics in Col-0.

Examples of BRXL2 dynamics (green, pBRXL2::BRXL2-YFP) during stomatal lineage asymmetric cell divisions (labeled by white arrowheads) and symmetric cell divisions (labeled by yellow arrows) on the abaxial surface of a 3 dpg wild-type Col-0 cotyledon. Cell membrane is labeled by pATML1::RCI2A-mCherry in magenta. White numbers in frames indicate hours:minutes relative to first frame. Scale bar, 10 µm.

We quantified the BPI of Col-0 and ctr1 cotyledons at 4 days post germination (dpg). As expected, ctr1G738R cotyledons showed a higher average BPI than Col-0 (Figure 1E), with fewer cells displaying low BPIs (highly polarized BRXL2). When we specifically analyzed the low BPI cells, we found no significant difference in crescent size or peak height between Col-0 and ctr1G738R (Figure 1—figure supplement 2B–D). Through additional time-lapse imaging, we demonstrated that, like ACDs in Col-0, stomatal lineage ACDs in ctr1G738R cotyledons were always preceded by the appearance of polarized BRXL2 in the precursor cell (Figure 1—figure supplement 2A, bottom-left panels; Video 2).

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BRXL2 dynamics in ctr1G738R.

Examples of BRXL2 dynamics (green, pBRXL2::BRXL2-YFP) during stomatal lineage asymmetric cell divisions (labeled by white arrowheads) and symmetric cell divisions (labeled by yellow arrows) on the abaxial surface of a 3 dpg ctr1G738R cotyledon. Cell membrane is labeled by pATML1::RCI2A-mCherry in magenta. White numbers in frames indicate hours:minutes relative to first frame. Scale bar, 10 µm.

These results raised a conundrum: if cells in ctr1G738R can polarize BRXL2, then how was ctr1G738R identified and mapped based on a disrupted BRXL2 polarity phenotype? Two potential explanations emerged, each reflecting the dynamic nature of polarity. First, because BRXL2 is polarized in cells undergoing ACDs and depolarized in cells undergoing SCDs, altering the ratios of these division types could decrease the proportion of cells with polarized BRXL2. This change might be detected as a population-level BRXL2 polarity decrease during the screen and suggests that the role of CTR1 is primarily to maintain stem-cell capacity. Second, we had also observed that BRXL2-polarized crescents did not persist as long after ACDs in ctr1G738R compared to Col-0 (Figure 6A), and this reduction of BRXL2 persistence would also result in the appearance of relatively fewer polarized cells when observing BRXL2 polarity at a single timepoint. This result suggests several possible models of CTR1 action in regulating BRXL2 polarity and ACD, but also raises questions about post-divisional functions of polarity factors and how polarity is maintained.

Disruption of CTR1 results in diminished stem-cell capacity

To dissect the relationship between BRXL2 behavior with stem-cell capacity and with stomatal cell fate determination, we calculated the stomatal index (SI, ratio of stomata to all epidermal cells) of fully developed cotyledons of Col-0 and ctr1 mutants at 14 dpg. Compared to Col-0, the SIs of different ctr1 mutants were significantly elevated (Figure 3A, B, E, Figure 3—figure supplement 1A–C). Because of the flexible trajectory of the stomatal lineage (Figure 1A), increased SI has several possible origins: (1) an increase in cells entering the stomatal lineage (entry division), (2) an increase in secondary entry via SLGC spacing divisions, or (3) a decrease in meristemoid self-renewal by ACDs (amplifying divisions). To evaluate the contributions of these possibilities, we developed a whole-leaf-based lineage tracing method. We tracked the developmental progression of all epidermal cells in Col-0 and ctr1G738R cotyledons within a 48 hr time window (3–5 dpg) and captured the developmental progression of more than 500 pairs of meristemoids and SLGCs per genotype (Figure 3F). Strikingly, the percentage of amplifying ACDs from ctr1G738R cotyledons was significantly reduced, while the percentage of spacing ACDs was similar to Col-0 (Figure 3G). When individual meristemoids were tracked, we found many underwent fewer rounds of amplifying ACDs during 48 hr in ctr1G738R compared to Col-0 (Figure 3H).

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Ethylene signaling modulates the symmetric cell division/asymmetric cell division balance during leaf epidermal development.

(A–D) Differential interference contrast (DIC) images of (A) Col-0, (B) ctr1G738R, (C) 10 μM 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Col-0, and (D) ein3eil1ebf1ebf2 14 dpg cotyledons grown on ½ Murashige and Skoog media without sugar. (E) Quantification of the stomatal index of Col-0, ACC-treated Col-0, and selected ethylene signaling mutants (n = 8/genotype). (F–H) Results of tracing epidermal cell lineages in Col-0 and ctr1G738R cotyledons. (F) Total numbers of different division types tracked. (G) Percentage of each division type among total divisions. (H) Quantification of number of divisions the tracked meristemoids undergo, accompanied by cartoon representation (n > 500 cells/genotype). (I) Total abaxial epidermal cell number in Col-0 and ctr1G738R cotyledons from 3 to 5 dpg (n = 3–5 cotyledons/genotype/day). All p-values are calculated by Student’s t-test due to the small sample sizes. Scale bars in (A–D), 10 μm.

Fewer amplifying ACDs should result in fewer epidermal cells; this was confirmed by comparing total cell numbers of whole Col-0 and ctr1G738R cotyledons at 3, 4, and 5 dpg. At 3 dpg, ctr1G738R and Col-0 cotyledons had similar numbers of cells, suggesting a similar level of stomatal entry divisions. However, by 5 dpg, Col-0 had accumulated about 30% more cells than ctr1G738R (Figure 3I). Together, these results suggest that meristemoids prematurely exit stem-cell divisions in ctr1G738R plants, thereby elevating the SCD/ACD ratio and ultimately generating fewer epidermal cells, a higher SI, and smaller leaves.

Ethylene signaling regulates polarity protein complex and stomatal lineage development

CTR1 is best known as a negative regulator of ethylene signaling, but it also is enmeshed in cross-talk with other signaling pathways. Ethylene’s ability to affect stomatal lineage development was previously noted (Serna and Fenoll, 1996), but the details of the regulation were not clear. To test whether ethylene, in general, affects stomatal lineage ACD/SCD decisions, we treated pBRXL2::BRXL2-YFP-expressing seedlings with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). A 10 μM ACC treatment significantly increased average BPI at 4 dpg and SI at 14 dpg relative to mock-treated controls (Figure 2G, J, Figure 3C, E). Upon ethylene reception, one of the core elements of the ethylene pathway, ETHYLENE INSENSITIVE 2 (EIN2), is cleaved, translocates to the nucleus, and activates the transcription factors EIN3 and its homolog ETHYLENE INSENSITIVE LIKE 1 (EIL1) to mediate ethylene signaling (Alonso et al., 1999; An et al., 2010; Chang et al., 2013; Chao et al., 1997; Qiao et al., 2012; Wen et al., 2012). CTR1 acts as a negative regulator by phosphorylating EIN2 and preventing its cleavage (Qiao et al., 2012; Wen et al., 2012). In plants overexpressing EIN3 (EIN3ox) (Chao et al., 1997), the average BPI and SI were higher than the average BPI and SI in Col-0 (Figure 2H, J, Figure 3E, Figure 3—figure supplement 1D). Conversely, a slight decrease of BPI and SI was observed in the ethylene-insensitive mutants ein2-5 (Alonso et al., 1999) and the quadruple mutant ein3eil1ebf1ebf2, where EBF (EIN3-binding F-BOX) proteins were eliminated to approximate complete lack of ethylene response (An et al., 2010, Figure 2I, J, Figure 3D, E). To test whether CTR1 acts primarily through the EIN2/EIN3 core pathway to regulate stomatal stem-cell divisions, we treated ein2-5 single mutants and ein3eil1ebf1ebf2 quadruple mutants with 10 μM ACC for 4 days. In these ethylene-insensitive backgrounds, ACC treatment did not affect BRXL2 polarity (Figure 2—figure supplement 1), suggesting that CTR1 acts via the canonical ethylene signaling pathway to modulate meristemoid division behaviors, regulate stomatal density, and limit leaf growth.

These genetic and pharmacological perturbations of ethylene signaling indicate that ethylene signaling disrupts BRXL2 polarity primarily through shifting cell identity from meristemoids (which polarize BRXL2) to GMCs (which do not). These results demonstrate how a systemic signal alters tissue-level development and suggest that BPI may be an easily scorable proxy for stem-cell potential, a phenotype that was tedious and labor-intensive to measure previously (Vatén et al., 2018).

Glucose signaling antagonizes ethylene signaling and enhances amplifying divisions

For BPI to be generally useful as an estimate of stomatal stem-cell potential, we needed to identify situations where stem-cell potential was enhanced to provide a counterpoint to the repression we observed in ctr1. Previous studies found that ethylene and sugar signaling can act in opposition, and so we hypothesized that if sugar signaling also acted in the stomatal lineage, altered availability or perception of sugar would be reflected in the BPI (Haydon et al., 2017; Karve et al., 2012; Yanagisawa et al., 2003; Zhou et al., 1998). Indeed, addition of sucrose to the media resulted in a dose-dependent decrease in average BPI in both ctr1G738R and Col-0 cotyledons (Figure 4A–D, K, Figure 4—figure supplement 1).

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Sucrose and glucose signaling increase BRXL2 polarity.

(A–J) BRXL2 localization pattern in 4 dpg cotyledons grown on ½ Murashige and Skoog (MS) plates with various sugars (pBRXL2::BRXL2-YFP in green). (A, C, E, G, and I) Col-0 and (B, D, F, H, and J) ctr1G738R. (A, B) No sugar, (C, D) 1% sucrose, (E, F) 1% fructose, and (I, J) 1% 3-O-methyl-D-glucose. (K) BRXL2 polarity index (BPI) quantification of Col-0 and ctr1G738R grown on ½ MS plates with different sucrose concentrations (n = 50 cells/genotype). (L) BPI quantification in 4 dpg Col-0 and ctr1G738R seedlings growing on ½ MS plates with various sugars (n = 50 cells/genotype). The same BPI measurements of Col-0 and ctr1G738R from no sugar and 1% sucrose treatment are included in (K) and (L) for easier visual comparison. All p-values are calculated by Mann–Whitney test. Scale bars in (A–J), 10 μm.

The effect of sucrose on BPI could be due to its role as a nutrient or a signal. In Arabidopsis, sucrose is the major transport form of sugar and the major energy source for sink tissues where it is further broken down into glucose and fructose to provide energy for cell metabolism, growth, and divisions. Glucose also acts as a signaling molecule during plant development and responds to environmental cues via a hexokinase (HXK)-mediated pathway (Eveland and Jackson, 2012). We therefore tested the ability of metabolizable, non-metabolizable, signaling-active, and signaling-inactive forms of sugars to change BPI in Col-0 and ctr1G738R. Addition of glucose, but not fructose, decreased average BPI (Figure 4E–H, L). The glucose signaling-inactive analog, 3-O-methyl-D-glucose (3-OMG) (Cortès et al., 2003), failed to alter BPI (Figure 4I, J, L), suggesting that it is glucose signaling, rather than cellular energy status, that affects stomatal lineage progression. To test whether these treatments specifically targeted BRXL2 or whether BRXL2-YFP reports a more general effect on cell polarity, we tested the effects of glucose and ethylene on BASL polarity using a pBASL::YFP-BASL reporter line (Figure 4—figure supplement 2A; Rowe et al., 2019). Like BRXL2 polarity, BASL polarity was significantly enhanced with 2% glucose treatment and reduced with 10 µM ACC treatment (Figure 4—figure supplement 2B–E). These results suggest that glucose and ethylene impact cell polarity in the stomatal lineage more generally.

We then tested whether the stomatal lineage response to ethylene was mediated by the polarity proteins themselves. In stomatal development, BRXL2 is genetically redundant with other BRX family members, and the BRX family appears to physically interact with and be mutually dependent on BASL (Rowe et al., 2019). Therefore, to most cleanly assay hormone response in the absence of polarity protein activity, we treated the BASL null mutant (basl-2) with ACC. 10 µM ACC can still increase the stomatal index in basl-2, although the degree of increase is less than that in Col-0 (Figure 4—figure supplement 2F). These results are consistent with polarity proteins playing a minor role in directly mediating ACC response.

To confirm our model that a decrease in BPI upon glucose treatment reflected an increase in stem-cell potential, we tracked division types of all epidermal cells in time courses. Consistent with its ability to decrease average BPI in treated plants, 2% glucose treatment promoted amplifying ACDs in both Col-0 and ctr1G738R (Figure 5A). Additionally, the SI of ctr1G738R grown in the presence of 2% glucose was shifted back to the wild-type level (Figure 5B, Figure 5—figure supplement 1), consistent with glucose-antagonizing ethylene signaling and boosting the ability of meristemoids to undergo amplifying ACDs.

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Glucose promotes amplifying asymmetric cell divisions independent of ethylene signaling and HXK1- or TOR-mediated glucose signaling pathways.

(A) Cartoon of types of divisions meristemoids undergo, and fraction of each type in Col-0, ctr1G738R, Col-0 with 2% glucose treatment, and ctr1G738R with 2% glucose treatment (n > 500 cells/condition). Data for the no sugar condition of Col-0 and ctr1G738R are also reported in Figure 3H. (B) Stomatal index of 14 dpg Col-0 and ctr1G738R growing on ½ Murashige and Skoog (MS) plates or 2% glucose ½ MS plates (n = 8/condition). (C) BRXL2 polarity index (BPI) quantification of 4.5 dpg abaxial cotyledons of Col-0, ein2-5, and ein3eil1ebf1ebf2 grown on ½ MS plates or 2% glucose ½ MS plates (n = 30 cells/genotype). (D) BPI quantification of 4 dpg abaxial cotyledons of Ler-0 and gin2 grown on ½ MS plates or 2% glucose ½ MS plates (n = 45 cells/genotype). (E) BPI quantification in 4 dpg Col-0 treated with TOR inhibitor AZD8055 and/or 2% glucose (n = 30 cells/genotype). (F) BPI quantification of true leaves in 9 dpg Col-0 seedlings 110 μE normal light condition for 7 days and then transferred to different low light intensity conditions for 48 hr (n = 30 cells/genotype). All p-values are calculated by Mann–Whitney test.

Glucose control of stomatal differentiation is independent of ethylene signaling, HXK1 signaling, or TOR signaling

The relationship between glucose and ethylene signaling has been explored in detail in other contexts, leading to the model that active HKX-1-mediated glucose signaling promotes EIN3 degradation and reduces ethylene signaling activity (Yanagisawa et al., 2003). To test if glucose regulates stomatal divisions through EIN3 inhibition, we compared BPIs in the ethylene-insensitive mutants ein2-5 and ein3eil1ebf1ebf2 grown with and without glucose in the media. Both mutants had significantly reduced BPIs with 2% glucose treatment at 4.5 dpg (Figure 5C, Figure 5—figure supplement 2A–F), suggesting that the influence of glucose in stomatal divisions is independent of the core components of ethylene signaling.

To test another mechanism by which glucose signaling is connected to stomatal lineage development, we quantified the BPI in previously established HXK1 loss-of-function mutants, hxk1-3 (Huang et al., 2015) and gin2 (Moore et al., 2003), in response to glucose treatment. When treated with 2% glucose, both mutants exhibited a decrease in average BPI similar to their corresponding wild-type controls, Col-0 and Ler-0 (Figure 5D, Figure 5—figure supplement 3A–J), suggesting that glucose’s regulation in stomatal development is not mediated by HXK1. Another glucose signaling pathway, the target of rapamycin (TOR) pathway, has been suggested to act downstream of sugar signaling as a major controller of plant growth-related processes, including meristem proliferation, leaf initiation, and cotyledon growth (Li et al., 2017; Rexin et al., 2015; Xiong et al., 2013). We treated pBRXL2::BRXL2-YFP-expressing seedlings grown with and without 2% glucose with 1 μM AZD-8055, an ATP-competitive inhibitor of TOR (Montané and Menand, 2013), for 2 days. Despite the presence of AZD-8055, glucose still decreased the average BPI (Figure 5E, Figure 5—figure supplement 4). Therefore, despite clear evidence that glucose signaling can influence stomatal lineage behaviors, we have been unable to link this regulation to known HXK1 or TOR-mediated sugar signaling pathways.

Stomatal BPI can respond to physiological depletion of sugars

By experimentally adding sugars, we could modulate BPI and stomatal divisions. The critical question then becomes whether this is biologically relevant—do Arabidopsis leaf epidermal cells sense endogenous levels of glucose or sucrose and adjust the stomatal lineage to create leaves of appropriate cellular composition? To test this, we adapted an approach used in Moraes et al., 2019 and reduced light intensity to limit the photosynthetic rate in seedlings. Because sugar is the primary product of photosynthesis, this experimental procedure serves to exhaust endogenous sugars in leaves. We transferred 7 dpg Col-0 seedings from our regular high-light intensity (110 μE) growth condition to low-light conditions (11 or 0 μE). After 48 hr, seedlings grown in low-light conditions showed increased BPIs in their leaves (Figure 5F, Figure 5—figure supplement 2G–I), indicating fewer ACDs took place. We could reverse the effect of low (11 μE) light on BPI in the true leaves of Col-0 by addition of 2% glucose, and this response also occurred in ein3eil1ebf1ebf2 plants (Figure 5—figure supplement 5). Together, these results are consistent with sugar in the leaf providing feedback to coordinate epidermal development with photosynthesis.

Post-division BRXL2 crescent is associated with meristemoid fate determinacy

In previous sections, we showed that CTR1, ethylene, and sugar signaling regulate the balance between stomatal lineage cells undergoing proliferative ACDs and differentiating SCDs, where the connection to BRXL2 polarity is largely indirect. However, in our examination of ctr1G738R, we also noticed that polarized BRXL2 was in fewer SLGCs than it was in Col-0 and suspected that BRXL2 was less persistent in these cells. We monitored persistence of the polarized BRXL2 crescent directly through time-lapse imaging. The BRXL2 crescent was significantly less persistent in ctr1G738R than in Col-0 (Figure 6A, B, Figure 6—figure supplement 1A), and glucose increased persistence of BRXL2 crescent in both ctr1G738R and Col-0 seedlings (Figure 6C). In light of the observation that meristemoids in ctr1G738R underwent fewer amplifying ACDs (Figure 3H) and glucose promoted amplifying ACDs in both Col-0 and ctr1G738R (Figure 5A), a positive correlation emerges between the persistence of the post-division BRXL2 polarity complex in the SLGC and the self-renewal capacity of the meristemoid (the SLGC sister derived from the previous ACD).

Figure 6 with 1 supplement see all
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Post-division persistence of BRXL2 polarity is associated with meristemoid fate determinacy.

(A) Time-lapse images of post-asymmetric cell division (ACD) BRXL2 dynamics from stomatal lineage ground cells (SLGCs) in Col-0 and ctr1G738R cotyledons (3 dpg). 00:00 (hours:minutes) marks cell plate formation. (B) Quantification of BRXL2 polarity dynamics after ACDs in Col-0 and ctr1G738R. Individual measurement per each cell (n = 3 cells per genotype) shown in thin lines and the respective trend per each genotype with 0.95 confidence interval is indicated as the thick line with gray band. (C) Persistence of BRXL2 post-ACD in SLGCs from 3 dpg Col-0 and ctr1G738R grown in ½ Murashige and Skoog (MS) media or ½ MS media with 2% glucose. (D) Relationship between persistence of BRXL2 in SLGCs and division behavior of their meristemoid sisters. (E) Examples of division behaviors quantified in (D). Time-lapse analysis of BRXL2 polarity in 3 dpg Col-0 cotyledons followed by lineage tracing. BRXL2 was imaged every 40 min for 16 hr, then plants returned to ½ MS plate for 48 hr, then reimaged to capture divisions and fate of the BRXL2-expressing cells. Different division types are marked with asterisks (GMC division), triangles (amplifying division), and arrows (spacing divisions). (F–I) Evidence that ethylene and glucose signaling does not affect SPCH level in individual stomatal lineage cells. (F) Quantification of gSPCH reporter fluorescence intensity at 4 dpg in (G) mock, (H) 10 μM 1-aminocyclopropane-1-carboxylic acid, and (I) 2% glucose-treated Col-0 cotyledons (n = 3 cotyledons/treatment; n > 120 cells/treatment). Lookup table Fire is used to false color gSPCH reporter intensity (color key in figure). All p-values are calculated by Mann–Whitney test. Scale bars in (A, E), 5 μm; (I), 10 μm.

Two plausible explanations for a non-cell-autonomous effect of BRXL2 persistence on its sister meristemoid’s behavior are (1) that these cells communicate or (2) that by monitoring BRXL2 we witnessed a differentiation event already specified in their mother cell; for example, a change in SPCH activity that distinguishes cells with higher self-renewal potential and cells that will undergo a final ACD.

SPCH initiates all the ACDs in the stomata lineage (MacAlister et al., 2007) and directly binds the promoters of BRXL2 and BASL in ChIP-Seq studies (Lau et al., 2014). BRXL2 crescent persistence, and the change of that persistence in response to ethylene and glucose treatment, therefore, might be directed by quantitative changes in SPCH expression in the cells undergoing ACD. We quantified SPCH protein levels in individual cells from 10 μM ACC, 2% glucose, and mock-treated Col-0 cotyledons by measuring the fluorescence intensity of a functional genomic SPCH reporter (pSPCH::gSPCH-YFP in spch3) (Lopez-Anido et al., 2020). We found no significant change in gSPCH-YFP levels upon treatment with either ACC or glucose (Figure 6F–I). We also examined gSPCH reporter dynamics during ACDs in these different treatment conditions and found no obvious changes in gSPCH reporter peak intensity, onset of expression, or post-divisional persistence (Figure 6—figure supplement 1C, D).

If BRXL2 crescent persistence in an SLGC is not coupled to the upstream (SPCH) transcriptional response, then this persistence is unlikely to be reflecting a decision made pre-ACD in the mother cell. To correlate BRXL2 crescent persistence post-ACD with the subsequent divisions and fates of the daughters, we performed detailed time-lapse imaging (16 hr, 40 min intervals) followed by a 48 hr time course in Col-0. We found that the post-ACD BRXL2 crescent persistence in SLGCs was predictive of sister meristemoid behavior. BRXL2 crescents persisted, on average, 2 hr longer when meristemoids underwent amplifying ACDs than when meristemoids divided symmetrically (Figure 6D, E). Interestingly, BRXL2 was not predictive of the behavior of the cell in which it resides. There was no significant difference between persistence in SLGCs that underwent spacing ACDs and those that did not (Figure 6—figure supplement 1B). Together, these results suggest that there must be communication between the sister cells resulting from an ACD. Hypotheses about the nature of this communication are presented in Figure 7 and will be discussed below.

Figure 7
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Models at organ and cell scales for connection between systemic signaling, cell polarity, stomatal stem-cell potential, and leaf growth.

(A) Schematic representation of the regulation of ethylene and glucose on amplifying divisions in meristemoids. Potential regulatory mechanisms used by these signals are illustrated in dashed box. (B) Schematic representation of modes of communication possible between stomatal lineage ground cells and meristemoids during polarity and cell division control.

Discussion

Plants respond to environmental stimuli by modifying their development. As the major organs of photosynthesis, leaves must regulate their size, position, and gas-exchange capacity to adapt and compete. Using genetics, live-cell imaging, lineage tracing, and quantitative image analysis of the simple, yet flexible, Arabidopsis stomatal lineage, we have been able to link ethylene and sugar signaling to the self-renewing capacity of epidermal stem cells. The immediate readout of ethylene and glucose antagonism is a shift in population of cells expressing polarized vs. depolarized BRXL2, and the ultimate readout is a change in the size and the cell-type composition of the leaf (Figure 7A). We also uncovered a surprising correlation between the ACD potential of meristemoids and the persistence of polarized BRXL2 in SLGCs (Figure 7B), suggesting active communication and coordination between these sister cells.

Finding CTR1 alleles in screens for ACD regulators was not expected, and we considered whether there was a true role for ethylene signaling or whether we were seeing the effect of altering CTR1 on other pathways. Genetic and pharmacological experiments (Figures 2 and 3), however, confirmed the participation of multiple ethylene signaling components in regulating stomatal lineage ACDs. Ethylene also inhibits stem-cell divisions in the shoot apical meristem of Arabidopsis (Hamant et al., 2002), revealing a common theme to regulation of stem-cell behaviors in the aerial tissues. Ethylene is considered an ‘aging’ and a ‘stress’ hormone (Iqbal et al., 2017; Schaller, 2012) and regulates many different aspects of plant development, often through its cross-talk with auxin (Muday et al., 2012; Strader et al., 2010; Vaseva et al., 2018; Wen et al., 2012). In both root and leaf epidermal cells, for example, ethylene promotes local auxin biosynthesis. Elevated auxin levels then inhibit cell expansion and regulate plant growth (Vaseva et al., 2018). Elevated auxin signaling can also induce expression of ethylene biosynthesis genes (Abel et al., 1995; Tsuchisaka and Theologis, 2004), creating a feedback loop between these two hormones. In our case, ethylene might regulate stomatal lineage cell divisions through its feedback with auxin. Auxin has also been shown to influence cell division and differentiation in the stomatal lineage, but there are some conflicting results, with Le et al., 2014 suggesting that auxin promotes meristemoid ACDs and Balcerowicz et al., 2014 concluding that auxin inhibits ACDs. The interplay of auxin and ethylene and the influence of auxin on all types of stomatal lineage divisions and fate transitions are beyond the scope of this paper, but will be an exciting future direction and may be answered more definitively by new tools like engineered TIR1 (Uchida et al., 2018) expressed in specific stomatal lineage cell types.

We did pursue the cross-talk between ethylene and sugar signaling, and found that higher levels of each resulted in opposite effects on BRXL2 persistence and stomatal lineage ACDs. Introduction of 2% glucose to growth media increased the stem-cell-like ACDs, even in ethylene-insensitive mutants, indicating that the epidermis is likely perceiving these signals independently. An effect of sugars on stomatal production was recently reported (Han et al., 2020). Their experimental conditions differ substantially from ours, however, making direct comparisons of results impossible, but both studies concur that sugars promote stomatal development. What information is sugar providing? Glucose and sucrose are the major products of photosynthesis produced in mesophyll cells, and recent work demonstrates multiple modes of communication between mesophyll and epidermis to coordinate these tissues for optimal growth and gas exchange (Baillie and Fleming, 2020; Dow et al., 2017; Sugano et al., 2010). It is attractive to consider mesophyll-derived sugar signaling as a way to promote growth and stomatal production, and future experiments could be designed to trace the source of sugar perceived by the stomatal lineage.

Perhaps our most interesting and surprising result was that temporal variations in BRXL2 persistence correlated with ACD potential. In particular, although BRXL2 is expressed in SLGCs, it was the behavior of the sister meristemoid that was affected. This made us consider what properties of the meristemoid are most important for that cell’s behaviors (Figure 7B). Previous work considered the expression of the transcription factor SPCH as the key to modulating stomatal ACDs (Lau et al., 2018; Simmons et al., 2019; Vatén et al., 2018; Zhang et al., 2016). However, while SPCH is necessary for divisions, neither glucose nor ethylene signaling had a dose-dependent effect on SPCH levels. On the other hand, meristemoids vary in cell size, and ethylene and sugar have opposite effects on cell expansion. Thus, one possibility, parallel to the situation in the C. elegans germline, is that smaller meristemoids undergo fewer rounds of ACDs before committing to terminal differentiation. This hypothesis could also explain the difference in ethylene’s effect on ACD potential in meristemoids and SLGCs. SLGCs are larger than meristemoids, thus ethylene-mediated repression of cell expansion will cause meristemoids to fall below the critical size threshold more often than SLGCs.

By what mechanisms could SLGCs influence their sister meristemoid, and how might persistent BRXL2 polarity drive this regulation? Signals from the meristemoid to the SLGC rely on the mobile peptide EPIDERMAL PATTERNING FACTOR2 (EPF2). Perception of EPF2 leads to higher MAPK activity in SLGCs (Lee et al., 2015). Elevated MAPK activity not only inhibits divisions (Bergmann et al., 2004; Lampard et al., 2009) but also enhances BASL polar localization (Zhang et al., 2015). Thus, BRXL2 polarity persistence in the SLGC could be a readout of the ACD potential of the sibling meristemoid. In this scenario, EPF2 secreted by the self-renewing meristemoid is perceived by the neighboring SLGC, stimulating the MAPK signaling pathway in that SLGC. Elevated MAPK activity then promotes nuclear export and cortical enrichment of BASL (Zhang et al., 2015), which in turn sustains BASL and BRXL2 polarity. When the meristemoid becomes a GMC, EPF2 is no longer produced and MAPK signaling is not activated in the neighboring SLGC. BASL is retained in the nucleus, and, without its partner, BRXL2 becomes depolarized.

We propose, however, that longer persistence of the polarity domain in the SLGC can also act as the source of a signal. This signal may be either chemical or mechanical (or both). For example, BASL and BRXL2 polar crescents precede local cell outgrowth (Bringmann and Bergmann, 2017; Mansfield et al., 2018) and differential expansion in the SLGCs could also serve as a division-promoting signal to the meristemoid. Such a mechanism has been shown to maintain tissue integrity among mechanically coupled cells (Hamant and Haswell, 2017). Polarity proteins BASL, BRX, and POLAR scaffold MAPKs, PAX, and BIN2 kinases, respectively (Houbaert et al., 2018; Marhava et al., 2018; Zhang et al., 2015). In many situations, including these three scaffold/kinase examples, scaffolding increases signaling output and creates extensive positive feedback on polarity (Houbaert et al., 2018; Marhava et al., 2018; Zhang et al., 2015). Additionally, higher signal accumulation in auxin (MAPK, PAX) or brassinosteroid (BIN2) pathways can also lead to the production of mobile signals, and such signals have been connected to coordination of growth and stomatal lineage progression (Houbaert et al., 2018; Kim et al., 2012; Le et al., 2014). Distinguishing among these mechanisms will require new tools to specifically alter polarity crescent persistence in an otherwise unperturbed background, but advances in inducible degradation systems (Faden et al., 2016; Sallee et al., 2018) may enable these experiments in the future.

Materials and methods

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Gene (Arabidopsis thaliana)CTR1TAIR (https://www.arabidopsis.org/)AT5G03730Receptor-coupled kinase involved in ethylene signaling
Gene (Arabidopsis thaliana)BRXL2TAIR (https://www.arabidopsis.org/)AT3G14000Polarity protein in the stomatal lineage
Gene (Arabidopsis thaliana)BASLTAIR (https://www.arabidopsis.org/)AT5G60880Polarity protein in the stomatal lineage
Gene (Arabidopsis thaliana)ATML1TAIR (https://www.arabidopsis.org/)AT4G21750Homeobox transcription factor
Gene (Arabidopsis thaliana)EIN2TAIR (https://www.arabidopsis.org/)AT5G03280Component of ethylene signaling
Gene (Arabidopsis thaliana)EIN3TAIR (https://www.arabidopsis.org/)AT3G20770Transcriptional factor in ethylene signaling
Gene (Arabidopsis thaliana)EIL1TAIR (https://www.arabidopsis.org/)AT2G27050Transcriptional factor in ethylene signaling
Gene (Arabidopsis thaliana)EBF1TAIR (https://www.arabidopsis.org/)AT2G25490F-box protein involved in ethylene signaling
Gene (Arabidopsis thaliana)EBF2TAIR (https://www.arabidopsis.org/)AT5G25350F-box protein involved in ethylene signaling
Gene (Arabidopsis thaliana)HXK1TAIR (https://www.arabidopsis.org/)AT4G29130Hexokinase in the glucose signaling network
Gene (Arabidopsis thaliana)SPCHTAIR (https://www.arabidopsis.org/)AT5G53210bHLH transcription factor involved in stomatal development
Gene (Arabidopsis thaliana)TMMTAIR (https://www.arabidopsis.org/)AT1G80080LRR receptor-like protein involved in stomatal development
Strain, strain background (Arabidopsis thaliana)Col-0ABRCCS28166Wild-type Arabidopsis ecotype used in this study
Strain, strain background (Arabidopsis thaliana)Ler-0ABRCCS20Wild-type Arabidopsis ecotype used in this study
Strain, strain background (Agrobacterium tumefaciens)GV3101OtherElectrocompetent
A. tumefaciens
Genetic reagent (Arabidopsis thaliana)ctr1-1Kieber et al., 1993; DOI:10.1016/0092-8674(93)90119-b
Genetic reagent (Arabidopsis thaliana)ctr1-btkIkeda et al., 2009; DOI:10.3389/fpls.2017.00475
Genetic reagent (Arabidopsis thaliana)ein3eil1ebf1ebf2Sugano et al., 2010; DOI: 10.1105/tpc.110.076588
Genetic reagent (Arabidopsis thaliana)ein2-5Alonso et al., 1999; DOI:10.1126/science.284.5423.2148
Genetic reagent (Arabidopsis thaliana)gin2Moore et al., 2003; DOI:10.1126/science.1080585
Genetic reagent (Arabidopsis thaliana)hxk1-3Huang et al., 2015; DOI:10.3389/fpls.2015.00851
Genetic reagent (Arabidopsis thaliana)EIN3oxChao et al., 1997; DOI:10.1016/s0092-8674(00)80300–1
Genetic reagent (Arabidopsis thaliana)ctr1G738RThis paperA ctr1 (in Col-0,Arabidopsis thaliana) mutant harboring a point mutation; first introduced in Figure 1 and fully described in Figure 1—figure supplement 1; request to DCB Laboratory, Stanford, USA
Genetic reagent (Arabidopsis thaliana)pTMM::amiRNA-CTR1This paperTransgenic line expressing an artificial microRNA (amiRNA) knockdown construct targeting CTR1 under the stomatal lineage-specific promoter TMM; first introduced in Figure 2; request to DCB Laboratory, Stanford, USA
Genetic reagent (Arabidopsis thaliana)pATML1::CTR1; ctr1G738RThis paperTransgenic line expressing CTR1 under the epidermal-specific promoter ATLML1 in the ctr1G738R mutant background; first introduced in Figure 1—figure supplement 1; request to DCB Laboratory, Stanford, USA
Genetic reagent (Arabidopsis thaliana)pBRXL2::BRXL2-YFP; pATML1::mCherry-RCI2ARowe et al., 2019; DOI: 10.1101/614636
Genetic reagent (Arabidopsis thaliana)pBASL::YFP-BASL; pATML1::mCherry-RCI2ARowe et al., 2019; DOI: 10.1101/614636
Genetic reagent (Arabidopsis thaliana)pSPCH::gSPCH-YFP; spch3Lopez-Anido et al., 2020; DOI: 10.1101/2020.09.08.288498
Chemical compound, drugPropidium iodideThermo FisherThermo Fisher: P3566Dye for staining intercellular space in Arabidopsis
Chemical compound, drugAZD-8055Fisher ScientificFisher Scientific: 50-101-5840ATP-competitive inhibitor of TOR
Chemical compound, drug1-Aminocyclopropane-1-carboxylic acidSigmaSigma: A3903-100MGEthylene precursor
Chemical compound, drugGlucoseSigmaSigma: G7021-1KG
Chemical compound, drug3-O-Methyl-D-glucopyranoseSigmaSigma: M4849-10GNon-metabolizable sugar
Chemical compound, drugSucroseSigmaSigma: S3929-1KG
Chemical compound, drugFructoseSigmaSigma: 1040071000
Commercial assay or kitRNeasy plant mini kitQIAGENQIAGEN: 74104
Commercial assay or kitiScript cDNA synthesis kitBio-RadBio-Rad: 170-8891
Commercial assay or kitSsoadvanced Universal SYBR Green SupermixBio-RadBio-Rad: 172-5274
Software, algorithmLeica Application Suite XLeicaVersion: 3.5.2.18963
Software, algorithmFIJISchindelin et al., 2012;
DOI:10.1038/nmeth.2019		Version: 2.0.0-rc-69/1.52 p
Software, algorithmPOMEGong et al., 2021; DOI:10.1111/nph.17165Version:1.0.0
Software, algorithmRhttps://www.R-project.org/Version: 4.0.1
Software, algorithmTrackMateTinevez et al., 2017; DOI: 10.1016/j.ymeth.2016.09.016Version: 6.0.1
Software, algorithmRStudiohttps://rstudio.comVersion: 1.3.959

Plant material and growth conditions

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All Arabidopsis lines used in this study are in Col-0 background except for gin-2 (Ler-0), and sources of previously reported mutants and transgenic lines are listed in the Key Resources Table. Newly generated lines include pTMM::CTR1amiRNA and pATML1::CTR1; ctr1G738R.

All Arabidopsis seeds were surface-sterilized by bleach or 75% ethanol and stratified for 2 days. After stratification, seedlings were vertically grown on ½ Murashige and Skoog (MS) media with 1% agar for 3–14 days under long-day condition (16 hr light/8 hr dark at 22°C) and moderate-intensity full-spectrum light (110 μE) unless noted otherwise.

Identification and map-based cloning of CTR1

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Col-0 seeds homozygous for the reporter pBRXL2::BRXL-YFP were mutagenized with ethylmethanesulfonate (EMS). Seedlings from M2 families were screened individually at 3–5 dpg for loss of polar YFP localization on a Leica SP5 confocal microscope. Candidate mutants were then backcrossed, and F2 progeny with and without the mutant phenotype (>50 plants of each type) were pooled, sequenced, and analyzed as in Wachsman et al., 2017 with the exception that identification of potentially causal single-nucleotide polymorphisms was done by sequence comparisons among the many mutants rather than relative to a reference line.

Vector construction and plant transformation

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To generate pTMM::amiRNA-CTR1, an artificial microRNA sequence targeting CTR1 was designed with the Web MicroRNA Designer platform (http://wmd3.weigelworld.org) (Schwab et al., 2006), engineered with the pRS300 plasmid with the TMM promoter, and cloned into the binary vector R4pGWB401 (Nakagawa et al., 2008). pATML1::CTR1 was generated by cloning CTR1 coding sequence from cDNA into pENTR and combining with ML1 promoter sequences in binary vector R4pGWB401. Primers used to generate these two constructs are listed in the primers section below. Transgenic plants were then generated by Agrobacterium-mediated transformation (Clough and Bent, 1998), and transgenic seedlings were selected on ½ MS plates with 50 μM kanamycin.

Drug treatments

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To assay the influence of different types and concentrations of sugar on BPI or SI, filter-sterilized 40% sucrose, 20% glucose, 20% fructose, or 20% 3-OMG water solutions were prepared as stock solutions and added to the sterilized ½ MS media with 1% agar to make ½ MS sugar treatment plates. Similarly, ACC was dissolved in water and filter-sterilized to create the 100 mM ACC stock solution. This ACC stock solution was then added to the sterilized ½ MS media with 1% agar to make ½ MS ACC treatment plates. Surface-sterilized and -stratified Arabidopsis seeds were then plated on these plates and vertically grown for 3–14 days under long-day condition for the respective experiments. For AZD-8055 experiments, AZD-8055 was dissolved in DMSO to create 1 mM stock solution. AZD-8055 stock solution or DMSO was then added to the sterilized ½ MS media with 1% agar to make ½ MS AZD-8055 or mock treatment plates. Surface-sterilized and -stratified Arabidopsis seeds were then plated on regular ½ MS plates and vertically grown for 2 days prior to being transferred to AZD-8055 or mock treatment plates and vertically grown two more days before image acquisition. For time-lapse experiments, ½ MS solution was supplemented with 2% glucose prior to loading seedling into the chamber, and all flow-through solution contained the same glucose concentration.

Microscopy and image acquisition

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All fluorescence imaging experiments were performed on a Leica SP5 or Leica SP8 confocal microscope with HyD detectors using 40× NA1.1 water objective with image size 1024*1024 and digital zoom from 1× to 2×.

For time-lapse experiments, 3 dpg seedlings were mounted in a custom imaging chamber filled with ½ MS solution (Davies and Bergmann, 2014). Laser settings for each reporter, except the membrane marker (pATML1::mCherry-RCI2A), were adjusted to avoid over-saturation. For the time-lapse experiments reported in this study, there was a 30–45 min interval between each image stack capture. For the reoccurring time-lapse experiments, seedlings were imaged in the time-lapse chamber for 16 hr with the protocol stated above. After imaging, seedlings were removed from the imaging chamber and returned to MS-agar plates (with appropriate supplements) for 8 hr under standard light and temperatures. The same epidermal surface from the same plant was reimaged using the same time-lapse protocol each successive day from 3 to 5 dpg. The three sets of time-lapse images were combined together, with the time between recordings noted, to create a time-lapse covering about 64 hr of development.

For time-course (lineage tracing) experiments, where images of the same whole epidermis were acquired every 24 hr from 3 to 5 dpg, each seedling was carefully mounted on a slide with vacuum grease outlining the border of the cover slide. In this setting, vacuum grease provides mechanical support to avoid crushing the seedlings. After each image acquisition, seedlings were carefully unmounted from the slide and returned to the ½ MS plate and normal growth condition until the next image acquisition. For the analysis of these time-course (lineage tracing) experiments, refer to the ‘Lineage tracing analysis’ section.

All raw fluorescence image Z-stacks were projected with Sum Slices in FIJI unless noted otherwise. For all time-lapse images, drift was corrected using the Correct 3D Drift plugin (Parslow et al., 2014) prior to any further analysis.

For SI counting, seedlings were collected at 14 dpg. Samples were cleared with 7:1 solution (7:1 ethanol:acetic acid), treated with 1 N potassium hydroxide solution, rinsed in water, and then mounted in Hoyer’s solution. Individual leaves were then imaged with a Leica DM6B microscope with 20× NA0.7 air objective in differential contrast interference mode.

Image quantification

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For POME measurements of BRXL2 polarity in different mutants and treatments, florescence images of BRXL2 and membrane marker or staining in these conditions were acquired with 40× water objective and 2× digital zoom. Three to five individual images were captured from the same region of cotyledon from different seedlings. For each individual image, relative brightness of BRXL2 reporter was used to select the 10 brightest cells, and POME was used to measure BRXL2 distribution along the cell membrane in each of the selected cells. This selection was made as a way to capture cells that had recently divided either symmetrically or asymmetrically and was the fairest comparison of BRXL2 polarity across different mutants and treatments. With POME, the cortex of each cell was divided into 63 portions, and the fluorescence intensity of BRXL2 reporter in each portion was measured and reported. Details of BRXL2 measurement with POME are available in Gong et al., 2021. BPI is defined as the fraction of measurements above the half maximum. To calculate the BPI for each cell, the maximum BRXL2 fluorescence intensity of all 63 measured portions was determined, and the fraction of all measurements with BRXL2 fluorescence intensity above half of this maximum was calculated as the BPI. BPIs of all the measured cells in the same conditions were then grouped, plotted, and compared with other conditions.

For the quantification of BRXL2 polarity persistence, the duration of BRXL2 polarity persistence was counted manually in different genotypes and treatments. For each cell, the beginning of post-ACD BRXL2 polarity was set as the image in which formation of the cell plate was first visible, while the end of post-ACD BRXL2 was set as the first time frame where BRXL2 polar crescent was no longer visually detectable. Post-ACD BRXL2 polarity dynamics from three individual cells of Col-0 and ctr1G738R (without added sugar) were measured with POME (Gong et al., 2021), and the dynamic of the BPI and the normalized crescent amplitude (peak amplitude divided by that at timepoint 0) were quantified and plotted in Figure 6B and Figure 6—figure supplement 1A.

For the quantification of SPCH reporter dynamics under mock, 10 µM ACC, or 2% glucose treatment, 10 random ACDs of each condition were chosen from respective time lapse of the genomic pSPCH::gSPCH-YFP reporter expressed in a spch3 null mutant background (taken with the same basic imaging settings). Each ACD was then analyzed with the TrackMate FIJI plugin (Tinevez et al., 2017) for automated nucleus segmentation and tracking. Mean fluorescence intensity of the gSPCH-YFP reporter of each nucleus at each timepoint was then extracted and plotted in Figure 6—figure supplement 1D.

Lineage tracing analysis

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In each lineage tracing experiment, whole leaf images of the same leaves from different days were grouped together. Each individual cell from the 3 dpg (or any starting timepoint) was then annotated for divisions and lineage relationship each day after the starting point. BRXL2 reporter presence and polarity level in each cell and in different days was also recorded to help determine the division type as GMC divisions display depolarized BRXL2 while amplifying and spacing ACDs harbor a polarized BRXL2 crescent. After all the cells on the leaf epidermis were annotated, division behaviors of all cells are summarized in a spreadsheet, from which the total number and the fraction of each division type, fraction of each meristemoid behavior, total number of cells at the starting and ending timepoints, and any other developmental behaviors of interest were calculated.

RNA extraction and qRT-PCR analysis

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Whole seedlings of Col-0 and hxk1-3 at 7 dpg were collected. Seven seedlings were grouped into a biological replicate, and two biological replicates per genotype were assayed. RNA of each biological replicate was then extracted with RNeasy plant mini kit (QIAGEN) with on-column DNAse digestion. Then, 1 μg of total RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). The qPCR reactions were performed on a CFX96 Real-Time PCR detection system (Bio-Rad) with the Ssoadvanced Universal SYBR Green Supermix (Bio-Rad). Three technical replicates were performed per biological replicate. Expression level of HXK1 was then calculated and normalized to the expression level of the reference gene UBC21 using the ΔΔCT method.

Statistical analysis

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All statistical analyses in this paper were performed in RStudio. Unpaired Mann–Whitney tests and Student’s t-test were conducted to compare two data samples with compare_means function from the ggpubr package (Kassambara, 2020). For all graphs, p-values from the unpaired Mann–Whitney tests or Student’s t-test were directly labeled on these graphs except in Figure 1E, where values are provided in Figure 1—figure supplement 3.

Primers

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Primer namePurposeSequence
CTR1_fwCloningCACCATGGAAATGCCCGGTAGAAG
CTR1_rvCloningCAAATCCGAGCGGTTGGGCGG
I miR-s CTR1CloningGATATTTGATTTGACGCACGCAGTCTCTCTTTTGTATTCC
II miR-s CTR1CloningGACTGCGTGCGTCAAATCAAATATCAAAGAGAATCAATGA
III miR-s CTR1CloningGACTACGTGCGTCAATTCAAATTTCACAGGTCGTGATATG
IV miR-s CTR1CloningGAAATTTGAATTGACGCACGTAGTCTACATATATATTCCT
hxk1-3_LPGenotypingTTGATTATTTCTTCTTTCTGGCTTG
hxk1-3_RPGenotypingAGAACAGAAAACTGACATCTGAACC
ein2-5_fwGenotypingGCTCTTGTTCTTCTCTAGTC
ein2-5_rvGenotypingGAAGCATCATTGCCACCAAG
ctr1G738R_fwGenotyping/sequencingCTAGGTCCTATTTCCAATGGAAG
ctr1G738R_rvGenotyping/sequencingGGATTTAAGTTACCCCATGGTTG
UBC21_qPCR_fwqRT-PCRTCCTCTTAACTGCGACTCAGG
UBC21_qPCR_rwqRT-PCRGCGAGGCGTGTATACATTTG
HXK1_5′_qPCR_fwqRT-PCRCTGAATCCAGGCGAACAGA
HXK1_5′_qPCR_rwqRT-PCRTGTATCGCCAAAGAAAGCAG
HXK1_3′_qPCR_fwqRT-PCRAGCTACGTTGATAATCTTCCTTCC
HXK1_3′_qPCR_rwqRT-PCRTGTTTAACAACACGCTCTTGC

Data availability

All data generated on analyzed during this study are include in the manuscript and supporting files.

References

  36. Software
    1. Kassambara A
    (2020) Ggpubr: 'Ggplot2' Based Publication Ready Plots
    Ggpubr: 'Ggplot2' Based Publication Ready Plots.
    https://CRAN.R-project.org/package=ggpubr
    1. Serna L
    2. Fenoll C
    (1996)
    Ethylene induces stomata differentiation in Arabidopsis
    The International Journal of Developmental Biology Suppl 1:123S–124.

Decision letter

  1. Hao Yu
    Reviewing Editor; National University of Singapore & Temasek Life Sciences Laboratory, Singapore
  2. Jürgen Kleine-Vehn
    Senior Editor; University of Freiburg, Germany
  3. Olivier Hamant
    Reviewer; Université de Lyon, ENS de Lyon, France
  4. Dolf Weijers
    Reviewer; Wageningen University, Netherlands

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

In this study, the authors identified a hitherto unknown connection between the polarity of BRXL2 in the stomatal lineage cells and ethylene signaling through a genetic screen. They quantified the polarity of individual cells during cell division across cell populations, and revealed that ethylene and glucose signaling, which are involved in environmental and nutritional pathways, respectively, regulate antagonistically the balance of asymmetric and symmetric cell divisions in the stomatal lineage. Furthermore, they discussed a potential link between BRXL2 stability in stomatal lineage ground cells and the behavior of their smaller meristematic sister cells (meristemoids). Overall, this study provides insights into understanding how environmental factors and nutritional status affect stem cell behavior in the stomatal lineage.

Decision letter after peer review:

Thank you for submitting your article "Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Hao Yu as the Reviewing Editor, and Christian Hardtke as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Olivier Hamant (Reviewer #1); Dolf Weijers (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

In this study, the authors identified a hitherto unknown connection between the polarity of BRXL2 in the stomatal lineage cells and ethylene signaling through a genetic screen. They quantified the polarity of individual cells during cell division across cell populations, and revealed that ethylene and glucose signaling, which are involved in environmental and nutritional pathways, respectively, regulate antagonistically the balance of asymmetric and symmetric cell divisions in the stomatal lineage.

Furthermore, they discussed a potential link between BRXL2 stability in stomatal lineage ground cells (SLGCs) and the behavior of their smaller meristematic sister cells (meristemoids). Overall, this study provides insights into understanding how environmental factors and nutritional status affect stem cell behavior in the stomatal lineage.

Essential revisions:

While there was a common interest about the topic and the relevant findings, some concerns on the underlying molecular mechanisms, quality of the experiments, and interpretation of results as listed below need to be addressed to support the conclusions.

1) Although there is evidence for the correlation between ethylene/glucose signalling and the degree of polarity of BRXL2 and stomatal production, the underlying mechanisms and the functional specificity of BRXL2 polarization/depolarization are unclear. We suggest the authors to examine other polarity proteins, such as BASL, in some key experiments, including analyzing their activities under ethylene/glucose influence and/or in the signaling mutants. In addition, investigation of the leaf response to ethylene, light, and sugar in various genetic backgrounds, such as brxl2 and basl mutants or overexpression lines, will also help to clarify the role of BRXL2 and other polarity proteins in linking systemic signaling with stomatal stem-cell potential and leaf growth. These proposed experiments are necessary for evaluating the significance of the mechanisms underlying the correlation between systemic information (ethylene/glucose signalling) and stomatal lineage stem cell behavior.

2) Examination of glucose effect in Figure 5 could be improved by increasing sample size and/or optimizing the growth/genetic conditions under which the BPI baseline is higher.

3) Further analysis and clarification of the effects of ethylene/glucose treatment on SPCH protein level or its behavior are needed to support the current results in the manuscript, which seems different from the observations in a recent publication (Han et al., 2020). Similarly, the authors should explain the difference in the stomatal index of sugar-treated seedlings between this study and the work published by Han et al.

4) Please include a discussion on the potential mechanism(s) for controlling BRXL2 persistence at the molecular level.

Please also take into consideration the other specific comments from the reviewers below to revise the manuscript.

Reviewer #1:

The authors reveal a role of ethylene and glucose in determining BRXL2 polarity, with implication for the balance between symmetric and asymmetric cell division in the stomatal lineage, and thus organ size. Beyond the integration of the stomatal genetic pathway with biochemical cues (reflecting environmental conditions), they also find that BRXL2 polarity is a two-step process and that these above-mentioned cues rather play a role in the second step (polarity maintenance). The relation between glucose and ethylene is explored but remains open-ended.

This is a very interesting work, with a solid genetic backbone, and a key finding (link with biochemical cues and sequential polarity). Some loose ends need to be fixed before publication though. This may help clarify the main message too. In fact, the title, impact statement and some of the Abstract becomes clear only after one reads the paper. I hope the suggestions below will be helpful.

1) How specific is the BRXL2 depolarization ? Have the authors investigated other polarly localized proteins ? It would be important to have such a control, at least for the main/simpler tests (e.g. ACC and sugar treatments).

2) "To test if glucose regulates stomatal divisions through EIN3 inhibition, we compared BPIs in the ethylene insensitive mutants ein2-5 and ein3eil1ebf1ebf2 grown with and without glucose in the media. Both mutants had significantly reduced BPIs with 2% glucose at 4.5 dpg (Figure 5C and Figure 5—figure supplement 2A-F), suggesting that the influence of glucose in stomatal divisions is independent of the core components of ethylene signaling.” In fact the baseline in these mutants is already low without sugar, and the reduction after sugar treatment is not that significant (Figure 5C : p-value : 0.023 and 0.0095). So I would be more cautious here. Maybe one way to go around this is to increase sample size (i.e. >30 cells per genotype). Based on the results obtained on TOR and HXK1, I would not exclude a possible response saturation here, meaning that one would need to find growth/genetic conditions in which the baseline is higher, e.g. ein2 in conditions X with a higher BPI, then treat with sugar – if the BPI remains high, sugar is acting via ethylene. Based on the authors' results, condition X could be low light actually. Of course it's too early to say, but if that works, it would also help showcase the result in Figure 5F which is a bit buried and would deserve more light.

3) The conclusion about a possible overarching signal (biochemical or mechanical) is consistent with the idea that ethylene and glucose may affect the whole cell physiology, that the maintenance of BRXL2 polarity is affected (not the initial trigger) and possibly that BRXL2 might only be one of the many targets for which an impact on polarity is found. This relates to comment 1 on the need to test at least one or two other polar proteins to check for specificity. This would not decrease the impact of the article if all proteins respond, it would simply be consistent with a more generic response (which is equally interesting). A possible "generic" scenario is that ethylene might promote wall differentiation (hence reduced cell size), thus reduce wall tension, thus hindering polarity. Glucose (maybe via its antagonistic role on ethylene, see comment 2) would have a counteracting effect. This actually would be very much in line with Brinkmann and Bergmann, 2017, from the same group. This could also be discussed in relation to turgor pressure (see Long 2020 Curr Biol) and tested (e.g. cellulose or isoxaben treatment would weaken the wall and might restore BRXL2 polarity in ctr1)

Reviewer #2:

Gong et al. describe a very careful analysis of BRXL2 polarization in wild-type and ctr mutant stomatal lineage cells. In doing so, they find a clear correlation between persistence of the polar BRXL2 crescent after asymmetric division and the fate of the daughter cell. Longer persistence of the crescent correlates with increased stemness. Through analyzing the ctr mutant, ethylene is identified as a trigger that influences stomatal index (and leaf size) by influencing post ACD stemness. Conversely, glucose signaling (through yet unidentified components) also signals to extend stemness by promoting persistence of BRXL2.

This work presents a very interesting paradigm of control of organ growth and cellular composition by (environmental) signals through tuning stemness. It is a bit disappointing however, that much is based on correlations, and no clear molecular mechanism is provided. Is tuning BRXL2 levels sufficient for this response? Is only BRXL2 controlled, or would BASL likely be a target of this regulation?

There are a few points that could help in addressing these questions:

1) Is the leaf growth response to ethylene, light, sugars altered in the various brxl2 and basl mutants or overexpression lines?

2) Given that BRXL2 and BASL are interdependent in their function and localization, it would be interesting to determine the behavior of BASL in a similar set of assays.

3) It is not clear to me how the authors envisage that BRXL2 persistence is controlled at the molecular level. Does this involve active stabilization (or active degradation in the case where BXRL2 does not persist)?

Reviewer #3:

In this manuscript from the Bergmann lab, the authors report the regulation of a polarity factor during stomatal asymmetric cell divisions (ACDs) and the involvement of ethylene and glucose signaling in the process. Through a genetic screen, they identified a surprising connection between the polarity of BRXL2 in the stomatal lineage cells and ethylene signaling. Lineage tracing of WT and the ctr1 mutant suggested that the amplifying ACDs are suppressed in the ethylene signaling mutant. They further showed that ethylene and glucose signalling can oppositely influence the degree of polarity of BRXL2 and stomatal production. Finally, they described a potential connection between BRXL2 stability in the stomatal lineage ground cells (SLGCs) and the behaviour of their smaller meristematic sister cells (meristemoids).

The correlation between the degree of polarity of a polarized factor and the stomatal stem cell activity, is novel, and could represent a broader phenomenon that was previously overlooked. The quantitative and lineage tracing approaches also represent powerful methods to describe polarity and dissect the activity of the stomatal lineage. However, there are some additional experiments that would help strengthen the authors' claims.

1) The authors exclusively used BRXL2 as the polarity protein in their analyses. What about the behaviour of BASL, the other critical component of the polarity complex? Analyzing the activity of BASL under ethylene/glucose influence or in the signaling mutants would provide a fuller picture of the phenomenon and may help dissect the underlying mechanism.

2) It is somewhat surprising that SPCH protein level or its behaviour does not seem to be influenced by ethylene/glucose treatment, especially since a recent study proposes that SPCH protein can be stabilized by sugar (Han et al., 2020). How many times were the time-lapse analyses of SPCH in Figure 6—figure supplement 1 conducted? Additional quantitative data on the persistence/peak levels of SPCH during ACD, similar to how BRXL2 was characterized, would provide better support of the authors' proposal. Further, during ACD, how is the protein level of BRXL2 in WT compared to ctr1?

3) Stomatal index is a useful but relatively indirect measure of the activity of the stomatal lineage, and thus, the lineage tracing approach that the authors employed is superior in this context. However, since SI still represents developmental outcome, it is again surprising to see that the sugar-treated seedlings here had a significantly lower SI compared to WT, while they were higher or indistinguishable in the previous study (Han et al., 2020). How can this be explained?

4) The authors may want to cite Serna and Fenoll, 1996, as an earlier study on the effect of ethylene on stomatal development.

5) For Figure 3G, I found it confusing to include GMC SCD here, as opposed to having only the two types of ACDs, which would represent the behavior of meristemoids. Also, the two types of ACDs can also be shown in Figure 3F to make it more useful.

6) What is the sample size for Figure 3I?

7) For Figure 7B, it would be more appropriate to include a "question mark" on arrows/text that do not have experimental support.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your revised article "Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Hao Yu as the Reviewing Editor, and Jürgen Kleine-Vehn as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Olivier Hamant (Reviewer #2); Dolf Weijers (Reviewer #3).

The reviewers were very enthusiastic about your work and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential Revisions:

1) For Figure 1—figure supplement 2B, the second and third BRXL2-YFP images of Col-0 seems to be mixed up. The authors should check these images and correct them if necessary.

https://doi.org/10.7554/eLife.63335.sa1

Author response

Essential revisions:

While there was a common interest about the topic and the relevant findings, some concerns on the underlying molecular mechanisms, quality of the experiments, and interpretation of results as listed below need to be addressed to support the conclusions.

1) Although there is evidence for the correlation between ethylene/glucose signalling and the degree of polarity of BRXL2 and stomatal production, the underlying mechanisms and the functional specificity of BRXL2 polarization/depolarization are unclear. We suggest the authors to examine other polarity proteins, such as BASL, in some key experiments, including analyzing their activities under ethylene/glucose influence and/or in the signaling mutants. In addition, investigation of the leaf response to ethylene, light, and sugar in various genetic backgrounds, such as brxl2 and basl mutants or overexpression lines, will also help to clarify the role of BRXL2 and other polarity proteins in linking systemic signaling with stomatal stem-cell potential and leaf growth. These proposed experiments are necessary for evaluating the significance of the mechanisms underlying the correlation between systemic information (ethylene/glucose signalling) and stomatal lineage stem cell behavior.

We thank the reviewers for the suggestion of expanding the study to ask not just whether BRXL2 polarity reported a response to external conditions, but whether it (and other polarity proteins) were part of the mechanism mediating that response. We have now added data on BASL polarity in stomatal lineage cells. These data are from seedlings harboring a BASLp::YFP-BASL reporter grown under two treatments (+ control): germination on ½ MS plate with and without 2% glucose or 10µM ACC. We found that ACC treatment significantly reduced tissue-level BASL polarity, while glucose treatment significantly enhanced it (Figure 4—figure supplement 2B-E). This was similar to the result with BRXL2 polarity. These results suggest that glucose and ethylene’s effect is not just on BRXL2, but stomatal cell polarity in general.

We then tested whether stomatal lineage development can be modulated by ethylene signaling pathway in the absence of the polarity module. BRXL2 is redundant with other BRX family members and our genetic data in Rowe et al., 2019 indicate that BRX-family and BASL work together, so to get the clearest readout of the effect of loss of polarity on the response to ACC, we looked at the basl null mutant (basl-2).

In this cell polarity mutant, we found that 10µM ACC treatment can still increase the stomatal index (SI) (Figure 4—figure supplement 2F). The degree of increase of SI in basl-2 upon ACC treatment is less than that in wild-type Col-0, but the starting SI is also higher. Given this result, it appears that part, but not all, of ethylene’s regulation on stomatal lineage development might be through the polarity protein complex. Our models at the end discuss how polarity proteins could be a driver or a reporter of cell fate decisions.

It was not clear to us whether the suggestion to look at overexpression of polarity factors was to test whether polarization in other cells could be modulated, or whether the phenotype of the OE lines with respect to stomatal production was to be measured. Because the temporal persistence of BRXL2 polarity in stomatal lineage cells appears to be the relevant behavior for division response, we felt that examining polarity in ectopic positions would not be biologically meaningful. OE (or expression of hyperactive) polarity proteins does not lead to noticeable phenotypes and/or we haven’t been able to observe the reported phenotypes in our hands. .

2) Examination of glucose effect in Figure 5 could be improved by increasing sample size and/or optimizing the growth/genetic conditions under which the BPI baseline is higher.

As noted by the reviewers, the BPI “baseline” in cotyledons from ethylene insensitive mutants ein2-5 and ein3eil1ebf1ebf2 is lower than for Col-0, and therefore it is possible that we would not be able to see a mild effect of glucose treatment.

We had this concern as well, and so we measured the ability of glucose to promote BRXL2 polarity in ein2-5, ein3eil1ebf1ebf2, and Col-0 cotyledons at a later developmental stage (Figure 5C, 4.5 dpg), than we did for most other measurements of BPI (4dpg, compare Col-0 BPI in Figure 1E or 2J to that in 5C). At this slightly later timepoint, BPI are no longer saturated (100% less than 0.3) in ein2-5 or ein3eil1ebf1ebf2 and we observed that glucose significantly promoted tissue level BRXL2 polarity (Figure 5—figure supplement 2A-F) and reduced BPI in both ein2-5 and ein3eil1ebf1ebf2 (Figure 5C).

To push the BPI baseline even higher, we imaged the BRXL2 reporter at 9 dpg in 11 uE low light condition (same experimental procedure as the experiment of Figure 5F) and found that the addition of 2% glucose significantly promoted tissue level BRXL2 polarity in both ein3eil1ebf1ebf2 and Col-0 true leaves (Figure 5—figure supplement 5). These new results reinforce the idea that the regulation of BRXL2 polarity and stomatal lineage development by glucose is unlikely through the cross talk between glucose signaling and ethylene signaling.

3) Further analysis and clarification of the effects of ethylene/glucose treatment on SPCH protein level or its behavior are needed to support the current results in the manuscript, which seems different from the observations in a recent publication (Han et al., 2020). Similarly, the authors should explain the difference in the stomatal index of sugar-treated seedlings between this study and the work published by Han et al.

The reviewers are correct that it appears that our findings on SPCH differ from those reported in Han et al., 2020. We have now stated explicitly that our experimental set-up and analysis differs significantly from Han et al., 2020. But, we also note that in the few places where we do similar experiments, our results trend in the same direction. The main differences are:

1) Different growth conditions

The majority of experiments conducted by Han et al., 2020, were from seedlings grown in MS liquid culture while our experiments were from seedlings grown on solidified agar-MS plates. Epidermal development is severely impacted when Arabidopsis seedlings are grown in the liquid culture (Author response image 1A), likely due to hypoxia and stress. To illustrate the difference between plants grown in liquid culture and on plates, we imaged the whole cotyledons from these two conditions, quantified the surface area of these cotyledons, and counted the number of cells on the abaxial epidermis from each cotyledon (Author response image 1B-C). We found that at the same developmental stages, cotyledons grown on plates are about three times bigger in leaf size and have about two times more cells on the abaxial epidermis, suggesting that both cell division and differentiation of stomatal lineage are strongly suppressed in seedlings grown in liquid culture.

Author response image 1
Download asset Open asset
Liquid culture significantly alters epidermal development.

(A) Confocal images of 4 dpg Col-0 cotyledons grown on regular ½ MS plates (top) and in ½ MS liquid culture (bottom). Cell outline is visualized with pATML1::RCI2A-mCherry (gray). (B-C) Quantification of cotyledon surface area (B) and total cell numbers (C) from cotyledons shown in A. All p-values are calculated by Student’s t- test. Scale bars in A, 50 μm.

2) Different SPCH reporter

We monitored a SPCH reporter that contains the full genomic regions (gSPCH, Lopez-Anido et al., 2020), that shows a greater dynamic range than the older SPCH CDS reporter (cSPCH) used in the Han paper. As a result, we believe our experiments are more sensitive in detecting the full picture of SPCH expression profile in the stomatal lineage.

3) Different measurements of SPCH, and different sugar treatments

It is also important to note that changes in SPCH levels could be measured as an increase in the number of cells expressing SPCH or in the intensity of SPCH in those cells that express it. Figure 1 from Han et al., 2020, showed a higher percentage of cells are expressing SPCH, not that SPCH levels are higher per cell, when 1% sucrose was added to the ½ MS liquid culture. Judging from the Figure 1 from Han et al., cSPCH reporter fluorescence intensity under mock and 1% sucrose treatment, they reach a similar conclusion as our findings from Figure 6 F-I.

To test whether sucrose and glucose have different effects on SPCH expression, we imaged and quantified the gSPCH reporter from Arabidopsis seedlings grown on ½ MS plate with no sugar, 2% glucose, 2% sucrose, and 10uM ACC (Author response image 2). Similar to glucose, sucrose didn’t increase the fluorescence intensity of gSPCH reporter in SPCH expressing cells.

Author response image 2
Download asset Open asset
SPCH reporter fluorescence intensity under different sugar and ethylene treatments.

Quantification of gSPCH reporter fluorescence intensity at 4 dpg in mock, 10 μM ACC, 2% glucose, and 2% sucrose treated Col-0 cotyledons (n = 8 cotyledons/treatment; n>200 cells/treatment).

To characterize SPCH dynamics during ACDs under different ethylene and sugar treatments, we performed time-lapse imaging of the gSPCH reporter in each of these conditions and quantified the changes of SPCH reporter intensity in individual ACDs (10 ACDs per condition). There is variation in SPCH expression in each measured cell, but no clear variation that correlates with ethylene or sugar treatment (Figure 6—figure supplement 1C-D).

Together, these results suggest SPCH itself is likely not the direct target of glucose or ethylene signaling during relatively unstressed conditions. Rather, we contend that SPCH provides a downstream readout of changes in meristemoid division behaviors, since SPCH is required for ACDs. With more meristemoids undergoing ACDs in glucose treated cotyledons and fewer meristemoids undergoing ACDs in ACC treated cotyledons, the number of SPCH expressing cells are expected to be different.

Stomatal index is the readout of the combination of stomatal lineage entry divisions in meristemoid mother cells, amplifying division in meristemoids, and spacing division in SLGCs, and it has been reported to be sensitive to environmental changes. Due to the strong inhibition of stomatal lineage development in liquid culture (Author response image 1), it is reasonable to see stomatal index to respond differently to sugar treatments.

4) Please include a discussion on the potential mechanism(s) for controlling BRXL2 persistence at the molecular level.

Please also take into consideration the other specific comments from the reviewers below to revise the manuscript.

We think BRXL2 persistence in SLGC could either be 1) a readout of meristemoids self-renewing capacity though the EPF2-MAPK-BASL polarity pathway or 2) an active source of intercellular signaling communication or mechanical force originated in SLGC that regulates meristemoids ability to undergo amplifying divisions. We added discussion of these points in the Discussion section of the manuscript and Figure 7 and briefly describe the models below.

In the first case, EPF2 secreted by the actively self-renewing meristemoid is perceived by the neighboring SLGC, elevating SLGC’s EPF2-MAPK signaling (Lee et al., 2015). This elevated MAPK activity then promotes export of nuclear BASL to the polar crescent, sustaining BASL/BRXL2 polarity (Zhang et al., 2016; Zhang et al., 2015). When the meristemoid transitions to a GMC, EPF2 is no longer produced, resulting in lower MAPK activity in the neighboring SLGC. Replenishment of cortical BASL from the nuclear pool is then lost, leading BASL/BRXL2 polarity to diminish.

In the second case, because of the intense cross-regulation between BASL/BRXL2 polar crescent and the kinases (MAPKs, PAX, BIN2, other AGC kinases etc.) they scaffold (Houbaert et al., 2018; Marhava et al., 2020; Zhang et al., 2015), BASL/BRXL2 polar crescent could act through these kinase pathways to regulate the transport and local distribution of auxin or brassinosteroids, Both auxin or brassinosteroid have been shown to regulate stem-cell division and lineage progression in the stomatal lineage (Houbaert et al., 2018; Kim et al., 2012; Le et al., 2014) and change in their local concentration is likely to alter the development of the surrounding cells.

Alternatively, the orientation of BASL/BRXL2 polar crescent has been linked to tissue level polarity and proposed to regulate local cell expansion (Bringmann and Bergmann, 2017; Mansfield et al., 2018). Prolonged BASL/BRXL2 could potentially trigger redistribution of regional mechanical force and affect the stem-cell divisions in the surrounding cells.

Reviewer #1:

The authors reveal a role of ethylene and glucose in determining BRXL2 polarity, with implication for the balance between symmetric and asymmetric cell division in the stomatal lineage, and thus organ size. Beyond the integration of the stomatal genetic pathway with biochemical cues (reflecting environmental conditions), they also find that BRXL2 polarity is a two-step process and that these above-mentioned cues rather play a role in the second step (polarity maintenance). The relation between glucose and ethylene is explored but remains open-ended.

This is a very interesting work, with a solid genetic backbone, and a key finding (link with biochemical cues and sequential polarity). Some loose ends need to be fixed before publication though. This may help clarify the main message too. In fact, the title, impact statement and some of the Abstract becomes clear only after one reads the paper. I hope the suggestions below will be helpful.

1) How specific is the BXL2 depolarization ? Have the authors investigated other polarly localized proteins ? It would be important to have such a control, at least for the main/simpler tests (e.g. ACC and sugar treatments).

Please see response to essential revision 1.

2) "To test if glucose regulates stomatal divisions through EIN3 inhibition, we compared BPIs in the ethylene insensitive mutants ein2-5 and ein3eil1ebf1ebf2 grown with and without glucose in the media. Both mutants had significantly reduced BPIs with 2% glucose at 4.5 dpg (Figure 5C and Figure 5—figure supplement 2A-F), suggesting that the influence of glucose in stomatal divisions is independent of the core components of ethylene signaling.” In fact the baseline in these mutants is already low without sugar, and the reduction after sugar treatment is not that significant (Figure 5C : p-value : 0.023 and 0.0095). So I would be more cautious here. Maybe one way to go around this is to increase sample size (i.e. >30 cells per genotype). Based on the results obtained on TOR and HXK1, I would not exclude a possible response saturation here, meaning that one would need to find growth/genetic conditions in which the baseline is higher, e.g. ein2 in conditions X with a higher BPI, then treat with sugar – if the BPI remains high, sugar is acting via ethylene. Based on the authors' results, condition X could be low light actually. Of course it's too early to say, but if that works, it would also help showcase the result in Figure 5F which is a bit buried and would deserve more light.

Please see response to essential revision 2.

3) The conclusion about a possible overarching signal (biochemical or mechanical) is consistent with the idea that ethylene and glucose may affect the whole cell physiology, that the maintenance of BRXL2 polarity is affected (not the initial trigger) and possibly that BRXL2 might only be one of the many targets for which an impact on polarity is found. This relates to comment 1 on the need to test at least one or two other polar proteins to check for specificity. This would not decrease the impact of the article if all proteins respond, it would simply be consistent with a more generic response (which is equally interesting). A possible "generic" scenario is that ethylene might promote wall differentiation (hence reduced cell size), thus reduce wall tension, thus hindering polarity. Glucose (maybe via its antagonistic role on ethylene, see comment 2) would have a counteracting effect. This actually would be very much in line with Bringmann and Bergmann, 2017, from the same group. This could also be discussed in relation to turgor pressure (see Long 2020 Curr Biol) and tested (e.g. cellulose or isoxaben treatment would weaken the wall and might restore BRXL2 polarity in ctr1).

Please see response to essential revision 4.

Reviewer #2:

Gong et al. describe a very careful analysis of BBRXL2 polarization in wild-type and ctr mutant stomatal lineage cells. In doing so, they find a clear correlation between persistence of the polar BRXL2 crescent after asymmetric division and the fate of the daughter cell. Longer persistence of the crescent correlates with increased stemness. Through analyzing the ctr mutant, ethylene is identified as a trigger that influences stomatal index (and leaf size) by influencing post ACD stemness. Conversely, glucose signaling (through yet unidentified components) also signals to extend stemness by promoting persistence of BRXL2.

This work presents a very interesting paradigm of control of organ growth and cellular composition by (environmental) signals through tuning stemness. It is a bit disappointing however, that much is based on correlations, and no clear molecular mechanism is provided. Is tuning BRXL2 levels sufficient for this response? Is only BRXL2 controlled, or would BASL likely be a target of this regulation?

There are a few points that could help in addressing these questions:

1) Is the leaf growth response to ethylene, light, sugars altered in the various brxl2 and basl mutants or overexpression lines?

Please see response to essential revision 1.

2) Given that BRXL2 and BASL are interdependent in their function and localization, it would be interesting to determine the behavior of BASL in a similar set of assays.

Please see response to essential revision 1.

3) It is not clear to me how the authors envisage that BRXL2 persistence is controlled at the molecular level. Does this involve active stabilization (or active degradation in the case where BXRL2 does not persist)?

Please see response to essential revision 4.

Reviewer #3:

In this manuscript from the Bergmann lab, the authors report the regulation of a polarity factor during stomatal asymmetric cell divisions (ACDs) and the involvement of ethylene and glucose signaling in the process. Through a genetic screen, they identified a surprising connection between the polarity of BRXL2 in the stomatal lineage cells and ethylene signaling. Lineage tracing of WT and the ctr1 mutant suggested that the amplifying ACDs are suppressed in the ethylene signaling mutant. They further showed that ethylene and glucose signalling can oppositely influence the degree of polarity of BRXL2 and stomatal production. Finally, they described a potential connection between BRXL2 stability in the stomatal lineage ground cells (SLGCs) and the behaviour of their smaller meristematic sister cells (meristemoids).

The correlation between the degree of polarity of a polarized factor and the stomatal stem cell activity, is novel, and could represent a broader phenomenon that was previously overlooked. The quantitative and lineage tracing approaches also represent powerful methods to describe polarity and dissect the activity of the stomatal lineage. However, there are some additional experiments that would help strengthen the authors' claims.

1) The authors exclusively used BRXL2 as the polarity protein in their analyses. What about the behaviour of BASL, the other critical component of the polarity complex? Analyzing the activity of BASL under ethylene/glucose influence or in the signaling mutants would provide a fuller picture of the phenomenon and may help dissect the underlying mechanism.

Please see response to essential revision 1.

2) It is somewhat surprising that SPCH protein level or its behaviour does not seem to be influenced by ethylene/glucose treatment, especially since a recent study proposes that SPCH protein can be stabilized by sugar (Han et al., 2020). How many times were the time-lapse analyses of SPCH in Figure 6—figure supplement 1 conducted? Additional quantitative data on the persistence/peak levels of SPCH during ACD, similar to how BRXL2 was characterized, would provide better support of the authors' proposal. Further, during ACD, how is the protein level of BRXL2 in WT compared to ctr1?

Please see response to essential revision 3.

3) Stomatal index is a useful but relatively indirect measure of the activity of the stomatal lineage, and thus, the lineage tracing approach that the authors employed is superior in this context. However, since SI still represents developmental outcome, it is again surprising to see that the sugar-treated seedlings here had a significantly lower SI compared to WT, while they were higher or indistinguishable in the previous study (Han et al., 2020). How can this be explained?

Please see response to essential revision 3.

4) The authors may want to cite Serna and Fenoll, 1996, as an earlier study on the effect of ethylene on stomatal development.

We added the citation in the “Ethylene signaling regulates polarity protein complex and stomatal lineage development” section.

5) For Figure 3G, I found it confusing to include GMC SCD here, as opposed to having only the two types of ACDs, which would represent the behavior of meristemoids. Also, the two types of ACDs can also be shown in Figure 3F to make it more useful.

The SCD/ACD ratio concept that we introduce early on and use throughout the text groups all observed ACDs together. Thus we think it’s important to give the reader a graphical representation of this information (Figure 3F). Figure 3G is designed to report the shift in types of divisions in the ctr1 mutant. We think it is useful to demonstrate that even with an overall reduction in the number of cell divisions (Figure 3F) not all division types are affected equally. At the stages we are able to follow, most entry divisions would have already occurred and thus they would represent a very small # of total divisions.

6) What is the sample size for Figure 3I?

N=3. We added this info to the figure legend of Figure 3I.

7) For Figure 7B, it would be more appropriate to include a "question mark" on arrows/text that do not have experimental support.

We added the question mark as suggested.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential Revisions:

1) For Figure 1—figure supplement 2B, the second and third BRXL2-YFP images of Col-0 seems to be mixed up. The authors should check these images and correct them if necessary.

Thank you for catching this. The images were indeed shifted. We have fixed this and made sure this (and other cells) are now correctly aligned.

Reference:

Bringmann, M., & Bergmann, D. C. (2017). Tissue-wide Mechanical Forces Influence the Polarity of Stomatal Stem Cells in Arabidopsis. Curr Biol,27(6), 877-883. https://doi.org/10.1016/j.cub.2017.01.059

Dow, G. J., Berry, J. A., & Bergmann, D. C. (2017). Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange. New Phytol, 216(1), 69-75. https://doi.org/10.1111/nph.14746

Han, C., Liu, Y., Shi, W., Qiao, Y., Wang, L., Tian, Y., Fan, M., Deng, Z., Lau, O. S., De Jaeger, G., & Bai, M. Y. (2020). KIN10 promotes stomatal development through stabilization of the SPEECHLESS transcription factor. Nat Commun, 11(1), 4214. https://doi.org/10.1038/s41467-020-18048-w

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., Eeckhout, D., Boeren, S., Karimi, M., Betti, C., Jacobs, T., Fenoll, C., Mena, M., de Vries, S., De Jaeger, G., & Russinova, E. (2018). POLAR-guided signalling complex assembly and localization drive asymmetric cell division. Nature, 563(7732), 574-578. https://doi.org/10.1038/s41586-018-0714-x

Kim, T. W., Michniewicz, M., Bergmann, D. C., & Wang, Z. Y. (2012). Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature, 482(7385), 419-422. https://doi.org/10.1038/nature10794

Le, J., Liu, X. G., Yang, K. Z., Chen, X. L., Zou, J. J., Wang, H. Z., Wang, M., Vanneste, S., Morita, M., Tasaka, M., Ding, Z. J., Friml, J., Beeckman, T., & Sack, F. (2014). Auxin transport and activity regulate stomatal patterning and development. Nat Commun, 5, 3090. https://doi.org/10.1038/ncomms4090

Lee, J. S., Hnilova, M., Maes, M., Lin, Y. C., Putarjunan, A., Han, S. K., Avila, J., & Torii, K. U. (2015). Competitive binding of antagonistic peptides fine-tunes stomatal patterning. Nature, 522(7557), 439-443. https://doi.org/10.1038/nature14561

Lopez-Anido, C. B., Vatén, A., Smoot, N. K., Sharma, N., Guo, V., Gong, Y., Anleu Gil, M. X., Weimer, A. K., & Bergmann, D. C. (2020). Single-Cell Resolution of Lineage Trajectories in the Arabidopsis Stomatal Lineage and Developing Leaf. bioRxiv, 2020.2009.2008.288498. https://doi.org/10.1101/2020.09.08.288498

Mansfield, C., Newman, J. L., Olsson, T. S. G., Hartley, M., Chan, J., & Coen, E. (2018). Ectopic BASL Reveals Tissue Cell Polarity throughout Leaf Development in Arabidopsis thaliana. Curr Biol, 28(16), 2638-2646 e2634. https://doi.org/10.1016/j.cub.2018.06.019

Marhava, P., Aliaga Fandino, A. C., Koh, S. W. H., Jelinkova, A., Kolb, M., Janacek, D. P., Breda, A. S., Cattaneo, P., Hammes, U. Z., Petrasek, J., & Hardtke, C. S. (2020). Plasma Membrane Domain Patterning and Self-Reinforcing Polarity in Arabidopsis. Dev Cell, 52(2), 223-235 e225. https://doi.org/10.1016/j.devcel.2019.11.015

Rowe, M. H., Dong, J., Weimer, A. K., & Bergmann, D. C. (2019). A Plant-Specific Polarity Module Establishes Cell Fate Asymmetry in the Arabidopsis Stomatal Lineage. bioRxiv, 614636. https://doi.org/10.1101/614636

Zhang, Y., Guo, X., & Dong, J. (2016). Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH. Curr Biol, 26(21), 2957-2965. https://doi.org/10.1016/j.cub.2016.08.066

Zhang, Y., Wang, P., Shao, W., Zhu, J. K., & Dong, J. (2015). The BASL polarity protein controls a MAPK signaling feedback loop in asymmetric cell division. Dev Cell, 33(2), 136-149. https://doi.org/10.1016/j.devcel.2015.02.022

https://doi.org/10.7554/eLife.63335.sa2

Article and author information

Author details

  1. Yan Gong

    Department of Biology, Stanford University, Stanford, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1329-7096

  2. Julien Alassimone

    Department of Biology, Stanford University, Stanford, United States
    Present address
    Plantpathology, Institute for Integrative Biology, ETH Zürich, Zürich, Switzerland
    Contribution
    Conceptualization, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8118-2605

  3. Rachel Varnau

    Department of Biology, Stanford University, Stanford, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3203-9597

  4. Nidhi Sharma

    Howard Hughes Medical Institute, Stanford University, Stanford, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9725-5338

  5. Lily S Cheung

    School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, United States
    Contribution
    Visualization, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8089-7783

  6. Dominique C Bergmann

    1. Department of Biology, Stanford University, Stanford, United States
    2. Howard Hughes Medical Institute, Stanford University, Stanford, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    bergmann@stanford.edu
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0873-3543

Funding

Howard Hughes Medical Institute

  • Yan Gong
  • Nidhi Sharma
  • Dominique C Bergmann

Swiss National Science Foundation (EPM-PBLAP3-142757)

  • Julien Alassimone

European Molecular Biology Organization (ALTF-878-2013)

  • Julien Alassimone

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Markus Grebe for providing the seeds of ctr1-btk, Dr. Wenrong He and Dr. Hongwei Guo for providing the seeds of ein3eil1ebf1ebf2 and EIN3ox, Dr. Jiaying Zhu and Dr. Zhiyong Wong for providing the seeds of gin2, and Dr. Jose Alonso for providing the seeds of ein2-5 and ctr1-1. We also thank Katelyn McKown, Gabriel Amador, and other members of the Bergmann lab for valuable feedback on the manuscript and Dr. José Dinneny for access to microscopy resources.

Senior Editor

  1. Jürgen Kleine-Vehn, University of Freiburg, Germany

Reviewing Editor

  1. Hao Yu, National University of Singapore & Temasek Life Sciences Laboratory, Singapore

Reviewers

  1. Olivier Hamant, Université de Lyon, ENS de Lyon, France
  2. Dolf Weijers, Wageningen University, Netherlands

Publication history

  1. Received: September 22, 2020
  2. Accepted: March 18, 2021
  3. Accepted Manuscript published: March 19, 2021 (version 1)
  4. Version of Record published: March 30, 2021 (version 2)

Copyright

© 2021, Gong et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Yan Gong
  2. Julien Alassimone
  3. Rachel Varnau
  4. Nidhi Sharma
  5. Lily S Cheung
  6. Dominique C Bergmann
(2021)
Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division
eLife 10:e63335.
https://doi.org/10.7554/eLife.63335

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