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STOMATA

Introduction to stomatal development

During postembryonic development, the plant epidermis generates several distinct cell types, including stomatal guard cells. Stomata act as valves through which atmospheric CO2 can enter the plant and O2 and water vapor can escape, and each consists of paired guard cells surrounding a central pore. Although the overall pattern of stomata on the leaf surface is variable among species, stomata are almost universally patterned according to a one-cell spacing rule, such that at least one intervening epidermal cell separates nearby stomata from one another. Both the patterned distribution of stomatal complexes and the differentiation of the guard cells themselves are associated with asymmetric, oriented divisions.

 

photo1

Arabidopsis stomatal development requires asymmetric entry divisions of MMCs (light green) to create meristemoids (M, green), which may self-renew via amplifying divisions or differentiate into GMCs (progression from green to aqua to blue) that divide symmetrically to form guard cells (GCs, purple). Spacing divisions of MMCs next to stomatal precursors are oriented as well as asymmetric. White cells are stomatal lineage ground cells (SLGCs) that typically become crenulated pavement cells as shown on top right.

 
Arabidopsis stomatal development

Arabidopsis stomatal precursors arise from asymmetric divisions of an apparently random subset of cells in the immature leaf epidermis. As of yet, no morphological or gene expression patterns have unambiguously marked this cell population, so these cells (meristemoid mother cells, MMCs) are defined retrospectively. An asymmetric entry division of the MMC creates a meristemoid and a stomatal lineage ground cell (SLGC) as its smaller and larger daughters, respectively.  The meristemoid has limited self-renewing capacity and may continue to undergo asymmetric amplifying divisions, with the smaller daughter of each division round retaining meristemoid identity and the larger becoming an SLGC. Eventually, the meristemoid will differentiate into a guard mother cell (GMC) that undergoes symmetric division to produce the paired guard cells of the stoma. The SLGCs produced at various stages of lineage progression may differentiate into large, lobed, pavement cells, or may also become MMCs, dividing asymmetrically to create secondary meristemoids.  These secondary entry divisions, called spacing divisions, lead to a sort of “fill in” pattern where new meristemoids arise among mature precursors and stomata. To maintain the one-cell spacing pattern, the spacing divisions creating secondary meristemoids are not only asymmetric, but are oriented such that the new meristemoid forms distal to the existing stoma/precursor.

 

Signaling during stomatal development

Cell divisions that generate secondary meristemoids are oriented relative suggesting that external signals might play a key role in stomatal development, and indeed, several receptors and receptor like-kinases are required for the maintenance of one-cell spacing. The LRR-receptor like protein TOO MANY MOUTHS (TMM) was the first component of this network to be identified (Nadeau and Sack, 2002) and has subsequently been joined by potential LRR-RLK signaling partners ERECTA, ERECTA-LIKE1 and ERECTA-LIKE2 (collectively referred to as the ERECTA family, or ERf) (Shpak et al., 2005). Loss of TMM or ERf function results in the production of excess stomata arranged in clusters, and these factors appear both to orient asymmetric division and repress stomatal fate at various stages of lineage progression.

 

Loss-of-function mutations in two related genes encoding putative ligands, EPIDERMAL PATTERNING FACTOR 1 (EPF1, Hara et al., 2007) and EPF2 (Hunt and Gray, 2009), also confer defects in stomatal patterning. EPF2, which is expressed in early stomatal lineage cells, appears to limit the number of cells that undergo lineage entry. EPF1, on the other hand, is expressed in relatively late stomatal precursors and primarily regulates orientation of spacing divisions. Interestingly, epistasis analyses indicate that EPF1 activity depends on both TMM and the ERf but that EPF2 possesses some TMM-independent functions, potentially reflecting specificity in ligand-receptor interactions (Hunt and Gray, 2009).   A third member of this ligand family, STOMAGEN, is a positive regulator of stomatal development (Sugano et al, 2010 and Kondo et al, 2010). How it acts in opposition to EPF1 and EPF2 is an area of active research.

Scheme of major signaling and transcriptional inputs into stomatal development; arrows and T-bars indicate positive and negative effects, respectively, and are directed to the stage in which they have been demonstrated to act.

Scheme of major signaling and transcriptional inputs into stomatal development; arrows and T-bars indicate positive and negative effects, respectively, and are directed to the stage in which they have been demonstrated to act.

 

Transcriptional regulation of stomatal development

Transcription factors also play a critical role in asymmetric division and cell fate establishment in the stomatal lineage (Kanaoka et al., 2008; Kutter et al., 2007; Lai et al., 2005; MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007). One set of these transcription factors belongs to the conserved bHLH family, which is found in animal as well as plant systems. Based upon loss and gain of function phenotypes, five bHLH transcription factors serve as major cell fate regulators in the stomatal lineage (Kanaoka et al., 2008; MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007), and three of these five (SPEECHLESS (SPCH), MUTE, and FAMA) display restricted expression patterns that correlate with specific stages of lineage progression (MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007).

 

SPCH and MUTE are responsible for the initiation and termination, respectively, of asymmetric divisions in the stomatal lineage. SPCH is expressed transiently in a subset of epidermal cells, many of which undergo entry (and later, spacing) divisions to produce meristemoids, and strong spch mutations block stomatal lineage initiation (MacAlister et al., 2007; Pillitteri et al., 2007). SPCH is a direct target of MAP kinase phosphorylation, and although overexpression of wild-type SPCH confers little phenotype, expression of SPCH variants lacking phosphorylation sites induces excess asymmetric divisions and meristemoid overproduction (Lampard et al., 2008). MUTE, which acts in late meristemoids to terminate amplifying division, is not subject to similar MAPK control and can, upon overexpression, generate an epidermis composed almost entirely of stomata by inducing all epidermal cell types to become GMCs, which then divide to become pairs of  guard cells (MacAlister et al., 2007; Pillitteri et al., 2007).

 

After the initial decision: how to execute an asymmetric division

The identification of signaling components and transcription factors (at least one of which is a direct target of the signaling pathways) as key regulators of asymmetric division in the Arabidopsis stomatal lineage fits well with what is observed in later rounds of meristemoid production, when new precursors must be intercalated with existing stomata in a patterned fashion. Identification of these factors, however, does not explain how the relatively high-level ‘decision’ process is translated into asymmetry at the cell biological level. BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), an unequally segregated protein, may offer insight into the problem of executing an asymmetric division (Dong et al., 2009).

 

Polarized expression pattern of BASL at cell periphery and in nuclei of asymmetrically dividing stomatal lineage cells

Polarized expression pattern of BASL at cell periphery and in nuclei of asymmetrically dividing stomatal lineage cells

 
In the absence of BASL, stomatal lineage divisions show reduced physical and fate asymmetry, generating daughter cells inappropriately similar in size, marker expression, and ultimate identity (Dong et al., 2009). BASL encodes a novel, plant-specific protein possessing no recognizable functional domains. BASL is expressed primarily in asymmetrically dividing stomatal lineage cells, and it is the dynamic behavior of BASL protein within these cells that is most informative.  Prior to a typical asymmetric division, BASL is found in the nucleus, but begins to accumulate in a cortical crescent; this crescent is always positioned so that it is inherited by the larger daughter. After division, the smaller daughter has BASL in the nucleus, while the larger has BASL both in the nucleus and at the cortex (and Dong et al., 2009). Time-lapse experiments tracing BASL dynamics in single cells revealed two possible developmental trajectories for each daughter. The smaller (meristemoid) can become a GMC, losing nuclear BASL in the process, or it can divide again asymmetrically after first establishing a new cortical crescent. The larger (SLGC) can differentiate into a non-stomatal epidermal cell, losing nuclear BASL (but sometimes retaining the cortical crescent), or it can divide asymmetrically to form a secondary meristemoid—a fate that correlated with retention of both nuclear and cortical BASL. Production of a secondary meristemoid requires that the SLGC reorient its axis of polarity in order to maintain one-cell spacing. This reorientation is reflected in the cortical BASL crescent’s relocation to the opposite side of the cell, ensuring that it is distal to the newly forming meristemoid.

 

Outstanding questions

In the past decade work from many groups has generated a developmental framework for Arabidopsis stomatal development and identified many of the crucial genes.  There are undoubtedly more regulators to be found; these may include new components of signaling pathways that modulate spacing and stomatal density, other transcriptional regulators that promote specific cell identities and certainly more components that generate the physical asymmetries of stomatal lineage divisions.  Once we have populated the lists of “stomatal genes”, however, it is critical to begin building connections among these genes.  Our group is taking a variety of experimental approaches to expand the repertoire of stomatal regulators and understand how they work together. Vignettes about some of our favorite current projects can be accessed by clicking the icons on left of the lab research page.

 

Relevant Publications

Bergmann lab publications are in bold. 

 

Bergmann, D. C., Lukowitz, W., Somerville, C. R. (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science. 304(5676), 1494-7.

 

Bergmann, D. C. and Sack, F. D. (2007). Stomatal development. Annu. Rev. Plant. Biol. 58,163-81.

 

Dong J, Macalister CA, Bergmann DC (2009) BASL Controls Asymmetric Cell Division in Arabidopsis. Cell. 2009 Jun 10. PMID: 19523675

 

Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C., Kakimoto, T. (2007). The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev. 21(14), 1720-1725.

 

Hunt, L  and  J. Gray. (2009) Signaling Peptide EPF2 Controls Asymmetric Cell Divisions During Stomatal Development. Curr Biol.

 

Kanaoka, M. M., Pillitteri, L. J., Fujii, H., Yoshida, Y., Bogenschutz, N. L., Takabayashi, J., Zhu, J. K., Torii, K. U. (2008). SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation. Plant Cell 20(7), 1775-1785.

 

Kutter, C., Schob, H., Stadler, M., Meins, F.,Jr, Si-Ammour, A. (2007). MicroRNA-mediated regulation of stomatal development in arabidopsis. Plant Cell 19(8), 2417-2429.

 

Lai, L. B., Nadeau, J. A., Lucas, J., Lee, E. K., Nakagawa, T., Zhao, L., Geisler, M., Sack, F. D. (2005). The arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell .

 

Lampard, G. R., Macalister, C. A., Bergmann, D. C. (2008). Arabidopsis stomatal  initiation is controlled by MAPK-mediated regulation of the bHLH, SPEECHLESS. Science 2008 Nov 14;322(5904):1113-6. PMID: 19008449

 

MacAlister, C. A., Ohashi-Ito, K., Bergmann, D. C. (2007). Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445(7127), 537-540.

 

Nadeau, J. A. and Sack, F. D. (2002). Control of stomatal distribution on the Arabidopsis leaf surface. Science 296(5573), 1697-1700.

 

Ohashi-Ito, K. and Bergmann, D. (2006). Arabidopsis FAMA controls the final Proliferation/Differentiation switch during stomatal development. Plant Cell 18(10), 2493-2505.

 

Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L., Torii, K. U. (2007). Termination of asymmetric cell division and differentiation of stomata. Nature 445(7127), 501-505.

 

Shpak, E. D., McAbee, J. M., Pillitteri, L. J., Torii, K. U. (2005). Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309(5732), 290-293.

 

Wang, H., Ngwenyama, N., Liu, Y., Walker, J. C., Zhang, S. (2007). Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19(1), 63-73.

SIGNALING

Local and regional signaling and pattern formation

Stomatal development requires coordination of cell fate, cell division and overall pattern. Stomata and their precursors exchange signals with neighbor epidermal cells, with underlying tissues and over long distances. The ultiple inputs and the discrete and easily scorable cell fate outputs combined with the ability to manipulate gene expression in specific cells makes the stomatal lineage an excellent in vivo system to test general principles of plant signal transduction.

 

MAPK signaling 

Mitogen-activated protein kinase (MAPK) signaling networks are found in all eukaryotic organisms and modulate a wide variety of biological processes including cell division, developmental pathways (including stomatal development) and responses to abiotic and biotic stresses. Plant genomes encode large gene families of MAPK pathway components, often making the precise functions of individual kinases difficult to ascertain. We have used the discrete stages and phenotypes of the stomatal lineage to assay the contributions of individual MAPK pathway members to cell fate and proliferation.

 
Former postdoc Diego Wengier is continuing this work in his new lab (University of Buenos Aires, Argentina)

 

Creating the appropriate cellular milieu for signal transduction

Signal transduction from a cell’s surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. In contrast to animals, where developmental and defense signals are mediated through many different signaling pathways (e.g., Hh, Wnt, Notch), plant signaling pathways are dominated by LRR-RLK receptors; in Arabidopsis there are more than 230 such proteins (Shiu and Bleecker, 2001). Recent progress on the behaviors of these LRR-RLKs makes it clear that there are multiple modes of ligand-receptor interaction as well as requirements for subcellular localization and dynamic trafficking. The ERECTA family receptors that have major roles in plant development, defense and homeostasis, yet little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry and live-cell imaging we showed the VST family of proteins is required for ERECTA-mediated signaling in growth and stomatal cell fate determination, and reveal subtle complexities in the ways different ERECTA family receptors behave. The VSTs are a small group of proteins with a passing resemblance to vesicle trafficking regulators, but we show that this is not their function. Instead, we find that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, forming regions where these two membrane systems can exchange. Such PM-ER contact sites have important roles in neuronal calcium signaling and for response to stress conditions, but this is the first report of a role promoting efficient and differential signaling from a specific receptor kinase family (ERECTA) to downstream intracellular targets.

 
Former Postdoc Chin-Min (Kimmy) Ho is continuing work on the cell biology of signal transduction in her new lab (Academia Sinica, Taiwan)

 

Cross talk among development/defense/hormone pathways

Crosstalk in pathways can occur at many levels. Work from Zhiyong Wang’s lab (Carnegie institution) has focused on Brassinosteroid signaling and in collaboration, we have found direct connections between upstream kinases in BR signaling and MAPK elements used for stomatal development.  How these and other hormone pathways interface with stomatal development and the core developmental regulators is a topic of significant current interest.

 
Former Postdoc On Sun Lau is continuing work on environmental signals into stomatal development in his new lab (National University of Singapore)

 

Cross talk in ligand/receptor signaling

Core signaling pathways function in multiple programs during multicellular development. The mechanisms that compartmentalize pathway function or confer process specificity, however, remain largely unknown. Starting from genetic screens to understand how the TMM receptor guided different responses in different tissues, we uncovered mutations in putative signaling elements downstream and, surprisingly, upstream of TMM. The upstream elements are members of the EPF family of secreted peptide ligands shown, in the case of EPF1, EPF2 and STOMAGEN, to inhibit or promote stomatal development by engaging the ERECTA family of receptor-like kinases, partners of TMM. We first showed that CHALLAH (EPFL6) could repress stomatal development, but only when TMM was not present. Later examination of EPFL genes EPFL6/CHALLAH (CHAL), EPFL5/CHALLAH-LIKE1, and EPFL4/CHALLAH-LIKE2 (CLL2) reveals that this family may mediate additional ER-dependent processes. chal cll2 mutants display growth phenotypes characteristic of er mutants, and genetic interactions are consistent with CHAL family molecules acting as ER family ligands. We propose that different classes of EPFL genes regulate different aspects of ER family function and introduce a TMM-based discriminatory mechanism that permits simultaneous, yet compartmentalized and distinct, function of the ER family receptors in growth and epidermal patterning.

 

Relevant lab publications

Ho CM, Paciorek T, Abrash E, Bergmann DC. (2016) Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases. Dev Cell. 2016 Aug 22;38(4):345-57. doi: 10.1016/j.devcel.2016.07.016 PMID: 27554856

 
Lampard GR, Wengier DL, Bergmann DC. (2014) Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity. Plant Cell. 2014 Aug;26(8):3358-71. doi: 10.1105/tpc.114.127415. PMID:25172143

 
Wengier DL, Bergmann DC. (2012) On fate and flexibility in stomatal development. Cold Spring Harb Symp Quant Biol. 2012;77:53-62. doi: 10.1101/sqb.2013.77.015883. PMID:23444192

 
Kim TW, Michniewicz M, Bergmann DC, Wang ZY.(2012) Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature. 2012 Feb 5;482(7385):419-22. doi: 10.1038/nature10794. PMID:22307275

 
Abrash EB†, KA Davies† and DC Bergmann; (2011) Generation of signaling specificity by spatially restricted buffering of ligand-receptor interactions, Plant Cell 23(8):2864-79 †equal contribution  PMID: 21862708

 
Katsir L†, KA Davies†, DC Bergmann; and T Laux‡ (2011) Peptide signaling in plant development, Current Biology,21(9):R356-364 PMID: 21549958

 
Rowe MH, Bergmann DC (2010) Complex signals for simple cells: the expanding ranks of signals and receptors guiding stomatal development. Curr Opin Plant Biol. 2010 Oct;13(5):548-55. PMID: 20638894

 

Abrash EB and Bergmann DC (2010) Regional specification of stomatal production by the putative ligand CHALLAH. Development. 2010 Jan 7; 10.1242/dev.040931. PMID: 20056678

 

Lampard GR, Lukowitz W, Ellis BE, Bergmann DC (2009) Novel and expanded roles for MAPK signaling in Arabidopsis stomatal cell fate revealed by cell type-specific manipulations. Plant Cell. 2009 Nov;21(11):3506-17. PMID: 19897669

 

Lampard GR*, Macalister CA*, Bergmann DC (2008) Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 2008 Nov 14;322(5904):1113-6. PMID: 19008449  * contributed equally

 

Bergmann DC, Lukowitz W, Somerville CR (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science. 2004 Jun 4;304(5676):1494-7. PMID: 15178800

 

Funded by: NIH and GBMF-HHMI

TRANSCRIPTION

Transcriptional control of stomatal development

Five bHLH transcription factors, the three stage-specific regulators SPCH, MUTE and FAMA and their potential partners, the more widely expressed genes ICE1/SCRM and SCRM2 play major roles in stomatal development. A major challenge is to understand how proteins with nearly identical bHLH domains control the different steps in stomatal development.  In animal neural and muscle development, members of the Acheate-Scute and MyoD classes of bHLHs normally specify specific cell fates; promoter-swap experiments indicate that the proteins are interchangeable if expressed in each other’s domains.  However, the same is not true for the stomatal bHLHs.  With the idea that elements of protein structure unique to each stomatal bHLH are critical, we focus on the regulation and activities of SPCH and FAMA, the bHLHs that control initial and final stomatal differentiation stages, respectively.

 

SPCH and asymmetric division

SPCH controls the symmetry-breaking event that leads to the creation of the stomatal lineage; in its absence, epidermal cells cannot create meristemoids. How does the presence of SPCH initiate asymmetric divisions? What are its downstream targets? We are pursing these questions by determining direct targets of SPCH and monitoring the cell-biological changes induced upon SPCH expression. By developing a highly sensitive ChIP assay (MOBE-ChIP, Lau et al., 2014), we found that SPCH is associated with a large number of regulatory regions. SPCH appears to directly regulate both the positive (stomatal promoting) genes in the stomatal pathway, as well as the negative (stomatal inhibiting) stomatal signaling pathways indicating that SPCH is part of extensive positive and negative feedback loops.
What controls the expression of SPCH itself? Although we know little about the transcriptional regulation of SPCH, we have shown that MAPK phosphorylation is critical for modulating SPCH protein expression and activity. The coupling of MAPK signaling to SPCH activity provides cell type specificity for MAPK output while allowing the integration of developmental and environmental signals into the production and spacing of stomata.

 

FAMA and the final differentiation step

FAMA appears to be absolutely required to allow stomatal development. To date, no mutations or treatments that promote stomatal formation (for example, loss of function mutations in YODA, TMM, and the ERECTA family or overexpression of positive regulators SPCH and MUTE) can overcome the need for FAMA in creating guard cells. If FAMA is the master regulator of guard cell differentiation, then identifying its targets will let us understand how these highly specialized cells are formed. In addition, we’ve recently shown that its position at the end of the pathway is important for preventing mature stomata from returning to earlier stem-cell states. This “permanent shut-down” requires an interaction between FAMA and the retinoblastoma related (RBR) proteins, and we’ve show that this interaction facilitates RBRs recruitment to regulatory regions of early stomatal genes (Matos et al., 2014)

 

How did SPCH, MUTE and FAMA obtain their current roles?

SPCH, MUTE and FAMA are very closely related, yet each has a unique and essential role in Arabidopsis stomatal development. What features of the proteins are responsible for their unique functions, and what roles do gene expression changes play? How might the system have evolved? Using fine-tuned assays of in vivo gene function and time-lapse imaging of expression we identified regions of these three bHLHs responsible for their unique functions and generate a plausible model for how the different activities could arise from modification and duplication of the original ancestral gene. Surprisingly, we found that regions of sequence conservation (e.g. the nearly identical DNA binding domains) differed in function among the three proteins.

 

Relevant lab publications

Lau OS, Bergmann DC. (2015) MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies. Plant J. 2015 Oct;84(2):443-50. doi: 10.1111/tpj.13010. PMID: 26332947

 

Simmons AR, Bergmann DC (2015) Transcriptional control of cell fate in the stomatal lineage. Curr Opin Plant Biol. 2015 Nov 6;29:1-8. doi: 10.1016/j.pbi.2015.09.008 PMID: 26550955

 
Matos JL, Lau OS, Hachez C, Cruz-Ramírez A, Scheres B, Bergmann DC (2014) Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module. Elife. 2014 Oct 10;3. doi: 10.7554/eLife.03271. PMID:25303364

 
Davies KA, Bergmann DC (2014) Functional specialization of stomatal bHLHs through modification of DNA-binding and phosphoregulation potential. Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):15585-90. doi: 10.1073/pnas.1411766111 PMID:25304637

 
Lau OS, Davies KA, Chang J, Adrian J, Rowe MH, Ballenger CE, Bergmann DC (2014) Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells. Science 2014 Sept; DOI: 10.1126/science.1256888 PMID:25190717

 
Matos JL, Bergmann DC (2014) Convergence of stem cell behaviors and genetic regulation between animals and plants: insights from the Arabidopsis thaliana stomatal lineage. F1000Prime Rep. 2014 Jul 8;6:53. doi: 10.12703/P6-53. eCollection 2014. PMID:25184043

 
Robinson SJ, P Barbier de Reuille, DC Bergmann, P Prusinkiewicz and E Coen (2011) Generation of spatial patterns through cell polarity switching. Science 333(6048):1436-40 PMID: 21903812

 

Hachez C†, K Ohashi-Ito†, J Dong and DC Bergmann (2011) Differentiation of Arabidopsis guard cells: analysis of the networks incorporating the basic helix-loop helix transcription factor, FAMA. Plant Physiology 155: 1458-1472.  PMID: 21245191

 

Lampard GR*, Macalister CA*, Bergmann DC (2008) Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 2008 Nov 14;322(5904):1113-6. PMID: 19008449* These authors contributed equally to this work

 

MacAlister CA, Ohashi-Ito K, Bergmann DC (2007) Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature. 2007 Feb 1;445(7127):537-40. PMID: 17183265

 

Ohashi-Ito K, Bergmann DC (2006) Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell. 2006 Oct;18(10):2493-505. PMID: 17088607

 

Funded by: NIH and GBMF-HHMI

EVOLUTION

Stomatal development in diverse species

Stomata are an ancient and nearly ubiquitous feature of land plants and can be found in fossil leaves as early as ~400 mya. Plants often exhibit species-specific distributions and numbers of stomata on their surfaces. In contrast to this diversity in pattern, guard cell morphology has remained quite constant–there are only two classes of stomatal guard cells: the kidney-shaped cells found most plant species and the dumbbell-shaped guard cells found in grasses. We are interested in identifying the genes and developmental rules for stomata across a wide range of species.  We have identified orthologs of many of the stomatal regulators from Arabidopsis and are characterizing their roles in other plants, concentrating primarily on the bHLHs FAMA, MUTE and SPCH in non-vascular plants and in the grasses.

 

Stomatal development in grasses

The grasses are true innovators in stomatal structure and function and the dumbbell shaped stomata with their associated subsidiary cells have measurably more sensitive responses to environmental cues. The arrangement of stomata in files along the leaf surface is also different from the pattern in Arabidopsis.  We have taken both targeted and non-biased approaches to uncover the basic of stomatal production and pattern in grasses.

 
Together these forward and reverse genetic approaches revealed that despite major differences in stomatal lineage initiation and stomatal structure between grasses and dicots, homologous bHLH transcription factors regulate these process in both groups. Using fluorescent reporters of gene activity, overexpression and alteration of function (protein and cis-regulatory swaps) tools, we dissected how the same transcription factors could yield different outcomes and found clade-specific differences in gene expression and post-translational modification of protein stability and activity. When considered as networks, regulatory interactions among genes also shift between Arabidopsis and Brachypodium.

 
In our targeted approaches in rice and maize, we found that FAMA, the gene most closely associated with guard cell differentiation, is conserved between Arabidopsis and grasses. Rice osfama mutants fail to complete the final stage of stomatal differentiation, and OsFAMA can complement the Arabidopsis mutants. This suggests that FAMA acts at a high level in a transcriptional regulatory cascade – in essence specifying the transition to guard cell fate, not the details of guard cell morphology.  In Arabidopsis, SPCH and MUTE control the formation and termination of the self-renewing meristemoids. In the expression and functional studies on the rice and maize homologs of these genes, we found some evidence for their conserved roles in stomatal development; however, there were also some significant differences.

 
Taken together, these studies provide an unprecedented level of mechanistic understanding of stomatal development in representatives of the most agriculturally important plant family, and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity.

 

Stomatal development in non-vascular plants

SPCH, MUTE and FAMA are highly similar, yet each is unique required for stomatal development. How did this gene family evolve? We analyzed the bHLH subclass to which these genes belong in representatives of the angiosperms, gymnosperms, lycopods and mosses.  Basal plant groups encode fewer proteins of this subclass, and the features unique to SPCH, MUTE and FAMA (eg. SPCH’s MAPK target domain) are lost. 

 
Moss and lycopod genes are hybrid in structure and in their function in complementation tests, suggesting that as stomatal pattern became more complex, a single stomatal-promoting bHLH duplicated to take on roles at multiple stages. A recent collaborative project revealed that indeed, the same moss bHLH sufficient to complement mute or fama in Arabidopsis is required to make stomata in its native context. Moreover, the partnership between this representative of the SPCH, MUTE and FAMA clade and the representative of the ICE1/SCRM clade is conserved in the moss Physcomitrella.

 

Relevant lab publications

Raissig MT, Abrash E, Bettadapur A, Vogel JP, Bergmann DC (2016) Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity. Proceedings of the National Academy of Sciences 2016 doi: 10.1073/pnas.1606728113 PMID: 27382177

 
Chater CC, Caine RS, Tomek M, Wallace S, Kamisugi Y, Cuming AC, Lang D, MacAlister CA, Casson S, Bergmann DC, Decker E, Frank W, Gray JE, Fleming A, Reski R, Beerling DJ (2016) Origin and function of stomata in the moss Physcomitrella patens, Nature Plants. Nov 2016, 2:16179 doi: 10.1038/nplants.2016.179 PMID: 27892923

 
Vatén A and DC Bergmann (2012) Mechanisms of stomatal development: an evolutionary view. Evodevo 3(1):11. PMID: 22691547

 
MacAlister CA and DC Bergmann (2011) Sequence and function of bHLHs required for stomatal development in Arabidopsis are deeply conserved in land plants. Evolution and Development 13:2, 182–192. PMID: 21410874

 

Liu T, Ohashi-Ito K, Bergmann DC (2009) Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses. Development. 2009 Jul;136(13):2265-76. PMID: 19502487

 

Funded by: GBMF-HHMI

SYSTEMS

Systems biology of stomatal development   

There are many outstanding questions about how cells transit through the stomatal development pathway and how the individual genes we have identified fit into a regulatory hierarchy.  Many of these questions can only be answered by understanding at a global scale what happens at each cell state transition. For example, what is the inventory of genes required to make a meristemoid? Which are required to be a meristemoid?  What are the direct targets of each of the stomatal bHLHs?  How can expression of such similar transcription factors generate such different outcomes?

 

Generating a map of stomatal lineage cells by FACs sorting 

Based on the pioneering work of Ken Birnbaum and Phillip Benfey using FACS-based sorting of GFP-positive cells to create an atlas of “cell type specific” information from roots, we have used markers expressed at different times in the progression of stomatal lineage cells from early proliferative stages, through differentiation.  By comparing these cells among themselves and in reference to the epidermis as a whole, we have identified signatures of individual cell types.  As well, these profiles are rich sources of genes for future study (for example, in the asymmetry section). In contrast to other datasets that focus on terminally differentiated cells or permanent populations, the stomatal lineage data track cells in transition, and comparison of these cells to other single cell transcriptomes has revealed surprises.

Data from these experiments are searchable online using a database that will be incorporated into the BAR eFP viewer upon publication of the major datasets. An example screenshot is below:

Stomatal Map

Creation of a “stomatal map” by isolating pure population of cells at different stages (each represented as a different color bar under cell types). Marked cells are protoplasted (left bottom panel), sorted and then used for RNA extraction and quantification on microarrays or by RNA-seq.

 

Modeling of stomatal development

As our cell-type-specific data increase, it is also imperative to develop a developmental framework in which to position these vast datasets. In collaboration with computational biologists and modelers in Enrico Coen’s group (John Innes Center), computational models were designed to develop a minimal set of rules to explain the complex patterns and behaviors of stomatal lineage cells. This work has already yielded basic rules for division timing and direction, and candidate genes (SPCH and BASL) that possess some of the required properties to enforce these rules. Equally important, these models posit additional regulatory requirements for which there are not yet obvious candidate genes. The details of stomatal-lineage biology, with its easily tracked, counted and manipulated self-renewing cells and the technical resources for generating and interpreting large scale datasets in Arabidopsis make this an excellent system to identify new stem-cell regulators.

 

Relevant Lab publications
Adrian J, Chang J, Ballenger CE, Bargmann B, Alassimone J, Davies KA, Lau OS, Matos JL, Hachez C, Lanctot Z, Vatén A, Birnbaum KD, Bergmann DC (2015) Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population. Dev Cell. 2015 Apr 6;33(1):107-18. doi: 10.1016/j.devcel.2015.01.025. PMID: 25850675

 
Kajala K, Ramakrishna P, Fisher A, Bergmann DC, De Smet I, Sozzani R, Weijers D, Brady SM (2014) Omics and modelling approaches for understanding regulation of asymmetric cell divisions in Arabidopsis and other angiosperm plants. Ann Bot. 2014 Jun;113(7):1083-1105. PMID:24825294

 
Bargmann BO, Vanneste S, Krouk G, Nawy T, Efroni I, Shani E, Choe G, Friml J, Bergmann DC, Estelle M, Birnbaum KD (2013) A map of cell type-specific auxin responses. Mol Syst Biol. 9:688. doi: 10.1038/msb.2013.40. PMID:24022006 ; PMCID: PMC3792342

 
Robinson SJ, P Barbier de Reuille, DC Bergmann, P Prusinkiewicz and E Coen (2011) Generation of spatial patterns through cell polarity switching. Science 333(6048):1436-40 PMID: 21903812

 

Funded by: NIH and GBMF-HHMI

ASYMMETRY

Asymmetric cell division and generation of polarity

Asymmetric cell divisions create diverse cell types and maintain stem-cell populations.  In many systems, asymmetric division relies on asymmetric protein localization within the mother cell and between the daughters. For example, the PAR proteins are conserved animal polarity regulators that are localized to restricted regions of the cell periphery and influence cell fate by guiding division planes and by localizing cell fate regulators so that that these determinants are segregated to one daughter.

 

Plants, like animals, use asymmetric divisions for development and self-renewal; however, plant genomes do not encode homologues of the animal asymmetry regulators and structural features of plant cells preclude many of the animal cell division mechanisms.  We have exploited the stereotyped asymmetric and oriented division pattern of Arabidopsis stomatal development to identify genes that serve as plant cells “asymmetry” regulators.

 

BASL is required for plant asymmetric divisions

BASL is required for the physical and cell fate asymmetry of stomatal lineage divisions. Moreover, BASL protein exhibits a unique and dynamic subcellular localization during asymmetric divisions; accumulating in the nucleus, in a crescent at the periphery, or in both locations, depending on the identity and division behavior of the stomatal lineage cell. In a reversal from the situation in animal stem-cell populations, BASL appears to be active at the cortex of the differentiating cell whereas nuclear localization serves a sequestration function.  To our knowledge, BASL is the first protein reported to have this localization in plants and the only stem-cell associated protein that functions in this manner.

 

BASL is normally expressed only in stomatal lineage cells. However, when expressed in other cells, BASL accumulates in a peripheral crescent indicating that most, if not all, plant cells have machinery for polarized protein trafficking and maintenance at the periphery.  BASL, therefore, provides an entry into the general problem of plant cell polarity.

 

We are currently asking: How is BASL trafficked and maintained at its polarized peripheral location? What is the function of BASL at the cell periphery? What is its function in the nucleus?

 

BASL is also not the only protein polarized in the stomatal lineage. The Torii lab recently reported another novel protein, POLAR, whose polarized expression at the periphery is dependent on BASL.  Using our “stomatal map” (see section on systems) we identified yet more polarized proteins in the stomatal lineage.  Excitingly, one such family appears to contain true partners of BASL as they interact physically with BASL and are mutually required for polarized localization and function.

 

Relevant lab publications

Kajala K, Ramakrishna P, Fisher A, Bergmann DC, De Smet I, Sozzani R, Weijers D, Brady SM (2014) Omics and modelling approaches for understanding regulation of asymmetric cell divisions in Arabidopsis and other angiosperm plants. Ann Bot. 2014 Jun;113(7):1083-1105. PMID:24825294

 
Axelrod JD, Bergmann DC (2014) Coordinating cell polarity: heading in the right direction? Development. 2014 Sept; 141(17):3298-3302. PMID:25139852

 

Dong J, Macalister CA, Bergmann DC (2009) BASL Controls Asymmetric Cell Division in Arabidopsis. Cell. 2009 Jun 10. PMID: 19523675

 

Abrash EB, Bergmann DC (2009) Asymmetric cell divisions: a view from plant development. Dev Cell. 2009 Jun;16(6):783-96.PMID: 19531350

 

Funded by:  NSF and HHMI-GBMF

ENVIRONMENT

Stomata and the environment

Stomata serve as the main conduits for gas exchange between plants and the atmosphere; they are critical for photosynthesis and exert a major influence on global carbon and water cycles. For a plant, an optimal balance of CO2 uptake and water vapor release depends on the number, position and behavior of stomatal complexes.  The properties of stomatal production and distribution are fine-tuned by signals operating across a wide range of spatial and temporal scales. For example, many (but not all) plants decrease their stomatal production in response to elevated [CO2].

 

How do plants sense the environment, and how do they alter their stomatal development in response? While identifying the components of the climate sensing apparatus is an extremely difficult problem, many of the genes we already study are very likely to be part of the response.  Understanding how these genes are regulated by local and long-range cues is a goal of this work.

 

In collaboration with Joe Berry, an ecophysiologist at the Carnegie Institution, Department of Global Ecology, we are also developing novel integrated approaches to measure the consequences of altered stomatal production from the molecular to the small plot scale. These experiments address major theoretical and practical questions about the effects of climate change on plants: how are processes at the leaf and individual level scalable to the community, landscape and global level?  How well do global models predict behavior of individual plants? What is the potential for mitigating the detrimental effects of climate change by altering stomatal characteristics?

 

Relevant lab publications

Dow GJ, Bergmann DC (2014) Patterning and processes: how stomatal development defines physiological potential. Curr Opin Plant Biol. 2014 Jul 21;21C:67-74. doi: 10.1016/j.pbi.2014.06.007. PMID:25058395

 
Kumari A, Jewaria PK, Bergmann DC, Kakimoto T (2014) Arabidopsis Reduces Growth Under Osmotic Stress by Decreasing SPEECHLESS Protein. Plant Cell Physiol. 2014 Dec;55(12):2037-46. doi: 10.1093/pcp/pcu159. PMID:25381317

 
Dow GJ, Bergmann DC, Berry JA (2013) An integrated model of stomatal development and leaf physiology. New Phytol. doi: 10.1111/nph.12608. PMID:24251982

 
Dow GJ, Berry JA, Bergmann DC (2013) The physiological importance of developmental mechanisms that enforce proper stomatal spacing in Arabidopsis thaliana. New Phytol. doi: 10.1111/nph.12586. PMID:24206523

 

Bergmann D (2006) Stomatal development: from neighborly to global communication. Curr Opin Plant Biol. 2006 Oct;9(5):478-83. PMID: 16890476

 

Funded by: Stanford BIO-X and GBMF-HHMI

STOMATA

SIGNALING

TRANSCRIPTION

EVOLUTION

SYSTEMS

ASYMMETRY

ENVIRONMENT