Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity

Abstract

Stomata, epidermal valves facilitating plant-atmosphere gas exchange, represent a powerful model for understanding cell fate and pattern in plants. Core basic helix-loop-helix (bHLH) transcription factors regulating stomatal development were identified in Arabidopsis, but this dicot's developmental pattern and stomatal morphology represent only one of many possibilities in nature. Here, using unbiased forward genetic screens, followed by analysis of reporters and engineered mutants, we show that stomatal initiation in the grass Brachypodium distachyon uses orthologs of stomatal regulators known from Arabidopsis but that the function and behavior of individual genes, the relationships among genes, and the regulation of their protein products have diverged. Our results highlight ways in which a kernel of conserved genes may be alternatively wired to produce diversity in patterning and morphology and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity.stomatal development | bHLH transcription factor | Brachypodium | grass S tomata are valves on the surface of plants with central roles in gas exchange and biosphere productivity. Stomata are both ancientthey appear on 400 million-year-old fossils-and nearly ubiquitously found in extant land plants. The diversity of stomatal morphologies and patterned distributions across different plant families coupled with rapidly advancing functional genomic resources offers a powerful opportunity to follow morphological innovation and gene regulatory network evolution simultaneously. In most plants, stomata consist of two kidney-shaped epidermal guard cells (GCs) surrounding a pore (Fig. 1A). Grass stomatal morphology is unique, featuring dumbbell-shaped GCs flanked by subsidiary cells (SCs) (Fig. 1A), and physiological measurements suggest this derived form is more efficient (1). The distribution of stomata on leaves is also species specific. Dicots such as Arabidopsis display a scattered distribution, with avoidance of direct contact being the most basic patterning rule; dispersed stem cell-like stomatal precursors divide throughout the leaf to produce this pattern and promote the typical "broadleaf" or radial growth characteristic of these plants (Fig. 1A). Grasses, in contrast, generate stomata, which are always oriented in the same direction, from specific cell files. These stomatal lineage files are established in a single zone at the leaf base with differentiation proceeding in a linear gradient toward the tip (Fig. 1A).Our understanding of the genetic underpinnings of stomatal fate and pattern is derived mostly from studies in the dicot Arabidopsis where the group Ia basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (AtSPCH), AtMUTE, and AtFAMA establish stomatal lineage identity, regulate the transition to terminal precursor fate, and promote the differentiation of GCs, respectively (2-4). The function of these stage-specific factors requires heterodimerization with one of ...

SHORTROOT-Mediated Increase in Stomatal Density Has No Impact on Photosynthetic Efficiency

“…However, ectopic expression of ZmSCR1 in rice does not perturb stomatal density or patterning, at least when expressed constitutively and at high levels (Wang et al, 2017). Therefore, stomatal specification must be mediated by interactions with alternative downstream targets, the most likely candidates being homologs of BdSPCH and BdICE, which have been shown to act in the earliest stages of specification in B. distachyon (Raissig et al, 2016). The role of OsSCR may be limited to later stages in the process, where asymmetric cell divisions are needed to form the stomatal complex (Kamiya et al, 2003).…”

“…As such, positional signals must trigger the specification of stomatal cell fate. Because stomatal cell files are specified in positions flanking procambial centers, it is conceivable that positional signals emanating from the developing vein either inhibit stomatal specification in epidermal cells directly above and below the procambium or induce specification in epidermal cell files at the flanks of the procambial center.In rice (Oryza sativa ssp japonica), maize, and the model grass Brachypodium distachyon, stomatal identity and differentiation appear to be regulated by the same group of basic helix-loop-helix (bHLH) transcription factors that operate in the well-established pathway that was discovered in the eudicot Arabidopsis (Arabidopsis thaliana; Liu et al, 2009;Raissig et al, 2016Raissig et al, , 2017. However, orthologous genes have been adopted into different roles in the two patterning networks.…”

“…However, orthologous genes have been adopted into different roles in the two patterning networks. In B. distachyon, the combined activity of the bHLH proteins SPEECHLESS (BdSPCH1 and BdSPCH2) and INDUCER OF CBF EXPRESSION1 (BdICE) leads to the specification of stomatal fate, with loss-of-function mutants in either BdSPCH1/BdSPCH2 or BdICE lacking the characteristically spaced files of small epidermal cells at the leaf base that are the first visible indication of stomatal specification (Raissig et al, 2016). In considering what might induce stomatal specification in those cell files, we reflected on known patterning mechanisms that operate around the vasculature.…”

“…In rice (Oryza sativa ssp japonica), maize, and the model grass Brachypodium distachyon, stomatal identity and differentiation appear to be regulated by the same group of basic helix-loop-helix (bHLH) transcription factors that operate in the well-established pathway that was discovered in the eudicot Arabidopsis (Arabidopsis thaliana; Liu et al, 2009;Raissig et al, 2016Raissig et al, , 2017. However, orthologous genes have been adopted into different roles in the two patterning networks.…”

Reducing Stomatal Density in Barley Improves Drought Tolerance without Impacting on Yield

“…In contrast to the Arabidopsis model system, our knowledge of stomatal development in crops is relatively limited (Raissig et al, 2016). Although the grasses include many of our major global crops, our molecular understanding of their transpirational control mechanisms remains extremely limited.…”