MYB16 expression in the stomatal lineage: Wrong place at the wrong time leads to stomata side-by-side

Plant leaves need to achieve a balance between restricting water loss from the cells while allowing CO₂ uptake for photosynthesis. Adaptation to life on the land led to the evolution of a solution: formation of the cuticle and the stomata. The cuticle—a layer of lipid polymers and wax—covers the leaf epidermis, sealing it from the environment and protecting leaf tissues from drying. Formation of stomatal pores in the epidermis ensures flexibility as they open and close in response to environmental cues and mediate CO₂ uptake and transpiration. Formation of the cuticle is linked with stomatal development (Bird and Gray, 2003; Yang et al., 2011) but it is largely unknown how these processes are coordinated.

Stomata are formed during leaf development through a series of controlled cell divisions (Lee and Bergmann, 2019). In developing leaf primordia, a subset of cells produces the transcription factor SPCH that is necessary for stomatal initiation. SPCH-expressing cells divide asymmetrically, yielding a smaller meristemoid and a larger stomatal lineage ground cell (SLGC). The SLGCs and meristemoids may undergo further divisions and form additional stomatal lineage cells, whereas meristemoids finally differentiate and divide to form pairs of guard cells that make up stomata. Polarity proteins ensure that asymmetric divisions in the stomatal lineage avoid formation of adjacent stomata, leading to the one-cell-spacing rule: there is at least one pavement cell between stomata. In this issue of The Plant Cell, Shao-Li Yang and colleagues (Yang et al., 2022) show that downregulation of the MYB16 transcription factor in the meristemoid step of the stomatal lineage is necessary to ensure appropriate polarity of cell divisions and formation of stomata according to the one-cell-spacing rule. Given that MYB16 is a known regulator of cuticle biosynthesis genes, this work links the regulation of cuticle formation with stomatal development.

Yang et al. (2022) analyzed the expression of MYB16 and SPCH in different stomatal lineage cell types and found that SPCH is preferentially expressed in meristemoids while MYB16 in SLGCs, hence confirming previous observations (Ho et al., 2021). However, time-lapse imaging showed that MYB16 was also expressed in meristemoids but was quickly replaced by SPCH expression before asymmetric cell divisions. SPCH suppressed transcription from the MYB16 promoter in vitro, suggesting that SPCH downregulates MYB16 expression in meristemoids in planta. MYB16 was expressed again in young guard cells, suggesting that suppression of its expression specifically in the meristemoid stage is important for normal stomatal development.

The authors analyzed the effects of altered MYB16 expression in stomatal lineage cells on stomatal patterning. MYB16-deficient plants had a slight reduction in stomatal numbers, whereas inducible and ectopic expression of MYB16 in either asymmetrically dividing cells or throughout the stomatal lineage produced higher numbers of stomata that more often occurred in clusters, violating the one-cell-spacing rule. Ectopic MYB16 expression throughout the stomatal lineage led to induction of cuticular biosynthesis-related genes, thicker cuticle in all epidermal cells and thinner cell walls in stomatal lineage cells. To test whether stomatal cluster formation was related to cutin accumulation in these plants, the authors expressed the cutinase CDEF1 in the epidermis of plants ectopically expressing MYB16. Epidermal CDEF1 expression reduced stomatal cluster formation, indicating that stomatal spacing is linked with cutin deposition.

Stomatal clustering can arise as a consequence of altered cell polarity in asymmetrically dividing stomatal lineage cells. As establishment of polarity depends on the mechanical properties of the epidermis, Yang et al. (2022) hypothesized that changes in the leaf cuticle in ectopic MYB16 expression lines affect the physical properties of the epidermis and thus modulate the establishment of polarity, potentially leading to stomatal clustering. Indeed, polarity patterns were altered in plants ectopically expressing MYB16 but these phenotypes together with the stomatal clustering were rescued when plants were grown on high percentage agar that affects pavement cell stiffness and cell wall tension. These data indicate that MYB16 mis-expression affects the composition of the cell wall and the cuticle, thus influencing the mechanical properties of the epidermis and leading to aberrant polarity setup in the stomatal lineage cells (see Figure).

Figure.

MYB16 expression in meristemoids leads to stomatal clustering. In wild-type epidermis, SPCH suppresses MYB16 expression in meristemoids, ensuring establishment of polarity and normal stomatal patterning. Ectopic expression of MYB16 in meristemoids leads to altered cuticular and cell wall properties, decrease in polarity protein, and formation of stomatal clusters. Adapted from Yang et al. (2022), Figure 8.

The work by Yang et al. (2022) brings to spotlight the yet poorly understood question on how the coordination between the biosynthesis of cuticle and the formation of stomata is achieved in leaf development. The results indicate that formation of cuticle is tightly regulated and integrated with stomatal lineage progression, thus ensuring efficient stomatal spacing while forming the protective cuticular layer.

Funding

This work was supported by the Estonian Research Council grant (PSG404).