Shaping a flexoskeleton: pectate lyase PLL12 facilitates stomatal movements

Plants control water loss and CO₂ uptake by adjusting stomatal aperture in response to environmental cues, such as light and dark transitions. Stomatal opening and closure are caused by the swelling or shrinking of guard cells brought about by the movement of ions and water across guard cell membranes. Stomatal aperture adjustments occur in minutes and stomata can open or close multiple times a day. How do guard cell walls tolerate and aid these fast and recurring changes?

Plant cell walls are made up of cellulose, hemicellulose, and pectin. Recent evidence suggests that pectin and its modifications are especially important for stomatal function, as plants deficient in pectin-modifying enzymes have impaired stomatal responses (Amsbury et al., 2016; Rui et al., 2017). Whether pectate lyases, a class of enzymes that cleave pectin, contribute to stomatal function was unknown. In the current issue of The Plant Cell, Yintong Chen and colleagues (Chen et al., 2021) show that PECTATE LYASE LIKE12 (PLL12) promotes stomatal opening and closure in Arabidopsis thaliana, likely by affecting pectic homogalacturonan abundance and cross-linking in the guard cell wall.

The authors identified PLL12 as a pectate lyase gene with a potential role in stomatal function due to an increase in its expression in plants overproducing the guard cell developmental regulator FAMA (Hachez et al., 2011). Chen et al. (2021) detected PLL12 promoter activity in guard cells but also in other cell types, prompting them to generate Arabidopsis lines where PLL12 expression was suppressed via miRNA silencing specifically in guard cells (PLL12kd), in addition to using a PLL12 null mutant (pll12-1) to address how PLL12 influences stomatal dynamics.

Analysis of stomatal opening and closure in pll12-1 and PLL12kd leaves showed that mutant stomata were initially more closed, and stomatal movements in both directions were slower and reduced in amplitude, in plants deficient in PLL12 function compared with the wild type. Chen et al. (2021) then asked whether PLL12 deficiency affected the biomechanical properties of guard cell walls. They used nanoindentation of guard cells that were induced to open, then close in response to a light–dark cycle to repeatedly measure the force associated with indenting the cells to different depths over a stomatal opening-closure cycle. Combining these data with finite-element modeling allowed the authors to estimate wall modulus and turgor pressure in wild-type and PLL12-deficient guard cells. In pll12-1 mutants, guard cell longitudinal wall modulus was higher and turgor pressure lower than in wild-type plants, both in steady-state conditions in the dark and during stomatal opening in the light. Thus, PLL12 is needed to establish normal turgor and wall mechanics in guard cells to ensure fast stomatal responses.

Chen et al. (2021) used antibody labeling to test whether guard cell wall composition depends on PLL12. They found higher levels of labeling with an antibody recognizing de-methylesterified homogalacturonan, a putative substrate for PLL12 that is able to form cooperative Ca²⁺ crosslinks, in pll12-1 mutants (see Figure). The authors suggest that increased pectin crosslinking might contribute to the higher wall modulus in guard cells and reduced stomatal movements in pll12-1 mutants. The lack of PLL12 also affected plant growth and cell proliferation: the pll12-1 mutants were stunted and had smaller guard cells and pavement cells in the epidermis than the wild type, resulting in increased stomatal density but normal stomatal index. The PLL12kd plants also had smaller leaf rosettes but no change in epidermal cell sizes or stomatal density, indicating that the altered epidermal cell sizes in pll12-1 knockouts were likely caused by PLL12 function in non-stomatal cells.

Figure.

Cross-linked homogalacturonan levels are higher in guard cells of pll12-1 than wild-type Col. Green signal shows immunolabeling with 2F4, an antibody for cross-linked homogalacturonan, and magenta shows S4B staining of cellulose in the cell wall, scale bar = 5 µm. Adapted from Chen et al. (2021), Figure 3.

For fast stomatal movements, guard cell walls need to accommodate rapid changes in cell volume and turgor pressure. The work by Chen et al. (2021) suggests that in Arabidopsis, pectin cleavage by PLL12 helps to achieve the flexible but strong cell walls needed for guard cells to actuate efficient stomatal opening and closure.