Cortical microtubules and fusicoccin response in clustered stomatal guard cells induced by sucrose solution immersion

ABSTRACT

We previously found that sucrose solution immersion treatment permitted ectopic guard cell differentiation, resulting in clustered stomatal guard cells. Using this system, we examined the effects of sucrose solution-induced stomatal clustering on guard cell cortical microtubules and the stomatal response to fusicoccin. Confocal observation revealed that the radial orientation of cortical microtubules was largely maintained in clustered guard cells. Outward movement of cortical microtubule plus-ends was also kept in the clustered guard cells. Fusicoccin treatment induced stomatal opening in both spaced and clustered stomata, although sucrose solution-treated guard cells had lower stomatal apertures. These results suggested that immersion treatment with sucrose solution perturbed the one-cell spacing of stomata but not the cortical microtubule organization required to open stomatal pores.

Stomata are important organs that have an essential role in transpiration, which promotes water uptake and nutrient absorption by the roots and gas exchange for photosynthesis and respiration. A pair of differentiated guard cells, which form a stoma, have radial cortical microtubules elongating outward from the ventral to dorsal side.^1,2 During stomatal movement, the cortical microtubules undergo dynamic reorganization.^3-7 Pharmacological and genetic perturbations of microtubules result in impaired stomatal movement in response to environmental cues.^3,6,8 These findings suggest that cortical microtubules are deeply involved in the regulation of stomatal movement. Various cellular functions have been proposed for cortical microtubules including regulation of ion channel activity, cell wall deposition, and signal transduction pathways; however, the molecular cell biological mechanisms of cortical microtubule-mediated stomatal regulation are still debated.⁹

In many dicotyledonous plants including Arabidopsis thaliana, guard cells do not appear to form adjacent to other guard cells. This one-cell spacing patterning is realized by ligand-receptor-based cell-to-cell communication in stomatal differentiation.¹⁰ Previously, we found that sucrose solution immersion treatment induced clustered stomata in A. thaliana cotyledons.¹¹ Sucrose solution treatment reduced callose deposition during the unequal cell division that separates stomatal lineage cells and nonstomatal lineage cells.¹¹ Guard cell lineage marker proteins leaked into the jigsaw puzzle-shaped pavement cells in sucrose solution, suggesting that sucrose solution treatment perturbed stomatal guard cell identities in the cotyledon epidermis.¹¹ However, it is unclear whether sucrose solution immersion treatment affects the radial orientation of cortical microtubules. In addition, we do not know whether the clustered guard cells can respond normally to environmental cues and control their stomatal apertures. In this study, we examined the organization of cortical microtubules and stomatal movement of clustered guard cells in A. thaliana treated with sucrose solution immersion.

To investigate the effects of sucrose solution immersion treatment on cortical microtubules in guard cells, we used transgenic A. thaliana expressing GFP-tagged Arabidopsis β-tubulin 6 isoform (TUB6).¹² Radial cortical microtubules were frequently observed in sucrose solution-induced clustered guard cells as in normally spaced guard cells in the sucrose-free control (Fig. 1a). To quantitatively evaluate the cortical microtubule orientation, we measured the mean angular difference as an indicator of the radial orientation of cortical microtubules using image analysis techniques, basically according to our previous report¹³ (see also supplementary materials). In the case of random orientations, the mean angular difference becomes approximately 45°. Values higher and lower than 45 degrees represents a radial and longitudinal orientation, respectively. The mean angular difference values of cortical microtubules were 57.0° and 55.1° in spaced and clustered guard cells, respectively (Fig. 1b). These values are comparable with radial actin microfilaments in A. thaliana guard cells.¹³ A significant difference between spaced and clustered guard cells was not detected (Fig. 1b), suggesting that radial cortical microtubules were maintained in clustered guard cells induced by sucrose solution immersion. We also monitored the movement of cortical microtubule plus-ends in guard cells using the microtubule plus-end marker GFP-EB1b¹⁴ and variable-angle epifluorescence microscopy¹⁵ (Fig. 2a). Outward movement of GFP-EB1b-labelled microtubule plus-ends was observed in both cases (Fig. 2a, b). Quantification of the frequency of outward movement revealed that more than 80% of GFP-EB1b-labelled microtubule plus-ends moved outward from the ventral to the dorsal side in clustered guard cells as in sucrose-free control guard cells (Fig. 2c). These results suggested that the outward orientation of cortical microtubule growth was kept in the clustered guard cells.

Figure 1.

Effects of sucrose solution immersion treatment-induced stomatal clustering on cortical microtubule orientation. (a) GFP-TUB6-labelled cortical microtubules in spaced guard cells in the sucrose-free control (left) and clustered guard cells in 3% sucrose solution (right). Scale bars = 10 μm. (b) Quantitative evaluation of cortical microtubule orientation in guard cells. Mean angular differences were measured using processed images of spaced guard cells in the sucrose-free control (white column) and clustered guard cells in 3% sucrose solution (black column) (see also supplementary materials). Values are arithmetic means SD from 9–12 independent pairs of guard cells. Significance was determined using Mann–Whitney's U-test. N.S. indicates not significant. The P-value was 0.169.

Figure 2.

Effects of sucrose solution immersion treatment-induced stomatal clustering on outward movement of microtubule plus-ends. (a) GFP-EB1b-labelled cortical microtubules plus-ends in spaced guard cells in the sucrose-free control (left) and clustered guard cells in 3% sucrose solution (right). Representative epifluorescence images (top) and variable-angle epifluorescence microscopic images (bottom) of the same guard cells are shown. The former and the latter were used to check stomatal clustering and monitor movements of GFP-EB1b-labelled microtubule plus-ends, respectively. Scale bars = 10 μm. (b) Kymographs along the v–d line and v′–d′ line as shown in (a). Note that the plus-ends of cortical microtubules moved outward from the ventral to dorsal side in both conditions. (c) Frequency of outward movement of cortical microtubule plus-ends. The percentages of outward movement of microtubule plus-ends in spaced guard cells in the sucrose-free control (white column) and clustered guard cells in 3% sucrose solution (black column) were measured. Values are arithmetic means SD from 7–12 independent guard cells. Significance was determined using Mann–Whitney's U-test. N.S. indicates not significant. P-value was 0.240.

Next, stomatal apertures were examined in the samples treated with or without sucrose solution immersion. Sucrose solution-treated guard cells had lower stomatal aperture (4.96 ± 1.34 μm (DMSO control, sucrose-free) vs. 0.992 ± 1.19 μm (DMSO control, 3% sucrose)) (Fig. 3a, b, DMSO control). These results might be due to differences in the osmotic pressure of the solutions. When we observed vacuoles using fluorescent dye BCECF, vacuole shrinkage was observed in sucrose solution-treated guard cells, supporting this hypothesis (see also supplementary materials). In addition, absence of subsidiary cells may also contribute to lower apertures in the clustered stomata because subsidiary cells function as a supplier and receiver of bulk water and ions, and assist stomatal opening.^16,17,18

Figure 3.

Effects of sucrose solution immersion treatment-induced stomatal clustering on fusicoccin-induced stomatal opening. (a) Representative bright field images of spaced stomata in the sucrose-free control (left) and clustered stomata in 3% sucrose solution (right). Stomata were treated with DMSO (top) or 10 μM fusicoccin (bottom) for 2 h. Scale bar = 5 μm. (b) Apertures of DMSO- and fusicoccin-treated stomata in the sucrose-free control (white columns) and 3% sucrose solution (black columns). Values are arithmetic means SD from 100 independent stomata. Significance was determined using Mann–Whitney's U-test. *p-value < 0.0001. (c) Apertures of DMSO- and fusicoccin-treated spaced stomata (angled stripe column) and clustered stomata (gray column) in 3% sucrose solution. Values are arithmetic means SD from 22–78 independent stomata. Significance was determined using Mann–Whitney's U-test. *p-value < 0.0001.

Although we found sucrose solution immersion treatment resulted in decrease in stomatal apertures, we observed normal radial cortical microtubule both in the sucrose-free control and 3% sucrose condition (Fig. 1). Therefore, we speculated that sucrose solution treatment-induced clustered guard cells could open in response to environmental cues. To evaluate the responses of the stomata, we used the fungal phytotoxin fusicoccin, which opens stomata via continuous activation of the plasma membrane H⁺-ATPase in guard cells.¹⁹ Stomatal apertures were significantly increased by fusicoccin treatment in both conditions (Fig. 3a, b, Fusicoccin treatment). To more directly examine the effects of the stomatal spatial distribution on fusicoccin-induced stomatal opening, we separately measured the stomatal apertures of spaced guard cells (27.0%) and clustered guard cells (73.0%) in sucrose solution-treated samples. In both cases (spaced and clustered distribution), fusicoccin treatment significantly induced stomatal opening (Fig. 3c), indicating that sucrose solution-induced clustered stomata can open in response to fusicoccin. These results suggest that the ectopically-differentiated and clustered guard cells have normal radial cortical microtubules and can open stomata at least in response to fusicoccin treatment.

Other intracellular structures besides microtubules, such as actin microfilaments, endoplasmic reticulum, and chloroplasts, are also suggested to contribute to stomatal movement.^5,13,20-22 Future cell biological studies will clarify their contributions to the stomatal movement of clustered guard cells induced by sucrose solution immersion treatment.

Funding Statement

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI to K.A. (26891006), S.H. (16H04802), and T.H. (17K19380).

Disclosure of potential conflicts of interests

The authors declare that they have no competing interests.

Acknowledgments

We thank Robbie Lewis, MSc, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.