Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Abstract

Background and aims: In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods: Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key results: Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions: This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.

Keywords: Arabinan; Funaria hygrometrica; LM13; LM19; LM6; homogalacturonan; immunolabelling; moss; pectin; plant cell wall; rhamnogalacturonan; stomata; ultrastructure.

Fig. 1.

Diagram of a Funaria stoma, which consists of guard cells with continuous cytoplasm. (A) Transverse section though polar end. (B) Transverse section through pore. DW, dorsal wall; gc, guard cell; IW, inner wall; OW, outer wall; VW, ventral wall.

Fig. 2.

Young stomata before forming the pore. (A) Guard cells were rounded before separation of ventral walls to form the pore. (B) The ventral wall did not reach the polar end of the stoma. (C) Dense cytoplasm at the edge of the polar end of the stoma. (D–F) Immunolocalization of pectins in guard cell walls of young stomata. Black dots in images are colloidal gold labels attached to specific antibodies. (D) LM19 in inner wall, (E) LM5 in outer wall and (F) LM6 in ventral wall. Notice the cuticle (*) in outer walls (E and I) but not in the inner walls (arrowhead in D and H). (G–H) Middle lamella between ventral walls before separation to form the pore. (G) LM19 labels in walls and the middle lamella. (H) LM6 labels in walls but not the middle lamella. (I) LM6 labels in outer wall and in vesicles near the wall; the outer wall was covered by a thin cuticle (*). Abbreviations: c, chloroplast; DW, dorsal wall; er, endoplasmic reticulum; g, Golgi body; IW, inner wall; m, mitochondrion; n, nucleus; OW, outer wall; r, ribosome; v, vacuole; VW, ventral wall. Scale bars: (A, B) = 2 µm; (C) = 500 nm; (D–I) = 100 nm.

Fig. 3.

Stomata after pore opening. (A) Guard cells were elliptical in transverse section. (B) Differential thickening of guard cell walls with outer, inner and ventral walls thicker than the dorsal wall. (C) The external wall layer (EWL) had a uniform electron density but the middle wall layer (MWL), added towards the inside of the cell, was blotchy; the cuticle (*) was thicker at this stage. (D) LM20 localized weakly in the external wall layer of inner walls. (E) Ventral walls did not label for LM5. (F) Ventral walls labelled for LM6 but not in the ledge (arrow). (G) LM13 labelled the outer walls but not the adjacent epidermal cells. Abbreviations: *, cuticle; c, chloroplast; DW, dorsal wall; ecw, epidermal cell wall; IW, inner wall; m, mitochondrion; n, nucleus; OW, outer wall; v, vacuole; VW, ventral wall. Scale bars: (A, B) = 2 µm; (C, E, F) = 100 nm; (D, G) = 500 nm.

Fig. 4.

Mature stomata. (A) Guard cells with layered walls. (B) Guard cell outer wall with differential LM19 label in external, middle and internal wall layers. (C) More homogeneous LM6 label in wall layers of outer wall. Asterisk denotes the cuticle. (D) LM13 label in guard cell wall but not in epidermal cell wall. (E) LM19 localized in outer ledge. (F) LM13 did not localize in epidermal cells. (G, H) LM20 (G) and LM6 (H) did not localize in the outer ledge except very sparsely (arrows). Scale bars: (A) = 2 µm; (B, C) = 100 nm; (D, F) = 500 nm; (E, G, H) = 100 nm. Abbreviations: c, chloroplast; DW, dorsal wall; ecw, epidermal cell wall; EWL, external wall layer; gcw, guard cell wall; IWL, inner wall layer; m, mitochondrion; MWL, middle wall layer; n, nucleus; OW, outer wall; v, vacuole.

Fig. 5.

(A) Average guard cell wall thickness at different developmental stages. Bars represent standard error. (B) Label ratio measured as the average number of dots per unit area divided by the average thickness of walls for each stage. (C) Changes in the ratio of arabinans (LM6 + LM13) to HG (LM19) during guard cell wall development. (D) Two-way ANOVA of significant interaction between stage and label type showing label frequency during wall development (P = 0·0002). Different letters represent means that are significantly different according to Tukey's test; bars are standard deviations. Only the internal wall layer was measured for mature stomata. Labelling for LM19 was significantly different in mature stomata, but LM6 and LM13 showed no differences across stages. IW, inner wall; Mature, mature stomata in expanded capsule; OW, outer wall; Pore, young stomata with pore; VW, ventral wall; Young, young stomata before pore opening.