The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers

Abstract

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants' physical defence barriers.

Keywords: abscisic acid; cuticle; glucosinolate-myrosinase system; microscopy; myrosin cells; myrosinase; plant defence; rosette leaves; stomatal guard cells; vacuoles.

Figure 1

Area of stomata complex and number of stomata (total, open, and closed) assessed by scanning electron microscopy on rosette leaves of wild type (WT), tgg1, tgg2, and tgg1 tgg2 double mutants of Arabidopsis [31]. (A). Area of stomata complexes. Bars represent the means ± SE (n = 60). Different letters above the bars indicate significant differences between WT, tgg1, tgg2, and tgg1 tgg2 as determined for stomata complex by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05). (B). Number of stomata (total, open and closed) per 0.1 mm² of abaxial surface of rosette leaf. Bars represent the means ± SE. (WT: n = 23), (tgg1: n = 34), (tgg2: n = 31), and tgg1 tgg2 (n = 29). Different letters (a, b and c) above the bars indicate significant differences between WT, tgg1, tgg2, and tgg1 tgg2 as determined separately for each of the three categories (total, open, closed) by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05).

Figure 2

Proportion of stomata with guard cell (GC) stained or not stained (GCs lacking staining in vacuoles) by toluidine blue in light microscopy (LM) semi-thin sections (longitudinal) from rosette leaves of WT, tgg single and double mutants of Arabidopsis after staining with toluidine blue. Bars represent the means ± SE. The average of number of stomata with stained or not stained GCs per genotype were counted from semi-thin sections (WT: n = 14), (tgg1: n = 7), (tgg2: n = 12), and (tgg1 tgg2: n = 8). Different letters (a and b) above the bars indicate significant differences between WT, tgg1, tgg2 and tgg1 tgg2 as determined separately for each of the two categories (stained, not stained) by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05).

Figure 3

LM images of stomata and GCs from leaf segments of semi-thin sections (longitudinal) of rosette leaves of WT, tgg single and double mutants of Arabidopsis stained with toluidine blue. (A). WT: GC where vacuoles showed toluidine staining, (B). tgg1 single mutant: GC where vacuoles lacked toluidine blue staining, (C). tgg2 single mutant: GC where vacuoles showed toluidine staining, (D). tgg1 tgg2 double mutant: GCs vacuoles showed no toluidine staining. (Scale bars = 10 μm).

Figure 4

Effect of abscisic acid (ABA) (100 µM) on stomatal pore length, width, and stomatal aperture of WT and tgg single and double mutants. Length (A) and width (B) of the stomatal pore in WT, tgg single and double mutants after mock treatment or treatment with ABA. Different letters (a, b and c) above the bars indicate significant differences between WT, tgg1, tgg2 and tgg1 tgg2 for each treatment as determined by a Kruskal-Wallis test followed by a Tukey test for pairwise comparisons (p < 0.05). (C) Stomatal aperture in WT, tgg single and double mutants after mock treatment or treatment with ABA. Bars represent the means ± SE (n = 200). Asterisks above bars indicate a significant effect of the ABA treatment on stomatal aperture (* p < 0.05).

Figure 5

TEM of stomata and GCs from rosette leaves of WT, tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis. (A,B). WT: GCs showing chloroplasts, open stomatal pore, nuclei, and small to big vacuoles in GCs. (C,D). tgg1 single mutant: Showing vacuoles with less electron-dense material and open stomatal pores, (E,F). tgg2 single mutant: (E). Stoma with slightly open stomatal pore, nucleus, with both small and big vacuoles, and chloroplasts with more prominent starch grains. (F). A bigger stoma with open stomatal pore; showing chloroplasts, nuclei, and big vacuoles in GCs with electron-dense granular material. (G,H). tgg1 tgg2 double mutant: Small sized stomata showing vacuoles with less electron-dense material, chloroplasts, nuclei. C = chloroplast, M = mitochondria, N = nucleus, SP = stomatal pore, and V = vacuole. (Bars = 2 μm).

Figure 6

Area of GCs and vacuoles from transmission electron micrographs of rosette leaves of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants of Arabidopsis. Bars represent the means ± SE (WT: n = 12), (tgg1: n = 15), (tgg2: n = 15), and tgg1 tgg2 (n = 18). Different letters (a, b and c) above the bars indicate significant differences in guard cell area and vacuole area between WT, tgg1, tgg2, and tgg1 tgg2 as determined by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05).

Figure 7

Chloroplast and starch grain area of guard cells from transmission electron micrographs of rosette leaves of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants of Arabidopsis. (A). Area of guard cell chloroplast in WT, tgg single and double mutants. Bars represent the means ± SE (WT: n = 49), (tgg1: n = 59), (tgg2: n = 35), and tgg1 tgg2 (n = 41). Different letters (a and b) above the bars indicate significant differences in guard cell chloroplast area between WT, tgg1, tgg2, and tgg1 tgg2 as determined by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05). (B). Area of guard cell starch grains in WT, tgg single and double mutants. Bars represent the means ± SE (WT: n = 105), (tgg1: n = 159), (tgg2: n = 90), and tgg1 tgg2 (n = 112). Different letters (a, b and c) above the bars indicate significant differences in guard cell starch grain area between WT, tgg1, tgg2, and tgg1 tgg2 as determined by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05).

Figure 8

TEM of stomata complex and GCs (transdermal sections; adaxial side) from rosette leaves of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants of Arabidopsis revealed variations for outer stomatal ledge and cell wall. (A). WT: Stomata complex showing nucleus, mitochondria, and vacuole in either of the GC. Cell wall is differentially thickened with thickest near the stomatal pore, and thin between the GCs. The stomatal ledge is attached at the end of stomatal pore. (B). tgg1 single mutant: Stomata complex showing vacuolated GC, with thick cell wall near the stomatal pore. GCs showing stomatal ledges at the end of stomatal pore. (C). tgg2 single mutant: Stomata complex showing thick cell wall and reduced stomatal ledges at the end of stomatal pore. (D). tgg1 tgg2 double mutant: Stomata complex showing very thick cell wall between the GCs and very thick stomatal ledges. CW = cell wall, M = mitochondria, N = nucleus, SL = stomatal ledge, and V = vacuole (Bars in A–C = 1 μm and in D =2 μm), (Magnification = 18,500× in A–C; and 11,000× in D).

Figure 9

Cell wall (CW) thickness of guard cells as measured from transmission electron micrographs of rosette leaves of WT, tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis. Error bars represent the means ± SE (WT: n = 17), (tgg1: n = 17), (tgg2: n = 8), and tgg1 tgg2 (n = 20). Different letters (a and b) above the bars indicate significant differences in guard cell CW thickness between WT, tgg1, tgg2 and tgg1 tgg2 as determined by a Kruskal-Wallis test followed by a Dunn’s method for pairwise comparisons (p < 0.05).

Figure 10

Confocal images of 17d old Arabidopsis rosette leaves (abaxial side) expressing TGG1-GFP, showing GC expression pattern (A) and subcellular localization (B,C), and 14-day old cotyledons marked with different fluorescent constructs labelling (D). (A). GCs (TGG1-GFP) shown in green and chloroplasts (autofluorescence) shown in red. Image is a maximum projection of ten optical slices in Z direction (Bar = 50 µm). (B). Twenty optical sections through a whole GC from top (1) to bottom (20), showing that the TGG1 fusion protein seem to localize within the GC tonoplasts/vacuoles (Bar = 10 µm) (C). High resolution, single sections from individual GC (Bar = 5 µm). (D). GCs expressing fluorescent markers for Actin (left), Endoplasmic reticulum (ER) (middle) and Tonoplast (right) (Bar = 10 µm). Actin was visualized by yellow fluorescent protein (YFP) fused to the actin-binding domain of mouse talin (mTalin) [53]. ER was visualized by YFP fused to a synthetic oligonucleotide encoding the ER retention signal HDEL (at C-terminus) and the signal peptide of AtWAK2 (A. thaliana wall-associated kinase 2; at the N-terminus) (ER-yk, CS16252) [53,54]. Tonoplast was visualized by YFP fused to the C-terminus of γ-TIP, an aquaporin of the vacuolar membrane (vac-yk; CS16258) [53,55]. Seeds of the transgenic Arabidopsis thaliana plants expressing the fluorescent markers for ER and tonoplast were obtained from Nottingham Arabidopsis Stock Centre (NASC).