Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways

Abstract

We isolated a total of 3 x 10(8) guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range two-dimensional (2D) gels, and 2D liquid chromatography-matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K(+) channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.

Figure 1.

Three Proteomic Methods Identified 1734 Unique Proteins of the Arabidopsis GC Proteome. (A) Images showing high-purity GCP preparations (×100; inset magnification ×400). (B) A total of 1712, 58, and 59 unique proteins were identified from 2D LC-MALDI MudPIT, BR, and NR methods, respectively; 19 proteins were identified by all three methods. For each method, two independent biological samples were analyzed. (C) A 2D gel image from the broad pH range method. The first dimension was run using a 24-cm, pH 3 to 10 IPG strip. In total, 138 protein spots were detected via Coomassie blue staining. Twelve spots were identified as TGG1. Identifications of numbered spots can be found in Supplemental Table 5 online. (D) 2D gel images from the narrow pH range method. Proteins were first fractionated into five fractions, and each protein fraction was separated on a narrow pH range IPG strip. From B1 to B5, the pH ranges are 3 to 6, 4.5 to 5.5, 5.3 to 6.3, 6.1 to 7.1, and 6 to 10 respectively. Thirty-seven spots were identified as TGG1. Identifications of numbered spots can be found in Supplemental Table 5 online.

Figure 2.

tgg1 Mutants Are Hyposensitive to Wounding Promotion of Stomatal Closure at 5 min. (A) At 5 min, tgg1-1 and tgg1-3 mutants are hyposensitive to wounding promotion of stomatal closure compared with Col. (B) tgg1-1 and tgg1-3 mutants show no significant difference compared with Col in wounding inhibition of stomatal opening at 30 min and 1 h. For (A) and (B), leaves were treated blindly and simultaneously with a slicker brush (Bailey et al., 2005) to ensure the same amounts of tiny holes were punched in all leaves. n = 4 independent experiments. At least 60 stomata were measured for each treatment per genotype per replicate. Data are presented as mean ± se. Data were analyzed by Student's t test, P < 0.05 was considered significant (*).

Figure 3.

Regulatory Effects of Glucosinolates and/or Myrosinases on Arabidopsis Stomatal Apertures and GC K⁺_in Currents of Col and tgg1 Mutants in the Presence and Absence of ABA. (A) tgg1-1 and tgg1-3 mutants respond similarly to the wild type (Col) in ABA induction of stomatal closure. (B) tgg1-1 and tgg1-3 mutants are hyposensitive to ABA inhibition of stomatal opening. For (A) and (B), n = 3 experiments. At least 60 stomata were measured for each treatment per genotype per replicate. Data are mean ± se. P < 0.05 (Student's t test) was considered significant (*). (C) Typical GC whole-cell K⁺ current traces of Col and tgg1 mutants under control, 50 μM glucosinolate (G), and/or 0.2 unit/mL myrosinase (M) application to the GC cytosol, with or without 50 μM ABA. Current and time scales are as shown. (D) K⁺ current density (mean ± se) at −219 mV of GC whole-cell inward K⁺ currents. n = 14, 7, 6, 10, 7, and 6 for Col under control, ABA, G, G+M, M, and ABA+M treatments, respectively; n = 13, 10, 11, 6, 7, and 7 for tgg1-1 under control, ABA, G, G+M, M, and ABA+M treatments, respectively; n = 16, 8, 13, 8, 6, and 12 for tgg1-3 under control, ABA, G, G+M, M, and ABA+M treatments, respectively. P ≤ 0.01 (Student's t test) was considered significant (*) for (D).

Figure 4.

Speculative Model of Interactions between Glucosinolates, Myrosinase (TGG1), and K⁺ Channels in GC ABA Signaling. Left and right GCs indicate events before and after, respectively, activation of signaling pathways. Abiotic stresses increase ABA delivery to GCs. ABA either repartitions glucosinolates (G) from the vacuole to the cytosol (arrow 1) or enhances myrosinase (M) activity or substrate affinity (arrow 2). Inhibition of inward K⁺ channel (K⁺_in) activity might result from channel modification by the resultant hydrolyzed products of glucosinolates (e.g., isothiocyanates [ITC]), as in mammalian cells (arrow 3) or might result from decreased ROS scavenging (arrow 4) by ascorbate (Asc). Hydrolyzed products of glucosinolates may diffuse through the stomatal pore (shaded arrows) and thus deter biotic invaders. Reported biotic (wound) induced increases in ABA (Schmelz et al., 2003) may also provide positive feedback to the glucosinolate-myrosinase system.