ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

Abstract

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses such as drought, partly by modulating growth through poorly understood mechanisms. Here, we show that ABA-triggered repression of cell proliferation in the Arabidopsis thaliana root meristem relies on the swift subcellular relocalization of SNF1-RELATED KINASE 1 (SnRK1). Under favorable conditions, the SnRK1 catalytic subunit, SnRK1α1, is enriched in the nuclei of root cells, and this is accompanied by normal cell proliferation and meristem size. Depletion of two key drivers of ABA signaling, SnRK2.2 and SnRK2.3, causes constitutive cytoplasmic localization of SnRK1α1 and reduced meristem size, suggesting that, under nonstress conditions, SnRK2s promote growth by retaining SnRK1α1 in the nucleus. In response to ABA, SnRK1α1 translocates to the cytoplasm, and this is accompanied by inhibition of target of rapamycin (TOR), decreased cell proliferation, and reduced meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and ABA-elicited inhibition of TOR. Furthermore, fusing SnRK1α1 to an SV40 nuclear localization signal leads to defective ABA-dependent TOR repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK1α1 subcellular localization are crucial for inhibiting TOR and root growth in response to ABA. Rapid relocalization of central regulators such as SnRK1 may represent a general strategy of eukaryotic organisms to respond to environmental changes.

Keywords: abscisic acid; energy signaling; growth control; root; target of rapamycin.

Fig. 1.

Impact of ABA and SnRK2s on SnRK1α1 subcellular localization and cell proliferation in the root apical meristem. (A) Meristems of Col-0, snrk2d, and snrk2d/1α1 7-d-old seedlings with or without ABA treatment (5 µM, 48 h). Arrowheads: regions used for violin plots. n = 23 or 24; P < 0.05, two-way ANOVA with Tukey’s honestly significant difference test. (Scale bar: 50 µm.) (B) Root apices of 4-d-old SnRK1α1-GFP#1 seedlings with or without ABA treatment (50 µM, 3 h). (Scale bar: 30 µm.) (C) Electron micrograph of meristematic epidermal cells with magnification (Lower) showing the cellular ultrastructure. C: cytoplasm; N: nucleus; No: nucleolus. (Scale bars: Upper, 5 µm; Lower, 1 µm.) (D) (Left) SnRK1α1-GFP in epidermal cells of 4-d-old root meristems. (Scale bar: 10 μm.) Dotted lines: nuclear boundary. (Right) SnRK1α1-GFP quantification and N/C ratios from two independent lines. n = 5 or 6; one-way ANOVA with Dunett’s test. (E) Nuclear SnRK1 activity in 8-d-old roots of the NLS-ACC reporter line with or without ABA treatment (50 µM, 3 h). (Left) Representative immunoblot and Ponceau-S staining as loading controls. (Right) Quantification of ACC phosphorylation [P(S79)-ACC/total ACC]. n = 3, two-tailed Student t test. (F) Impact of the presence (control, SnRK1α1-GFP#2) or absence (SnRK1α1-GFP#2; snrk2d) of SnRK2s on SnRK1α1-GFP localization assessed as in D. n = 8; two-tailed Student t test. (Scale bar: 10 μm). (G) SnRK2.2-GFP in epidermal cells of 4-d-old root meristems treated and quantified as in D. n = 5; two-tailed Student t test. (Scale bar: 10 μm). PI, propidium iodide; ns, nonsignificant; a.u., arbitrary units.

Fig. 2.

Impact of SnRK1α1 subcellular localization on TOR signaling and root growth. (A and B, Upper) Representative immunoblots of RPS6^S240 phosphorylation (phospho-RPS6/total-RPS6) in Col-0 (A) or control-α1 and NLS-α1 (B) seedlings treated with or without ABA (50 µM, 3 h) or LMB (2.5 µM, 3 h or 1 h prior to ABA addition). Ponceau-S staining serves as loading control. Same gel images were cropped for showing α1 and NLS-α1 contiguously. (A and B, Lower) Phospho-RPS6/total-RPS6 quantification. (A) n = 5 or 6; (B) n = 3; error bars, SEM; two-tailed Student t test. (C) Under favorable conditions, SnRK1α1 is sequestered in the nucleus by complexes containing SnRK2 [and a PP2C (3)], enabling TOR activity in the cytoplasm, cell proliferation, and root growth. Dissociation of these complexes by ABA-bound PYR/PYL/RCAR receptors releases SnRK1α1, which exits the nucleus and inhibits TOR and growth. TORC1, TOR Complex 1; N, nucleus; C, cytoplasm; ns, nonsignificant. Created with BioRender.com.