Low ABA concentration promotes root growth and hydrotropism through relief of ABA INSENSITIVE 1-mediated inhibition of plasma membrane H+-ATPase 2

Abstract

The hab1-1abi1-2abi2-2pp2ca-1 quadruple mutant (Qabi2-2) seedlings lacking key negative regulators of ABA signaling, namely, clade A protein phosphatases type 2C (PP2Cs), show more apoplastic H⁺ efflux in roots and display an enhanced root growth under normal medium or water stress medium compared to the wild type. The presence of low ABA concentration (0.1 micromolar), inhibiting PP2C activity via monomeric ABA receptors, enhances root apoplastic H⁺ efflux and growth of the wild type, resembling the Qabi2-2 phenotype in normal medium. Qabi2-2 seedlings also demonstrate increased hydrotropism compared to the wild type in obliquely-oriented hydrotropic experimental system, and asymmetric H⁺ efflux in root elongation zone is crucial for root hydrotropism. Moreover, we reveal that Arabidopsis ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of Arabidopsis plasma membrane H⁺-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr⁹⁴⁷), whose dephosphorylation negatively regulates AHA2.

Fig. 1. Effects of root growth in mutants from ABA signaling pathway and exogenous ABA application on root growth of Arabidopsis WT Col-0 seedlings upon a water potential gradient assay with mRNA-seq analysis.

Growth phenotype of Col-0, aba1-1, aba2-1, 112458, cyp707a2-1, and Qabi2-2 seedlings grown on one-half–strength Hoagland medium [normal medium (NM)] (A) or one-half–strength Hoagland medium containing 0.3% (v/v) glycerol and 0.06% (w/v) alginic acid [water stress medium (WSM)] (B) for vertical growth lasting 5 days after which germinated for 5 days. (C) Measurement of primary root (PR) length of Col-0, aba1-1, aba2-1, 112458, cyp707a2-1, and Qabi2-2 seedlings shown in (A) and (B). Means ± SD (n ≥ 12). *P < 0.05 and **P < 0.01 as determined by a Student’s t test. (D) Number of DEGs in Col-0 (normal medium) versus Col-0 (water stress medium). Up-regulated and down-regulated genes are shown in red and green colors, respectively. (E) Pie chart showing the percentages of significant enrichment for biological process (BP) gene ontology terms in Col-0 (normal medium) versus Col-0 (water stress medium). (F) Bar charts showing the percentages of phytohormone-related DEGs in Col-0 (normal medium) versus Col-0 (water stress medium). (G) Col-0 seedlings [5 days after germination (dag)] grown on one-half–strength Murashige and Skoog (MS) medium were transferred and grown vertically on one-half–strength Hoagland medium without (left) or with 0.3% (v/v) glycerol and 0.06% (w/v) alginic acid (right) in the absence or presence of 0.1, 3, or 10 μM ABA for other 5 days. Scale bar, 10 mm. (H) Measurement of primary root length of Col-0 seedlings shown in (G). Error bars indicate ±SD (n = 4 in three independent experiments). *P < 0.05 and **P < 0.01 as determined by a Student’s t test. Photo credit: Rui Miao, Fujian Agriculture and Forest University.

Fig. 2. Effects of sodium vanadate on root growth and curvature of Col-0 WT and pp2cs quadruple (Qabi2-2) mutant seedlings under water stress medium and obliquely oriented hydrotropic experimental system.

(A) Five-dag Col-0 and Qabi2-2 seedlings grown on one-half–strength MS were transferred and grown vertically on normal medium or water stress medium without or with 10 μM sodium vanadate (VA) for other 5 days. Scale bar, 10 mm. (B) Measurement of primary root length of Col-0 and Qabi2-2 seedlings shown in (A). VA, a PM H⁺-ATPase inhibitor. (C) Photographs show that 5-dag Col-0 and Qabi2-2 seedlings were transferred to split agar plates including 0.4 M d-sorbitol in the bottom with lower water potential [obliquely oriented hydrotropic experimental system (OHES)] for 16 hours in the absence or presence of 20 μM VA. (D) Root curvature angle shown in (C). Data are averages from three independent experiments. Error bars represent SD (n = 6). The asterisks indicate a significant difference based on the Student’s t test (*P < 0.05 and **P < 0.01). Values with the same letters (a, b, or c) were not significantly different from one another [one-way analysis of variance (ANOVA), P < 0.05). Photo credit: Rui Miao, Fujian Agriculture and Forest University.

Fig. 3. H⁺ efflux in roots of Col-0 WT and Qabi2-2 seedlings.

(A) H⁺ efflux in primary root elongation zone (EZ) of Col-0 WT, Col-0 (0.1 μM ABA), Col-0 (10 μM ABA), and Qabi2-2 mutant of these Arabidopsis plants was grown in normal medium. The values shown are the means, and error bars represent ±SD of six replicates from two independent experiments. The asterisks indicate a significant difference based on the Student’s t test (**P < 0.01). (B and C) H⁺ efflux in primary root elongation zone of Col-0 WT and Qabi2-2 mutant of these Arabidopsis plants was grown in normal medium, water stress medium (B), or obliquely oriented hydrotropic experimental system (C). The values shown are the means, and error bars represent ±SD of six replicates from two independent experiments. (D) Arabidopsis PM AHA activity was measured in Col-0 WT and Qabi2-2 seedlings in the absence or presence of 20 μM VA. PM vesicles were isolated from 10-dag Col-0 WT and Qabi2-2 seedlings. The values shown are the means, and error bars represent ±SD of three replicates from two independent experiments. (E) Five-dag Col-0 WT roots transferred to normal medium or obliquely oriented hydrotropic experimental system lasting 2 or 16 hours were stained with 1 mM HPTS solution supplemented with 0.01% (v/v) Silwet L-77 for 30 min, and the root cells of elongation zone were visualized using the confocal (405- and 458-nm excitation wavelengths). The y axis represents the mean 458/405 values of the roots grown in normal medium or obliquely oriented hydrotropic experimental system lasting 2 or 16 hours. Error bars represent ±SD (n = 4). Values with the same letters (a, b, c, or d) were not significantly different from one another (one-way ANOVA, P < 0.05).

Fig. 4. Interaction of ABI1 with AHA2.

(A) Split-ubiquitin Y2H assay showing the interaction between ABI1 and AHA2. QDO, quadruple dropouts; X, X-galactosidase; DIC, differential interference contrast. (B) Bimolecular fluorescence complementation (BiFC) assays showing in vivo interaction of ABI1 with AHA2 in tobacco (N. benthamiana) leaf epidermal cells.

Fig. 5. ABI1 binds to C terminus of AHA2 and dephosphorylates Thr⁹⁴⁷.

(A) Representative cartoon image showing the interaction of cytosolic ABI1 with homology modeling putative C terminus of AHA2 that located in PM. TM, transmembrane. (B) Y2H assay to detect the interaction of ABI1 with A domain, N domain, P domain, and AHA2 (C100). OD₆₀₀, optical density at 600 nm. AD, the activation domain; Lam, lamin; T, T-antigen; BD, the DNA binding domain. (C) The LCI images of N. benthamiana leaves coinfiltrated with the agrobacterial strain GV3101 containing the indicated fusion proteins. (D) In vivo dephosphatase assay examining the phosphorylation level of Thr⁹⁴⁷ in the presence of ABI1–green fluorescent protein (GFP) or GFP, respectively. AHA2 (C100)–FLAG with ABI1-GFP or GFP were coexpressed in N. benthamiana leaves. The isolated PM fractions were mixed with extracts from GRF6–overexpressing (OE) transgenic Arabidopsis plants (AT5G10450), a member of the 14-3-3 family, to preserve pThr⁹⁴⁷ during the analysis. The phosphorylation level of the Thr⁹⁴⁷ in the C-terminal R domain of AHA was determined by immunoblot using anti–pThr⁹⁴⁷ antibody. The phosphorylation level of AHA was quantified as the ratio of the signal intensity from the phosphorylated AHA to that from the amount of AHA2 (graph at middle). These assays were repeated at least three times with similar results. Values with the same letters (a and b) were not significantly different from one another (one-way ANOVA, P < 0.05).

Fig. 6. Inhibition of phosphatase activity at low ABA concentrations leads to increased Thr⁹⁴⁷ phosphorylation of AHA2.

(A) In vivo phosphorylation level of Thr⁹⁴⁷ in the C-terminal R domain of AHA2 in Col-0 WT either in the absence or presence of 0.1 μM ABA, and Qabi2-2 plants grown in normal medium. The phosphorylation level of the Thr⁹⁴⁷ in the C-terminal R domain was determined by immunoblotting using pThr⁹⁴⁷ antibody. The phosphorylation level of AHA was quantified as the ratio of the signal intensity from the phosphorylated AHA to that from the amount of AHA (graph at middle). These assays were repeated at least three times with similar results. (B to D) In vitro phosphatase assay showed inhibition of ABI1 activity by PYL8 at low ABA concentration. (B) The half-maximal inhibitory concentration (IC₅₀) for ABI1 in the presence of PYL8 was calculated using different ABA concentrations. (C) Kinetic analysis of ABI1 activity for 20 min in the presence of PYL8 and the indicated ABA concentrations. Abs₄₀₅, absorbance at 405 nm. (D) End point activity of ABI1 after 30-min incubation. The asterisks indicate a significant difference based on the Student’s t test (*P < 0.05). (E) Model of ABI1-mediated PM H⁺ extrusion through interaction with AHA2. AHA2 represented as a transmembrane protein, which located into PM. In the absence of ABA (left), apo-PYL1 receptor [Protein Data Bank (PDB) code: 3KAY] does not interact with ABI1. Therefore, ABI1 interacts with and dephosphorylates the C-terminal R domain of AHA2. In the presence of low concentrations of ABA (right), the PYL1 receptor is in a complex with ABI1 phosphatase (PDB code: 3JRQ). Hence, ABI1 is unable to interact with and dephosphorylate AHA2. Then, AHA2 maintains in the phosphorylated state and promotes root growth and hydrotropism through apoplastic acidification.