Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis

Abstract

Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including seed germination. Although phosphorylation has been suggested to be important, the protein kinases required for ABA signaling during seed germination and seedling growth remain elusive. Here, we show that two protein kinases, SNF1-RELATED PROTEIN KINASE2.2 (SnRK2.2) and SnRK2.3, control responses to ABA in seed germination, dormancy, and seedling growth in Arabidopsis thaliana. A snrk2.2 snrk2.3 double mutant, but not snrk2.2 or snrk2.3 single mutants, showed strong ABA-insensitive phenotypes in seed germination and root growth inhibition. Changes in seed dormancy and ABA-induced Pro accumulation consistent with ABA insensitivity were also observed. The snrk2.2 snrk2.3 double mutant had a greatly reduced level of a 42-kD kinase activity capable of phosphorylating peptides from ABF (for ABA Response Element Binding Factor) transcription factors. ABA-induced expression of several genes whose promoters contain an ABA response element (ABRE) was reduced in snrk2.2 snrk2.3, suggesting that the mechanism of SnRK2.2 and SnRK2.3 action in ABA signaling involves the activation of ABRE-driven gene expression through the phosphorylation of ABFs. Together, these results demonstrate that SnRK2.2 and SnRK2.3 are redundant but key protein kinases that mediate a major part of ABA signaling in Arabidopsis.

Figure 1.

Seed Germination and Dormancy Assays. (A) Diagrams of SnRK2.2 and SnRK2.3 showing positions of the T-DNA insertions. (B) RT-PCR analysis with SnRK2.2, SnRK2.3, and Tubulin primers using total RNA extracted from seedlings of the wild type (Columbia [Col-0]) and snrk2.2 snrk2.3 as the template. (C) and (D) Photographs of Col-0, snrk2.2, snrk2.3, and snrk2.2 snrk2.3 seedlings on control (Murashige and Skoog [MS]) medium with 3% sucrose (C) or 0.6 μM ABA medium (D) at 9 d after the end of stratification. (E) Quantification of the percentage of seedlings with green cotyledons after 6 d on the indicated concentrations of ABA (means ± se; n = 3). Each measurement consisted of at least 30 seeds. For symbols, see (F). (F) Quantification of radicle emergence of each genotype at 3 d after the end of stratification (means ± se; n = 3). Medium used was MS medium without sucrose. (G) Germination of nonstratified seeds at 7 d after sowing on MS medium with 3% sucrose. (H) Germination time course (measured by appearance of green cotyledons) for nonstratified seeds of each genotype (means ± se; n = 3). For symbols, see (F).

Figure 2.

ABA Inhibition of Seedling Growth. (A) Photographs of seedlings at 14 d after transfer to control medium (MS medium with 3% sucrose) or medium containing 50 μM ABA. Seedlings were 4 d old at the time of transfer and had equal root lengths at that time. (B) Quantification of root length and seedling fresh weight for seedlings treated as described for (A). For fresh weight determination, seven seedlings were weighed at one time and the result was divided by seven. Data are means ± se (n = 28 for root length and n = 4 for fresh weight). (C) Quantification of root length for seedlings at 14 d after transfer to control medium or medium containing 0.5 μM ABA. Data are means ± se (n = 21). Col-0, snrk2.2, snrk2.3, and snrk2.2 snrk2.3 are indicated by C, 2, 3, and 2/3, respectively.

Figure 3.

Leaf Water Loss Assay. Water loss was measured using detached leaves of Col-0, snrk2.2, snrk2.3, and snrk2.2 snrk2.3 (top panel) or Landsberg erecta (Ler) and snrk2.6/ost1 (bottom panel). Data are means ± se (n = 4 to 8). FW, fresh weight.

Figure 4.

Expression of SnRK2.2 and SnRK2.3. (A) RT-PCR analysis of tissue distribution of SnRK2.2 and SnRK2.3 expression. Numbers at right indicate the number of PCR cycles performed. (B) to (D) GUS staining of plants with promoter:GUS expression driven by 2-kb promoter fragments of SnRK2.2, SnRK2.3, and SnRK2.6 (arranged from left to right in each row of photographs). Bars in (B), (C), and (D) = 1 mm, 1 mm, and 20 μm, respectively.

Figure 5.

In-Gel Kinase Assay. In-gel kinase assay with proteins extracted from the wild type (Col-0), snrk2.2, snrk2.3, and snrk2.2 snrk2.3 seedlings under control conditions or 30 min after 100 μM ABA treatment. The ABF2 fragment (amino acids Gly-73 to Gln-119 [A]), ABI5 fragment (Arg-132 to Gln-190 [B]), and ABF1 fragment (Gly-83 to Glu-131 [C]) were used as substrates. The graphs at bottom indicate relative radioactivity (mean ± se; n = 4 for ABF2, n = 3 for ABI5 and ABF1) of the 42- and 45-kD bands after ABA treatment normalized relative to the 42-kD activity of the wild type. In the graphs, Col-0, snrk2.2, snrk2.3, and snrk2.2 snrk2.3 are indicated by C, 2, 3, and 2/3, respectively.

Figure 6.

Expression of ABA-Regulated Genes and ABA-Induced Pro Accumulation. (A) Expression of the ABA-upregulated genes RD29B, RAB18, RD22, RD29A, NCED3, and P5CS1 assayed by quantitative RT-PCR in Col-0 (C), snrk2.2 (2), snrk2.3(3), and snrk2.2 snrk2.3 (2/3) seedlings under control conditions or after 3 h of exposure to 100 μM ABA. Data are means ± se (n = 3). In some cases, such as with RD29B, expression in control seedlings was too low to be visible on the graphs. (B) Expression of RD29B and RAB18 in seeds imbibed for 24 h (means ± se; n = 3). (C) Pro contents of seedlings after transfer to plates containing 10 μM ABA for 96 h. Data are means ± se (n = 6 to 8). FW, fresh weight.