Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis

Abstract

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.

Figure 1.

Expression Patterns of the RPK1 Gene and RPK1 Protein. (A) ABA-induced expression of RPK1. Total RNAs were extracted from wild-type Arabidopsis (ecotype Columbia) treated with distilled water or one of several plant hormones: 1 mM ethephon or 100 μM ABA, SA, MeJA, GA₃, BA, or 2,4-D. The blot was hybridized with a ³²P-labeled RPK1 probe. (B) RPK1 protein expression is upregulated by ABA treatment. Crude proteins were extracted from wild-type plants or suspension cells treated with 100 μM ABA and blotted on phenylmethylsulfonyl fluoride membranes after SDS-PAGE. RPK1 protein was detected with anti-RPK1 antibody. (C) Tissue-specific expression of the RPK1 promoter–GUS fusion. Activity was higher in the plant treated with 100 μM ABA for 10 h (1) than in the untreated plant (2). High GUS activity was detected in the embryo of the mature seed (3). (D) Detection of fluorescence of RPK1-GFP under a confocal microscope in the root of a transgenic plant expressing 35S:RPK1-GFP. RPK1-GFP was localized on the cell surface (1). The image was distorted and localized on the cell membrane when cells were plasmolyzed by treatment with 0.8 M mannitol (2). Arrows indicate individual cells. GFP was localized in whole cells of the 35S:GFP transgenic plant (3).

Figure 2.

Phenotypes of RPK1 Knockouts Plants. (A) Expression levels of the RPK1 gene and protein in knockout plants. Total RNAs extracted from RPK1 knockout plants (rpk1-1 and rpk1-2) and wild-type (Ws) plants were blotted and hybridized with a cDNA probe or a strand-specific probe of RPK1. Protein gel blot analysis of RPK1 knockout (rpk1-1 and rpk1-2) and wild-type (Ws) plants using anti-RPK1 antibody. (B) T-DNA insertion site of rpk1-1 and rpk1-2. T-DNA was inserted 504 and 989 bp, respectively, downstream of the ATG. (C) and (D) Seed germination rates of RPK1 knockouts. (C) rpk1-1, rpk1-2, 35S:RPK1 rpk1-1 (complemented), and wild-type plants were grown with (closed symbols) or without (open symbols) 0.5 μM ABA. (D) Seed germination rates on day 4 at various concentrations of ABA. sd values were calculated from three individual experiments. n = 30 seeds per experiment. (E) Root growth inhibition by ABA. Young seedlings of controls and rpk1-1 were transferred to ABA-containing medium and incubated for 10 d. The relative root length was measured and is shown as a percentage of the root length grown without ABA. sd values were calculated from three individual experiments. n = 10 seedlings per experiment.

Figure 3.

Phenotypes of Antisense-RPK1 Transgenic Plants. (A) Expression levels of RPK1 in antisense transgenic plants. Total RNAs extracted from transgenic plants carrying the 35S vector (vector control [VC]) or from four transgenic lines carrying 35S:antisense-RPK1 (L1 to L4) (Columbia) were blotted and hybridized with a cDNA probe or a strand-specific probe of RPK1. (B) Protein gel blot analysis of the VC and antisense-RPK1 transgenic suspension cells (LA and LB) using anti-RPK1 antibody. (C) Seed germination of antisense-RPK1 plants. VC and transgenic plants (L1 to L4) were grown with (closed symbols) or without (open symbols) 0.5 μM ABA. sd values were calculated from three individual experiments. n = 30 seeds per experiment. (D) ABA sensitivity of cell growth rate of antisense-RPK1 transgenic lines. Suspension cells were cultured in 100 μM ABA medium (open symbols) or in ABA-free medium (closed symbols). LA and LB, antisense-RPK1 transgenic lines. sd values were calculated from three individual experiments.

Figure 4.

Plant Growth and Stomatal Closure of RPK1 Knockout Plants in Response to ABA. (A) Plants were grown for 2 weeks on medium containing ABA. WT, the wild type (Ws). On ABA-containing media, RPK1 knockout plants grew and greened faster than did the wild type. (B) Stomatal closure of guard cells as a result of ABA treatment. Leaves of wild-type and RPK1 knockouts were treated with/without ABA for 2 h, and the stomatal aperture was measured. sd values were calculated from three individual experiments. n = 10 guard cells from two leaves of each plant per experiment. (C) Guard cells of the wild type and rpk1-1 treated with/without 5 μM ABA for 2 h.

Figure 5.

RNA Gel Blot Analysis of RPK1 Target Genes. Total RNAs were extracted from wild-type (Ws), VC, rpk1-1, and antisense-RPK1 transgenic suspension cells treated with or without 10 μM ABA for 5 h. (A) RNA gel blot analysis of rpk1-1 and antisense-RPK1 transgenic plants. cDNA fragments of ADH1 (alcohol dehydrogenase), RD22, AtMYC2, AT2g15970 (similar to cold acclimation protein), SPE2 (Arg decarboxylase, AT4g34710), ERDs, AREB1, xyloglucan β1,4-glucanase (AT4g30270), COR15A, SAMDC (S-adenosylmethionine decarboxylase, AT3g02470), and β-tubulin genes were used as probes. (B) RNA gel blot analysis of antisense-RPK1 transgenic suspension culture cells. cDNA fragments of LEA-like protein (AT4g02380), putative POX (peroxidase, AT2g18950), alternative oxidase 1a (AT3g22370), caltractin-like protein (AT2g46600), RD26, DnaJ-like protein (AT4g36040), cytochrome p450 (AT4g19230), copper homeostasis factor (AT3g56240), SOD (superoxide dismutase, AT4g25100), ERD11, and β-tubulin genes were used as probes.

Figure 6.

Protein Constructions and Dominant-Negative Phenotypes of Overexpressor of RPK1-LRR. (A) Constructions of the RPK1 protein, the RPK1-LRR domain, and the BRI1-KD chimeric protein (RL-BK). RPK1-LRR contains a signal peptide (SP), LRR, and transmembrane domain (TM). RL-BK contains the RPK1 N-terminal region, including the juxtamembane domain (JM) and BRI1-KD. (B) and (C) RPK1-LRR ox phenotypes. (B) Plant growth on media with/without ABA for 2 weeks. (C) Germination rates of the VC and the RPK1-LRR overexpressing lines (La and Lb) on media with/without ABA (0 or 1 μM).

Figure 7.

Phenotypes of the RL-BK Overexpressing Transgenic Plants. (A) RL-BK overexpressor (line L3; 1) shows normal growth plants (2) and the BR-insensitive phenotype plants (3, weak phenotype; 4, strong phenotype). 2 to 4, RL-BK L3 plants in higher magnification. (B) Left panel: RNA gel blot analysis of the RL-BK overexpressing (RL-BK ox) lines using a ³²P-labeled RL-BK chimeric gene or a BR-responsive cyclinD3 gene as probes. Right panel: BRI1 expression levels in the RL-BK transgenic lines, L2 and L3, and bri1-4. Total RNA from dwarf (d) and normal plants (n) of the same RL-BK line were isolated separately and used for quantitative RT-PCR. Experiments were repeated three times. Data represent means ± sd. (C) Proportions of dwarf plants in the RL-BK overexpressing lines (L1 to L3), which were named in order of the strength of expression levels of RL-BK. (D) Germination rates of the VC and the RL-BK overexpressing lines (L2 and L3) on media with/without ABA (0 or 1 μM).