The Arabidopsis A4 subfamily of lectin receptor kinases negatively regulates abscisic acid response in seed germination

Abstract

Abscisic acid (ABA) is an important plant hormone for a wide array of growth and developmental processes and stress responses, but the mechanism of ABA signal perception on the plasma membrane remains to be dissected. A previous GeneChip analysis revealed that a member of the A4 subfamily of lectin receptor kinases (LecRKs) of Arabidopsis (Arabidopsis thaliana), At5g01540 (designated LecRKA4.1), is up-regulated in response to a low dose of ABA in the rop10-1 background. Here, we present functional evidence to support its role in ABA response. LecRKA4.1 is expressed in seeds and leaves but not in roots, and the protein is localized to the plasma membrane. A T-DNA knockout mutant, lecrka4.1-1, slightly enhanced ABA inhibition of seed germination. Interestingly, LecRKA4.1 is adjacent to two other members of the A4 subfamily of LecRK genes, At5g01550 (LecRKA4.2) and At5g01560 (LecRKA4.3). We found that loss-of-function mutants of LecRKA4.2 and LecRKA4.3 exhibited similarly weak enhancement of ABA response in seed germination inhibition. Furthermore, LecRKA4.2 suppression by RNA interference in lecrka4.1-1 showed stronger ABA inhibition of seed germination than lecrka4.1-1, while the response to gibberellic acid was not affected in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines. Expression studies, together with network-based analysis, suggest that LecRKA4.1 and LecRKA4.2 regulate some of the ABA-responsive genes. Taken together, our results demonstrate that the A4 subfamily of LecRKs has a redundant function in the negative regulation of ABA response in seed germination.

Figure 1.

Expression and protein subcellular localization patterns of LecRKA4.1. A, RT-PCR analysis of LecRKA4.1 expression in different organs. Shown are shoots (Sh) and roots (R) of 7-d-old seedlings, mature leaves (L), stems (St), and flowers (F). ACT2 was used as an internal control. RT was prepared as described elsewhere (Gao et al., 2008). B to H, Expression patterns of LecRKA4.1 as revealed by P_LecRKA4.1:GUS. Shown are a 5-d-old seedling (B), an enlarged view of a root segment immediately from the root-hypocotyl junction (C), a mature leaf (D), an enlarged view of guard cells (E), an inflorescence with several flowers (F), and a germinating seed with and without seed coat (G and H). I, Transient expression of LecRKA4.1-GFP and LecRKA4.1^351–682-GFP in leaf pavement cells. GFP images are shown at left. An mCherry version of RFP was used as a control (middle). The merged GFP and RFP images are shown at right. The arrows indicate the position of the nucleus. Bars = 20 μm.

Figure 2.

The lecrka4.1-1 knockout mutant slightly enhances the ABA response in seed germination inhibition. A, RT-PCR analysis of LecRKA4.1 mRNA expression in seedlings of the wild type (WT), lecrka4.1-1, and two transgenic lines (7-2 and 11-4) expressing the 2X35S:LecRKA4.1 transgene in lecrka4.1-1. ACT2 was used as an internal control. B, Functional complementation of lecrka4.1-1 by 2X35S:LecRKA4.1. The transgenic lines 7-2 and 11-4 showed similar seed germination percentages as the wild type in the presence of 0.6 μm ABA. Germination was scored at 3 d after the cold treatment. Data represent averages and sd of germination percentages for three replicates, each with about 40 to 50 seeds.

Figure 3.

Mutants of LecRKA4.2 and LecRKA4.3 slightly enhance ABA response in seed germination inhibition. A, RT-PCR analysis of LecRKA4.1, LecRKA4.2, and LecRKA4.3 transcript levels in seedlings of the wild type (WT) and various mutants. ACT2 was used as an internal control. B and C, lecrka4.2 and lecrka4.3 mutants showed similar seed germination kinetic profiles with 0.3 μm ABA (B) and ABA dose-response patterns (C) as lecrka4.1-1. Germination for dose response was scored at 4 d after cold treatment. Data represent averages and sd of germination percentage for three replicates, each with about 40 to 50 seeds.

Figure 4.

The lecrka4.1-1; LecRKA4.2 (RNAi) lines enhance the ABA response in seed germination inhibition. A, RT-PCR analysis of LecRKA4.2 and LecRKA4.3 mRNA expression in seedlings of the wild type (WT), lecrka4.1-1, and two LecRKA4.2 RNAi lines (S2 and F9) in the lecrka4.1-1 background. ACT2 was used as an internal control. B and C, S2 and F9 lines showed a stronger ABA seed germination inhibition response than lecrka4.1-1. Seed germination kinetic profiles (B) were determined using 0.3 μm ABA, and ABA dose response (C) was measured at 4 d after cold treatment. Each data point represents the average and sd of three replicates, each with about 40 to 50 seeds. D and E, S2 and F9 lines showed smaller green cotyledon percentages at 6 d (D) and 9 d (E) under ABA stress. Each point represents the average and sd of three replicates, each with about 40 to 50 seeds.

Figure 5.

Real-time PCR analysis of gene expression in response to ABA. Four-day-old wild-type (WT), lecrka4.1-1, and F9 seedlings grown in liquid MS medium were treated for 12 h with 0, 0.3, and 0.9 μm ABA prepared in fresh MS medium. Expression of ABI1 (A), ABI2 (B), At5g27420 (C), and two genes, AZF1 (D) and WRKY53 (E), which were selected from the assembled protein-protein interaction network (Fig. 6), was quantified using real-time PCR analysis. The mRNA levels for each of these genes were normalized with ACT2, and relative mRNA levels in nontreated wild-type seedlings for each gene were set at 1. Average and se values for three biological repeats are shown. The wild type, lecrka4.1-1, and F9 are designated in all panels as shown in A.

Figure 6.

A LecRKA4.1 and LecRKA4.2 gene interaction network. Three members of LecRKA4 genes were mapped to the protein-protein interaction database, with only LecRKA4.1 and LecRKA4.2 present in the database. This analysis revealed a total of 70 unique genes (Supplemental Table S1) that showed 149 interactions, and a network was then assembled based on these interactions. Arrowheads indicate the two genes (WRKY53 and AZF1) whose transcript levels were analyzed using real-time PCR (Fig. 5, D and E).