The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein alpha-subunit GPA1 and regulates seed germination and early seedling development

Abstract

Heterotrimeric G proteins are implicated in diverse signaling processes in plants, but the molecular mechanisms of their function are largely unknown. Finding G protein effectors and regulatory proteins can help in understanding the roles of these signal transduction proteins in plants. A yeast two-hybrid screen was performed to search for proteins that interact with Arabidopsis G protein alpha-subunit (GPA1). One of the identified GPA1-interacting proteins is the cupin-domain protein AtPirin1. Pirin is a recently defined protein found because of its ability to interact with a CCAAT box binding transcription factor. The GPA1-AtPirin1 interaction was confirmed in an in vitro binding assay. We characterized two atpirin1 T-DNA insertional mutants and established that they display a set of phenotypes similar to those of gpa1 mutants, including reduced germination levels in the absence of stratification and an abscisic acid-imposed delay in germination and early seedling development. These data indicate that AtPirin1 likely functions immediately downstream of GPA1 in regulating seed germination and early seedling development.

Figure 1.

Interaction between AtPirin1 and GPA1. (A) Reconstitution of the yeast two-hybrid interaction between AtPirin1 and GPA1. The indicated combinations of the bait (pLexA-NLS) and prey (pACT) constructs were transformed into the yeast reporter strain. Transformants were examined for β-galactosidase activity in the presence of 5-bromo-4-chloro-β-d-galactoside (+X-gal) and for growth in the absence of His (−His). (B) Coomassie blue staining of the GST-GPA1 fusion protein and GST (positions of the GST-GPA1 and GST proteins are indicated by arrows). MW, molecular mass. (C) In vitro protein interaction between AtPirin1 and GPA1. GST-GPA1 was overexpressed in E. coli, immobilized on glutathione-Sepharose 4B beads, and incubated with ³⁵S-labeled AtPirin1 protein obtained by coupled in vitro transcription/translation. AtPirin1 binds to GST-GPA1 but not to GST alone.

Figure 2.

AtPirin1 Amino Acid Sequence Analysis, AtPirin1 Gene Structure, and Locations of the T-DNA Insertions. (A) Alignment of the deduced amino acid sequences of AtPirin1, AtPirin2, AtPirin3, AtPirin4, tomato pirin (TOMpirin), and human pirin (HUMpirin). The alignment was generated using the CLUSTAL X program with default parameters. Identical amino acids are highlighted with a black background, and similar residues are highlighted with a gray background. Regions corresponding to motifs 1 and 2 of the cupin domain are indicated. The single and double asterisks mark residues corresponding to the beginning of the longest and shortest GPA1-interacting AtPirin1 clones, respectively, identified in the two-hybrid screen. Arrows indicate the positions of T-DNA insertions in the atpirin1-1 and atpirin1-2 mutants. (B) Scheme of the AtPirin1 gene. Relative positions of potential cis elements mentioned in the text are shown. The positions of the atpirin1-1 and atpirin1-2 T-DNA insertions are indicated. Boxes represent exons, and motifs 1 and 2 of the cupin domain are marked by vertical and horizontal hatching, respectively.

Figure 3.

AtPirin1 Transcript Levels Are Regulated by ABA and Low-Fluence Red Light. (A) Transcript levels of the AtPirin1 gene are induced by ABA. AtPirin1 transcript levels were examined by relative quantitative RT-PCR in 9-day-old light-grown Arabidopsis plants sprayed with 100 μM ABA (+) or distilled water (−). The ABA-responsive Rab18 transcript (Lang and Palva, 1992) served as a positive control for the assay. (B) Transcript levels of the AtPirin1 gene are induced by red light. AtPirin1 transcript levels were examined by relative quantitative RT-PCR in seedlings grown for 6 days in the dark and treated with a mock pulsed light (d) or irradiated with a single pulse of low-fluence blue light (b) or low-fluence red light (r). 18S rRNA was used as an endogenous RT-PCR standard. Representative experimental replicates are shown.

Figure 4.

Seed Germination of atpirin1 and gpa1 Mutants. (A) Germination of wild-type Col and atpirin1-2 mutant seeds. (B) Germination of wild-type Ws, atpirin1-1, gpa1-1, and gpa1-2 mutant seeds. Matched seed lots on premoistened filter paper were placed directly at 22°C under continuous white light for germination. Values presented are mean percentages of germination from three independent experimental replicates (each replicate included 70 to 150 seeds per line) with standard errors. Heterozygous and homozygous mutants are indicated by +/− and −/−, respectively, next to the corresponding mutant genotype. Germination data for stratified wild-type Col seeds (wild type Col str.) and stratified wild-type Ws seeds (wild type Ws str.) are included in (A) and (B), respectively. Upon stratification, there was no statistically significant difference in germination rates between wild-type and corresponding mutant seeds.

Figure 5.

Seed Germination and Early Seedling Development of atpirin1 and gpa1 Mutants Are Hypersensitive to ABA. Matched seed lots on half-strength Murashige and Skoog (1962) agarose medium supplemented with ABA were stratified for 48 h at 4°C before being placed at 22°C under continuous white light for germination. Heterozygous and homozygous mutants are indicated by +/− and −/−, respectively, next to the corresponding mutant genotype. (A) Germination of wild-type Col seeds and atpirin1-2 mutant seeds in the presence of the indicated concentrations of ABA at day 10 after stratification. (B) Germination of wild-type Ws seeds and atpirin1-1, gpa1-1, and gpa1-2 mutant seeds in the presence of the indicated concentrations of ABA at day 10 after stratification. (C) Germination of wild-type Col seeds and atpirin1-2 mutant seeds in the presence of 800 nM ABA over time (days after stratification). (D) Germination of wild-type Ws seeds and atpirin1-1, gpa1-1, and gpa1-2 mutant seeds in the presence of 800 nM ABA over time (days after stratification). Values shown in (A) to (D) are mean percentages of germination from three independent experimental replicates (each replicate included 70 to 150 seeds per line) with standard errors. (E) Early seedling development of atpirin1-2 mutants is inhibited by ABA. No ABA (−) or 500 nM ABA (+) was added to the growth medium in phytatrays. Note that >50% of the mutant seeds shown are germinated as judged by radicle emergence (cf. with [A]). (F) Early seedling development of atpirin1-1, gpa1-1, and gpa1-2 mutants is inhibited by ABA. No ABA (−) or 800 nM ABA (+) was added to the growth medium in phytatrays. Note that ∼30 to 40% of the mutant seeds shown are germinated as judged by radicle emergence (cf. with [D]). Photographs for (E) and (F) were taken on day 10.

Figure 6.

Complementation of the atpirin1-1 ABA-Hypersensitive Phenotype in Plants Carrying the AtPirin1 Transgene. Matched seed lots on half-strength Murashige and Skoog (1962) agarose medium supplemented with ABA were stratified for 48 h at 4°C before being placed at 22°C under continuous white light for germination. The atpirin1-1 heterozygous mutant genotype is indicated by +/−. (A) Germination of two independent complementation lines, 1-1PorAp1 and 1-1PorAp2, carrying AtPirin1 cDNA under the control of the PorA promoter in the atpirin1-1 mutant background. Germination was tested in the presence of the indicated concentrations of ABA at day 10 after stratification. Wild-type Ws seeds and atpirin1-1 mutant seeds were included for comparison. (B) Germination of two independent complementation lines, 1-1PorAp1 and 1-1PorAp2, wild-type Ws seeds, and atpirin1-1 mutant seeds in the presence of 800 nM ABA over time (days after stratification). Values presented in (A) and (B) are mean percentages of germination from three independent experimental replicates with standard errors. (C) Early seedling development of the 1-1PorAp1 and 1-1PorAp2 complementation lines. Wild-type (Ws) and atpirin1-1 mutant seeds were included for comparison. No ABA (−) or 800 nM ABA (+) was added to the growth medium in phytatrays.

Figure 7.

Bolting and Flowering Time of atpirin1-1 and atpirin1-2 Plants in Long-Day Growth Conditions. (A) At day 17, atpirin1-1 plants have longer bolts and start flowering, whereas the majority of wild-type Ws plants are just initiating bolting. (B) Results are plotted as a percentage of plants in the wild-type Ws and atpirin1-1 heterozygous populations that flower on a particular day. (C) Results are plotted as a percentage of plants in the wild-type Col and atpirin1-2 homozygous populations that flower on a particular day. Flowering was scored when the first flower opened. Values presented in (B) and (C) are mean percentages of germination from three independent experimental replicates (each replicate included 25 to 40 plants per line) with standard errors. Number of days was counted from the day when planted seeds were shifted to long-day conditions after stratification. Heterozygous and homozygous mutants are indicated by +/− and −/−, respectively, next to the corresponding mutant genotype.

Figure 8.

Schemes of AtPirin1 Modes of Action. Together with GPA1, AtPirin1 positively regulates seed germination and early seedling development by overcoming the negative effects of ABA and/or activating germination-promoting pathway(s). AtPirin1 mRNA levels are upregulated by ABA, suggesting the existence of a negative feedback regulatory loop of the ABA signaling pathway. Independent of GPA1, AtPirin1 is involved in the regulation of flowering time. Thick arrows indicate either known pathways or pathways established in this study.