PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction

Abstract

Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsis mutant, pat (phytochrome A signal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1 mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncated PAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.

Figure 1

FR light responses in wild-type and mutant seedlings. (A) Seedlings grown for 4 days under continuous FR light (6 μmoles/m²/sec) (top); enlargements of the cotyledons (bottom). (B) After the FR light treatment the same seedlings were transferred for 1 day into white light. (C) Response of the hypocotyl elongation to different fluences of FR light. Wild-type (Col; ⧫), phyA-211 (phyA; █), and pat1-1 (▴) seedlings were grown under the indicated fluences for 4 days. Error bars, s.e.m.

Figure 1

FR light responses in wild-type and mutant seedlings. (A) Seedlings grown for 4 days under continuous FR light (6 μmoles/m²/sec) (top); enlargements of the cotyledons (bottom). (B) After the FR light treatment the same seedlings were transferred for 1 day into white light. (C) Response of the hypocotyl elongation to different fluences of FR light. Wild-type (Col; ⧫), phyA-211 (phyA; █), and pat1-1 (▴) seedlings were grown under the indicated fluences for 4 days. Error bars, s.e.m.

Figure 1

FR light responses in wild-type and mutant seedlings. (A) Seedlings grown for 4 days under continuous FR light (6 μmoles/m²/sec) (top); enlargements of the cotyledons (bottom). (B) After the FR light treatment the same seedlings were transferred for 1 day into white light. (C) Response of the hypocotyl elongation to different fluences of FR light. Wild-type (Col; ⧫), phyA-211 (phyA; █), and pat1-1 (▴) seedlings were grown under the indicated fluences for 4 days. Error bars, s.e.m.

Figure 2

Structure of the PAT1 gene and the encoded protein. (A) A schematic representation of the PAT1 gene with its intron/exon structure, its encoded protein, and the insertion of the T-DNA. Structural domains of the protein are indicated. Y(Tyr-379). (B) Deduced amino acid sequence of PAT1 (GenBank accession number AF153443). Leucines in the two L-rich domains are indicated in bold, the VHIID motif is double underlined, putative tyrosine phosphorylation site is underlined [[RK]-x (2,3)-[DE]-x (2,3)-Y] (Patschinsky et al. 1982), and ▾ indicates the disruption of the coding sequence by the T-DNA insertion. (C) Alignment of the VHIID domain of PAT1 compared to those of several GRAS proteins [AF210731 (SCL1), AF036302 (SCL5), AF036308 (SCL13), AF210732 (SCL21); Pysh et al. 1999; Di Laurenzio et al. 1996; Peng et al. 1997; Silverstone et al. 1998; Schumacher et al. 1999]. Residues identical with PAT1 are shown in reverse contrast, identical amino acids are marked with a star. The VHIID motif is indicated with a line. Numbers give residue of first amino acid as referred to in the GenBank. (†) Partial clone.

Figure 2

Structure of the PAT1 gene and the encoded protein. (A) A schematic representation of the PAT1 gene with its intron/exon structure, its encoded protein, and the insertion of the T-DNA. Structural domains of the protein are indicated. Y(Tyr-379). (B) Deduced amino acid sequence of PAT1 (GenBank accession number AF153443). Leucines in the two L-rich domains are indicated in bold, the VHIID motif is double underlined, putative tyrosine phosphorylation site is underlined [[RK]-x (2,3)-[DE]-x (2,3)-Y] (Patschinsky et al. 1982), and ▾ indicates the disruption of the coding sequence by the T-DNA insertion. (C) Alignment of the VHIID domain of PAT1 compared to those of several GRAS proteins [AF210731 (SCL1), AF036302 (SCL5), AF036308 (SCL13), AF210732 (SCL21); Pysh et al. 1999; Di Laurenzio et al. 1996; Peng et al. 1997; Silverstone et al. 1998; Schumacher et al. 1999]. Residues identical with PAT1 are shown in reverse contrast, identical amino acids are marked with a star. The VHIID motif is indicated with a line. Numbers give residue of first amino acid as referred to in the GenBank. (†) Partial clone.

Figure 3

Specificity of mutants disrupted in GRAS family members for their respective pathway. (A) Photographs of Col, pat1-1, scr2, rga1-24, and gai. Seedlings were grown for 4 days under continuous FR light (4.5 μmoles/m²/sec) and then transferred into white light for 1 day. (B) Photographs of three-week-old scr2 and pat1-1 seedlings grown on vertical tissue culture medium under white light conditions with their respective background ecotypes, Landsberg (Ler) and Columbia (Col).

Figure 4

PAT1 gene expression analysis. (A) PAT1 expression level under different light conditions detected by RT–PCR. Total RNA was extracted from 5-day-old seedlings grown for 4 days in dark and then transferred into the following light regimes: (1) white light; (2) FR light (3 hr); (3) FR light (18 hr); (4) dark; (5) total RNA was extracted from 3-week-old white-light grown plants. The upper panel shows the RT–PCR reaction performed with PAT1-specific primers, the lower panel with actin-specific primers as a control. FR fluence rate: 4.5 μmoles/m²/sec. (B) Poly(A)⁺ RNA Northern blot using a 5′ fragment of the PAT1 cDNA as a probe. (1) wild type, (2) pat1-1. Plants for RNA extraction were grown in white light. To confirm equal loading, the Northern was rehybridized with an actin probe.

Figure 5

Overexpression of 35S–ΔCPAT1 in wild type phenocopies pat1-1. Seedlings were grown for 4 days under continuous FR light (1.2 μmoles/m²/sec) and subsequently transferred into white light (WL) for 3 days. Three independent transgenic lines are shown with Col and pat1-1 as controls.

Figure 6

Expression studies of three different phyA-regulated genes CHS, CAB, and PET E. WT, phyA, pat1-1, complemented pat1-1 (pat1-1/35S-PAT1) and WT overexpressing a 35S–ΔCPAT1 transgene (WT/ΔCPAT1) seedlings were grown under continuous dark for 4 days (1) and induced by 3 hr of FR light (2). Seven μg of total RNA were loaded on each lane and equal loading was confirmed using an 18S DNA probe.

Figure 7

Subcellular localization of PAT1 in transgenic plants. A 35S–PAT1–GFP fusion gene (PAT1–GFP) and a control 35S-GFP gene (GFP) were introduced into wild type by Agrobacterium tumefaciens-mediated vacuum-infiltration. (A) The root-tips of transgenic plants were analyzed for GFP signal. (B) Nuclei in the same cells as in A were stained with DAPI (4′,6′-diamidino-2-phenylindole).