A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis

Abstract

Previously, we described the AtGRF [Arabidopsis thaliana growth-regulating factor (GRF)] gene family, which encodes putative transcription factors that play a regulatory role in growth and development of leaves and cotyledons. We demonstrate here that the C-terminal region of GRF proteins has transactivation activity. In search of partner proteins for GRF1, we identified another gene family, GRF-interacting factor (GIF), which comprises three members. Sequence and molecular analysis showed that GIF1 is a functional homolog of the human SYT transcription coactivator. We found that the N-terminal region of GIF1 protein was involved in the interaction with GRF1. To understand the biological function of GIF1, we isolated a loss-of-function mutant of GIF1 and prepared transgenic plants subject to GIF1-specific RNA interference. Like grf mutants, the gif1 mutant and transgenic plants developed narrower leaves and petals than did wild-type plants, and combinations of gif1 and grf mutations showed a cooperative effect. The narrow leaf phenotype of gif1, as well as that of the grf triple mutant, was caused by a reduction in cell numbers along the leaf-width axis. We propose that GRF1 and GIF1 act as transcription activator and coactivator, respectively, and that they are part of a complex involved in regulating the growth and shape of leaves and petals.

Fig. 1.

AtGRF proteins show transactivation activity in the yeast GAL4 system. DNA fragments encoding the depicted portions of GRF1 (1F-1ΔF), GRF2 (2F-2ΔF), and GRF5 (5C) were fused to the DNA sequence encoding GAL4-DBD and introduced into yeast cells. Two independent transformants for each fusion construct were tested for their ability to activate the expression of the HIS3 and LacZ reporter genes. -Trp and -His/Trp denote the colonies grown on plates lacking Trp and His/Trp, respectively; LacZ denotes β-galactosidase activities on filters after a colony-lift assay.

Fig. 2.

Characterization of the AtGIF gene family. (A) Protein-protein interaction studies between GRF1 and GIF1 proteins in the yeast two-hybrid system. The 1ΔF or 2ΔF construct (see Fig. 1) was introduced into yeast cells together with the construct for GIF1 fused to GAL4-AD. As control, empty vectors containing inserts only for DBD or AD were also introduced with each of the 1ΔF, 2ΔF, and GIF1 constructs. -TL and -TLH denote the colonies grown on plates lacking Trp/Leu and His/Leu/Trp, respectively; and LacZ indicates β-galactosidase activity. (B) Schematic comparison of the domain structures of the GIF and SYT proteins. (C) Tissue-specific expression of GIF genes. Lanes: 1, root tips of seedlings; 2, uppermost stem; 3, internode; 4, fully grown leaves; 5, young leaves; 6, shoot tips containing the shoot apical meristem and early flower buds; 7, mature flowers. A phylogenetic tree derived from amino acid sequence shows the relatedness between GIF proteins.

Fig. 3.

In vitro column-binding assay. (A and B) HA-tagged full-length GRF1 protein (A) or HA-tagged GRF1 fragments (B) were applied to a GST or GST-GIF1 column, as indicated. After extensive washing with the binding buffer to remove nonspecifically bound protein, HA-tagged GRF1 or its fragments retained on the column were eluted and subjected to immunoblot analysis with anti-HA antibody. Q, W, and C above the lanes indicate the GRF1 fragments containing the QLQ, WRC, and C-terminal regions, respectively. (C) The full-length HA-GRF1 protein was applied to a GST-SNH or GST-QG fusion column followed by immunoblot analysis with anti-HA antibody. “Input” indicates the signal elicited by 1% of applied protein as a control.

Fig. 4.

Molecular activities of GIF1 proteins. (A) Transactivation assay in the yeast GAL4 system. DNA encoding full-length GIF1 or its segments, as depicted, was fused to DNA encoding GAL4-DBD and introduced into yeast cells. See Fig. 1 legend for procedures and labeling. (B) GIF1-EYFP fusion proteins form nuclear speckles. Onion epidermal cells were transformed transiently with the plasmid construct for the GIF1-EYFP fusion protein or empty vector for EYFP alone, as indicated. Fluorescence of cells was observed 24 h after incubation. Arrows indicate nucleus, and the numbers at the bottom denote the number of cells exhibiting nuclear speckles per the number of cells examined.

Fig. 5.

The phenotypes of Atgif1 mutant and AtGIF1-RNAi transgenic plants. (A) Representation of the gif1 T-DNA insertional mutation. Black boxes, exons; solid line, introns; inverse triangle, T-DNA integration site. The ruler is a scale of base pairs. (B) RT-PCR determination of GIF1 mRNA content in the mutant. Amplification of actin mRNA was used as control. (C) Col and gif1 plants (Left) and rosette leaves from the first to fourth leaf 20 d after germination (Right). (D) Dimensional parameters of the first two rosette leaves from Col and gif1 plants at 20 d. (E and F) Col and gif1 flowers and petals (E) and their dimensional parameters (F). (G and H) Dimensional parameters of the first two rosette leaves (G) and dimensional parameters of petals (H) from Ws and GIF1-RNAi plants. C, Col; g, gif1; W, Ws; R, GIF1-RNAi line 7; T, the grf1-2 grf2 grf3 triple mutant; and 5, 7, 8, GIF1-RNAi lines. Error bars indicate standard error.

Fig. 6.

Cooperative effects of Atgif1 and Atgrf triple mutations. (A) Seed production. Bars: 1, Col; 2, gif1; 3, Ws; 4, GIF1-RNAi; 5, grf1-2 grf2 grf3; 6, grf1-2 grf2 grf3 gif1/GIF1;7, grf1-2 grf2/GRF2 grf3 gif1;8, grf1-2 grf2/GRF2 grf3/GRF3 gif1;9, grf1-2 grf3 gif1. (B) Leaf growth. (C) Bars 1, 2, and 3, are as in A. Bar 4, grf1-2 grf2 grf3; bar 5, grf1-2 grf2 gif1. Error bars indicate standard error.

Fig. 7.

Number and area of cells in a transverse line at maximum width of leaves and petals. (A) Subepidermal palisade cells of the first two rosette leaves. (B) Abaxial epidermal cells of petals were analyzed. Bars: 1, Col; 2, gif1; 3, Ws; 4, GIF1-RNAi; 5, grf1-2 grf 2 grf3; 6, grf1-2 grf3 gif1.