GIGANTUS1 (GTS1), a member of Transducin/WD40 protein superfamily, controls seed germination, growth and biomass accumulation through ribosome-biogenesis protein interactions in Arabidopsis thaliana

Abstract

Background: WD40 domains have been found in a plethora of eukaryotic proteins, acting as scaffolding molecules assisting proper activity of other proteins, and are involved in multi-cellular processes. They comprise several stretches of 44-60 amino acid residues often terminating with a WD di-peptide. They act as a site of protein-protein interactions or multi-interacting platforms, driving the assembly of protein complexes or as mediators of transient interplay among other proteins. In Arabidopsis, members of WD40 protein superfamily are known as key regulators of plant-specific events, biologically playing important roles in development and also during stress signaling.

Results: Using reverse genetic and protein modeling approaches, we characterize GIGANTUS1 (GTS1), a new member of WD40 repeat protein in Arabidopsis thaliana and provide evidence of its role in controlling plant growth development. GTS1 is highly expressed during embryo development and negatively regulates seed germination, biomass yield and growth improvement in plants. Structural modeling analysis suggests that GTS1 folds into a β-propeller with seven pseudo symmetrically arranged blades around a central axis. Molecular docking analysis shows that GTS1 physically interacts with two ribosomal protein partners, a component of ribosome Nop16, and a ribosome-biogenesis factor L19e through β-propeller blade 4 to regulate cell growth development.

Conclusions: Our results indicate that GTS1 might function in plant developmental processes by regulating ribosomal structural features, activities and biogenesis in plant cells. Our results suggest that GIGANTUS1 might be a promising target to engineer transgenic plants with higher biomass and improved growth development for plant-based bioenergy production.

Figure 1

Tissue specific expression profile and Phylogenetic analysis of GTS1. A). GENEVESTIGATOR-Microarray data showing highly expressed GTS1 gene in embryo, root apical meristem, root tip, abscission zone and shoot apex [24]. B) Experimental expression analysis of GTS1 showing increased transcript accumulation in germinated seed, young and developed rosette leaves and developed flower. C). Phylogenetic relationships between plant genes containing WD40 repeat domains. The GTS1 genes of Arabidopsis thaliana, Oryza sativa, and Zea mays (shown in red), belong to a subclade of the dominate clade (gray branches) containing most of the plant genes listed. Genebank accession numbers were used for all genes with the exception of Arabidopsis thaliana and Oryza sativa for which the Gene ID number from the SALK database were used.

Figure 2

Physical map of GTS1 knockout gene and phenotypic characterization of gts1 mutant. (A) The GTS1 gene with the positions of exons (numbered black rectangles) and introns (thick lines) are represented. The 5’ and 3’ untranslated regions are depicted in white rectangles. The location of the gts1 T-DNA insertion is shown using an inverted black triangle. The names and locations of primers used for RT-PCR experiments are also indicated. Bar = 0. 5 kb. (B) The T-DNA insertion causes a knockout expression of the gene. The quality of the RNA and the loading control was assayed by monitoring ACTIN gene expression. (C-F) GTS1 negatively controls seed germination. gts1 mutant germinated faster at 1 and 3 days after incubation in water (D, F) than the wild type (C, E). (G-H) GTS1 controls biomass accumulation and growth development in Arabidopsis. (H), Growth rate of gts1 is faster than that of WT (G) at 15 DAG. gts1 shows larger leaf area (biomass) (H) than WT (G). DAG = Days after germination.

Figure 3

Mutation in GTS1 gene promotes early flowering, growth development and biomass accumulation. A) a faster growth of gts1 mutant compared to WT is depicted with gts1 displaying a taller phenotype than WT. B)gts1 mutant flowers earlier than WT as depicted by a reduced number of gts1 rosette leaves compared to WT at bolting time. Gts1 mutant accumulates higher cell biomass than WT as shown by a bigger overall gts1 rosette leaf area compared to WT (B).

Figure 4

Correlation of expression pattern between GTS1 and the ribosomal protein. Samples whose contribution is more than 1.0 are outputted. The probe pair giving highest correlation is selected from all combination between the probes for the two loci, 266466_at (GTS1) and 258410_at (L19e) respectively. Sample contribution score is calculated as a product of z-scored expression values. The average of the score is the pearsons correlation coefficient.

Figure 5

Structure of GTS1, a WD40 repeat protein. A) Top, bottom and side views of the seven-bladed β-propeller structure (most stable form) made by using PyMol software (http://www.pymol.org/), with the N-terminal and C-terminal regions in blue and red color, respectively. A depicted model is included to show the basic WD40 β-sheet structures conformation of the cylinder structure with a tunnel-like structure in the centre that communicate both top and bottom sides. B) 180° rotated views of the electrostatic potential representation on the GTS1 protein surface. The surface colors are clamped at red (-10) or blue (+10).

Figure 6

Conservational and ligand binding domain analysis of GTS1, a WD40 repeat protein. A) Consurf-conservational analysis of GTS1 protein showed in two individual views rotated 180°. The conserved and variable residues are presented as space-filled models and colored according to the conservation scores. The interacting area of the protein with ribosomal counterparts has been highlighted by a red discontinue line. B) Detailed view of the ligand-binding area of GTS1 with a peptide and the spatial distribution of the interacting residues in a detailed view.

Figure 7

Conservational and ligand binding domain analysis of ribosomal Nop16 and L19e proteins. A) Consurf-conservational analysis of Nop16 protein showed in two individual views rotated 45°. The conserved and variable residues are presented as space-filled models and colored according to the conservation scores. The interacting areas of the protein with GTS1 protein and ribonucleic acid have been highlighted in red and blue color, respectively. B) Detailed views of the ligand-binding area of Nop16 protein with a chain of ribonucleic acid and the spatial distribution of the interacting residues depicted in both detailed views. C) Consurf-conservational analysis of L19e protein showed in two individual views rotated 45°. The conserved and variable residues are presented as space-filled models and colored according to the conservation scores. The interacting areas of the protein with GTS1 protein and ribonucleic acid have been highlighted in red and blue color, respectively. D) Detailed views of the ligand-binding area of L19e protein with ribonucleic acid and the spatial distribution of the interacting residues depicted in both detailed views.

Figure 8

Analysis of the interaction between GTS1, a WD40 repeat and Nop16 proteins. A) The complex between GTS1 (blue surface and cartoon representation) and Nop16 (white/gray surface and cartoon representation) from the top view. B) Surface/cartoon structure rotated 90° in blue (GTS1) and white/gray (Nop16) are depicted, and highlight the large interacting surface between both proteins. C) Electrostatic potential depicted in both interacting partners, where has been highlighted both areas involved in the interaction by light-blue discontinue arrows. The surface colors are clamped at red (-10) or blue (+10).

Figure 9

Analysis of the interaction between GTS1, a WD40 repeat and L19e proteins. A) The complex between GTS1 (green surface and cartoon representation) and L19e (white/gray cartoon representation) from a lateral view. B) Surface/cartoon structure rotated 45° in green (GTS1) and white/gray (L19e) are depicted, and highlight the two interacting surfaces between both proteins. C) Electrostatic potential represented in both interacting partners, where has been highlighted both areas involved in the interaction by black discontinue arrows. A detailed view of this interaction has been depicted. The surface colors are clamped at red (-10) or blue (+10).