GIGANTEA is a nuclear protein involved in phytochrome signaling in Arabidopsis

Abstract

In a genetic screen of available T-DNA-mutagenized Arabidopsis populations for loci potentially involved in phytochrome (phy) signaling, we identified a mutant that displayed reduced seedling deetiolation under continuous red light, but little if any change in responsiveness to continuous far-red light. This behavior suggests disruption of phyB, but not phyA signaling. We have cloned the mutant locus by using the T-DNA insertion and found that the disrupted gene is identical to the recently described GIGANTEA (GI) gene identified as being involved in control of flowering time. The encoded GI polypeptide has no sequence similarity to any known proteins in the database. However, by using beta-glucuronidase-GI and green fluorescent protein-GI fusion constructs, we have shown that GI is constitutively targeted to the nucleus in transient transfection assays. Optical sectioning by using the green fluorescent protein-GI fusion protein showed green fluorescence throughout the nucleoplasm. Thus, contrary to previous computer-based predictions that GI would be an integral plasma membrane-localized polypeptide, the data here indicate that it is a nucleoplasmically localized protein. This result is consistent with the proposed role in phyB signaling, given recent evidence that early phy signaling events are nuclear localized.

Figure 1

gi mutant seedlings are selectively hyposensitive to red light. Hypocotyl lengths of wild-type (Col) and gi mutants with different alleles (gi-1, gi-2, gi-100) grown for 3 days in the dark (D) or a range of Rc (A) or FRc (B) fluence rates. Hypocotyl length expressed as percentage of dark value is shown in the insets. Data are expressed as mean ± SEM. (C) Seedlings grown either in dark (D), or in Rc (3.84 μmolm⁻²s⁻¹) or FRc (6.8 μmolm⁻²s⁻¹) for 3 days.

Figure 2

Expression of GI and neighboring genes is altered in gi-100 mutant seedlings. (A) Diagrammatic representation of the insertionally tagged gi-100 gene (g6) and neighboring genes (g4, g5, g7, g8) within the sequenced BAC T22J18. The inverted triangle shows the location of the T-DNA insert in the gi-100 mutant. g4 = T22J18.4; g5 = T22J18.5; g6 = T22J18.6 (GI); g7 = T22J18.7; g8 = T22J18.8. (B) Northern blots of GI and neighboring-gene transcripts in both wild-type and gi-100 mutant seedlings. An 18S rDNA probe was used to reprobe each blot to show the amount of RNA loaded in each lane. Approximate marker sizes are shown on the left. (C) Quantification of transcript levels using a PhosphorImager. Signals for each transcript were normalized with the 18S rDNA signal and expressed as a percentage of the highest value obtained for each gene. Data are expressed as mean ± SEM (n ≥ 2).

Figure 3

Complementation of gi-100 mutant with wild-type GI locus. (A) Fluence-rate response curves for hypocotyl lengths of two complemented lines (C33 and C23) along with wild-type (Col) and gi-100 mutant seedlings grown in darkness (D) or a range of Rc fluence rates for 3 days. Hypocotyl length expressed as percentage of dark value is shown in the inset. Data are expressed as mean ± SEM. (B) Visual phenotypes of the two complemented lines (C33 and C23), gi-100, and wild type grown either in D or in Rc (3.84 μmolm⁻²s⁻¹). (C) Complementation of the gi-100 late flowering phenotype with the wild-type GI locus. Rosette leaf number (RLN#) or days were counted when the plants bolted under SD (Left; 9 h white light of 128 μmolm⁻²s⁻¹ and 15 h dark) or LD (Right; 18 h white light of 62 μmolm⁻²s⁻¹ and 6 h dark) at 21°C. Flowering time for the wild type (Col), gi-100 mutant and the two complemented lines (C33 and C23) are shown. Data are expressed as mean ± SEM.

Figure 4

GI localizes to the nucleus. (A) Schematic representations of GUS-GI and GFP-GI fusion constructs used in transient assays. The hatched boxes represent computer-predicted putative membrane spanning domains, and the black bars represent clusters of residues in which three of four are basic amino acids. (B) Transient transfection assay in onion epidermal cells using GUS-GI constructs. Top pair of panels show GUS staining for (i) GUS only control (construct I) and (ii) GUS-GI (construct II). Middle panels (iii and iv) show 4′,6-diamidino-2-phenylindole (DAPI) staining for nuclei of the cells in the above panels. Bottom panels (v and vi) show superimposition of the above two panels in each case to show colocalization. Arrows in ii and vi indicate position of GUS-stained nuclei. (Bar = 100 μM.) (C) Bar graphs showing quantitation of the subcellular localization of each GUS-GI construct shown in A. Panel numbering corresponds to the construct designated in A. Individual onion cells transiently expressing each construct were visually scored for GUS distribution after 3 h incubation and assigned to one of five categories: N, completely nuclear; C, completely cytosolic; N/C, 50% nuclear and 50% cytosolic; N/c, 75% nuclear and 25% cytosolic; n/C, 25% nuclear and 75% cytosolic. (D) 2 μm optical sections of top (i, iv, and vii), middle (ii, v, and viii) and bottom (iii, vi, and ix) regions of a nucleus transformed with GFP-GI (construct VII) scanned at 1024 × 1024 pixel resolution with a 25× oil objective. Upper row of panels shows GFP fluorescence (i, ii, and iii), middle row of panels shows propidium iodide (PI) staining of the nuclear DNA (iv, v, and vi), and the bottom row of panels shows merging of the corresponding upper two panels to show colocalization (vii, viii, and ix). (Bar = 10 μM.) (E) Amino acid sequence from residue 543 to 783 of GI showing the clusters of basic amino acids (reverse contrast). Arrow indicates the junction between amino acids 749 and 750.