The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo--cytoplasmic partitioning

Abstract

Low temperature is one of the most important environmental stimuli that control gene transcription programs and development in plants. In Arabidopsis thaliana, the HOS1 locus is a key negative regulator of low temperature-responsive gene transcription. The recessive hos1 mutation causes enhanced induction of the CBF transcription factors by low temperature as well as of their downstream cold-responsive genes. The hos1 mutant plants flower early, and this correlates with a low level of Flowering Locus C gene expression. The HOS1 gene was isolated through positional cloning. HOS1 encodes a novel protein with a RING finger motif near the amino terminus. HOS1 is ubiquitously expressed in all plant tissues. HOS1--GFP translational fusion studies reveal that HOS1 protein resides in the cytoplasm at normal growth temperatures. However, in response to low temperature treatments, HOS1 accumulates in the nucleus. Ectopic expression of HOS1 in wild-type plants causes cosuppression of HOS1 expression and mimics the hos1 mutant phenotypes.

Figure 1

The CBF transcription factor genes are expressed at higher levels in hos1 mutant plants in response to cold treatments. Total RNA (20 μg per lane) was isolated from wild-type and hos1 mutant seedlings treated with cold (0°C for the indicated time periods), ABA (100 μM, 3 h), NaCl (300 mM, 5 h), or PEG (30%, 5 h). The RNA blot was probed with ³²P-labeled RD29A, CBF2, CBF3, and actin cDNA probes, with the actin used as a loading control.

Figure 2

The early flowering phenotype and reduced expression of the vernalization regulator FLC in hos1 mutant plants. (A) Flowering time of C24 wild-type and hos1 mutant plants with and without 2 or 4 wk of vernalization treatment at 0°C. Flowering time is indicated by the total number of rosette leaves at time of flower bud emergence. Shown are the average and standard deviation (n = 15). (B) FLC expression. Total RNA (20 μg per lane) was isolated from wild-type and hos1 mutant seedlings grown in agar plates that were incubated at 0°C for 0, 2, and 4 wk. The tubulin gene was used as a loading control.

Figure 3

Positional cloning of the HOS1 gene. (A) Physical mapping of HOS1. Genetic mapping delimited HOS1 to the BAC clone T5I7. The hos1 mutation was identified by sequencing and comparing all predicted genes on this BAC from hos1 mutant and wild-type plants. (B) Structure of HOS1 and the position of the hos1 mutation. Positions are relative to the initiation codon. Filled boxes indicate the open reading frame, and lines between boxes indicate introns. The premature stop codon created by the hos1 mutation is boxed.

Figure 4

Complementation of hos1 mutant by the wild-type HOS1 gene. Ten-day-old wild-type seedlings, hos1, and homozygous T₃ progenies of a hos1 plant containing the wild-type HOS1 transgene (designated as hos1 + HOS1) grown in agar medium were incubated at 0°C for 24 h before the bioluminescence image was taken. (A) A picture of wild-type, hos1, and T₃ progenies of hos1 plants containing the HOS1 complementation construct. (B) Bioluminescence image of plants shown in A. (C) Quantification of the luminescence intensities in B.

Figure 4

Complementation of hos1 mutant by the wild-type HOS1 gene. Ten-day-old wild-type seedlings, hos1, and homozygous T₃ progenies of a hos1 plant containing the wild-type HOS1 transgene (designated as hos1 + HOS1) grown in agar medium were incubated at 0°C for 24 h before the bioluminescence image was taken. (A) A picture of wild-type, hos1, and T₃ progenies of hos1 plants containing the HOS1 complementation construct. (B) Bioluminescence image of plants shown in A. (C) Quantification of the luminescence intensities in B.

Figure 5

. The HOS1 cDNA sequence and the conceptual translational product of its longest ORF (GenBank accession no. AAB87130). The RING finger domain is underlined, and a putative nuclear localization signal is double-underlined.

Figure 6

Alignment of the RING finger domain of HOS1 with those of animal IAPs. Amino acid residues in black indicate identical matches, and those in gray indicate conserved substitutions. The conserved Cys and His residues of the RING finger are indicated by asterisk marks. A missing Cys residue in HOS1 is indicated by a question mark. GeneBank accession numbers for various IAPs are as follows: Human IAP, AAC83232; Pig IAP, AAC39271; Mouse IAP, AAC42078; Rat IAP, AAG22971; HOS1, AAB87130.

Figure 7

HOS1 transcript levels in wild-type and hos1 mutant plants subjected to various stress treatments. Wild-type and hos1 mutant 3-week-old seedlings were incubated at 0°C for 0 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, or 48 h. Seedlings were also treated with ABA (A: 100 μM, 3 h), NaCl (N: 300 mM, 5 h), and PEG (P: 30%, 5 h). Total RNA was extracted, and 20 μg RNA was loaded into each lane. The blots were hybridized with ³²P-labeled HOS1, RD29A, CBF2, and β-tubulin cDNA probes. The β-tubulin gene was used as a loading control.

Figure 8

Ubiquitous expression of HOS1 in Arabidopsis seedlings. Fifteen-day-old transgenic plants containing the HOS1 promoter–GUS construct were analyzed for GUS expression. (A) Whole seedling, (B) roots, (C) leaf, and (D) close-up of part of a leaf.

Figure 9

Cold treatment alters HOS1–GFP localization in Arabidopsis seedlings. (A) Cytoplasmic localization of HOS1–GFP fusion protein in root cells without cold treatment. T₂ 10-day-old seedlings containing the HOS1–GFP translational fusion construct grown on MS agar plates in the dark were analyzed for GFP expression under confocal microscope. The arrow points to a cell with strong cytoplasmic green fluorescence. (B) Nuclear localization of HOS1–GFP after cold treatment. Seedlings were incubated at 0°C for 48 h before GFP analysis. The spots emitting green fluorescence correspond to nuclei as confirmed by propidium iodide staining (red stain in insert). Arrows point to nuclei. (C) Those cells expressing strong cytoplasmic GFP at normal growth temperatures show strong nuclear GFP after cold treatment. Arrows point to nuclei. (D) Cells in C were stained with propidium iodide. Arrows point to nuclei. (E,F) Cytoplasmic GFP localization in the roots of transgenic Arabidopsis expressing an acidic ribosomal protein–GFP fusion (Cutler et al. 2000) either before (E) or after 48 h of cold treatment (F).

Figure 10

Ectopic expression of HOS1 causes silencing of HOS1 expression and mimics the hos1 mutant phenotypes. Ten-day-old wild-type seedlings (WT), hos1, and T₂ progenies of a wild-type plant containing the 35S–HOS1 construct (designated as line #4) were incubated at 0°C for 24 h before the bioluminescence image was taken. (A) Picture of wild-type, hos1, and T₂ progenies of transgenic line #4. (B) Bioluminescence image of plants in A. (C) Quantification of the luminescence intensities in B. Shown are the averages and standard deviations (n = 15). T₂ progenies of the #4 line were segregated into two groups, which are designated #4-high and #4-low, corresponding to those exhibiting high expression and low expression of RD29A–LUC, respectively. (D) Reduced HOS1 transcript abundance in the #4 cosuppression plants, as determined by quantitative RT–PCR.

Figure 10

Ectopic expression of HOS1 causes silencing of HOS1 expression and mimics the hos1 mutant phenotypes. Ten-day-old wild-type seedlings (WT), hos1, and T₂ progenies of a wild-type plant containing the 35S–HOS1 construct (designated as line #4) were incubated at 0°C for 24 h before the bioluminescence image was taken. (A) Picture of wild-type, hos1, and T₂ progenies of transgenic line #4. (B) Bioluminescence image of plants in A. (C) Quantification of the luminescence intensities in B. Shown are the averages and standard deviations (n = 15). T₂ progenies of the #4 line were segregated into two groups, which are designated #4-high and #4-low, corresponding to those exhibiting high expression and low expression of RD29A–LUC, respectively. (D) Reduced HOS1 transcript abundance in the #4 cosuppression plants, as determined by quantitative RT–PCR.