An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway

Abstract

To investigate essential components mediating stress signaling in plants, we initiated a large-scale stress response screen using Arabidopsis plants carrying the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter. Here we report the identification and characterization of a mutant, hos9-1 (for high expression of osmotically responsive genes), in which the reporter construct was hyperactivated by low temperature, but not by abscisic acid or salinity stress. The mutants grow more slowly, and flower later, than do wild-type plants and are more sensitive to freezing, both before and after acclimation, than the wild-type plants. The HOS9 gene encodes a putative homeodomain transcription factor that is localized to the nucleus. HOS9 is constitutively expressed and not further induced by cold stress. Cold treatment increased the level of transcripts of the endogenous RD29A, and some other stress-responsive genes, to a higher level in hos9-1 than in wild-type plants. However, the C repeat/dehydration responsive element-binding factor (CBF) transcription factor genes that mediate a part of cold acclimation in Arabidopsis did not have their response to cold altered by the hos9-1 mutation. Correspondingly, microarray analysis showed that none of the genes affected by the hos9-1 mutation are controlled by the CBF family. Together, these results suggest that HOS9 is important for plant growth and development, and for a part of freezing tolerance, by affecting the activity of genes independent of the CBF pathway.

Fig. 1.

The RD29A::LUC expression was hyperinduced in hos9-1 mutant plants in response to low temperature. RD29A::LUC expression was quantitatively measured as luminescence intensity (counts/seedling). The scale bar at the right shows the luminescence intensity from dark blue (lowest) to white (highest). (A) Wild-type (on the left) and hos9-1 (on the right) seedlings grown on an agar plate. (B) Luminescence of A after low-temperature treatment at 0°C for 24 h. (C) Wild-type (on the left) and hos9-1 (on the right) seedlings grown on an agar plate. (D) Luminescence of C after treatment with 100 μM ABA for 3 h. (E) Wild-type (on the left) and hos9-1 (on the right) seedlings on filter paper saturated with 300 mM NaCl. (F) Luminescence of E after treatment with 300 mM NaCl for 4 h. (G) Quantification of the luminescence intensity in B (Cold), D (ABA), and F (NaCl). Also shown are data for untreated plants (Control). (H) Time course of RD29A::LUC expression in hos9-1 and wild-type plants in response to low temperature (0°C). [Bars (G and H) represent standard deviation (n = 20).]

Fig. 2.

Freezing tolerance of hos9-1 plants. (A) Tolerance of hos9-1 plants at different temperatures below freezing under long-day photoperiod. The photographs were taken 10 d after freezing treatments. (B) Leakage of electrolytes in hos9-1 and wild-type plants when treated at temperatures below freezing. Wild-type (cold) and hos9-1 (cold), cold-acclimated wild-type, and hos9-1 plants, respectively. [Bars are standard deviation (n = 8).]

Fig. 3.

Gene regulation in hos9-1 and wild-type plants. (A) Expression of stress-responsive gene in hos9-1 and wild-type plants. Plants were subjected to low temperature (0°C) for the indicated time periods (h). ABA, 100 μM, for 3 h; NaCl, 300 mM, for 4 h. Tubulin gene was used as loading control. (B) Steady-state transcript levels of CBF genes in hos9-1 and wild-type plants. The plants were subjected to the same treatments as stated in A. Actin gene was used as loading control. (C) RT-PCR analysis of three of the genes that were tested in the microarray analysis. The two cold-stimulated genes with higher expression in the hos9-1 than wild type in the microarray analysis encode a WRKY family transcription factor (At2g46400) and an expressed protein (At2g32210). The hos9-1 target gene with lower expression than wild type in the microarray analysis encodes a plant defensin protein (At5g44420). The tubulin gene was used as loading control.

Fig. 4.

Growth defects of hos9-1 plants. (A and B) The growth as indicated by shoot and root fresh and dry weights. Shoots or roots were separated from wild-type and hos9-1 plants grown in soil under long-day photoperiod at indicated developmental stages. The fresh weight of shoot or root was determined, and the samples were then dried in an oven at 65°C for 48 h and dry weight was measured. [Bars represent standard deviation (n = 15).] (C) Three-week-old of wild-type (Left) and hos9-1 (Right) plants grown under long-day photoperiod. (D) Trichomes on the fifth leaf of the wild-type (Left) and the hos9-1 (Right) plants. (E) Quantification of trichome numbers on rosette leaves of wild-type and hos9-1 plants. The number of trichome on different leaves was counted on a Nikon Optiphot microscope (Nikon). [Bars represent standard deviation (n = 20).] (F) hos9-1 plant (Right) flowers later than wild-type plant (Left) under normal conditions (16 h light/8 h dark). (G) Vernalization responses of wild-type and hos9-1 plants (as indicated by flowering time/total leaf number at flowering) under long-day photoperiod. [Bars represent standard deviation (n = 20).]

Fig. 5.

HOS9 encodes a putative homeobox protein. (A) The structure of HOS9 gene and the position of the T-DNA insert in hos9-1 mutant genome. Positions are relative to the transcription start site. Filled boxes represent exons and lines between filled boxes represent introns. (B) Luminescence image after low-temperature treatment (0°C for 48 h) of wild-type, hos9-1, and hos9-1 transformed with pCAMBIA1200 empty vector [hos9-1 (vector)] and hos9-1 transformed with pCAMBIA1200 containing the HOS9 genomic fragment (hos9-1+HOS9). The plants weregrown on an agar plate. (C) Freezing tolerance of 1-mo-old plants shown in B. The treatment was done at -5°C for 5 h, and the photograph was taken 7 d after treatment. (D) Comparison of HOS9 with its homologs. Compared proteins are: HOS9 (AAC67326) from Arabidopsis; PRS (PRESSED FLOWER, BAB79446) from Arabidopsis; WUS (WUCHEL, CAA09986) from Arabidopsis.

Fig. 6.

Expression of the HOS9 gene and subcellular localization of HOS9 protein. (A) The HOS9 expression level was determined by RT-PCR. Actin gene was used as loading control. Control, no treatment; Cold, 0°C for 48 h; ABA, 100 μM for 3 h; NaCl, 300 mM for 5 h. (B) Expression of HOS9 under different photoperiods at room temperature (22°C) or low temperature (4°C, 7 d) by RT-PCR. Three micrograms of total RNA was used to synthesize the first-strand cDNA. Actin gene was used as loading control. L, long-day photoperiod; S, short-day photoperiod. (C) Histochemical staining of transgenic plants expressing HOS9 promoter::GUS.(D) The GFP-HOS9 fusion protein is localized in the nucleus. Confocal image of root cell in plants transformed with empty vector only (Left) and confocal image of root cell in GFP-HOS9 transgenic plants (Right).