RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

Abstract

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance.

Fig 1.

The los4-1 mutation reduces RD29A-LUC expression in response to cold but not to ABA or hyperosmotic stress. (A) RD29A-LUC expression was indicated by luminescence images and quantitatively measured as luminescence intensity. Wild-type (Left) and los4-1 (Right) seedlings, and their luminescence images taken after treatment at 4°C, or with 100 μM ABA for 3 h or 300 mM NaCl for 5 h. The color scale at right shows the luminescence intensity from dark blue (lowest) to white (highest). (B) Time courses of luminescence intensities of wild-type and los4-1 plants under cold, ABA, or NaCl treatment. Data represent means and standard errors (n = 20). Black square, wild type; open square, los4-1.

Fig 2.

Transcript levels of cold-regulated genes in los4-1 and wild-type plants. Plants were treated at 4°C for the indicated times. The TUBULIN gene was used as a loading control. WT, wild type.

Fig 3.

Chilling and freezing sensitivity of los4-1 mutant plants. (A) Wild-type and los4-1 mutant plants grown at 22°C, without chilling treatment, or treated at 4°C for 2 weeks or 2 months in light, or 2 weeks in dark. (B) Electrolyte leakage. Electrolyte leakage assays were done by using wild-type and los4-1mutant leaves from 2-week-old plants grown in soil and treated at 4°C in the dark. (C) Wild-type and los4-1 mutant plants grown at 22°C for 2 weeks were cold-acclimated at 4°C for 4 days in light and then treated at −8°C for 6 h. (D) Sucrose and proline contents in los4-1 mutant and wild-type plants treated at 4°C in light or dark for 5 days.

Fig 4.

Overexpression of the CBF3 gene reverses the chilling-sensitive phenotype of los4-1 mutant plants. (A) RD29A-LUC luminescence images of two CBF3 transgenic lines (line 1 on the right, heterozygous; line 2 on the left, homozygous), compared with los4-1 mutant and wild-type plants. The plants were grown at 22°C and not treated with any stress. (B) Expression of CBF3 in one transgenic line (line 2) without cold treatment. (C) los4-1 mutant plants transformed with the CBF3 gene were chilling-resistant. Transgenic line 2, wild-type and los4-1 mutant plants were treated at 4°C in dark for 14 days. (D). Freezing tolerance test of transgenic line 2 plants. After cold acclimation at 4°C for 4 days in light, wild-type (WT), los4-1 mutant, and los4-1 overexpressing CBF3 plants were treated at −8°C for 6 h.

Fig 5.

Positional cloning and characterization of the LOS4 gene. (A) Genetic mapping of LOS4. Genetic mapping delimited LOS4 between the bottom of BAC clone F8J2 and the top of BAC clone F4P12. The los4 mutation was identified by sequencing and comparing all predicted genes in this region from los4-1 mutant and wild-type plants. Structure of LOS4 and the position of the los4 mutation are indicated. Positions are relative to the initiation codon. Filled boxes indicate the ORF, and lines between boxes indicate introns. (B) Complementation of los4-1 mutant by the wild-type LOS4 gene. los4-1 mutant, wild-type, and los4-1 mutant transformed with the wild-type LOS4 gene (indicated as los4-1+LOS4) were treated at 4°C in dark for 14 days. The picture was taken 3 days after the plants were removed from 4°C to 22°C. (C) Alignment of LOS4 amino acid sequence with other RNA helicases. Amino acid residues in black indicate identical matches, and those in gray indicate conserved substitutions. The specific motifs conserved in DEAD-box RNA helicases are underlined. The mutation in los4-1 mutant is marked by a star. GenBank accession numbers for the RNA helicases are as follows: Dbp5-Human, ; DD19-Mouse, ; Dbp5-Yeast, NP 014689. (Da) A GFP-LOS4 fusion protein is localized in both nuclei and cytoplasm in the cells in transgenic Arabidopsis. Cells were stained with propidium iodide to indicate the nuclei (inset in red). (Db) A transgenic plant (line N7) expressing a GFP fused with a transcriptional factor as a positive control for nuclear localization (29). (Dc) Line Q1 expressing GFP fused with an acidic ribosomal protein as positive control for cytoplasmic localization (29). (Dd) A plant without GFP is shown as negative control.