Ectopic overexpression of SsCBF1, a CRT/DRE-binding factor from the nightshade plant Solanum lycopersicoides, confers freezing and salt tolerance in transgenic Arabidopsis

Abstract

The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193-228 (SsCBF1(193-228)). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

Figure 1. Low-temperature resistance test of S. lycopersicoides and CM.

(A–D) Phenotypes of S. lycopersicoides and CM before (A, B) and after (C, D) 8 h of 4°C treatment. Seedlings of S. lycopersicoides and Solanum lycopersicum (tomato cv. Castlemart) were grown under standard conditions and transferred to a climate chamber for the low-temperature stress. (E–F) Close-up shots of S. lycopersicoides and Solanum lycopersicum (tomato cv. Castlemart) leaves.

Figure 2. Sequence analysis of SsCBF1.

(A) Amino acid sequence alignment between SsCBF1 and other known CBF1s. The alignment was performed using ClustalX 2.0 and DNAMAN software. Black background indicated conserved residues among all the proteins selected. The AP2 DNA-binding domain and other signature motifs were indicated by solid lines. (B) Phylogenetic relationships between SsCBF1 and other CBFs from various species. The phylogenetic tree was generated by the neighbor-joining method using MEGA 5.0. Organisms were abbreviated as follows: St, Solanum tuberosum; Sc, Solanum commersonii; Sl, Solanum lycopersicum; Ss, Solanum lycopersicoides; Ca, Capsicum annuum; At, Arabidopsis thaliana. GenBank accession numbers of the CBFs are listed as follows: AtCBF1 (AEE85066), AtCBF2 (AEE85064), AtCBF3 (AEE85065), AtCBF4 (ABV27186), SsCBF1 (ACY79412), SlCBF1 (AAS77820), SlCBF2 (AAS77821), SlCBF3 (AAS77819), ScCBF1 (ACB45093), ScCBF2 (ACB45094), ScCBF3 (ACB45092), ScCBF4 (ACB45084), StCBF1 (ABI74671), StCBF2 (ABI94367), StCBF3 (ACB45095), StCBF4 (ACB45083), StCBF5 (ACB45082), CaCBF1 (AAZ22480), CaCBF3 (ADM73296).

Figure 3. Southern blot analysis of the SsCBF1 gene.

S. lycopersicoides genomic DNA was digested with restriction enzymes BamH I, Hind III, and Xba I. The hybridization was performed using the full-length SsCBF1 as a probe labeled with Digoxin.

Figure 4. Subcellular localization of SsCBF1-eGFP fusion protein.

(A) Schematic representation of 35S_pro:SsCBF1-eGFP construct used for transient expression in N. benthamiana leaves. (B) Subcellular localization assay of SsCBF1-eGFP fusion protein. N. benthamiana leaves transiently expressing eGFP alone (upper) and SsCBF1-eGFP (bottom) proteins were observed with a confocal microscope. Bars  = 50 µm.

Figure 5. Transactivation of CRT/DRE cis-element containing promoter by SsCBF1 and AtCBF1.

(A) Combinations of reporter and effector constructs used in the transient expression assays. Agrobacterium strain LBA4404 was used as a negative control. The 35S_pro:eGFP construct was used as a positive control. The reporter eGFP gene was driven by the wild-type or mutant COR15A promoter. The effectors SsCBF1 and AtCBF1 genes were driven by the CaMV35S promoter. (B) Semi-quantitative PCR detection of SsCBF1, AtCBF1 and ACTIN1 expression in tobacco leaves infiltrated with different combinations of constructs as shown in (A). (C) qRT-PCR analysis of eGFP expression. qRT-PCR procedure was as described in Methods. The results are representative of three independent experiments.

Figure 6. Transactivation of promoters with CRT elements only.

(A) Transient expression assays showing that SsCBF1 and AtCBF1 specifically bind to the CRT elements and activate the expression of LUC reporter gene. The bottom panel indicates the combination of reporter and effector plasmids infiltrated. The LUC reporter gene is driven by a promoter with or without four tandem CRT elements fused upstream of a minimal (−46) 35S promoter sequence (min35S_pro). The effector CBF1 genes are under the control of the CaMV35S promoter. Representative images of N.benthamiana leaves 72 h after infiltration are shown. (B) Quantitative analysis of luminescence intensity in (A). Five independent determinations were assessed. Error bars represent SD. Asterisks denote Student's t-test significance levels compared with the control: ***P<0.001. (C) qRT-PCR analysis of SsCBF1 and AtCBF1 expression in the infiltrated leaf areas shown in (A). Total RNAs were extracted from leaves of N. benthamiana coinfiltrated with the constructs. Five independent determinations were assessed. Error bars represent SD.

Figure 7. Functional dissection of the SsCBF1 C-terminal activation domain in yeast.

(A) Schematic protein structure of SsCBF1. Full-length SsCBF1 and its derivatives (SsCBF1^0–121, SsCBF1^122–156, SsCBF1^157–192, and SsCBF1^193–228) were tested for the transactivation activity (see Methods for details). SsCBF1 derivatives were symbolized by filled color boxes. NT, N-terminal; CTAD, C-terminal activation domain; DBD, DNA-binding domain. Bar  = 20 amino acids (aa). (B) Mapping the putative transcriptional activation domain of SsCBF1 in yeast. Based on the schematic protein structure of SsCBF1, SsCBF1 and its derivatives including the mutant version of SsCBF1^{193–228(W224A)} were fused to the Gal4-DBD (pGBKT7). The empty pGBKT7 vector was used as a negative control. Survival of yeast cells on the selective media needs the presence of functional activator peptides. X-a-Gal was used to test the expression of α-galactosidase.

Figure 8. SsCBF1 transcript levels in response to various abiotic stresses.

(A) Low-temperature induced expression pattern of SsCBF1 in S. lycopersicoides. S. lycopersicoides seedlings grown under standard conditions were transferred to a climate chamber set at 4°C for a 24 time course under constant light. The aerial parts were harvested for RNA extraction and qRT-PCR analysis. Zero time samples were taken prior to treatment. Transcript levels of SsCBF1 were normalized to the ACTIN2 expression. (B) Drought induced expression pattern of SsCBF1 in S. lycopersicoides. Detached young leaves of S. lycopersicoides were placed on a dry filter paper for drought treatment. Samples were collected according to the time course. The SsCBF1 mRNA levels were analyzed as in (A). (C) High-salinity induced expression pattern of SsCBF1 in S. lycopersicoides. Detached young leaves of S. lycopersicoides were placed on the filter paper soaked with NaCl solution (250 mM, 0.02% Tween-20) for a high-salinity treatment. Samples were collected at the indicated time points. The SsCBF1 mRNA levels were analyzed as in (A). Data shown are average and SD of triplicate reactions. Shown are representative data from one biological replicate and three biological replicates were conducted with similar results.

Figure 9. Freezing tolerance and delayed flowering phenotype of 35S:SsCBF1 transgenic Arabidopsis.

(A) Schematic representation of the construct used for Arabidopsis transformation of the SsCBF1 gene. (B) Relative expression of SsCBF1 in Col-0 and two T₃ generation transgenic lines (#11 and #18). Total RNA was extracted from 10-day-old seedlings, then analyzed by semi-quantitative RT-PCR. ACTIN7 gene was used as an internal control. (C) Comparison of freezing tolerance between Col-0 and two transgenic lines. See Methods for details. The right diagram indicates different genotypes used in the assay. (D) Late flowering phenotype of transgenic Arabidopsis overexpressing SsCBF1. Col-0 and two SsCBF1 OE lines were grown under the same conditions as described in Methods. Four-week-old plants were photographed.

Figure 10. Seed germination of transgenic Arabidopsis in response to NaCl.

(A–B) Seed germination ratio of Col-0 and two OE lines in the absence or presence of NaCl. Seeds from different genotypes were germinated on 1/2 MS agar plates (control, A) or supplemented with 150 mM NaCl (B), respectively. Germination was defined as the obvious sign of radicle tip emergence and scored daily until the indicated day. Data shown are average and SD of four independent experiments (each with 100 seeds for each genotype). (C) Increased tolerance of SsCBF1 transgenic lines to salt stress. Pictures were taken 3 days after stratification.

Figure 11. Effects of high salinity on root lengths of Col-0 and SsCBF1 transgenic lines.

(A) Representatives of Col-0 and two OE lines treated with 150 mM NaCl. Seeds of each genotype were germinated and grown vertically on MS medium for 4 d and then transferred to fresh MS medium containing 150 mM NaCl. Photographs were taken after 11d of growth. (B) Measurements of primary root lengths of plants shown in (A). All values are average and SD (n = 15). Asterisks denote Student's t test significance compared with Col-0 plants: **P<0.01. (C) High-salinity tolerance of Col-0 and two transgenic adult plants. 3-week-old plants (before treatment, upper panel) were irrigated with 300 mM NaCl solution every 3 d for 3 weeks. Bottom panel was photographed 3 weeks after the onset of irrigation.

Figure 12. qRT-PCR analysis of stress responsive genes in Col-0 and transgenic Arabidopsis seedlings in response to cold or salt stress.

The induction of stress-responsive genes (COR15A, RD29A, KIN2 and AtCBF1) was investigated by qRT-PCR. The experiments were repeated three times with similar results and the data shown are from one representative experiment. Error bars are SD of triplicate reactions.