Ectopic Overexpression of <i>SsCBF1</i>, a CRT/DRE-Binding Factor from the Nightshade Plant <i>Solanum lycopersicoides</i>, Confers Freezing and Salt Tolerance in Transgenic <i>Arabidopsis</i>

<div><p>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 (<i>SsCBF1</i>) was isolated from the cold-hardy plant <i>Solanum lycopersicoides</i>. 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 <i>SsCBF1</i> could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT <i>cis</i>-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of <i>SsCBF1</i> conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic <i>Arabidopsis</i>. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic <i>Arabidopsis</i>. 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.</p></div

Southern blot analysis of the <i>SsCBF1</i> gene.

<p><i>S. lycopersicoides</i> genomic DNA was digested with restriction enzymes <i>Bam</i>H I, <i>Hin</i>d III, and <i>Xba</i> I. The hybridization was performed using the full-length <i>SsCBF1</i> as a probe labeled with Digoxin.</p

qRT-PCR analysis of stress responsive genes in Col-0 and transgenic <i>Arabidopsis</i> seedlings in response to cold or salt stress.

<p>The induction of stress-responsive genes (<i>COR15A</i>, <i>RD29A</i>, <i>KIN2</i> and <i>AtCBF1</i>) 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.</p

Sequence analysis of SsCBF1.

<p>(<b>A</b>) 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>B</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, <i>Solanum tuberosum</i>; Sc, <i>Solanum commersonii</i>; Sl, <i>Solanum lycopersicum</i>; Ss, <i>Solanum lycopersicoides</i>; Ca, <i>Capsicum annuum</i>; At, <i>Arabidopsis thaliana</i>. 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).</p

<i>SsCBF1</i> transcript levels in response to various abiotic stresses.

<p>(<b>A</b>) Low-temperature induced expression pattern of <i>SsCBF1</i> in <i>S. lycopersicoides</i>. <i>S. lycopersicoides</i> 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 <i>SsCBF1</i> were normalized to the <i>ACTIN2</i> expression. (<b>B</b>) Drought induced expression pattern of <i>SsCBF1</i> in <i>S. lycopersicoides</i>. Detached young leaves of <i>S. lycopersicoides</i> were placed on a dry filter paper for drought treatment. Samples were collected according to the time course. The <i>SsCBF1</i> mRNA levels were analyzed as in (A). (<b>C</b>) High-salinity induced expression pattern of <i>SsCBF1</i> in <i>S. lycopersicoides</i>. Detached young leaves of <i>S. lycopersicoides</i> 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 <i>SsCBF1</i> 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.</p

Up-regulated genes in <i>35S:SsCBF1</i> transgenic plants (Q-value <0.001; fold change >2).

<p>Up-regulated genes in <i>35S:SsCBF1</i> transgenic plants (Q-value <0.001; fold change >2).</p

Effects of high salinity on root lengths of Col-0 and <i>SsCBF1</i> transgenic lines.

<p>(<b>A</b>) 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>B</b>) Measurements of primary root lengths of plants shown in (A). All values are average and SD (n = 15). Asterisks denote Student's <i>t</i> test significance compared with Col-0 plants: **<i>P</i><0.01. (<b>C</b>) 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.</p

Low-temperature resistance test of <i>S. lycopersicoides</i> and CM.

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

Freezing tolerance and delayed flowering phenotype of <i>35S:SsCBF1</i> transgenic <i>Arabidopsis</i>.

<p>(<b>A</b>) Schematic representation of the construct used for <i>Arabidopsis</i> transformation of the <i>SsCBF1</i> gene. (<b>B</b>) Relative expression of <i>SsCBF1</i> 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. <i>ACTIN7</i> gene was used as an internal control. (<b>C</b>) 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. (<b>D</b>) Late flowering phenotype of transgenic <i>Arabidopsis</i> overexpressing <i>SsCBF1</i>. Col-0 and two <i>SsCBF1</i> OE lines were grown under the same conditions as described in Methods. Four-week-old plants were photographed.</p

Transactivation of promoters with CRT elements only.

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