Heterologous expression of wheat VERNALIZATION 2 (TaVRN2) gene in Arabidopsis delays flowering and enhances freezing tolerance

Abstract

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.

Figure 1. TaVRN2 structure analysis.

A) Gene structure showing the two exons and one intron. Exon1 contains the zinc finger domain and exon2 contains the CCT domain. B) The alignment of TaVRN-A2 and TaVRN-B2 proteins. Regions of putative zinc finger highlighted in red, CCT domains highlighted in blue and difference of nine amino acids highlighted in yellow. C) The alignment of CCT domain of the two copies of TaVRN2 showing their different SNPs (in white and red). The sequence highlighted in yellow in the TaVRN-A2 corresponds to NcoI restriction site. D) Proteins alignment of CCT domain, Alignments are done by using Clustal W program from the web.

Figure 2. Transcripts level of TaVRN-B2 and TaVRN-A2 in both spring and winter wheat during cold acclimation.

A) TaVRN-B2 and TaVRN-A2, in cold acclimated plants. Wheat plants were grown for 14 days at 20°C under a long day (16 h) photoperiod, transferred to 4°C under identical photoperiods, and then sampled at regular intervals. B) TaVRN-B2 in cold acclimated plants under short day and long day conditions. Winter wheat plants were grown for 14 days at 20°C under either a long day (16 h) or a short day (8 h) photoperiod, transferred to 4°C under identical photoperiods, and then sampled at regular intervals. qRT-PCR were done using total reverse-transcribed RNA isolated from wheat aerial part. C) TaVRN-B2 relative transcripts abundance in different tissues. Winter wheat were grown for 7 days at 20°C. Non-acclimated control plants (NA) were maintained at 20°C for 6 days. Cold-acclimated plants (CA) were transferred at 4°C for 36 days. Total RNA was isolated from leaves, crown and roots, reverse-transcribed and subjected to qRT-PCR. Data shown represent mean values obtained from independent amplification reactions (n = 4), and the error bars indicate the range of possible RQ values define by the SE of the delta threshold cycles (Cts). Experiment was repeated three times with similar results.

Figure 3. Promoter analysis and transcripts level of TaVRN2 in response to various abiotic stresses.

A) Putative regulatory cis-elements in TaVRN2 promoter (MBS: MYB binding site involved in drought-inductibilty, ABRE: cis-acting element involved in abscisic acid responsiveness, HSE: cis-acting element involved in heat stress responsiveness, ACE: cis-acting element involved in light responsiveness, DRE: cis-acting element involved in cold stress and TATA-box). The plant CARE and PLACE programs were used for the promoter analysis. B) Transcripts abundance in winter wheat exposed to various stresses. Seven days old plants were exposed to different stresses: Non-acclimated (NA), cold acclimated (CA), wounding (W), heat shock (HS), salt (NaCl), water stress (RWC), abscisic acid (ABA). Data shown represent mean values obtained from independent amplification reactions (n = 4), experiment was repeated three times with similar results. A statistical difference between each sample and the expression observed in non acclimated plants is indicated by an asterisk on top of each histogram columns. The threshold for statistical significance was: *: p<0.05; **: p<0.01; ***: p<0.001; ns: P>0.05.

Figure 4. Analysis of transgenic Arabidopsis plants overexpressing TaVRN-B2.

A) Effect of TaVRN-B2 expression on flower initiation and development during growth under long day conditions at 20°C. The number of plants is expressed as means SEM (n = 99 plants). A statistical difference measurement from three independent experiments. The threshold for statistical significance is indicated by an asterisk on top of each histogram columns; in vertical, comparison between GUS and the three LINES and in horizontal, comparison between Line1 and two others lines; *: P<0.05, **: P<0.01, ***: P<0.001, ns: P>0.05 and sv: same value, statistical analysis is not possible. B) Phenotypic effect of TaVRN-B2 overexpression in Arabidopsis. Control plants 35S::GUS (GUS) and transgenic plants 35S::TaVRN-B2 (Line1) were grown under long day conditions at 20°C. Pictures were taken at 7, 14, 28, 42 and 50 days (d). C) Number of rosette leaves at the time of bolting in control plants (35S::GUS) and 3 transgenic lines 35S::TaVRN-B2 (L1–L3). Plants were grown under long-day conditions at 20°C and leaves were counted when the first bolt became visible. The number of leaves is expressed as means SEM (n = 18 plants). A statistical difference measurements from three independent experiments (n = 18). The threshold for statistical significance is indicated by an asterisk on top of each histogram columns; comparison between GUS and the three LINES; *: P<0.05, **: P<0.01, ***: P<0.001, ns: P>0.05. D) Transcript level of TaVRN2 in controls (wild type (WT) and 35S::GUS) and transgenic lines (L1–L3). Total RNA was extracted from leaves of 15 day-old plants grown under LD conditions at 20°C. TaVRN-B2 transcript levels were measured by RT–PCR. Each experiment was repeated three times with three biological replicas.

Figure 5. Effect of TaVRN-B2 overexpression on Arabidopsis flowering genes.

A) Expression pattern of flowering genes under unvernalized conditions in control (35S::GUS) and transgenic 35S::TaVRN-B2 plants. Seven and 14 days (d) old plants were grown at 20°C under LD conditions. Relative transcript abundance is normalized in relation to the level of each gene at 7 days of 35S::GUS control sample. B) Expression pattern of FLC under cold acclimation conditions in control (35S::GUS) and transgenic 35S::TaVRN-B2 plants (Line 1). Fifteen days old plants were grown at 4°C for 5 weeks. Relative transcript abundance in normalized to the 35S::GUS control sample at zero time. C) Total number of leaves (n = 32) of cold acclimated control (35S::GUS) and transgenic 35S::TaVRN-B2 plants (Line 1) at bolting stage.

Figure 6. TaVRN-B2 enhances freezing tolerance in Arabidopsis.

A) Control and transgenic plants after freezing test. Plants were grown for 3 weeks at 20°C (NA) or grown for 3 weeks at 20°C then transferred to 4°C for 7 days (CA7). Controls plants wild type (WT) and 35S::GUS), transgenic 35S::TaVRN-B2 lines (Line1–Line3). Plants were subjected to freezing test (NA frozen to −6.5°C and CA7 frozen to −10.5°C). Pictures were captured for the same plants before the freezing and after a recovery period of 2 weeks. B) Survival rate after freezing stress expressed as a percent of surviving plants. A statistical difference measurements from three independent experiments (n = 33). The threshold for statistical significance is indicated by an asterisk on top of each histogram columns; comparison between GUS and the three LINES; *: P<0.05, **: P<0.01, ns: P>0.05.

Figure 7. Effect of TaVRN-B2 over-expression on the accumulation of cold-regulated transcripts.

A) Transcript level of CBFs genes. Control (35S::GUS) and transgenic 35S::TaVRN-B2 plants (line 1) grown under long day conditions and exposed to 4°C for 3 and 6 hours. B) Transcript level of CORs genes. Control (35S::GUS) and transgenic 35S::TaVRN-B2 plants (line 1) grown under long day conditions at 4°C for 1 and 2 weeks. Transcripts levels were measured by RT-PCR. Each experiment was repeated three times using RNA prepared from three biological samples with similar results.