Regulation of freezing tolerance and flowering in temperate cereals: the VRN-1 connection

Abstract

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Figure 1.

qRT-PCR analysis of transcript levels of the CBF genes present at the FR-2 locus relative to the ACTIN endogenous control. Samples were collected from leaves of 4-week-old mvp-2/mvp-2 and Mvp-2/− plants (20°C) and again 1 d later at the same time following an 8-h cold treatment at 4°C. Values on the y axis were normalized and calibrated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). The same calibrator was used for all genes, so scales are comparable across genes. Values are averages of eight biological replications ± se. The inset shows CBF14 transcript levels, which were significantly higher than the other genes at this locus. P values for the differences between mvp/mvp and Mvp/− after the cold treatment were calculated using ANOVA and are indicated by asterisks: * P < 0.05, ** P < 0.01.

Figure 2.

qRT-PCR transcript levels of COR14b relative to TEF1 endogenous control. Plants were 4 weeks old at the beginning of the experiment and were exposed to 4°C for 12 d. Values on the y axis were normalized and calibrated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Homozygous mvp-2/mvp-2 plants (null VRN-1) are indicated by black squares and lines, and Mvp-2/− plants (one or two VRN-1 copies) are indicated by gray triangles and lines. Values are averages of eight biological replications in the untransformed scale ± se. P values were calculated using ANOVA of log_{(n + 1)}-transformed values for each time point: ** P < 0.01.

Figure 3.

Time courses of VRN-1 and COR genes COR14b, DHN5, and DHN8 in mvp-2/mvp-2 plants homozygous for a VRN-1 deletion (m) and Mvp-2/− plants with one or two functional VRN-1 copies (M). The presence of a faint VRN-1 hybridization in the 87-d mutant sample is suspected to be due to cross-contamination.

Figure 4.

VRN-1 and COR14b transcript levels in a set of T. monococcum lines differing in VRN-1 expression under short days. Abbreviations are as follows: W, warm conditions; C, decrease from 18°C to 6°C (occurring at daybreak); 2W, 2 weeks old; 6W, 6 weeks old; SD, short day; LD, long day; vrn-1, wild type; Vrn-1f, allele with a 1-bp deletion in the CArG box coupled with the VRN-1 intron 1 insertion; Vrn-1g, allele with a 34-bp deletion encompassing the CArG box; Vrn-1h, allele with an insertion in VRN-1 intron 1. The three accessions with the Vrn-1f allele carry the dominant Vrn-2 allele, whereas all the other accessions carry nonfunctional vrn-2 alleles. All the accessions have a spring growth habit. A, mRNA-blot analyses of three genotypes per promoter class (indicated by different PI numbers). Arrows and arrowheads identify the presence and absence, respectively, of COR14b transcripts in 6-week-old plants in the Vrn-1f and Vrn-1g natural mutants under short days and long days. B and C, qRT-PCR validation of VRN-1 and COR14b transcript levels relative to the ACTIN endogenous control at 4°C. Values on the y axes were normalized and calibrated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Lines carrying the wild-type allele (vrn-1) or Vrn-1h (spring, not induced in short days) are indicated by black bars, and lines carrying the Vrn-1f and Vrn-1g alleles (spring, induced in short days) are indicated by gray bars. Values are averages of five biological replications ± se. P values were calculated using contrasts between either vrn-1 or Vrn-1h and the average of the lines carrying the Vrn-1f and Vrn-1g alleles. Samples were collected when the plants were 2 weeks old (B) and 6 weeks old (C).