Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2

Abstract

Wheat is usually classified as a long day (LD) plant because most varieties flower earlier when exposed to longer days. In addition to LD, winter wheats require a long exposure to low temperatures (vernalization) to become competent for flowering. Here we show that in some genotypes this vernalization requirement can be replaced by interrupting the LD treatment by 6 weeks of short day (SD), and that this replacement is associated with the SD down-regulation of the VRN2 flowering repressor. In addition, we found that SD down-regulation of VRN2 at room temperature is not followed by the up-regulation of the meristem identity gene VRN1 until plants are transferred to LD. This result contrasts with the VRN1 up-regulation observed after the VRN2 down-regulation by vernalization, suggesting the existence of a second VRN1 repressor. Analysis of natural VRN1 mutants indicated that a CArG-box located in the VRN1 promoter is the most likely regulatory site for the interaction with this second repressor. Up-regulation of VRN1 under SD in accessions carrying mutations in the CArG-box resulted in an earlier initiation of spike development, compared to other genotypes. However, even the genotypes with CArG box mutations required LD for a normal and timely spike development. The SD acceleration of flowering was observed in photoperiod sensitive winter varieties. Since vernalization requirement and photoperiod sensitivity are ancestral traits in Triticeae species we suggest that wheat was initially a SD-LD plant and that strong selection pressures during domestication and breeding resulted in the modification of this dual regulation. The down-regulation of the VRN2 repressor by SD is likely part of the mechanism associated with the SD-LD regulation of flowering in photoperiod sensitive winter wheat.

Figure 1

A) Days to heading in winter T. monococcum accession G3116 under continuous LD or six weeks SD followed by LD. “Ver.” indicates plants vernalized for 6 weeks at 4 ºC whereas “Not ver.” indicates non-vernalized plants. Values are averages of 5 plants ± SE of the means. LD non-vernalized plants were discarded after 120 days without flowering. B) Days to heading in RNAi::VRN2 transgenic plants of hexaploid wheat ‘Jagger’ (TR) and the non-transgenic controls (NT). LD and SD indicate similar conditions to those described in ‘A’. C) VRN2 transcript levels in the same plants described in ‘B’. D) Comparison of VRN1 transcript levels in vernalized and unvernalized plants of T. monococcum G3116 grown under SD. E) Time course transcription profiles of VRN1 (red line, filled diamonds) and VRN2 (blue line, open diamonds). Initial samples (0w) were collected immediately before transferring unvernalized G3116 plants grown under LD for 7 weeks to SD (20°C). Plants were grown under SD for 6 weeks and samples were collected every week (1w to 6w). Finally, plants were transferred to LD for five weeks (1w to 5w). Note the delay in VRN1 transcription until the transfer to LD. C–G) Units for the Y-axis are linearized values using the 2^{(Δ ΔC_T)} method, where C is the threshold cycle (Livak and Schmittgen, 2001). ACTIN was used as endogenous control in C (SYBR GREEN) and UBIQUITIN in D and E (TaqMan).

Fig. 2

Sequence differences among VRN1 alleles. Top: 74-bp segment of the VRN1 promoter showing the location of the putative CArG box in blue and the CAP signal for transcriptional initiation underlined. Middle: schematic representation of the VRN1 gene in T. monococcum. Bottom: repetitive element inserted in VRN1 first intron in PI 306540 (Vrn1h) and PI 503874 (Vrn1f).

Fig. 3

A) VRN1 transcript levels in T. monococcum spring lines carrying Vrn1f (PI 503874, 1-bp deletion), Vrn1g (PI 349049, 34-bp deletion), Vrn1h (PI 306540, intron one insertion), and recessive vrn1 alleles combined with recessive vrn-2b (PI 323437) and vrn2a (DV92) alleles. B) Comparison of homozygous vrn1 and Vrn1 F2 plants from the cross between DV92 (vrn1 vrn2a) with PI 266844 (Vrn1f, Vrn2) or PI 326317 (Vrn1g vrn2b). We first selected plants homozygous for the recessive vrn2 allele using molecular markers to avoid differences in growth habit. C) VRN1 transcript levels in additional T. monococcum lines carrying different vernalization alleles. A–C) Units for the Y-axis are linearized values using the 2^{(−Δ ΔC_T)}method, where C is the threshold cycle (Livak and Schmittgen, 2001). TaqMan system for ACTIN (A) and UBIQUITIN (B and C) were used as endogenous controls. D–E) Apex development in plants of T. monococcum F2 plants from the cross between PI 266844 (Vrn1f) x DV92 (vrn1). Five homozygous lines with and without the 1-bp deletion in the CArG-box were selected within the homozygous vrn2a lines (spring growth habit). Apexes were observed D) after six weeks under SD (8 h of light) and E) two weeks after transferring the plants back to LD.

Fig. 4

Comparison of apex development in T. monococcum plants. A–B and E) Winter G3116 (vrn1); C and F) PI 306540 (Vrn1h); D & G) PI 266844 (Vrn1f). A) Continuous LD, vegetative stage. Under the same conditions lines with the dominant Vrn1h and Vrn1f alleles had already headed (not shown). B, C, D) Continuous SD. E, F, G) 10 weeks at SD and 4 weeks at LD (SD-LD). Note the more advanced development of the inflorescence in Vrn1f relative to vrn1 and Vrn1h in both SD and SD-LD. White bars represent 0.5 mm.