The wheat VRN2 gene is a flowering repressor down-regulated by vernalization

Abstract

Plants with a winter growth habit flower earlier when exposed for several weeks to cold temperatures, a process called vernalization. We report here the positional cloning of the wheat vernalization gene VRN2, a dominant repressor of flowering that is down-regulated by vernalization. Loss of function of VRN2, whether by natural mutations or deletions, resulted in spring lines, which do not require vernalization to flower. Reduction of the RNA level of VRN2 by RNA interference accelerated the flowering time of transgenic winter-wheat plants by more than a month.

Fig. 1

A. Genetic map of the VRN2 region on chromosome 5A^m of T. monococcum based on 5,698 gametes. Numbers of crossovers in the critical recombinant plants are indicated in boxes. B. Physical map of the wheat VRN2 region in T. monococcum and in colinear regions from barley and rice. BAC clones indicated in red have been sequenced (438,828-bp, AY485644). The order of BAC clones from left to right is: 374A18, 94E8, 304H18, 258C22, 301G15, 615O6, 650N20, 405L8, 271O11, 275P20, 157P20, 455C17, 322L23, 702K8, 32A1, 533H16 and 324G2 (bold letters indicate sequenced BACs). Additional information for the markers used in this figure has been deposited in the SOM.

Fig. 2

A. RT-PCR from leaves of unvernalized (U) or vernalized (V) G3116 winter wheat plants. RNA samples from the vernalized plants (6 weeks at 4°C) were collected 5 days after returning the plants to room temperature. Among the three genes completely linked to VRN2, RNA levels of ZCCT1 and ZCCT2 were down regulated by vernalization and those of AY485644.3 were not affected by vernalization. In the same cDNA samples, AP1 RNA levels were up regulated by vernalization. M indicates molecular weight DNA marker. B-C) Quantitative PCR. B. Leaves: Transcript levels of ZCCT1 (red scale) and AP1 (blue scale) relative to UBIQUITIN in G3116 (averages of 5 plants ± SE): 0: before 4°C; 2w, 4w, 6w: weeks at 4°C; 2w out: 2 weeks at room temperature after vernalization. C. Apices: Transcript levels of ZCCT1, ZCCT2 and AP1 (=VRN1) relative to ACTIN in G3116 (averages of 3 pools of apices from 5 plants each ± SE). U= unvernalized, V= 3–5 days at room temperature after 6 weeks of vernalization. Units are linearized values using the 2^(−ΔΔC_T) method, where CT is the threshold cycle.

Fig. 3

A. Transgenic Jagger T₁ plants segregating for the presence and absence of the RNA interference construct for ZCCT1 and for flowering time. B. Average heading date of T₁ plants carrying the transgene (31 plants) and without the transgene (11 plants). C-F. Average RNA level of 8 positive and 8 negative T₁ plants from the progeny of the early flowering T₀ plant. Units are linearized values using the 2^(−ΔΔC_T) method, where CT is the threshold cycle. C. RNA level of the RNAi construct; D-F. Endogenous RNA levels of D. ZCCT1, E. ZCCT2, F. AP1. Error bars indicate one standard error of the mean.