Interaction of photoperiod and vernalization determines flowering time of Brachypodium distachyon

Abstract

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Figure 1.

Vernalization and photoperiod interact to determine flowering time in a range of Brachypodium spp. accessions. A and B, Imbibed seeds of accessions were cold treated at 5°C in soil in an 8-h photoperiod for the indicated length of time (weeks), followed by outgrowth in a growth chamber set to 20-h light/4-h dark (A) or 16-h light/8-h dark (B). Flowering time is measured as the number of days to spike emergence (Zadoks scale = 50) from the end of cold treatment. Arrows indicate treatments where plants did not flower within the 120 d of the experiment. For TR8n in A and B, only flowering data for 12, 14, and 16 weeks of cold is presented (see Supplemental Fig. S1 for all flowering data for this accession.) C, Photographs of representative plants from each of the six flowering classes (described in the text) after 60 d of outgrowth in a 16-h photoperiod after vernalization. Number in parentheses below class name represents the number of accession(s) evaluated in this study that fall within that particular flowering class. ERF, Extremely rapid flowering; RF, rapid flowering; IRF, intermediate rapid flowering; IDF, intermediate delayed flowering; DF, delayed flowering; EDF, extremely delayed flowering. Bar = 5 cm.

Figure 2.

Effect of different photoperiods during and after vernalization on flowering time. A, Imbibed seeds sown on plates were exposed to 5°C for 3 weeks (Bd21, Bd21-3) or 7 weeks (Bd1-1) in either no light (DD), 8-h light/16-h dark (SD), 16-h light/8-h dark (LD), or constant light (LL). Nonvernalized controls were exposed to a similar light regime to an equivalent stage of development that the vernalized seedlings reached by the end of the cold treatment. Plants were grown in a 16-h photoperiod following vernalization after transplanting to soil. B, Imbibed seeds sown in soil were exposed to 5°C for 3 weeks (under a 16-h photoperiod) before shifting to one of four photoperiods: 12-h light/12-h dark, 14-h light/10-h dark, 15-h light/9-h dark, or 16-h light/8-h dark. For A and B, flowering time reflects the number of days to spike emergence (Zadoks scale = 50) from the end of cold treatment. Arrows indicate treatments where plants did not flower during the 120 d of the experiment. Asterisk indicates some plants within a treatment did not flower after 120 d. C, Photos of 60-d-old plants from B. NV, Nonvernalized; V, vernalized. Bar = 5 cm.

Figure 3.

Temperature range that satisfies the vernalization requirement. Imbibed Bd21-3 or Bd1-1 seeds were sown in soil and allowed to germinate to Zadoks stages 07 to 10. Seedlings were then transferred to different vernalization temperatures under a 16-h photoperiod for 3 weeks (Bd21-3) or 7 weeks (Bd1-1) before shifting to outgrowth in a 16-h photoperiod. Outgrowth conditions averaged 22°C during the light period and 18°C during the dark period under a 16-h photoperiod. During vernalization, temperatures fluctuated diurnally in a 16-h photoperiod (data not shown). A, Representative images of Bd21-3 plants after different temperatures during cold treatment (all plants are the same age). Flowering time of Bd21-3 (B) or Bd1-1 (C) plants measured as the number of primary leaves produced by the main shoot at the time of flowering. Arrows indicate plants did not flower during the 100 d of the experiment. NV, Nonvernalized. [See online article for color version of this figure.]

Figure 4.

VRN1, VRN2L, and FT expression in different accessions and photoperiods without cold treatment. A,C, and E, RT-qPCR of VRN1 (A), FT (C), and VRN2L (E) expression in the second leaf of Brachypodium spp. accessions. Plants were grown nonvernalized in a 20-h photoperiod and harvested at leaf stage 2 (Zadoks stages = 12–13). B,D, and F, RT-qPCR of VRN1 (B), FT (D), and VRN2L (F) expression in the second leaf of Brachypodium spp. accessions (Bd21, Bd21-3, and Bd1-1) grown nonvernalized in 8-, 16-, or 20-h photoperiods and harvested at leaf stage 2 during the middle of the photoperiod. Bars represent the average of three biological replicates ± 1 sd. DNF, Did not flower during 120 d of growth.

Figure 5.

VRN1 and FT overexpression cause rapid flowering. A, Representative photos of Bd21-3 wild-type, nontransgenic sibling plants (sibling), empty pANIC10A vector transformed plants (control), and UBI:VRN1 and UBI:FT plants grown nonvernalized in a 16-h photoperiod. Bar = 4 cm. B, Flowering time of Bd21-3 wild-type compared with independent UBI:VRN1 transgenic lines. Dark gray indicates plants carrying UBI:VRN1, and light gray indicates sibling plants segregating from the same primary transformant mother plant that do not carry the transgene. C, Flowering time averages of Bd21-3 wild-type and empty vector control plants (control) compared with flowering time of individual UBI:FT transgenic plants from different transgenic lines. For B and C, plants were grown nonvernalized in a 16-h photoperiod, and bars represent the average ± 1 sd. D to I, RT-qPCR data of leaf 2 from two-leaf stage UBI-VRN1, UBI-FT, or Bd21-3 control plants grown nonvernalized in a 16-h photoperiod. VRN1 expression (D and G), FT expression (E and H), and VRN2L expression (F and I). For D to I, bars represent the average of three biological replicates ± 1 sd. Single asterisk indicates P value < 0.01, and two asterisks indicate P value < 0.05. NT sib, Nontransgenic sibling plant. [See online article for color version of this figure.]

Figure 6.

VRN1, FT, and VRN2L gene expression with and without cold treatment of imbibed seeds. Imbibed seeds were given a subsaturating (short cold) vernalization treatment (1 week at 5°C for Bd21 and Bd21-3; 3 weeks at 5°C for Bd1-1) or a saturating (long cold) vernalization treatment (3 weeks at 5°C for Bd21 and Bd21-3; 7 weeks at 5°C for Bd1-1). Leaf 2 was harvested 11 d after removing from cold (B–D) or from emerging shoot tissue at the end of the cold treatment (A). Tissue was harvested at a similar stage of development from nonvernalized controls. Plants were grown in a 16-h photoperiod during and after vernalization. For C, Bd1-1 was treated for 6 weeks of cold. A, RT-qPCR of VRN1 in shoot tissue of Bd21, Bd21-3, and Bd1-1 during cold treatment. B, RT-qPCR of VRN1 in leaf 2 of Bd21, Bd21-3, and Bd1-1 after cold treatment. C, RT-qPCR of FT in leaf 2 of Bd21, Bd21-3, and Bd1-1 after cold treatment. D, RT-qPCR of VRN2L in leaf 2 of Bd21, Bd21-3, and Bd1-1 after cold treatment. Bars represent mean of three biological replicates ± se. Black indicates Bd21, gray indicates Bd21-3, and white indicates Bd1-1. NV, Nonvernalized; V, vernalized.

Figure 7.

VRN2L gene expression in leaves of vernalized plants during and after cold treatment. Seedlings were grown to the two-leaf stage (Zadoks stage = 12) before transferring to 5°C for 3 weeks. After cold treatment, plants were grown in a 16-h photoperiod. A, RT-qPCR of VRN2L in leaf 2 of nonvernalized (black) versus vernalized (gray) Bd21-3 at the end of a saturating vernalization treatment conducted in short days (8-h photoperiod) or long days (16-h photoperiod). B, RT-qPCR of VRN2L in leaf 2 of nonvernalized (black) versus vernalized (gray) Bd21-3 after cold treatment performed in 16-h photoperiod. Bars represent the average of three biological replicates ± 1 sd.

Figure 8.

VRN2L expression in response to cold. A, RT-qPCR of VRN2L in leaf 2 in plants that received the indicated lengths of cold treatments (5°C) in a 16-h photoperiod. Expression values of each treatment are shown relative to noncold-treated plants harvested at the same time of day. Bars represent an average of three biological replicates ± 1 sd.