Low-temperature and daylength cues are integrated to regulate FLOWERING LOCUS T in barley

Abstract

Interactions between flowering time genes were examined in a doubled haploid barley (Hordeum vulgare) population segregating for H. vulgare VERNALIZATION1 (HvVRN1), HvVRN2, and PHOTOPERIOD1 (PPD-H1). A deletion allele of HvVRN2 was associated with rapid inflorescence initiation and early flowering, but only in lines with an active allele of PPD-H1. In these lines, the floral promoter FLOWERING LOCUS T (HvFT1) was expressed at high levels without vernalization, and this preceded induction of HvVRN1. Lines with the deletion allele of HvVRN2 and the inactive ppd-H1 allele did not undergo rapid inflorescence initiation and were late flowering. These data suggest that HvVRN2 counteracts PPD-H1 to prevent flowering prior to vernalization. An allele of HvVRN1 that is expressed at high basal levels (HvVRN1-1) was associated with rapid inflorescence initiation regardless of HvVRN2 or PPD-H1 genotype. HvFT1 was expressed without vernalization in lines with the HvVRN1-1 allele and HvFT1 transcript levels were highest in lines with the active PPD-H1 allele; this correlated with rapid apex development postinflorescence initiation. Thus, expression of HvVRN1 promotes inflorescence initiation and up-regulates HvFT1. Analysis of HvVRN1 expression in different genetic backgrounds postvernalization showed that HvVRN2, HvFT1, and PPD-H1 are unlikely to play a role in low-temperature induction of HvVRN1. In a vernalization responsive barley, HvFT1 is not induced by low temperatures alone, but can be induced by long days following prolonged low-temperature treatment. We conclude that low-temperature and daylength flowering-response pathways are integrated to control expression of HvFT1 in barley, and that this might occur through regulation of HvVRN2 activity.

Figure 1.

Loss of HvVRN2 causes rapid inflorescence initiation and increased HvFT1 expression in plants with active PPD-H1 genotype. A, Heading date (average days to head emergence) of doubled haploid lines deleted for HvVRN2 (HvVRN1;ΔHvVRN2) and Halcyon grown in long-day glasshouse conditions. A, Active PPD-H1 allele. I, inactive ppd-H1 allele. Error bars represent se. B, Average length of the shoot apex in 14-d-old plants from the same lines. C, The shoot apices of representative lines carrying PPD-H1 or ppd-H1 alleles compared to Halcyon at different time points (days). DR, Double ridge, the first visible marker of inflorescence initiation. D, Expression levels of HvFT1 and HvVRN1 assayed by RT-PCR (40× cycles) in 7- and 14-d-old plants. Expression of ACTIN is shown as a loading comparison (25× cycles). E, Relative expression levels of HvFT1 and HvVRN1 in plants deleted for HvVRN2 but with different PPD-H1 genotypes. RNA from was pooled from four PPD-H1 lines or four ppd-H1 lines (same lines as in D) and expression levels were assayed by qRT-PCR and normalized to ACTIN. Asterisks indicate P value of Student's t tests: **, P < 0.01. ***, P < 0.001. Error bars represent se of triplicate reactions. [See online article for color version of this figure.]

Figure 2.

Overexpression of HvVRN2 down-regulates HvFT1 and delays flowering. A, Heading date (average days to head emergence) in long-day glasshouse conditions for transgenic plants homozygous for the HvVRN2 overexpression construct (Ubi:VRN2) and sibling null controls (Null) in the T2 generation. Asterisks indicate P values of Student's t tests: ***, P < 0.001. B, Development of the shoot apex in transgenic plants homozygous for the HvVRN2 overexpression construct (Ubi:VRN2) and sibling null controls (Null), in the T2 generation grown in long-day glasshouse conditions. Apices were sampled at various time points (days). DR, Double ridge, the first visible marker of inflorescence initiation. C, Relative expression levels of HvVRN2, HvVRN1, and HvFT1 assayed by qRT-PCR and normalized to ACTIN in RNA from a transgenic plant homozygous for the HvVRN2 overexpression construct (Ubi:VRN2) and a sibling null control (Null), in the T2 generation grown in long-day glasshouse conditions. Error bars represent se of triplicate reactions. ND, No expression was detected; NS, no significant difference between lines according to Student's t tests. [See online article for color version of this figure.]

Figure 3.

Expression of HvVRN1 promotes rapid inflorescence initiation independently of PPD-H1 genotype. A, Heading date (average days to head emergence) of doubled haploid lines with an allele of HvVRN1 that is expressed to high levels without vernalization (HvVRN1-1;HvVRN2) grown in long-day glasshouse conditions. A, Active PPD-H1 allele. I, Inactive ppd-H1 allele. Error bars represent se. B, Average length of the shoot apex in 14-d-old plants from the same lines. C, The shoot apices of representative lines carrying PPD-H1 or ppd-H1 alleles at different time points (days). DR, Double ridge, the first visible marker of inflorescence initiation. D, Expression levels of HvFT1, HvVRN1, and HvVRN2 assayed by RT-PCR in 14-d-old plants (40× cycles). Expression of ACTIN is shown as a loading comparison (25× cycles). E, Relative expression levels of HvFT1 and HvVRN1 in plants with the HvVRN1-1 allele but with different PPD-H1 genotypes and relative expression levels of HvVRN2 in nonvernalized plants with the HvVRN1 allele and the HvVRN1-1 allele. RNA from was pooled from five PPD-H1 lines or five ppd-H1 lines (same lines as in D), then expression levels were assayed by qRT-PCR and normalized to ACTIN. Asterisks indicate P value of Student's t tests: **, P < 0.01. NS, Not significant. Error bars represent se of triplicate reactions. [See online article for color version of this figure.]

Figure 4.

PPD-H1 genotype influences flowering time and HvFT1 expression levels in vernalized plants. A, Heading date (average days to head emergence) in long-day glasshouse conditions for nonvernalized (NV) or vernalized (+V) plants from 10 vernalization-requiring doubled haploid lines (HvVRN1;HvVRN2). A, Active PPD-H1 allele. I, Inactive ppd-H1 allele. Error bars represent se. B, Average length of the shoot apex in vernalized plants from the same lines. Plants were grown in long-day glasshouse conditions postvernalization, and were sampled after 1 week. C, The shoot apices of vernalized plants from representative lines. D, Expression levels of HvFT1, HvVRN1, and HvVRN2 (40× cycles) assayed by RT-PCR in nonvernalized plants after 2 weeks in long-day glasshouse conditions and vernalized plants after 1 week in long-day glasshouse conditions. Expression of ACTIN is shown as a loading comparison (25× cycles). E, Relative expression levels of HvFT1 and HvVRN1 in nonvernalized and vernalized plants (HvVRN1;HvVRN2). RNA from was pooled from seven PPD-H1 lines or three ppd-H1 lines (same lines as in D), then expression levels were assayed by qRT-PCR and normalized to ACTIN. Asterisks indicate P value of Student's t tests: ***, P < 0.001. NS, Not significant. Error bars represent se of triplicate reactions. [See online article for color version of this figure.]

Figure 5.

HvVRN1 is induced by low temperatures in the absence of HvVRN2. A, Heading date (average days to head emergence) in long-day glasshouse conditions for nonvernalized (NV) and vernalized (+V) plants from lines deleted for HvVRN2 and carrying an inactive ppd-H1 allele (HvVRN1;ΔHvVRN2;ppd-H1). B, Average length of the shoot apex in the same lines, in 14-d-old nonvernalized plants, compared to vernalized plants that had grown in long-day glasshouse conditions for 1 week postvernalization to reach a similar stage of development (leaf number). C, Apices from representative nonvernalized and vernalized plants at the same time points. DR, Double ridge, the first visible marker of inflorescence initiation. D, Expression levels of HvFT1 and HvVRN1 assayed by RT-PCR in vernalized plants (HvVRN1;ΔHvVRN2;ppd-H1; expression levels of HvFT1 and HvVRN1 in nonvernalized plants at the same developmental stage [Zadoks scale 12] are presented in Figure 1; 40× cycles). Expression of ACTIN is shown as a loading comparison (25× cycles). [See online article for color version of this figure.]

Figure 6.

Single nucleotide polymorphisms in the first intron of HvFT1 do not influence the vernalization requirement in the Sloop × Halcyon population. HvFT1 genotype, scored by CAPS marker, in 10 vernalization-requiring lines (HvVRN1;HvVRN2; see “Materials and Methods”). H, Halcyon allele; S, Sloop allele. Flowering time (days to head emergence) for nonvernalized (NV) and vernalized (+V) plants in long days in glasshouse conditions.

Figure 7.

HvFT1 is not induced by low temperatures alone. A, Relative expression levels of HvFT1 assayed by qRT-PCR and normalized to ACTIN in RNA from nonvernalized plants (Sonja, minus roots) that were grown for 2 weeks in short days then the following: maintained in short days for 2 weeks (SD), shifted to long days for 2 weeks (LD), vernalized for 9 weeks in short days and harvested directly from vernalization chamber (SDV), vernalized in short days for 9 weeks then returned to glasshouse temperatures for 2 weeks in short days (V+SD), and vernalized in short days for 9 weeks then returned to glasshouse temperatures in long days for 2 weeks (V+LD). The shoot apex of all plants was vegetative. Error bars represent se of triplicate reactions. B, Expression levels of HvVRN1 and HvFT1 assayed by RT-PCR in the same samples (40× cycles). Expression of HvVRN1 is shown as a positive control for the vernalization treatment. Expression of ACTIN is shown as a loading comparison (25× cycles).

Figure 8.

Interactions between genetic pathways controlling seasonal induction of flowering in temperate cereals. VRN2 represses FT1 to counteract PPD1 dependent long-day induction of FT1 prior to winter. Prolonged exposure to low temperatures up-regulates VRN1, which promotes inflorescence meristem identity at the shoot apex, accelerating inflorescence initiation independently of the long-day response pathway. VRN1 also represses VRN2 in the leaves to allow the long-day induction of FT1. FT1 can further accelerate inflorescence initiation and also subsequent stages of inflorescence development. According to this model, VRN1 acts in a low-temperature response pathway, FT1 acts in a daylength response pathway, and VRN2 integrates the low-temperature and daylength responses. In varieties where VRN2 is deleted, or where dominant VRN3 alleles are present, increased FT1 expression accelerates inflorescence initiation. This induces expression of VRN1 (dotted line).