In Arabidopsis, CONSTANS (CO) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS1 (CO1) and CONSTANS2 (CO2). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 (co1 co2) result in a small (3 d) but significant (P<0.0001) acceleration of heading time both in PHOTOPERIOD1 (PPD1) sensitive (Ppd-A1b, functional ancestral allele) and insensitive (Ppd-A1a, functional dominant allele) backgrounds. Under short days (SD), co1 co2 mutants headed 13 d earlier than the wild type (P60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1, CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1. Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat. An understanding of the mechanisms involved in the regulation of wheat heading time is required to engineer more productive varieties better adapted to new or changing environments. A large proportion of wheat’s natural variation in heading time is determined by differences in genes controlling the photoperiodic response. In this study, we show that the wheat PHOTOPERIOD1 (PPD1) gene has a stronger effect on heading time than CONSTANS1 (CO1) and CO2 in the regulation of the photoperiodic response, and that complex genetic interactions among these genes are important to fine-tune heading time. Using loss-of-function mutants for both CO1 and CO2, we demonstrate that these genes are not required for PPD1 to perceive differences in photoperiod and regulate heading time. Similarly, we show that in the absence of PPD1, CO1 can accelerate heading time more than 60 days in response to long days. Our results indicate that each of these two wheat photoperiod pathways can respond to differences in photoperiod even in the absence of the other one. Differences in the relative importance of these two pathways and in their epistatic interactions have contributed to the diversity of photoperiodic responses observed in different grass species.

Winter wheats require a long exposure to cold temperatures (vernalization) to accelerate flowering. However, varieties differ in the length of the period of cold required to saturate the vernalization response. Here we show that single nucleotide polymorphisms (SNP) at the binding site of the GRP2 protein in the VRN-A1 first intron (henceforth, RIP3) are associated with significant differences in heading time after a partial vernalization treatment. The ancestral winter VRN-A1 allele in ‘Triple Dirk C’ has one SNP in the RIP3 region (1_SNP) relative to the canonical RIP3 sequence, whereas the derived ‘Jagger’ allele has three SNPs (3_SNPs). Both varieties have a single VRN-A1 copy encoding identical proteins. In an F₂ population generated from a cross between these two varieties, plants with the 3_SNPs haplotype headed significantly earlier (P < 0.001) than those with the 1_SNP haplotype, both in the absence of vernalization (17 days difference) and after 3-weeks of vernalization (11 days difference). Plants with the 3_SNPs haplotype showed higher VRN-A1 transcript levels than those with the 1_SNP haplotype. The 3_SNPs haplotype was also associated with early heading in a panel of 127 winter wheat varieties grown in three separate controlled-environment experiments under partial vernalization (36 to 54 days, P < 0.001) and one experiment under field conditions (21 d, P < 0.0001). The RIP3 polymorphisms can be used by wheat breeders to develop winter wheat varieties adapted to regions with different duration or intensity of the cold season.

The photoperiodic response is critical for plants to adjust their reproductive phase to the most favorable season. Wheat heads earlier under long days (LD) than under short days (SD) and this difference is mainly regulated by the PHOTOPERIOD1 ( PPD1 ) gene. Tetraploid wheat plants carrying the Ppd-A1a allele with a large deletion in the promoter head earlier under SD than plants carrying the wildtype Ppd-A1b allele with an intact promoter. Phytochromes PHYB and PHYC are necessary for the light activation of PPD1 , and mutations in either of these genes result in the downregulation of PPD1 and very late heading time. We show here that both effects are reverted when the phyB mutant is combined with loss-of-function mutations in EARLY FLOWERING 3 ( ELF3 ), a component of the Evening Complex (EC) in the circadian clock. We also show that the wheat ELF3 protein interacts with PHYB and PHYC, is rapidly modified by light, and binds to the PPD1 promoter in planta (likely as part of the EC). Deletion of the ELF3 binding region in the Ppd-A1a promoter results in its upregulation at dawn, similar to PPD1 alleles with intact promoters in the elf3 mutant background. The upregulation of PPD1 is correlated with the upregulation of the florigen gene FLOWERING LOCUS T1 ( FT1 ) and early heading time. Loss-of-function mutations in ppd1 result in the downregulation of FT1 and delayed heading, even when combined with the elf3 mutation. Taken together, these results indicate that ELF3 operates downstream of PHYB as a direct transcriptional repressor of PPD1 , and that this repression is relaxed both by light and by the deletion of the ELF3 binding region in the Ppd-A1a promoter. In summary, the regulation of the light mediated activation of PPD1 by ELF3 is critical for the photoperiodic regulation of wheat heading time. Author Summary: The coordination of reproductive development with the optimal season for seed production is critical to maximize grain yield in crop species. Plants can perceive the length of the day or night (photoperiod) and use this information to anticipate seasonal changes. In most eudicot plants, CONSTANS plays a central role in the perception of photoperiod, but in wheat the main photoperiod gene is PHOROPERIOD1 ( PPD1 ). In this study, we show that the clock gene EARLY FLOWERING 3 ( ELF3 ) regulates the phytochrome-mediated light activation of PPD1 . Loss-of-function mutations in ELF3 result in the upregulation of PPD1 at and dawn, and in early heading under both long and short days, even in the absence of PHYB . A deletion in the PPD1 promoter including an ELF3 binding region also results in earlier heading under short days, indicating that ELF3 acts as a direct transcriptional repressor of PPD1 . This study shows that ELF3 plays a critical role in the wheat photoperiod pathway by regulating the light signal between the phytochromes and PPD1 . ELF3 provides an additional entry point to engineer heading time in wheat, an important trait for the development of better adapted varieties to a changing environment.

In Arabidopsis, CONSTANS ( CO ) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS 1 ( CO1 ) and CONSTANS 2 ( CO2 ). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 ( co1 co2 ) result in a small (3 d) but significant ( P <0.0001) acceleration of heading time both in PHOTOPERIOD1 ( PPD1 ) sensitive ( Ppd-A1b , functional ancestral allele) and insensitive ( Ppd-A1a , functional dominant allele) backgrounds. Under short days (SD), co1 co2 mutants headed 13 d earlier than the wild type ( P 60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1 , CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1 . Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat. Author summary: An understanding of the mechanisms involved in the regulation of wheat heading time is required to engineer more productive varieties better adapted to new or changing environments. A large proportion of wheat’s natural variation in heading time is determined by differences in genes controlling the photoperiodic response. In this study, we show that the wheat PHOTOPERIOD1 ( PPD1 ) gene has a stronger effect on heading time than CONSTANS1 ( CO1 ) and CO2 in the regulation of the photoperiodic response, and that complex genetic interactions among these genes are important to fine-tune heading time. Using loss-of-function mutants for both CO1 and CO2 , we demonstrate that these genes are not required for PPD1 to perceive differences in photoperiod and regulate heading time. Similarly, we show that in the absence of PPD1 , CO1 can accelerate heading time more than 60 days in response to long days. Our results indicate that each of these two wheat photoperiod pathways can respond to differences in photoperiod even in the absence of the other one. Differences in the relative importance of these two pathways and in their epistatic interactions have contributed to the diversity of photoperiodic responses observed in different grass species.