DIE NEUTRALIS and LATE BLOOMER 1 Contribute to Regulation of the Pea Circadian Clock

Abstract

The DIE NEUTRALIS (DNE) locus in garden pea (Pisum sativum) was previously shown to inhibit flowering under noninductive short-day conditions and to affect a graft-transmissible flowering signal. In this study, we establish that DNE has a role in diurnal and/or circadian regulation of several clock genes, including the pea GIGANTEA (GI) ortholog LATE BLOOMER 1 (LATE1) and orthologs of the Arabidopsis thaliana genes LATE ELONGATED HYPOCOTYL and TIMING OF CHLOROPHYLL A/B BINDING PROTEIN EXPRESSION 1. We also confirm that LATE1 participates in the clock and provide evidence that DNE is the ortholog of Arabidopsis EARLY FLOWERING4 (ELF4). Circadian rhythms of clock gene expression in wild-type plants under constant light were weaker in pea than in Arabidopsis, and a number of differences were also seen in the effects of both DNE/ELF4 and LATE1/GI on clock gene expression. Grafting studies suggest that DNE controls flowering at least in part through a LATE1-dependent mobile stimulus, and dne mutants show elevated expression of a FLOWERING LOCUS T homolog under short-day conditions. However, the early flowering of the dne mutant is not associated with altered expression of a previously described CONSTANS-like gene. Collectively, our results characterize the clock system and reveal its importance for photoperiod responsiveness in a model legume.

EARLY FLOWERING3 Redundancy Fine-Tunes Photoperiod Sensitivity

“…Although clear rhythms of TOC1a expression in constant darkness were not apparent, the overall level of expression in ppd-3 remained lower than the wild type. Collectively these results are similar to those previously reported for the sn-4 (Liew et al, 2014) and dne-1 mutations (Liew et al, 2009), indicating a small but clear effect of the ppd-3 mutation on rhythmic expression of several key circadian clock components, which suggests that the primary role of PPD may also be related to clock function.…”

“…Trough expression levels for the evening genes LATE1 and PRR59a were also significantly higher in ppd-3 than in the wild type, a difference also apparent under SDs. In contrast, there was no clear evidence of any effect of ppd-3 on expression of PRR37a, consistent with earlier reports on sn and dne mutants (Liew et al, 2009(Liew et al, , 2014.…”

“…Both of these parental lines carry the same hr mutation (Weller et al, 2012). Figure 1 and Supplemental Figure S1 confirm that in contrast to the photoperiod-responsive wild type (P , 0.001), the ppd-3 allele confers early flowering (P , 0.001) regardless of photoperiod, equivalent to ppd-1 and ppd-2 (Taylor and Murfet, 1996) and to sn and dne mutants (Murfet, 1971;King and Murfet, 1985;Liew et al, 2009Liew et al, , 2014. As all three ppd mutations appeared similar in their phenotypic effects, we used the ppd-3 mutant to explore the roles of PPD, as it was generated in the same genetic background as other relevant mutants, including sn and dne.…”

“…Interestingly, in both crop groups, mutations in ELF3 genes have been shown to contribute to the expansion in range of species through alteration of photoperiod responsiveness and the associated impact on yield (Faure et al, 2012;Matsubara et al, 2012;Weller et al, 2012;Zakhrabekova et al, 2012;Alvarez et al, 2016;Lu et al, 2017). In crop species, a role for ELF4 function has been clearly established only in the legume species pea (Pisum sativum; Liew et al, 2009), but mutations in LUX orthologs have also been shown to cause early flowering and impaired photoperiod response in pea and in the cereals einkorn wheat (Triticum aestivum) and barley (Hordeum vulgare; Mizuno et al, 2012;Campoli et al, 2013;Liew et al, 2014), suggesting that the EC genes are likely to be intimately linked to the photoperiod response mechanism in both crop groups.…”

“…Naturally occurring and induced mutations in the LUX ortholog SN completely eliminate responsiveness to photoperiod (Liew et al, 2014), whereas a mutant for the ELF4 ortholog DNE enhances the effect of the hr mutation but has little effect when functional HR is present (Liew et al, 2009(Liew et al, , 2014. In addition to these three EC homologs, mutations at two other loci confer early, photoperiod-insensitive flowering in pea: a dominant mutation in the PHYTOCHROME A (PHYA) gene (Weller et al, 2004) and recessive mutations at the PHOTOPERIOD (PPD) locus (Arumingtyas and Murfet, 1994;Taylor and Murfet, 1996;Murfet and Taylor, 1999), whose molecular identity is not yet known.…”