FHY3 and FAR1 Act Downstream of Light Stable Phytochromes

Abstract

FHY3 and FAR1 are positively acting transcription factors that directly regulate expression of a number of target genes in Arabidopsis thaliana. Here, we looked at the regulation of one specific target gene, ELF4. We demonstrate that the action of FHY3 and FAR1 in upregulation of ELF4 is light dependent. Furthermore, although FHY3 and FAR1 have been exclusively characterized as components of the phytochrome A signaling pathway because of their importance in regulating expression of phyA nuclear importers, we show that, as transcription factors in their own right, FHY3 and FAR1 act downstream of light stable phytochromes, phyB, phyD, and phyE. We demonstrate that light stable phytochrome acts in a red/far-red reversible manner to regulate the level of FHY3 protein. We also observed that ELF4 shows specific FHY3 and FAR1-mediated light induction in the evening and we show that regulation by light stable phytochromes at this time is important as it allows the plant to maintain normal ELF4 expression beyond dusk when the day length shortens, something which would not be possible through light labile phytochrome action. Without FHY3 and FAR1, ELF4 expression falls rapidly at dusk and in short days this results in an early drop in ELF4 expression, accompanied by a de-repression of an ELF4 target gene later in the night. Our results, therefore, demonstrate an important role for FHY3 and FAR1 as mediators of light stable phytochrome signaling.

Keywords: Arabidopsis; light; phytochrome; signal transduction; transcription.

FIGURE 1

(A) The action of FHY3 and FAR1 in regulation of ELF4 expression is light dependent. Wild-type (WT), fhy3, far1, and fhy3 far1 mutant seedlings were grown in 12 h white light:12 h dark cycles for 1 week before transfer to constant red light for a 4 days at of a range of different intensities. Mean ELF4 expression relative to IPP2 measured by RT-qPCR for two independent replicates + standard error. (B) FHY3 and FAR1 directly regulate acute light responsive ELF4 expression in the early part of the subjective night. WT and fhy3 far1 seedlings, both expressing ELF4::LUC, as well as WT seedlings expressing an ELF4::LUC construct in which the FHY3/FAR1 binding site was mutated (mFBS), were grown in 12 h white light/12 h dark cycles for 1 week prior to transfer to constant darkness at dawn (Circadian Time 0). Seedlings were then given a 30 min pulse of 100 μmol m^–2 s^–1 red light at one of four time points over a circadian cycle. Left hand axis (black labels): percentage increase in bioluminescence recorded 30 min after the pulse at the four time points indicated. Right hand axis (red labels): mean relative bioluminescence of untreated WT seedlings, shown by red circles, and measured at a number of points over the time course to show the background pattern of ELF4 expression. Dotted line represents trend line based on third order polynomial. Light and dark bars above the figure indicate the times of subjective day and night. N = at least 19 seedlings + standard error.

FIGURE 2

ELF4 expression in fhy3 far1 mutant seedlings is not buffered against changes in day length. WT and fhy3 far1 seedlings, both expressing ELF4::LUC, as well as WT seedlings expressing an ELF4::LUC construct in which the FHY3/FAR1 binding site was mutated (mFBS), were grown in 12 h white light/12 h dark cycles for 1 week before either (A) transfer to long days (16 h white light/8 h dark cycles); (B) maintenance in 12 h white light/12 h dark cycles; or (C) transfer to short days (8 h white light/16 h dark cycles). Values shown represent mean bioluminescence normalized to WT values at time zero of at least 17 seedlings + standard error.

FIGURE 3

Maintenance of the ELF4 expression peak beyond dusk in WT seedlings is observed following red light but not blue light. WT and fhy3 far1 seedlings, both expressing ELF4::LUC were grown in 12 h white light/12 h dark cycles for 1 week before transfer to either (A) 12 h red light/12 h dark cycles; or (B) 12 h blue light/12 h dark cycles. Values shown represent mean bioluminescence normalized to WT values at time zero of at least 19 seedlings + standard error.

FIGURE 4

Maintenance of the ELF4 expression peak beyond dusk in WT seedlings is red/far red reversible. (A) WT seedlings expressing ELF4::LUC, (B) fhy3 far1 seedlings expressing ELF4::LUC, and (C) WT seedlings expressing an ELF4::LUC construct in which the FHY3/FAR1 binding site was mutated (mFBS), were grown in 12 h white light/12 h dark cycles for 1 week before transfer to short days (8 h white light/16 h dark cycles). Seedlings were either treated with a 15 min end of day far red pulse (EODFR) or maintained in short days without EODFR treatment (Control). Values shown represent mean relative bioluminescence of at least 15 seedlings + standard error.

FIGURE 5

Maintenance of the ELF4 expression peak beyond dusk in WT seedlings involves phyB, phyD, and phyE. Seedlings were grown in 12 h white light/12 h dark cycles for 1 week before transfer to short days (8 h white light/16 h dark cycles). Seedlings were either treated with a 15 min EODFR or maintained in short days without EODFR treatment (Control). Expression of ELF4 in WT phyB, phyB phyD, and phyB phyE mutant seedlings 4 h following dusk. Values shown represent mean expression of ELF4 relative to WT level at dusk, normalized to IPP2 for minimum two replicates + standard error. (^∗p < 0.05 using a heteroscedastic t-test).

FIGURE 6

Maintenance of FHY3 protein beyond dusk is red light-specific and shows red/far red reversibility. Seedlings of the fhy3 mutant expressing an FHY3::FHY3-LUC translational-fusion reporter construct were grown in 12 h white light/12 h dark cycles for 1 week before transfer to either (A) red light/dark cycles; (B) blue light/dark cycles; or (C) 8 h white light/16 h dark cycles with a 15 min EODFR or without (Control). Arrows represent times of EODFR treatment. Values shown represent mean relative bioluminescence of at least 20 seedlings + standard error.

FIGURE 7

Growth of fhy3 far1 mutant seedlings in short days reveals an early release of PIF4 repression. Expression of PIF4 relative to UBQ10 as measured by RT-qPCR in WT and fhy3 far1 seedlings grown in 12 h white light/12 h dark cycles for 1 week before transfer to either long days (16 h white light/8 h dark cycles; A); or short days (8 h white light/16 h dark cycles; B). Values shown represent mean expression normalized to UBQ10 for three replicates ± standard error.

FIGURE 8

FHY3 and FAR1 act downstream of phyB, phyD, and phyE Pfr to maintain ELF4 expression into the early part of the night following a short day. An additional pathway acts to compensate for the loss of FHY3 and FAR1 at this same time in long days. This additional pathway can be activated by either red or blue light but is triggered only in the continued presence of light, suggesting the involvement of phyA as a photoreceptor.