Time

dependent repression is required for the seasonal flowering response in Arabidopsis

Evan Groover1, Greg Goralogia1, Takato Imaizumi1
1University of Washington - Department of Biology, Seattle, WA, 98105
egroover@uw.edu

Introduction TOPLESS is required for CDF-dependent Summary

The shift from vegetative growth to flowering in response to seasonal change is a well
documented behavior in plants. The transition to flowering is directly associated with the activity
of the endogenous circadian clock and occurs when the plant is stimulated by its optimal
regulation of flowering time Weve established that there is an interaction between TPL protein and the N-terminus of
CDF1. CDFs stand as repressors of the flowering response and serves as a link between the
endogenous circadian clock and the photoperiodic output. A phloem companion-specific
duration of light during the day (photoperiod). In the photoperiodic flowering pathway, dominant/negative mutation to TPL function has a similar effect on flowering time as cdf
To understand the effect of TPL on CO and FT regulation we created a series of TPL mutants that used the promoter of sucrose
transcriptional and posttranslational regulation of the transcription factor CONSTANS (CO) plays mutation. Its our hypothesis that CDFs recruit TPL in a time-dependent manner at the
symporter 2 (SUC2) to create TPL inactivation in phloem companion cells. Systemic tpl-1 mutation leads to pleiotropic effects that would
a critical role in day length-dependent regulation of the flowering gene, FLOWERING LOCUS T promoter to facilitate repressive activity. This is the mechanism through which CDFs are able
disrupt embryonic development and stem cell maintenance. Our approach to avoid this was to express the tpl-1 mutation in a cell-specific
(FT). to seasonally repress CO and FT transcription.
manner to better characterize the flowering phenotype. In addition to transforming wild type Arabidopsis (Col-0), we transformed
pSUC2:HA-tpl-1 into a series of flowering pathway mutant backgrounds that exhibited late flowering (35S:HA-CDF1, gi-2, fkf1-2). We
CO and FT expression is regulated in the cell by day length and temperature, as well as by
performed gene analysis on CO and FT by isolating mRNA, translating to cDNA and assaying via qPCR. We also took phenotype data for
transcriptional repression via a series of transcription factors called CYCLING DOF FACTORS
each line by recording leaf number at time of bolting.
(CDFs). CDF transcription and degradation is tightly regulated on a daily basis by outputs of the
circadian clock. In the afternoon of long days, CDF repression of the CO and FT promoters is (A) (B)
released by the action of the complex of GI and FKF1 proteins, both outputs of the circadian
clock. As FKF1 is a blue light photoreceptor, blue light is needed at a specific time point relative
to endogenous GI/FKF1 expression to induce this complex, such that its activity is dependent on
long day conditions. We studied the mechanism by which CDFs repress transcriptional activity,
specifically seeking to understand to what degree their activity is facilitated by the protein (B)
TOPLESS (TPL).

Proposed mechanism for TPL recruitment by CDFs at target loci

Phloem companion cell TPL knockout phenocopies
loss of cdf function Two different transgenic pSUC2:HA-
tpl-1 lines were grown alongside wild type and cdf mutant
lines in long day conditions. qPCR assay was performed
on young plants grown in long day conditions (16 hrs.
dark/8 hrs. light) for 10 days after germination to assay for
CO (A) and FT (B) activity.
Future Research
Currently, our research is aimed at better understanding how the number and
orientation of DOF binding sites in the presence or absence of TPL change the
sensitivity of the promoter to photoperiodic sunlight inputs. We hypothesize that the
removal of TPL will increase early-day CO expression regardless of daylength, but CO
expression will be unaffected by the amount of binding sites. We also expect that loss
of TPL function attenuates CDF1-dependent repression of flowering, such that
differences in CDF1 repression between different binding site architectures types will
be unchanged in the absence of the corepressor.

Repression by CDFs plays a regulatory role in the photoperiodic flowering pathway

The sequence of the cis-regulatory CDF1-binding motif at the CO promoter (red bases),
shows the variation in motif repeat number in A. thaliana. Number of DOF binding sites
might be responsible for ecotypical variation in photoperiod sensitivity. Understanding how
promoter architecture influences CO expression will strengthen our model of transcriptional
repression in the pathway.

Acknowledgements
WT: Short days: WT: Long days:
8hr light/ 16hr dark 16hr light/ 8hr dark Id like to thank my mentor Greg Goralogia for his constant support and for allowing me to be
involved with his research, Dr. Takato Imaizumi for giving me the opportunity to work with his
A. Thaliana flowering is dependent of photoperiod group, and all of the members of the Imaizumi lab for their assistance and perspective. Im
incredibly grateful to be able to learn from all of you.
TOPLESS interacts with CDFs Col-0 (wild
type)
pSUC2:HA-
tpl-1 in Col-0
pSUC2:HA-tpl-1
in 35S:HA-
CDF1
pSUC2:HA-
tpl-1 in gi-2
pSUC2:HA-
tpl-1 in fkf1-2

TOPLESS (TPL) is a globally expressed pleiotropic gene that plays an important role in development and
embryonic stem cell identity. Its local expression in phloem companion cells allows it to play a role in
regulating the photoperiodic flowering pathway.
70
tpl-1 mutants show earlier flowering phenotype in long day conditions

70
References
1. Golembeski, G.S., and Imaizumi, T. (2015) Photoperiodic regulation of florigen function
Prey 60 60
inArabidopsis thaliana. Arabidopsis Book 11:e0178
2. Song, Y.H., Smith, R.W., To B.J., Millar, A.J., and Imaizumi, T. (2012) FKF1 conveys timing
Empty
Empty

50 50
TPL
TPL

information for CONSTANS stabilization in photoperiodic flowering. Science 336: 1045-

GI
GI

Total Leaf Number

Empty 40 40 1049
3. Imaizumi, T., Schultz T.F., Harmon, F.G., Ho, L.A., and Kay, S.A. (2005) FKF1 F-box protein
CDF1 30 30
mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 309:293-
297
N-CDF1 20 20

Bait 4. Imaizumi, T., Tran, H.G., Swartz, T.E., Briggs, W.R., and Kay, S.A. (2003) FKF1 is essential for
CDF1 photoperiodic-specific light signaling in Arabidopsis. Nature 426: 302-306
nMut-CDF1 10 10
5. Fornara, Fabio, et al. "Arabidopsis DOF transcription factors act redundantly to reduce
TPL 0 0
CONSTANS expression and are essential for a photoperiodic flowering
Col-0 35S:HA-CDF1 gi-2 fkf1-2 LD SD response." Developmental cell 17.1 (2009): 75-86.
YFP H2B-RFP Merge
Col-0 pSUC2:HA-tpl-1 Col-0 pSUC2:HA-tpl-1 6. Sawa, Mariko, and Steve A. Kay. "GIGANTEA directly activates Flowering Locus T in
Arabidopsis thaliana." Proceedings of the National Academy of Sciences 108.28 (2011):
CDF proteins contain conserved N-terminus TPL binds to N-terminus of CDF1 pSUC2:HA-tpl-1 mutation decreases flowering time in late flowering 11698-11703.
mutants tpl-1 mutation was transformed onto late flowering transgenic lines, pSUC2:HA- tpl-1 mutant significantly reduces flowering time in short day
residues that contain a binding site for TPL Y2H reveals that CDF1 forms a conditions Wild type and pSUC2:HA-tpl-1 lines were grown in long day (16h 7. Rosas, Ulises, et al. "Variation in Arabidopsis flowering time associated with cis-regulatory
plants were grown in long day conditions and evaluated by leaf number
repressor Each of the 6 CDFs contain a complex TPL. Alteration and excision phenotype at flowering. TOPLESS-deficient lines showed earlier flowering
light/8h dark) and short day (8hr light/16hr dark) conditions. TOPLESS-deficient variation in CONSTANS." Nature communications5 (2014).
conserved residue sequence that presumably line flowered earlier in long and significantly earlier in short day.
of CDF1 N-terminus negates TPL phenotype than background and wild type lines. Background phenotype data
enables the formation of the repressor complex interaction. GI protein was used as a from references 2, 5 and 6.
with TPL positive control for all constructs.