2016 Class Slides Floral Transition

Entrained daily rhythms that persist in the absence of environmental input

‘circa’ - approximately ‘dies’ – day

Organisms on earth evolved under a regimen of long and short days thatvaried with season. Light quantity, quality and duration allow a plant to ‘know’ where it is relative to daily cycles of light (L), darkness (D). This information is integrated with temperature to assist in detection of season.

‘Circadian’ vs. ‘Diurnal’

Both circadian and diurnal rhythms persist in L/D cycles, yet only circadian rhythms persist under free run conditions.

CIRCADIAN RHYTHMS -- General

diurnal

circadian

OUTPUT FROM THE CLOCK-

Jean Jacques d’Ortous de Mairan identifies persistent leaf movements in heliotropic plants moved to constant darkness.

Probably Mimosa pudica

(He also identified the Orion Nebula)

de Mairan, J. 1729, Observation botanique: Hist. Acad. Roy. Sci. 35–36.

Interesting Evidence from Cyanobacteria

Why would something with a lifespan of a few hours have a 24 h clock?

Photosynthesis evolves oxygen, yet oxygen potently inhibits nitrogenase. How do nitrogen-fixing bacteria do it? Studies showed it was accomplished at night, indicating that cyanobacteria had an internal (collective) oscillator.

Circadian clocksconfer fitness!Ouyama, et al., 1998

Biological Autoregulatory Negative Feedback Loops

LUCcab2

LUCccr2

TOOLS– promoters from circadian/light-regulated genes were fused to theluciferase transgene.

peaks in morning

peaks in evening

Performed by phytochromesand cryptochromes, no evidence for phototropin 1 (+/-).

Input to the Plant ClockPhytochromeaffects the amplitude, butnot the periodof cab geneexpression

Devlin and Kay, 2000

CCA1, TOC1 and LHY1 Participate in a Negative-Feedback Loop

TOC1 and ELF4 activate transcription of CCA1 and LHY

CCA1 and LHY levels peak at dawn due to phy activation, likely interaction through the G-box with PIF3.

CCA1 and LHY repress TOC1through the ‘Evening Element’

Positive regulators repressed=lower levels of CCA1 and LHY

The simplified skeleton story:

acute phy induction

Light

Short-Day Plants Long-Day Plants

Photoperiodic Control of FloweringShort-day plants (SDP) flower when day lengths are short.

Long-day plants (LDP) flower when day lengths are long.

A ‘night break’ is sufficient to interrupt flowering regime.

The Hourglass Model

Light regulates the accumulation of aregulatory product (yellow line). When athreshold is surpassed, the plant flowers.

The External Coincidence Model

The circadian oscillator dictates accumulationof a regulatory product that also requires acute activation of photoreceptors when above a threshold (dotted line).

The Internal Coincidence Model

Inductive responses mediated by convergentinput from multiple oscillators.

Yanovsky and Kay, 2003

What are the internal cues?

CONSTANS (CO) – identified as a defect in flowering in LD.

Encodes a DNA-binding protein that induces flowering on LD when overexpressed.

CO is nuclear localized

images from Coupland Lab website.

CO expression is regulated by integration between internal oscillator information and photoperiod; peaks occur in daytime on LD, but not on SD.

TOC1

CO activates flowering whentwo conditions are satisfied;1. LD conditions produce high levels of CO.2. Light activates phyA and cry2 receptors

Flowering Locus T (FT) is induced, leading to flowering.FT encodes a small globular protein

The activation of FT in the leaves induces changes meristem identity.

Which model does this support?

Coordination between CO and FT transcripts are necessary for correct flowering, but CO only induces FT when cry2 is activated.

Yanovsky and Kay, 2003

Regulation of Flowering in a LD and SD Plant

In the LD plant (Arabidopsis) CO activates FTleading to flowering, in SD CO represses FT.

In the SD plant (rice) CO represses FT and activates FT on short days.

LD SD

Yanovsky and Kay, 2003

The differences are not due to polymorphisms betweenCO, as the can be functionally interchanged.

CO partitioning and stability are determined by light sensors and dictate when flowering will initiate Valverde et al., 2004

what we know…

CO levels must be high to promote flowering, yet they must coincide withlight input. This suggests some additional factor or post-translational modification of CO must be occurring to regulate flowering.

What is happening and how can it be determined experimentally?

Figure 1. -- 35S::CO plants were generated and include a FTpromoter::LUCfusion. This allows the internal flowering cue (CO level) to remain constant. FT levels can be measured in response to different environmental cues.

Results Figure 1. – Blue light induces the response better than others. Luminescence is higher in BL + FR treated plants (A), CO transcript levels areunaffected (B), so it is likely BL/FR control of post-translational activity.

alternative interpretations?hypotheses?

dark

dark

in diurnal conditions LUCactivity is higher and peaksat dusk in LD conditions

NEXT QUESTION: Is FT induced due to re-localization of CO?

TO TEST: Build GFP::CO fusion and see when/where it accumulates in response to different light qualities.

(A) Accumulation in nucleiof WL, BL or FR-treatedplants

(B) Nuclear extracts wereprobed with a CO-specificantibody. CO only found inthe nucleus with W, BL or FRtreatment.

(C) CO only found in nucleusWT or OX plants grown in constantBL, not in co mutants

wt plants

35S::GFP::CO on LD

35S:: GFP::CO on SD

CO accumulates in nucleus differentially in LD/SD

The anti-CO antibody confirms the presence of CO in nuclear extracts in amounts consistent withGFP signal / photoperiod.

take-home message: CO LEVELS FALL RAPIDLYin DARKNESS look carefully- do GFP data match westerns?

do CO data match northerns? What about microarrays?

F. There is a transientpeak of CO right afterdawn, correlates with observed FT::LUC peak.

G. In WT plants CO could not be detected under WL conditions. Plants were grownunder LD or SD of BL.

This shows that the 35S:CO accumulation in WL is similar to the WT accumulation in BL.

CLEARLY, there is post-translational control of CO levels. Might this involveproteolytic regulation via the proteosome?

Figure 3.

A. CO levels decrease during the first 4 h of darkness.

Addition of MG132 stabilizes CO abundance in nuclear extracts derived

from L or D plants.

HIS::CO was produced and introducedto extracts containing the inhibitor and/or ATP. The sensitivity to degradationin the absence of this inhibitor showsthat CO is likely degraded by the proteosome. The second panel testsextracts with an anti-UBQ antibody.

It tells us that more stuff is ub-ated in light, but degraded in darkness.

Previous figures show accumulation of CO (and hence FT) in BL and FR.

The next test is the genetic test– do BLand FR receptors affect accumulation of CO/FT.

FT mRNA levels are affected by cry and phyA mutation under the appropriate wavebands

Protein levels are similarly affected.

phyB mutation makes CO more stable

Diurnal regulation of CO levels depends on the stability conferred throughcry’s and phyA.

phyB destabilizes CO, asmutants accumulate moreprotein.

Cry’s stabilize CO and more CO accumulates under BLbecause phyB is minimally here where cry’s are active.

can you make predictions about flowering time in phyB mutants? cry mutants? phyA mutants?

cry/phyA STABILIZE CO, phyB DESTABILIZES CO. This is consistent with effect of mutations on flowering time; phyB mutants flower early, cry2’s flowervery late.

QUESTION: cry2 and phyA are unstable in light. phyB is very stable. cry1 may not have much of a role (if any) because cry1 does not affect flowering time.

Can phyA and cry2 be detected when they are presumed to be active?

Spalding and Folta, 2005

Just when you thought you understood flowering time…

--CO activates flowering in a LD plant (Arabidopsis)but represses flowering in an SD plant (rice).

--conserved components that substitute for each other in monocots and dicots

How does this work in species that contain SD and LD varieties (like strawberry)?

makes a good test question.

Integration of TemperatureSome Arabidopsis ecotypes discriminate between spring and fall daylengthby incorporating temperature information.

Mechanism– the outputs of CO are repressed by Flowering Locus C

(FLC), a MADS-box transcription factor.

High FLC levels, such as in summer or fall,antagonize CO’s ability to affect

AGL20/SOC1 and FT.

no flowering if germinated in summerYanovsky and Kay, 2003

HOWEVER, if germinated in springthe cold represses FLC levels and COhas a greater influence on floral transition genes.

These also are influenced by VRN1,VRN2 and VRN3

VRN1 and VRN2 are involved in chromatin remodeling, so they epigenetically silence FLC expression. FLC levels are suppressed by cold treatment in the presence of VRN1&VRN2.

Take home message– some changes that confer seasonality are “hard-wired” physical changes.

Why is this un-plant-like?

Yanovsky and Kay, 2003

How does this fit biology?

CO mRNA abundance is clock regulated, and post-translationalmechanisms govern its stability.

This allows more accumulation on long days– as days grow progressively longer CO levels peak and induce FT.

Shade avoidance– when plants are grown in canopies or nearneighbors there is a low R/FR ratio. This means less activationthrough phyB. Think about this and how it meshes with the described results. (Cerdan/Chory 2004)

Spectral shifts…

Wigge et al., 2005

Mechanism of integration– CO and FT are producedin leaves, yet they act at meristem…. ?

three genes identified in microarray

CONCLUSION: FT is the major target of CONSTANS

FT interaction screen identifies FD, a bZIP proteinthat is expressed at the shoot apex

veg transition flowering

Mechanism of integration– CO and FT are producedin leaves, yet they act at meristem…. ?

where and when is FD expressed?

time after transfer to LD conditions

Fd

p=floral primordia, a=floral anlagen

Ap1

fd mutantAp1 transcript

FRUITFUL promoter::GUS

AP1 promoter::GUS

only expressed in litpart of leaf

Interaction at the promoter? Is FD just a spatial cue?

no CO, no FT CO and FTwhere do promoter deletions affectnormal response? Ap1::GUS

FT inducedFD expressedin shoot apex

mobilized

FT and FDallow integration of temporal and spatial signals

GOOD QUESTIONS—

Explain the three models proposed to regulate transition to flowering andthe experimental evidence that supports one of them.

BL, acting through cry2, stabilizes CO. How does what you knowabout cry’s role in early photomorphogenic development fit with this mechanistically?