the end of innocence: flowering networks explode in complexity

Available online at www.sciencedirect.com

The end of innocence: flowering networks explode in complexityDavid Pose1,a, Levi Yant2,a and Markus Schmid1

Substantial recent advances in genome-scale transcription

factor target mapping have provided a fresh view of the gene

networks governing developmental transitions. In particular,

our understanding of the fine-scale spatial and temporal

dynamics underlying the floral transition at the shoot apex has

seen great advances in the past two years. Single transcription

factors are regularly observed to act in complex manners,

directly promoting the expression of particular targets while

directly repressing the expression of others, based at least

partly on defined heterodimerization patterns. For single

regulators this behavior reaches into distinct physiological

processes, providing compelling evidence that particular

transcription factors act to directly integrate diverse processes

to orchestrate complex developmental transitions.

Addresses1 Max Planck Institute for Developmental Biology, Department of

Molecular Biology, Spemannstrasse 37-39, D-72076 Tubingen, Germany2 Department of Organismic and Evolutionary Biology, Harvard

University, 22 Oxford Street, Cambridge, MA 02138, USA

Corresponding author: Schmid, Markus

([email protected])a Equal contribution.

Current Opinion in Plant Biology 2012, 15:45–50

This review comes from a themed issue on

Growth and Development

Edited by Xuemei Chen and Thomas Laux

Available online 3rd October 2011

1369-5266/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2011.09.002

IntroductionThe recent influx of in vivo transcription factor binding

studies facilitated by chromatin immunoprecipitation

paired with genome wide readouts (ChIP-seq or ChIP-

chip) has radically augmented the complexity built into

our picture of gene regulatory networks (GRNs). As a

result, in the past two years, the network controlling

flowering in Arabidopsis thaliana has grown up from being

understood as simply a very complex set of pathways

controlled by a large number of genes: now it is seen as a

fantastically intricate web of crosstalk, feedback, and

redundancy, bound tightly with other developmental

processes by common transcription factors (TFs) that

act as sophisticated ‘process integrators’.

Extensive reviews on flowering have been published

recently [1,2]. Hence, we only briefly contextualize the

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genetic pathways involved in regulating flowering before

turning our focus to the integration of signals at the shoot

apical meristem (SAM), the spatial–temporal regulation

of flower development, and finally functions that floral-

related genes have in processes other than flowering time

or flower development in A. thaliana.

Flowering pathway integrators: at the gatewayof a tangled bankSignaling pathways responding to endogenous (gibberellic

acid (GA), autonomous and age) and environmental

(photoperiod and temperature) signals converge on genes

that then activate floral homeotic genes (Figure 1a). These

integrators include the mobile signal FLOWERING

LOCUS T (FT), which is thought to promote flowering

together with the meristem-specific bZIP TF FD [3,4],

even though the existence of this complex at the SAM has

yet to be confirmed. FT is expressed in response to the

output of the photoperiod pathway, CONSTANS (CO) [4–6] and is also up-regulated by autonomous, temperature,

vernalization and GA cues through the inhibition of the

repression of the FLOWERING LOCUS C–SHORT

VEGETATIVE PHASE (FLC–SVP) complex (reviewed

in [1]). At the SAM, the FT–FD complex activates the

MADS domain TF SUPRESSOR OF OVEREXPRES-

SION OF CONSTANS1 (SOC1), which, in a complex with

AGAMOUS-LIKE 24 (AGL24), promotes the expression

of the floral meristem identity genes LEAFY (LFY) directly

and APETALA1 (AP1) indirectly through LFY. AP1 is a

direct target of FT–FD as well, constituting a critical feed-

forward loop [4]. SOC1 expression is promoted by the

photoperiod through CO, in an age-dependent manner

through the microRNA-targeted SQUAMOSA BINDINGFACTOR-LIKE (SPL) gene products and by the GA path-

way. Repression of SOC1 is achieved by the FLC–SVP

complex, with SVP regulated by the autonomous, tempera-

ture and vernalization pathways [7]. Aside from its function

as a pathway integrator, SOC1 also regulates flower pat-

terning and floral meristem determinacy [7–9,10��].

Spatial and temporal dynamics govern floralpatterning regulation at the shoot apexWhen flowering is induced in A. thaliana, the vegetative

meristem (VM) first acquires inflorescence meristem

(IM) identity. Then the IM gives rise to the reproduc-

tive organs after generating the floral meristem (FM) on

its flank. These identity changes begin when signals

from floral-promoting pathways reach the vegetative

meristem. In A. thaliana, the IM remains indeterminate

due to the FT homolog TERMINAL FLOWER1 (TFL1),

which antagonizes LFY and AP1 expression (Figure 1a)

[11,12].


mailto:[email protected]

http://dx.doi.org/10.1016/j.pbi.2011.09.002

46 Growth and Development

Figure 1

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Current Opinion in Plant Biology

Dynamic genetic networks control flower development. (Central panel) Scanning electron micrograph of the main flowering apex of Arabidopsis

thaliana. Asterisk indicates the IM. Numbers refer to the developmental stage of buds [23]. (a) Before the floral transition, LFY and AP1 expression is

repressed by TFL1 in the VM. Signaling pathways converge in the integrators FT and SOC1 to initiate the floral transition. These factors activate AP1

and LFY, acting in a complex in the developing IM with FD and AGL24 respectively. (b) In stages 1 and 2 of FM, SVP acts redundantly with the SOC1–

AGL24 complex to down-regulate SEP3 expression, preventing precocious activation of the class B and C genes AP3, PI, and AG [10��]. AP1 and AP2

contribute directly to this repression as well [26,34]. (c) FM in early stage 3. Left panel: LFY and AP1 directly up-regulate SEP3, which, together with

LFY actives class B and C genes [10��]. AP1 is also up-regulated in positive-feedback loop [29�] (SEP3–LFY complex formation has not been

confirmed). Another key positive-feedback loop is the down-regulation of SOC1 and SVP by SEP3 [29�]. Right panel: AG expression is repressed in the

two outer whorls by SEP3–AP1 complex and AP2, the latter down-regulating miR172 [31,38]. Conversely, AP1 and AP2 are repressed in the two inner

whorls by SEP3-AG and miR172 respectively [32,35]. This temporally dynamic and spatially specific regulation ensures the proper organ formation

during the floral development. Arrows and block lines denote activation and repression respectively. Dotted arrows and block lines indicate a lower

effect of this regulation. Size of fonts indicates abundance. Squares represent groups of factors under the same regulation. Rounded rectangles

indicate dimers and shaded blue rounded rectangles, co-regulators.

The developmental changes that occur in the FM were

codified in the ABCE model, which postulates that four

regulatory functions (A, B, C and E) work combinatorially

for the proper organ formation in each whorl (Figure 1c)


[13,14]. A-class proteins AP1 and AP2 confer sepal iden-

tity in the first whorl. Their activity overlaps in the second

whorl with the B-class proteins AP3 and PISTILLATA

(PI), resulting in petal identity. Stamens are formed in the


Flowering networks explode in complexity Pose, Yant and Schmid 47

third whorl due to the combined activity of the B-class

proteins together with the C-class protein AGAMOUS

(AG), the latter specifying carpel development in the

fourth whorl. The E-class proteins SEPALLATA1–4

(SEP1–4) play a crucial role as co-regulators in all four

whorls.

Recent studies employing direct TF binding assays to

define this network at higher resolution have refined the

picture of gene interactions at the SAM. FM determi-

nation begins with the up-regulation of the plant-specific

TF LFY, and the MADS domain protein AP1 (Figure 1b)

[15]. Dramatic up-regulation of LFY in the floral anlagen

on the IM flank is often considered the first event in the

VM-IM transition and has been shown to be directly

promoted by the SOC1–AGL24 complex [16], and also

by SPL3 [17] and GAMYB33 [18], the latter expressed in

the apex in response to GAs. It was recently proposed that

the SOC1–AGL24 dimer activates LFY expression on the

flank of the developing IM in association with a hetero-

dimer formed by the KNOTTED-LIKE homeobox

protein SHOOT MERSTEMLESS (STM) and the

BELL-like homeobox (BLH) proteins PENNYWISE

(PNY) and POUND-FOOLISH (PNF) [19,20]. LFYexpression is also up-regulated indirectly by FT, which

promotes SOC1 [21]. Later, AP1 and LFY up-regulate

each other in a positive feedback loop that functions to

maintain flower meristem identity. AP1 expression is also

induced by FT–FD, in cooperation with STM-PNY/

PNF, and by SPL3 and SPL9 [17,20,22��].

The development of newly emerging flowers progresses

in stereotypic stages [23]. During stage 1 and 2 of flower

development, the primordium proliferates without differ-

entiating [23]. Differentiation starts in stage 3 with the

formation of the outer whorl, sepals, by the combined

action of A- and E-class genes (Figure 1). The lack of

differentiation in the first stages is achieved by maintain-

ing SEP3 in a silenced state. SEP3 expression is redun-

dantly down-regulated by the flowering time TFs SVP,

SOC1 and AGL24 (Figure 1b). Of these three TFs, SVPappears to be the most strongly expressed in stage 1 or 2

flowers [8], whereas expression of SOC1 and AGL24 is not

easily detectable at these stages in flower development

[8,24]. Nevertheless, genetic analyses clearly indicate

that SOC1 and AGL24 do contribute to the repression

of SEP3 in the youngest flower buds and ectopic expres-

sion of SEP3 was only observed in agl24-1 svp-41 double

and soc1-2 agl24-1 svp-41 triple mutants [10��]. SVP

indirectly causes trimethylation of histone H3 lysine 27

by interacting with TERMINAL FLOWER2/LIKE

HETEROCHROMATIN PROTEIN1 while SOC1

and AGL24 interact with SAP18, a member of Sin3/

histone deacetylase complex, preventing histone H3

acetylation, a mark associated with active transcription

[10��,25]. However, B- and C-class genes are not con-

trolled by SEP3 exclusively. Rather, their precocious


expression is safeguarded against by the heterodimers

AP1-AGL24 and AP1-SVP, which recruit the SEUSS–LEUNIG (SEU–LUG) co-repressor complex (Figure 1b)

[26]. Later, in early stage 3, AP1 promotes SEP3 expres-

sion both directly and indirectly through LFY (Figure 1c)

[27�,28�].

There is mounting evidence of negative feedback of floral

homeotic TFs onto flowering time integrators. This feed-

back ensures a rapid journey through the VM, IM, and

FM fate changes and also safeguards against floral rever-

sion. Direct repression of SVP, SOC1 and AGL24 by AP1

has been described [8], revealing an alternative, indirect

and gradual mode of AP1 promotion of SEP3 expression

(Figure 1c). AP1 also directly represses two other flower-

ing integrator genes, FD and its paralog FDP [27�] to

ensure a sharp transition and reinforcement to the com-

mitment to flower organ morphogenesis.

Once SEP3 is expressed, it functions together with LFY

to activate B (AP3 and PI) and C (AG) class genes in the

three inner whorls [10��]. Moreover, SEP3 and LFY

generate a positive feedback loop through the up-regu-

lation of AP1 [12,29�]. Another contribution to this loop is

the direct down-regulation of SOC1 and SVP by SEP3

(Figure 1c) [29�]. Because SEP3-SOC1 and SEP3-SVP

heterodimers can form, these flowering time TFs may

contribute to their negative auto-regulation in FMs [30].

Induction of SEP3 leads to the formation of SEP3-AP1

heterodimers that recruit the SEU–LUG co-repressor

complex and repress AG expression in the outer two

whorls [31]. This repression between AP1 and AG is

bidirectional: AP1 disappears from the center of the

flower during stage 3 due to the repressor activity of

AG [32], which may function in a complex with SEP3

(Figure 1c) [29�,30]. However, the relation between AG

and another A-class gene, AP2, seems to be uni-

directional. AP2 contributes to the down-regulation of

AG in the meristem through direct binding to the second

AG intron [33,34], although AG does not appear to

antagonize AP2 at the transcriptional level (Figure 1c).

AP2 expression is more clearly regulated by a microRNA,

miR172, through mRNA cleavage and translational inhi-

bition, although it is difficult to assess the precise bio-

logical importance of this due to strong negative feedback

of AP2 upon its own expression and that of related AP2-

like miR172 targets (Figure 1c) [35–37]. In situ hybridiz-

ation revealed that AP2 and miR172 expression are

complementary, with AP2 in the outer whorls from stage

2 onwards and miR172 in the inner whorls, resembling

the AG expression pattern [38]. However, there is some

overlap between AP2 and miR172 transiently in the

boundary between the perianth and reproductive organs

in the third whorl, consistent with miR172 not being

sufficient to fully repress AP2 [35,36,38]. What determines

the inner whorl-specific expression of MIR172 remains



unknown, but this expression pattern, together with the

presence of negative factors and/or lack of positive factors

that activate AP2 expression, explains why AP2 is absent in

the center of the young flower. On the contrary, AP2

maintains its specific function in the outer whorls directly

down-regulating MIR172 expression through the recruit-

ment of the SEU–LUG co-repressor complex (Figure 1c)

[34,39].

Regulator function: a picture of everincreasing complexityThis relationship of miR172 with its AP2-clade targets

serves as a cautionary tale: temporal and spatial dynamics

cause complexity to rapidly emerge from a small number

of players. It is becoming clear that another dimension

is broadly at play in developmental GRNs: single TFs

have the capacity to act either as direct transcriptional

activators or repressors of distinct suites of targets

[27�,28�,29�,34,40].

Recent TF target mapping studies have furnished a

wealth of illustrations: AP2 itself acts as a bifunctional

TF, directly repressing the expression of its repressor,

MIR172b in a feedback loop, but reinforcing this loop by

directly activating MIR156e expression, an indirect

repressor of MIR172b. AP2 also reinforces its function

by directly inducing the expression of another floral

repressor, AGL15, while directly repressing the partially

redundant floral promoters SOC1 and FRUITFUL (FUL)

[34]. The other A-class protein, AP1, directly represses

the floral repressor TFL1, while directly activating LFYand the floral homeotic genes AP2, AP3 and SEP3 [27�].While LFY directly activates homeotic genes in all four

whorls it also directly represses the expression of the

shoot identity gene TFL1 [28�]. Finally, SEP3 appears

to act bifunctionally, repressing the flowering time genes

SVP and SOC1 but promoting the expression of floral

organ identity genes AP1, AP3 and AG [29�]. While the

many examples are tantalizing, the underlying mechan-

isms are not yet clear. A dominant hypothesis evokes

dynamic combinations of co-repressor/co-activator com-

plexes governing these functional switches, such as has

been suggested for AP1 [23], FT–FD–TFL1 [41], and

the SEU–LUG–AP2 complexes [34,39].

A major consequence of the recent flurry of ChIP-seq and

ChIP-chip studies is that we not only know that TFs

directly target many loci, but importantly, a given TF can

directly promote development/differentiation of specific

organs by activating or repressing different suites of genes

depending on particular developmental contexts, exogen-

ous cues, or tissue types. Disentangling the regulatory

bases of these functional shifts is an important focus of

ongoing work in order to understand precisely how com-

plex developmental programs function. Meticulous,

stage-specific atlases of TF target maps for candidate


processes and TFs will prove invaluable to understand

the depth of this complexity.

Process integration, not simply pathwayintegrationRecent genome-scale TF target mapping studies have

revealed that flowering TFs have functions in processes

other than flowering time and/or flower development,

strictly speaking. In fact, a clear picture is emerging of

single TFs orchestrating the interactions of multiple

developmental processes, with the result that processes

not generally considered related are now directly linked.

New roles reaching outside of flowering have been sub-

stantiated for two of the most intensely studied flowering

integrators, FLC and LFY. Long established as a master

regulator of the response to vernalization in A. thaliana,

FLC has also been shown to work bifunctionally, directly

repressing FT, SOC1 and SEP3, but promoting the expres-

sion of floral repressors SMZ and TOE3 [42]. Perhaps more

surprisingly, FLC directly regulates genes that effect

juvenile to adult phase change, such as SPL15 [42]. This

is consistent with recent work that points to a role for FLC

in the transition from juvenile to adult phase [43]. Com-

bined with evidence that natural variation at the FLC locus

governs temperature-dependent germination through

genetic pathways previously considered to control only

flowering time [44], this points to FLC as a common

process integrator of several major events in the A. thalianalife cycle. It is no surprise that FLC regulation is proving to

be an extraordinarily rich topic with diverse noncoding

RNAs regulating FLC expression, which is also tied closely

to a dynamic state of chromatin remodeling [45–47].

Perhaps the most striking example of process integration

comes from genome-wide mapping of LFY targets [48�].While numerous studies have effectively explained pleio-

tropy by TF target mapping, this study discovered a wholly

new role. Unexpectedly, LFY coordinates the floral tran-

sition with defense responses, directing resources toward

flower and fruit development and away from defense in

order to maximize reproductive fitness at the critical

reproductive period [48�]. LFY bound directly to the

promoters of the MAMP (microbe-associated molecular

pattern) recognition receptor FLAGELLIN-SENSITIVE 2(FLS2) and ABC transporter PLEIOTROPIC DRUGRESISTANCE 8 (PDR8/PEN3) and controlled their

mRNA expression. These gene products are key players

in the basal plant immune response pathway leading to

callose deposition at the cell wall. Functional confirmation

came from testing the response to flg22 in a lfy mutant,

which exhibited a significant increase in the number of

flg22-induced callose deposits relative to wildtype [48�].

Concluding remarksUp to now, genome-wide TF mapping studies have been

largely isolated, with single groups choosing no more than


Flowering networks explode in complexity Pose, Yant and Schmid 49

a pair of related TFs. The next phase for GRN mapping

studies of the floral transition will be to assess multiple

TFs at several developmental stages and defined regions

in incipient flower primordia. That the advent of ChIP-

seq has already brought about such a deluge of interesting

data suggests that further technological development

probing GRN structure at a finer scale will well pay

off. To this end, the continued development of advanced

cell-sorting techniques will be required to determine the

binding sites of each TF and the expression profile of

targets in a particular subset of cells and developmental

stage. The application of Laser Capture Microdissection

(LCM), or the recent method for individual cell type

isolation INTACT (isolation of nuclei tagged in specific

cell types) [49], coupled to RNAseq will also allow precise

determination of gene expression changes in tightly

defined cell populations. With increasing resolution, we

can expect to observe more dynamic and variable roles for

each TF vis-a-vis the same targets, underscoring the

importance of the participation of co-factors and dynamic

local chromatin conditions in defining TF function.

AcknowledgementsWe thank J. Berger for assistance with scanning electron microscopy. Workin the M.S. lab on the regulation of the floral transition is supported by theDeutsche Forschungsgemeinschaft and the Max Planck Institute forDevelopmental Biology.

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� of special interest

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the end of innocence: flowering networks explode in complexity

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