Developmental Cell

Article

A Regulatory Framework for Shoot Stem CellControl Integrating Metabolic, Transcriptional,and Phytohormone SignalsChristoph Schuster,1 Christophe Gaillochet,1 Anna Medzihradszky,1 Wolfgang Busch,2,4 Gabor Daum,1 Melanie Krebs,3

Andreas Kehle,2 and Jan U. Lohmann1,*1Department of Stem Cell Biology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany2Max Planck Institute for Developmental Biology, 72076 Tubingen, Germany3Department of Plant Developmental Biology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany4Present address: Gregor Mendel Institute, 1030 Vienna, Austria

*Correspondence: jlohmann@meristemania.org

http://dx.doi.org/10.1016/j.devcel.2014.01.013

SUMMARY

Plants continuously maintain pluripotent stem cellsembedded in specialized tissues called meristems,which drive long-term growth and organogenesis.Stem cell fate in the shoot apical meristem (SAM) iscontrolled by the homeodomain transcription factorWUSCHEL (WUS) expressed in the niche adjacentto the stem cells. Here, we demonstrate that thebHLH transcription factor HECATE1 (HEC1) is atarget of WUS and that it contributes to SAM functionby promoting stem cell proliferation, while antago-nizing niche cell activity. HEC1 represses the stemcell regulators WUS and CLAVATA3 (CLV3) and,like WUS, controls genes with functions in meta-bolism and hormone signaling. Among the targetsshared by HEC1 and WUS are phytohormoneresponse regulators, which we show to act as mobilesignals in a universal feedback system. Thus, ourwork sheds light on the mechanisms guiding meri-stem function and suggests that the underlying reg-ulatory system is far more complex than previouslyanticipated.

INTRODUCTION

Despite the independent evolution of multicellularity in plants

and animals, the basic organization of their stem cell niches is

remarkably similar (Scheres, 2007). However, in contrast to

most animals, plants maintain continuously active pluripotent

stem cells during their entire life cycle. These cells are embedded

into specialized tissues, called meristems, which provide an

appropriate environment for long-term self-renewal (Weigel

and Jurgens, 2002). Because cell proliferation outside the meri-

stems is limited and plant cells are immobile because of their

rigid walls, it follows that stem cell behavior has to be continu-

ously coordinated with signals from outside the niche that relay

information about the developmental program and environ-

mental conditions. The shoot apical meristem (SAM) is the

438 Developmental Cell 28, 438–449, February 24, 2014 ª2014 Elsev

source of all above-ground tissue of a plant and therefore repre-

sents an excellent model to study stem cell homeostasis and

niche maintenance (Weigel and Jurgens, 2002).

SAM function in Arabidopsis is built around two core systems

acting in parallel: first, a suitable tissue environment is provided

by suppression of cell differentiation, and second, stem cell fate

is induced locally. Differentiation is repressed by the homeodo-

main transcription factor SHOOTMERISTEM-LESS (STM), which

is active throughout the entire meristem and stimulates the

biosynthesis of the phytohormone cytokinin (Jasinski et al.,

2005; Long et al., 1996; Yanai et al., 2005). In contrast, local

stem cell induction and maintenance in the central zone (CZ) is

based on the non-cell-autonomous activity of WUS expressed

specifically in the organizing center (OC), located in adjacent

deeper SAM layers (Laux et al., 1996; Mayer et al., 1998). Stem

cells in turn secrete the small CLAVATA3 (CLV3) peptide that

limits WUS expression through the CLAVATA1 (CLV1),

CLAVATA2 (CLV2), and CORYNE (CRN) receptors (Brand et al.,

2000; Clark et al., 1993, 1995, 1997; Fletcher et al., 1999; Muller

et al., 2008; Schoof et al., 2000; reviewed in Perales and Reddy,

2012). The non-cell-autonomous activity of WUS might be

attributed to movement of WUS protein into the stem cells (Fig-

ures S1A and S1B available online; Yadav et al., 2011); however,

the mechanisms that translate its function into cell behavior

only begin to emerge (Busch et al., 2010; Leibfried et al., 2005;

Lohmann et al., 2001; Yadav et al., 2013).

One important example is the local modulation of cytokinin

sensitivity by WUS, through the direct transcriptional repression

of type-AARABIDOPSIS RESPONSEREGULATOR (ARR) genes

that play important roles in dampening cytokinin responses

(Leibfried et al., 2005). Uncoupling of type-A ARRs from the

negative input provided by WUS leads to stem cell termination

phenotypes, whereas reducing their function causes the meri-

stem to expand (Leibfried et al., 2005; Zhao et al., 2010). Cyto-

kinin signaling is not only important downstream of STM and

WUS but also serves as a positive input for WUS expression

(Chickarmane et al., 2012; Gordon et al., 2009), suggesting

that the integration of transcriptional and hormonal systems un-

derlies SAM function. Indeed, local biosynthesis of cytokinin as

well as the activity of the type-A ARR cytokinin response genes

are indispensable for proper SAM activity in diverse plant spe-

cies (Giulini et al., 2004; Kurakawa et al., 2007). Consistently,

ier Inc.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

experimental modulation of cytokinin levels by ectopic expres-

sion or mutation of genes coding for cytokinin-inactivating en-

zymes, as well as loss of function in the three known cytokinin re-

ceptors ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and

AHK4/CYTOKININ RESPONSE1 (CRE1), lead to substantial

changes in SAM structure (Bartrina et al., 2011; Higuchi et al.,

2004; Nishimura et al., 2004; Riefler et al., 2006; Werner et al.,

2003).

Cytokinin signal transduction is based on a phosphorelay

cascade similar to bacterial two-component systems (Hwang

and Sheen, 2001): after signal perception by the AHK2, AHK3,

and AHK4/CRE1 transmembrane receptors, which mostly

localize to the endoplasmic reticulum, the cytoplasmatic histi-

dine kinase domain of the receptor dimer is phosphorylated

(Caesar et al., 2011; Hwang and Sheen, 2001; Inoue et al.,

2001; Mahonen et al., 2000; Suzuki et al., 2001; Wulfetange

et al., 2011; Yamada et al., 2001). The phosphoryl group is

then transferred to the nucleus via ARABIDOPSIS HISTIDINE

PHOSPHOTRANSFER (AHP) proteins, where it is passed on to

type-B ARRs, which in turn act as DNA-binding transcriptional

regulators of cytokinin response genes (Argyros et al., 2008;

Hutchison et al., 2006; Hwang and Sheen, 2001; Punwani

et al., 2010; Sakai et al., 2001; Suzuki et al., 1998). Among these

primary cytokinin response genes are the type-A ARRs coding

for small nuclear proteins, which are phosphorylated and stabi-

lized by AHPs in a cytokinin-dependent manner (Brandstatter

and Kieber, 1998; Hwang et al., 2012; Imamura et al., 1999;

Suzuki et al., 1998; To et al., 2007). Interestingly, most of the

ten members of the type-A ARR gene family act as negative-

feedback regulators of cytokinin signaling (Hwang and Sheen,

2001; To et al., 2004). In addition to cytokinin, the promoters of

the type-A ARRs ARR7 and ARR15 also respond to auxin,

another phyotohormone with essential roles for meristem activ-

ity, making them important integrator hubs in plant stem cell

control (Muller and Sheen, 2008; Zhao et al., 2010). The design

and function of the cytokinin signaling system has recently

been reviewed in El-Showk et al. (2013).

In the framework of the SAM, auxin is instrumental for speci-

fying the sites of organ initiation (Reinhardt et al., 2000; Vernoux

et al., 2000), which can be either leaves or flowers depending on

the developmental stage of the plant. Flowers contain four basic

organ types with stamens and carpels representing themale and

female reproductive organs, respectively. After fertilization, em-

bryo development ensues within the central gynoecium, which

will give rise to the fruit containing the seeds. The bHLH tran-

scription factors of the HECATE (HEC) subclade play important

roles during the development of the female reproductive organ

system, and loss of function of HEC1, HEC2, and HEC3 causes

severe defects in the septum, transmitting tract and stigma of the

gynoecium (Gremski et al., 2007). These defects are also found

in plants carrying a mutation in SPATULA (SPT), coding for

another bHLH transcription factor (Heisler et al., 2001), and

HECATE1 (HEC1) and SPT physically interact in yeast, suggest-

ing that the combinatorial activity of these regulators might be

required for correct patterning of the Arabidopsis fruit (Gremski

et al., 2007).

Despite recent advances in unraveling the mechanisms guid-

ing plant stem cell proliferation and differentiation (O’Maoileidigh

et al., 2013; Perales and Reddy, 2012), our understanding of cell-

Developm

cell communication and control of cell behavior within the stem

cell system is still fairly limited. Consequently, niche character-

ization after perturbation by environmental variation along with

modeling has suggested that so far unknown feedback systems

must exist in addition to the canonicalWUS-CLV loop to control

stem cell number and proliferation rate (Geier et al., 2008).

RESULTS

HECATE1, a Relevant WUSCHEL TargetTo uncover SAM regulatory mechanisms, we have undertaken

genome-wide analyses of WUS function (Busch et al., 2010;

Leibfried et al., 2005) and identified At5g67060, which codes

for the bHLH transcription factor HEC1, as a direct target of

WUS. By using posttranslational induction of ectopic WUS pro-

tein activity (Leibfried et al., 2005), we found that HEC1 RNA

levels were reduced by WUS in the absence of protein synthesis

(Figure 1A). Furthermore, WUS exhibited in vivo binding to chro-

matin of the HEC1 regulatory region that harbors both known

WUS binding sequences (Figures 1B and S1C–S1E). These re-

sults were also consistent with HEC1 RNA expression patterns

in the SAM: HEC1 transcripts accumulated throughout the

SAM and early flower primordia but were excluded from the

WUS domain and reduced in stem cells (Figures 1C, S1F, and

S1G). Furthermore, HEC1 expression was strongly repressed

in meristems with ectopic WUS activity (Figures S1H–S1K).

This potent negative regulation proved to be essential for meri-

stem function, because SAMs of plants carrying a pWUS:HEC1

transgene terminated and displayed phenotypes reminiscent of

weak wus mutants (Figure 1D). Expression levels of WUS and

CLV3were reduced even in pWUS:HEC1 plants with milder phe-

notypes (Figures 1E–1H), demonstrating that HEC1 function

potently interfered with OC activity, which in turn caused stem

cell exhaustion followed by a loss of CLV3 expression. Thus,

repression of HEC1 in the OC is an essential function of WUS

in SAM maintenance.

Roles of HECATE1 in Meristem RegulationHEC1 groups into a small subclade of bHLH transcription factors

and was previously shown to play important, but redundant,

roles in female reproductive tract development together with

the highly related HEC2 and HEC3 proteins (Gremski et al.,

2007). Consistently, we did not observe phenotypic alterations

in SAMs of hec1 single mutants. However, SAMs of hec1,2,3 tri-

ple mutants were significantly reduced in size (Figures 2A, 2D,

2E, 2H, and 2I) and showed aberrant organ initiation patterns,

demonstrating that HEC function is essential for proper meri-

stem development. To elucidate the underlying regulatory

changes in the SAM, we recorded WUS and CLV3 mRNA pat-

terns: whereas CLV3 expression was increased and expanded

in hec1,2,3 (Figures 2B, 2F, and 2J), WUS transcripts were

decreased compared to wild-type (WT) (Figures 2C, 2G, and

2K). Because it is thought that WUS is required to induce stem

cell fate and thus CLV3 expression (Brand et al., 2000; Mayer

et al., 1998; Schoof et al., 2000), these modifications highlight

the capacity of HEC factors to uncouple CLV3 expression from

the stem cell-activating input ofWUS and to modify the ratio be-

tween stem cell number and meristem size. Having shown that

HEC1 is necessary for proper meristem function, we asked

ental Cell 28, 438–449, February 24, 2014 ª2014 Elsevier Inc. 439

Figure 1. HEC1 Is a Relevant Direct Target

of WUS

(A) Quantification of HEC1 mRNA expression in

inflorescence apices of p35S:WUS-GR plants

after overnight mock and cycloheximide control

(C + M) or dexamethasone induction in the pres-

ence of cycloheximide (C + D) by qRT-PCR. Four

independent experiments were performed, each

assayed in triplicates and normalized to the

respective control.

(B) Enrichment of HEC1 promoter fragments

after ChIP-PCR and normalization to a control

sequence downstream of HEC1. Crosses denote

fragment with WUS consensus binding sequence.

(C) HEC1 mRNA expression pattern in the inflo-

rescence meristem as recorded by in situ hybrid-

ization. CZ, central zone; OC, organizing center.

(D) Phenotypes of WT and pWUS:HEC1 plants;

inset shows wus mutant apex. Arrow, arrested

SAM.

(E–H) In situ hybridization experiments on 21-day-

old WT (E and G) and pWUS:HEC1 plants with

intermediate phenotype (F and H). Upper panels

showWUS expression (E and F), and lower panels

show CLV3 expression (G and H). Scale bars,

1.5 cm (D); 50 mm (C and E–H).

See also Figure S1.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

whether it is also sufficient to modulate stem cell behavior.

Intriguingly, uncoupling HEC1 expression from the negative

input of WUS using the stem cell-specific CLV3 promoter led

to massive SAM expansion analogous to clv3 mutants (Figures

2M, 2Q, and 2P). Consistent with a redundant function of HEC

genes, we identified similar phenotypes when we expressed

HEC2 and HEC3 from the CLV3 promoter, but not when we

used PIF3, a more distantly related bHLH (Figure S2).HEC1 spe-

cifically stimulated proliferation of stem cells, because expres-

sion in the surrounding transient amplifying cells of the peripheral

zone (PZ) by the UFO promoter did not cause any phenotypes

(Figure 2L). Whereas SAM expansion in clv3 plants is caused

by increased and ectopicWUS expression (Figure 2S), we found

that both CLV3 and WUS RNA levels were strongly reduced in

pCLV3:HEC1 apices (Figures 2N and 2O). Because of the dy-

namic feedback organization of the stem cell control system,

we explored its behavior during the development of the

pCLV3:HEC1 phenotype and found that 21 days after germina-

tion when meristem expansion was already apparent, expres-

sion of both WUS and CLV3 expression was still detectable.

The CLV3 signal was lost only after 28 days, whereasWUS tran-

scription was strongly reduced and almost fully suppressed by

day 35 (Figures S3A and S3B). The non-cell-autonomous repres-

sion ofWUS in the absence ofCLV3 suggested the presence of a

so far unknown stem cell-derived molecule with CLV3-like

signaling functions. Interestingly, the expression ofSTM, another

homeodomain transcription factor with essential roles in

SAM function (Long et al., 1996), remained unchanged in

pCLV3:HEC1 apices (Figure S3C), demonstrating the regulatory

specificity of HEC1. In addition, we observed similar morpholog-

ical and molecular phenotypes in plants expressing HEC1 from

promoters that are independent of SAM function, such as p16,

which is highly active in proliferating cells (Figure S4). Impor-

tantly, stem cell overproliferation in pCLV3:HEC1 plants was

440 Developmental Cell 28, 438–449, February 24, 2014 ª2014 Elsev

not a mere consequence of CLV3 suppression, because

pCLV3:HEC1 and clv3mutant phenotypes were not only distinct

at the level of SAM organization (Figures S5A–S5C) but also ad-

ditive when genetically combined (Figure 2T). Thus, HEC1 activ-

ity in the central zone is able to uncouple stem cell fate from

CLV3 expression and to stimulate cell proliferation largely inde-

pendent of WUS and other known meristem regulators. To test

this further, we introduced the pCLV3:HEC1 transgene into a

wus mutant background, which still resulted in a substantial in-

crease in organ formation (Figures S5D–S54F), confirming the

autonomous proliferative potential of HEC1.

Mechanisms of HECATE1 FunctionHow can the HEC1 transcription factor promote such distinct

behavior, ranging from termination to proliferation in highly

similar cells of the meristem? Because bHLH factors tend to

act as dimers, we hypothesized that the functional output of

HEC1 might be dictated by its interacting partners. Gremski

et al. (2007) had shown that HEC1 is able to interact in yeast

with the distantly related SPATULA (SPT) bHLH transcription

factor, which plays important roles in fruit patterning (Heisler

et al., 2001). Because SPTmRNA also accumulates in the center

of the SAM (Figure 3A; Heisler et al., 2001), partially overlapping

with HEC1 transcripts (Figure 1C), we used fluorescence reso-

nance energy transfer (FRET)- fluorescence lifetime imaging mi-

croscopy (FLIM) to establish that HEC1 andSPT interact in nuclei

of plant cells (Figures 3B and 3C). Interestingly, the stem cell

overproliferation phenotypes caused by pCLV3:HEC1 were fully

suppressed in a sptmutant background, and the resulting plants

were almost indistinguishable fromWT (Figures 3D and 3F), sug-

gesting that HEC1 function is dependent on interaction with

other bHLH transcription factors with cell-type-specific expres-

sion, including SPT. In contrast, the stem cell proliferative poten-

tial of HEC1 is independent of HEC2 and HEC3 in stem cells and

ier Inc.

Figure 2. HEC1 Has Context-Dependent

Stem Cell Control Functions

(A–K) Wild-type plants (A–D) and hec1,2,3 mutant

plants (E–H). Inactivation ofHEC gene function led

to infertile plants (E, arrow) with smaller shoot

apical meristems (D, H, and I; p < 0.0001; WT: n =

26; hec1,2,3: n = 19), an enlarged CLV3 domain

with increased CLV3 expression (B, F, and J), and

a reduction of WUS expression (C, G, and K).

(L–T) pCLV3:HEC1 apices (L and M; n = 105) with

overproliferation phenotypes similar to clv3 mu-

tants (Q). Neither pUFO:HEC1 (n = 50) nor GUS

control plants (pCLV3:GUS: n = 80; pUFO:GUS:

n = 20) showed phenotypic alterations (L). WUS

and CLV3 expression was suppressed in

pCLV3:HEC1 apices (N and O, compare to B and

C), whereas WUS expression was expanded in

clv3 mutants (S, compare to C). SAMs of

pCLV3:HEC1 were massively enlarged compared

to pCLV3:GUS controls (p < 0.0001; pCLV3:GUS:

n = 15; pCLV3:HEC1: n = 23) (P); this phenotype

was further enhanced in the clv3 background (p <

0.0001; pCLV3:HEC1 in clv3: n = 42; pCLV3:GUS

in clv3: n = 36) (T). Error bars, SD. Asterisks indi-

cate statistical significance (*p < 0.05, ***p <

0.0001). Scale bars, 1 mm (A, E, M, and Q), 50 mm

(B, C, F, G, N, O, R, and S), and 20 mm (D and H).

See also Figures S2–S5.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

HEC1, HEC2, and HEC3 in other tissues, because we observed

meristem expansion phenotypes also in plants with stem cell-

specific HEC1 complementation in a hec1,2,3 triple mutant

background (Figure 3F). The relevance of the HEC1-SPT interac-

tion was further supported by the finding that spt single mutants

had mildly reduced SAMs, whereas meristems of hec1,2,3 spt

quadruple mutants were not significantly different from those

of hec1,2,3 triple mutants (Figure 3E). These results implied

that under regular growth conditions HEC function is limiting,

whereas after elevation of HEC1 levels, such as in CLV3:HEC1

apices, SPT becomes the restricting element of the regulatory

system. To test this experimentally, we expressed SPT from

the CLV3 promoter either alone or in combination with

pCLV3:HEC1. Whereas pCLV3:SPT plants did not develop

abnormal meristems, coexpression of both factors not only

Developmental Cell 28, 438–449,

increased the frequency of SAM expan-

sion but also caused a new class of

phenotypes with substantially fasciated

and split meristems (Figures 3F and S6).

Taken together, our results suggest that

apical stem cell proliferation is regulated

by interacting bHLH transcription factors,

including HEC1 andSPT, and that relative

levels of these factors dictate the prolifer-

ative potential.

Regulatory Potential of Plant StemCell RegulatorsTo elucidate the mechanisms translating

HEC1 activity into cell behavior, we per-

formed transcriptome profiling on inflo-

rescence apices of hec1,2,3 triple mutants, as well as plants

that carried an inducible HEC1 allele and identified response

genes by Z score-based meta-analysis (Busch et al., 2010).

Tracing their functions by gene ontology (GO) revealed four ma-

jor classes: (1) response to stimulus, (2) metabolic process, (3)

regulation of metabolic process, and (4) transport, with the first

class being activated and the last three classes being exclusively

repressed by HEC1, as well as a small group of cell-cycle genes

induced by HEC1 (Figure 4A). Strikingly, WUS response genes

showed a very similar functional signature (Busch et al., 2010),

suggesting that the orchestration of metabolic processes with

environmental and hormonal stimuli are essential activities of

plant stem cell regulators. The similarities extended to the level

of individual target genes, and we were able to identify 80 tran-

scripts that robustly responded to both transcription factors,

February 24, 2014 ª2014 Elsevier Inc. 441

Figure 3. HEC1 Interacts with the SPATULA bHLH Transcription

Factor In Vivo

(A) SPT expression in WT.

(B) Representative images from a control p35S:GFP-HEC1/p35S:mCherry-

NLS and a p35S:GFP-HEC1/p35S:mCherry-SPT infiltration into Nicotiana

benthamiana mesophyll cells.

(C) GFP lifetime of p35S:GFP-HEC1/p35S:mCherry-NLS and p35S:GFP-

HEC1/p35S:mCherry-SPT as measured by FRET-FLIM.

(D) SAM expansion driven by pCLV3:HEC1 was supressed in spt mutants. P,

flower primordia.

(E) SAM size of WT (n = 15), spt (n = 10), hec1,2,3 (n = 12), and hec1,2,3, spt

(n = 8) mutants.

(F) Phenotypic analysis of pCLV3:HEC1-spt (n = 16), pCLV3:HEC1-hec1,2,3

(n = 7), pCLV3:SPT (n = 34), pCLV3:GUS-pCLV3:HEC1 (n = 31), and

pCLV3:SPT-pCLV3:HEC1 (n = 34) T1 plants. Error bars, SD. Asterisks indicate

statistical significance (*p < 0.05, ***p < 0.0001). Scale bars, 50 mm.

See also Figure S6.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

significantly more than was expected by chance (p < 0.001) (Fig-

ure 4B). Intriguingly, 68 of these 80 genes showed opposing

response to WUS and HEC1, in line with the antagonistic

behavior of these two stem cell regulators in the SAM (Figure 4C;

Table S3). Furthermore, mapping HEC1 targets onto the

AtGenExpress expression atlas (Schmid et al., 2005) revealed

an exquisite context dependency ofHEC1 at the regulatory level,

and among the 463 response genes (p < 0.01; Tables S1 and S2),

we identified several clusters with tissue-specific activity pat-

terns. Most notably, among the genes with high expression in

the apex, whose activity was enhanced by HEC1, we found

several with functions in the cell cycle (Figures 4D and 5A).

This suggested that HEC1 stimulates cell proliferation in the

442 Developmental Cell 28, 438–449, February 24, 2014 ª2014 Elsev

apex by orchestrating the expression of cell-cycle regulators.

To test this functionally, we traced cell division patterns by using

HISTONE H4 mRNA to detect cells undergoing S phase in

SAMs with reduced or enhanced HEC1 function. Consistent

with a role of HEC1 in modulating cell proliferation, we found

substantial deviations from the WT pattern. Cell-cycle activity

was reduced in SAMs of hec1,2,3, but the developing flowers

of these plants exhibited a normal S phase pattern (Figures

5B and 5C), again confirming that HEC1 function is strongly

context dependent. In pCLV3:HEC1 apices, where HEC1 activ-

ity was uncoupled from the inhibition by WUS, cell-cycle activity

was dramatically enhanced and S phase cells could be de-

tected in large clusters (Figures 5E and 5F), likely coinciding

with HEC1 activity. By using image analysis to quantify the

results of the HISTONE H4 in situ hybridizations, we found

that the differences in mitotic index were statistically significant

(Figure 5D), confirming our hypothesis that one important

aspect of HEC1 function in the SAM is the stimulation of stem

cell proliferation.

A Stem Cell Feedback SystemIn addition to genes with function related to the cell cycle, we

also identified ARABIDOPSIS RESPONSE REGULATOR7

(ARR7) and ARR15 among the HEC1 response genes with

apex-specific expression (Figure 4D). Because ARR7 and

ARR15 are canonical cytokinin response genes of the type-A

ARR class that play essential roles in meristem control by inte-

grating auxin and cytokinin signals (Buechel et al., 2010; Giulini

et al., 2004; Leibfried et al., 2005; Werner and Schmulling,

2009; Zhao et al., 2010), we further investigated their response

to HEC1. By using qRT-PCR on inflorescence apices with

enhanced or reduced HEC1 function, we were able to confirm

a significant regulatory interaction with ARR5, ARR7, and

ARR15 (Figures 6A and 6B). Because type-AARRs encode small

phosphoproteins, which are able to repress WUS expression

(Leibfried et al., 2005) and to interfere with cytokinin signaling

(To et al., 2004), we hypothesized that these molecules could

represent mobile stem cell signals. To test this idea, we first re-

corded their mRNA patterns in SAMswith localizedHEC1misex-

pression and observed an accumulation of ARR7 and ARR15

transcripts in the corresponding domain (Figures 6C–6H),

demonstrating a cell-autonomous activation by HEC1. Because

type-A ARRs have been shown to antagonize cytokinin function

(To et al., 2004), this result suggested thatHEC1might also inter-

fere with cytokinin signaling. Consistent with this idea, we

observed a dramatic suppression of cytokinin signaling output

in SAMs of pCLV3:HEC1 plants (Figures 7A and 7B) using the

synthetic two-component sensor pTCS:3xYFP-NLS that mea-

sures cytokinin-dependent activation of B-type ARRs (Muller

and Sheen, 2008; Zurcher et al., 2013). Because this suppres-

sion was also observed in cells outside the CLV3 domain and

because HEC1 non-cell-autonomously suppressed WUS

expression (Figure 2O), we tested whether any of these proteins

would be able to move from cell to cell. Consistent with the cell-

autonomous activation of ARR7 and ARR15 by HEC1, we found

that HEC1-GFP was not able to move out of the L1 layer when

expressed from the epidermis-specific ML1 promoter, whereas

cell-to-cell movement was easily observed for ARR7-GFP or

GFP-ARR7 (Figures 7C–7G). Because type-A ARR protein

ier Inc.

Figure 4. Genome-wide Regulatory Potential of HEC1

(A) Overrepresented GO categories among HEC1 response genes (FDR p < 0.05): (1) response to stimulus, (2) metabolic process, (3) regulation of metabolic

process, and (4) transport. Ind., genes induced by HEC1; repr., genes repressed by HEC1.

(B) Venn diagram of HEC1 and WUS response genes.

(C) Response of shared target genes to WUS and HEC1 (Pearson correlation test: R = �0.68; p < 0.0001).

(D) Expression of apex-specific HEC1 target genes during Arabidopsis development. Yellow indicates increased expression, and blue indicates reduced

expression compared to the mean of all samples.

See also Tables S1–S3.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

activity and stability is regulated by cytokinin-dependent phos-

phorylation via AHP proteins (To et al., 2007), the status of the

cytokinin signaling system could have a substantial influence

on ARR7 mobility. To test this, we compared inflorescence

meristems expressing ARR7-GFP from the ML1 promoter after

Developm

2 and 4 hr of mock or cytokinin (100 mM BA) treatment. In the

cytokinin-treated samples, we observed a substantial increase

in nuclear GFP accumulation in the L1 providing evidence for

ARR7 stabilization with subcellular resolution (Figures 7H, 7I,

and S7A). We could rule out transcriptional activation of the

ental Cell 28, 438–449, February 24, 2014 ª2014 Elsevier Inc. 443

Figure 5. HEC1 Controls Cell-Cycle Genes

and Cell Division Patterns in the SAM

(A) Relative expression of cell-cycle genes from

apex cluster in hec1,2,3 and the induced pAlcA:

HEC1 plants. Expression levels are linearly trans-

formed gcRMA expression estimates from dupli-

cate Affymetrix Ath1 hybridizations.

(B and C) Histone H4 expression in SAMs of

hec1,2,3 (B) and wild-type plants (C).

(D) Mitotic index in SAMs of hec1,2,3 (n = 12), WT

(n = 11), and pCLV3:HEC1 (n = 9) plants.

(E and F) Histone H4 expression in SAMs of WT (E)

and pCLV3:HEC1 plants (F). Error bars, SD. As-

terisks indicate statistical significance (*p < 0.05,

**p < 0.01). Scale bars, 50 mm.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

pML1:ARR7-GFP transgene, because plants cotreated with

cytokinin and cycloheximide showed similar accumulation and

distribution of ARR7-GFP (Figure S7A). Interestingly, ARR7-

GFP was hardly detectable further than the L2 despite the

increase in protein abundance (Figure 7I), suggesting that activa-

tion of ARR7 protein by cytokinin-dependent phosphorylation

does not facilitate ARR7 movement. Quantification of GFP fluo-

rescence levels in L1 and L2 of treated and mock apices

confirmed this notion and showed that ARR7 mobility might

even be decreased in cells with high cytokinin signaling activity

(Figures 7J and S7B).

To test the functional relevance of type-A ARRs as down-

stream elements of HEC1, we expressed HEC1 in stem cells,

while reducing ARR7 and ARR15 activity in these cells by an arti-

ficial microRNA (Schwab et al., 2006). We not only observed

enhanced overproliferation phenotypes but also observed a

substantial increase in WUS RNA levels, when compared to

pCLV3:HEC1 SAMs (Figures 7K and 7L), consistent with the

function of ARR7 and ARR15 in WT SAMs (Zhao et al., 2010).

Conversely, WUS expression was reduced when we replaced

HEC1 by the constitutively active ARR7 (D85E; Leibfried et al.,

2005) (Figures 7M and 7N), demonstrating that type-A ARR

activity downstream of HEC1 constitutes as a potent non-cell-

autonomous signal affecting WUS expression and stem cell

proliferation.

Having shown that components of the cytokinin signaling

circuit represent important executors of HEC1 activity, we

wonderedwhetherHEC1 expressionmight also be under control

of cytokinin. Thus, we analyzed HEC1 mRNA levels in seedlings

that had been treated with cytokinin (5 mMBA) or mock for 0.5, 1,

or 3 hr, respectively. Although ARR7 mRNA abundance was

significantly increased by cytokinin at all three time points, we

were unable to detect any changes inHEC1 expression following

cytokinin treatment (Figure S7C). Taken together, our experi-

444 Developmental Cell 28, 438–449, February 24, 2014 ª2014 Elsevier Inc.

ments show thatHEC1 potently interferes

with cytokinin signaling via the transcrip-

tional activation of type-A ARRs, which

in turn can function as short-rangemobile

signals. Conversely, HEC1 gene expres-

sion does not seem to be under the con-

trol of cytokinin, suggesting that HEC1

acts as a negative upstream regulator of

cytokinin signaling outputs by activating inhibitory feedback

components of the pathway.

DISCUSSION

Based on the groundbreaking work of many labs and the exper-

imental data presented here, we have laid out a regulatory frame-

work for shoot stem cell control (Figure 7O), in which mobile

signals in the form of nuclear proteins, hormones, or secreted

ligands are arranged in intricate feedback communication loops,

which provide plasticity and stability to the dynamic shoot apical

meristem.

HEC1 Has Cell-type-Specific Functions in the SAMOur work highlights the dual role of the homeodomain transcrip-

tion factorWUS in regulating SAMactivity:WUSorchestrates cell

behavior, on the one hand, by providing spatial information and

locally influencing cytokinin signaling (Leibfried et al., 2005)

and, on the other hand, by tuning cell proliferation via the direct

transcriptional repression of HEC1. HEC1 in turn has cell-type-

specific functions in the SAM and strongly antagonizes WUS

expression in the OC. Although we cannot exclude other path-

ways, we hypothesize that this negative regulation is mediated

via type-A ARRs. ARR7 and ARR15 are transcriptionally acti-

vated by HEC1 in the SAM and have been shown to interfere

with WUS expression (Leibfried et al., 2005). Because WUS can

be transcriptionally activated by cytokinin (Buechel et al., 2010;

Gordon et al., 2009), it is tempting to speculate that the capacity

of type-A ARR to interfere with cytokinin signaling could be suffi-

cient to explain the loss ofWUS expression following HEC1 acti-

vation. Alternatively, loss-of-WUS activity might be an indirect

consequence from the interference of HEC1 with the basic tran-

scriptional programof theOC, becausemanyof the sharedHEC1

and WUS targets are regulated in opposing fashion. Prominent

Figure 6. HEC1 Locally Controls Cytokinin Feedback Regulators

(A and B) ARR5, ARR7, and ARR15 mRNA levels in apices of pAlcA:GUS and

pAlcA:HEC1 plants after overnight induction (A) and in WT and hec1,2,3

mutants (B) measured by qRT-PCR.

(C–H) mRNA expression patterns of ARR7 (C–E) and ARR15 (F–H) in SAMs of

WT (C and F), pWUS:HEC1 with intermediate phenotype (D and G), and

pCLV3:HEC1 (E and H) plants. Arrowheads indicate local accumulation of ARR

mRNAs at sites of elevated HEC1 activity. Error bars, SD. Asterisks indicate

statistical significance (*p < 0.05, **p < 0.01). Scale bars, 50 mm.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

examples are again ARR7 and ARR15, which are repressed by

WUS but are activated by HEC1, and thus could represent key

targets for a competitive regulation by HEC1 andWUS.

Within the stem cells, HEC1 has at least three important and

partially overlapping functionswith both positive and negative in-

fluences on stem cell activity: first, HEC1 antagonizes CLV3

expression, and reducing this negative stem cell signal will pro-

mote meristem enlargement (Fletcher et al., 1999). However, ge-

netic analysis demonstrated that the loss of CLV3 function

cannot fully explain phenotypes resulting from modifying HEC1

activity in the SAM. Second, via the activation of ARR7 and

ARR15, HEC1 is able to non-cell-autonomously interfere with

WUS expression, which in turn will lead to a reduction in SAMac-

tivity. We hypothesize that depending on the relative levels of

CLV3 and type-A ARRs in the stem cells, either the meristem

enlarging or reducing effect might be dominating, suggesting

thatHEC1 could act as a buffer of SAM function. Finally, the third

function of HEC1 in stem cells is the activation of cell-cycle reg-

ulators, which may explain the potential of HEC1 to stimulate

stem cell proliferation even in the absence of the WUS-CLV

system. It will be interesting to study the relative role of these reg-

ulatory pathways in diverse growth conditions and genetic back-

grounds in which the contributions of the three functions might

be exposed.

Developm

A Feedback System for Plant Stem Cell ControlAlthough the highly dynamic yet stable nature of the SAM has

long suggested that multiple feedback systems might act in

parallel to control the balance between cell proliferation and dif-

ferentiation, molecular evidence had been fairly limited (Geier

et al., 2008; Gordon et al., 2009). Despite the fact that cytokinin

has been implied to be an essential signal at multiple levels

(Buechel et al., 2010; Chickarmane et al., 2012; Gordon et al.,

2009; Leibfried et al., 2005), the cellular mobility of the hormone

is still unclear, especially because the relevant receptors mostly

locate to the endoplasmic reticulum (Caesar et al., 2011; Wulfe-

tange et al., 2011). Our finding that nuclear type-A ARRs can act

as mobile signals by non-cell autonomously interfering with

cytokinin signaling now defines a universal feedback system

acting both up- and downstream of key stem cell regulatory

nodes, such as the ones defined by WUS, cytokinin, or auxin

activities (Giulini et al., 2004; Leibfried et al., 2005; Muller and

Sheen, 2008; Zhao et al., 2010). Interestingly, ARR7 cell-to-

cell mobility appears restricted to one or two cell diameters

and to be largely independent of cytokinin signaling activity.

However, it seems conceivable that non-cell-autonomous

type-A ARR signaling based on cellular relay mechanisms could

achieve information transport over significantly longer dis-

tances, because interference with cytokinin signaling could in

turn affect type-A ARR expression in adjacent cells. However,

whereas the importance of ARR7 and ARR15 activity down-

stream of HEC1 and WUS has been established, the contribu-

tion of type-A ARR mobility to meristem function still remains

to be elucidated. The recent finding that a gradient of the nega-

tive cytokinin signaling factor, AHP6, is required to stabilize

phyllotaxis (Besnard et al., 2014) supports the idea that non-

cell-autonomous inhibition of cytokinin responses by mobile

intracellular regulators is an important feature of the signaling

network. Considering the role of cytokinin in sustaining SAM

function (Kurakawa et al., 2007; Werner et al., 2003) and acti-

vating WUS (Buechel et al., 2010; Chickarmane et al., 2012;

Gordon et al., 2009), our results further highlight the importance

of a dynamic interpretation of local transcriptional and global

hormonal signals for plant stem cell control.

Plant Stem Cell Regulators Share Functional SignaturesEven at the global gene-regulatory level, the integration of hor-

monal and environmental signals but also the regulation of meta-

bolic processes appear to be essential activities for plant stem

cell regulators, because the functions are significantly overrepre-

sented amongWUS andHEC1 targets. These findings are in line

with an important functional influence of the metabolic state on

meristem activity, which has been reported for the root and

shoot meristem (Wu et al., 2005; Xiong et al., 2013). Interestingly,

a close relative of WUS, namely, STIMPY/WOX9, has been

shown to mediate sugar signaling in the SAM, and meristem

arrest phenotypes of stipmutants are fully rescued by the exog-

enous application of sucrose to the growth medium (Wu et al.,

2005). In addition to sugar, STIP integrates cytokinin signals to

sustain meristematic cell proliferation, and ectopic STIP activa-

tion can partially rescue mutants with defects in cytokinin

sensing (Skylar et al., 2010). Apart from identifying candidate

genes for detailed functional analysis, the availability of sets of

downstream effectors for two unrelated stem cell regulatory

ental Cell 28, 438–449, February 24, 2014 ª2014 Elsevier Inc. 445

Figure 7. A Feedback System for Plant Stem Cell Control

(A and B) pTCS:GFP reporter activity in WT (A) and pCLV3:HEC1 (B) inflorescence meristems. P, flower primordia; arrowhead, CZ.

(C–E) GFP fluorescence in pML1:HEC1-GFP (C) or ARR7-GFP (D and E) under the control of ML1 promoter. Arrows mark the L2.

(F and G) GFP mRNA localization in SAMs carrying pML1:ARR7-GFP and pML1:GFP-ARR7 transgenes, respectively.

(H and I) GFP fluorescence in pML1:ARR7-GFP plants upon 2 hr of mock and Cytokinin (BA) treatment. The color-code depicts low (blue) to high (yellow-white)

fluorescence intensity.

(J) Quantification of the normalized fluorescence intensity. Cytokinin does not influence the mobility of the ARR7-GFP protein.

(K–N) WUS mRNA expression in pCLV3:GUS-pCLV3:HEC1 (K), pCLV3:AM7/15-pCLV3:HEC1 (L), pCLV3:GUS (M), and pCLV3:ARR7(D > E) (N) inflorescence

meristems. Of seven independent pCLV3:ARR7(D > E) T1 lines, five showed SAM phenotypes.

(O) Hypothetical model of HEC network connectivity in stem cell control. Error bars, SD. Asterisks indicate statistical significance (***p < 0.001). Scale bars, 50 mM

(A, B, K, L, M, N), 20 mM (C, D, E, H, I).

See also Figure S7.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

factors now opens exciting avenues to delineate the core genetic

requirements of plant stem cells.

Our experiments showed strictly cell-type-specific functions

of HEC1, and whereas in the CZ increased HEC1 activity

causes dramatic SAM expansion, expression in the OC leads

to meristem termination. In contrast, we were unable to detect

any SAM defects in plants expressing HEC1 in the transient

amplifying cells of the PZ. Because bHLH proteins tend to

act as dimers, a straightforward explanation could be the dif-

ferential interaction of HEC1 with other bHLH factors ex-

pressed in the SAM, including HEC2 and HEC3. Gremski

et al. (2007) have shown previously that HEC1 is able to

interact with SPT, but not with any of the HEC proteins in

yeast, and we have now confirmed a physical interaction of

HEC1 with SPT in plant cells. Our genetic analysis also indi-

446 Developmental Cell 28, 438–449, February 24, 2014 ª2014 Elsev

cated that HEC1 function is critically dependent on SPT and

that the relative levels of these transcription factors dictate

the proliferative potential of stem cells. Conversely, removing

HEC2 and HEC3 from stem cells did not impair the ability of

HEC1 to drive stem cell proliferation, suggesting that the inter-

play of HEC1 and SPT is indeed essential. Although we have

no direct evidence that the observed cellular behavior in

response to HEC1 and SPT is mediated by a complex of these

two proteins, it seems the most likely scenario to explain our

experimental data.

The idea that combinatorial activities of related bHLH tran-

scription factors can define functional domains within stem cell

systems and guide appropriate cell behavior is not new. Classic

work on animal stem cell niches has shown that the bHLH tran-

scription factor Myc is an essential regulator of cell proliferation

ier Inc.

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

and that this function is conserved throughout metazoan evolu-

tion (Eilers and Eisenman, 2008). Furthermore, c-Myc, along with

Sox2, Klf4, and Oct-4, is one of the classic factors required for

induction of pluripotent stem cells in vitro (Takahashi and Yama-

naka, 2006).Whether these similarities between plant and animal

stem cell systems are pure coincidence, the result of convergent

evolution, or whether they point to a common ancestral regula-

tory network remains to be elucidated.

EXPERIMENTAL PROCEDURES

Plant Material and Treatments

Plants were of Columbia background and grown at 23�C in long days. Ethanol

vapor inductions were performed overnight by placing a tip-box filled with

95% ethanol into the plant tray. For inductions with dexamethasone, 10-

day-old seedlings were incubated for 4 hr in 1/2 Murashige and Skoog (MS)

medium containing 15 mM dexamethasone, 0.3% ethanol, and 0.015% Silwet

L-77; 0.3% ethanol and 0.015% Silwet L-77 in 1/2 MS were used as control.

Cycloheximide was used at 10 mM. Cytokinin treatments were performed as

described in Gordon et al. (2009). 6-benzyladenine (BA) (Sigma) was used at

5 or 100 mM. The wus allele used corresponds to wus-4 in Col (provided by

Martin Hobe and Rudiger Simon), the clv3 allele was the transposon insertion

clv3-7, and the spt allele corresponds to spt-12 (Gremski et al., 2007; Ichihashi

et al., 2010). The hec1,3 double mutant previously described (Gremski et al.,

2007; Long et al., 1996) was crossed with hec2 T-DNA insertion line

(SM_3_17339).

Transgenes

The p35S:WUS-GR line was previously described (Busch et al., 2010;

Leibfried et al., 2005). The HEC1 coding sequence was amplified using

Gateway-tailed primers and cloned into pGEM-T easy (Promega) for

sequencing. For generating constitutive overexpression constructs, it was

then recombined into pDONR221 using Gateway Technology (Invitrogen)

and further recombined into pGREEN destination vectors carrying tissue-

specific promoters. Same procedures were used for making constructs of

GUS control, HEC2, HEC3, SPT, and PIF3 expression. For generating the

ethanol-inducible HEC1 line, HEC1 was recombined into a pGREENII vector

carrying the p35S:AlcA cassette, the AlcA promoter, and the OCS terminator.

p16 corresponds to a 0.5 kb promoter region upstream of the ribosomal gene

AT3G60245. To assess cytokinin signaling activity, we used the synthetic TCS

promoter driving the expression of 3xYFP-NLS (Muller and Sheen, 2008;

Zurcher et al., 2013); ARR7 or HEC1 coding sequences were fused in-frame

with GFP and driven under the AtML1 promoter. SPATULA was fused in-

frame with mCherry.

qPCR

qRT-PCR was performed on dissected apices of 21-day-old plants as previ-

ously described (Busch et al., 2010; Maier et al., 2009). Twenty plants were

used for each treatment and pooled. Amplification of TUBULIN served as

control.

Expression Profiling

Inflorescence shoot apices of 20 plants were microdissected 21 days after

germination, and RNA was extracted from pooled apices. For induction of

plants carrying the inducible pAlcA:HEC1 allele, ethanol vapor inductions

were performed overnight by placing a tip-box filled with 95% ethanol into

the plant tray. All experiments were conducted in duplicates. Expression esti-

mates were calculated by gcRMA (Busch et al., 2010; Wu et al., 2004) imple-

mented in R. Z score-based meta-analysis to identify HEC1 response genes

was done as described (Busch et al., 2010; Schmid et al., 2005), and a

HEC1 regulation score (HRS) was created for every gene. Array data are avail-

able at EBI under accession number E-MTAB-2193.

In Situ Hybridization and Microscopy

In situ hybridization, confocal laser scanning microscopy, and scanning elec-

tron microscopy were performed in accordance with standard protocols

Developm

(Maier et al., 2009). All in situ experiments were done on inflorescence apices

of 21-day-old plants grown under long day conditions at 22�C,with control and

mutant/treatment of the same age, grown at the same time. Meristem size was

measured by the maximumwidth between primordia. The quantification of the

WUS, CLV3, and HIS4 expression area and the calculation of the mitotic index

were done as described (Geier et al., 2008). For WT and hec1,2,3, the mean

expression domain of the three most central sections was calculated; for

pCLV3:HEC1, up to eight consecutive sections were averaged, because the

SAM is greatly enlarged.

FRET-FLIM

p35S:GFP-HEC1, p35S:mCherry-SPT, and p35S:mCherry-NLS were infil-

trated into Nicotiana tabacum leaves and imaged after 2 days on a Leica

TCS SP5II microscope equipped with a PicoHarp 300 TCSPC (PicoQuant)

system and an integrated FLIM-PMT (Hamamatsu). Lifetime images at 256 x

256 resolution were recorded using a 63x water immersion objective. The

donor was excited with a 470 nm pulsed laser with a laser repetition rate of

40 MHz. Donor fluorescence between 495 and 550 nm was collected over

100 frames. GFP lifetimes were calculated with a two-component decay

model using Symphotime software (PicoQuant).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed as described (Busch

et al., 2010; Leibfried et al., 2005; Zhao et al., 2010) with minor modifications.

Apices of 10-day-old wusmutants and moderately expressing p35S:WUS:GR

plants induced with dexamethasone for 4 hr were used as control and exper-

iment. Chromatin was solubilized with a Misonix S-4000 sonicator with cup

horn (10 s on, 15 s off, amplitude 10, for 4 min).

Statistical Analysis

For statistical analysis, data were first tested for normality using a Shapiro-Wilk

test. Then means were compared pairwise using either Welch’s t test or a Wil-

coxon rank-sum test. The significance level of shared response genes was

determined by random sampling. Correlation analysis was done using Pearson

correlation test. All calculations were performed in R.

ACCESSION NUMBERS

The EBI accession number for the array data reported in this paper is E-MTAB-

2193.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, and three tables and can be found with this article online at

http://dx.doi.org/10.1016/j.devcel.2014.01.013.

AUTHOR CONTRIBUTIONS

C.S., C.G., W.B., A.M., G.D., M.K., and A.K. performed experiments. C.S.,

C.G., and J.U.L. designed the experiments and wrote the paper.

ACKNOWLEDGMENTS

We thank Ingrid Haußer-Siller for help with REM, Tomi Bahr-Ivacevic for array

hybridizations, Zhong Zhao for discussion, and Klaus Harter, Marcus Heisler,

Thomas Holstein, Alexis Maizel, Kay Schneitz, Karin Schumacher, Detlef

Weigel, Jochen Wittbrodt, and members of the Lohmann laboratory for criti-

cally reading our manuscript. This work was supported by a grant from the

Deutsche Forschungsgemeinschaft (German Research Foundation; SFB873

to J.U.L.).

Received: August 12, 2013

Revised: November 25, 2013

Accepted: January 13, 2014

Published: February 24, 2014

ental Cell 28, 438–449, February 24, 2014 ª2014 Elsevier Inc. 447

Developmental Cell

A Regulatory Framework for Plant Stem Cell Control

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