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# WUSCHEL in the shoot apical meristem: old player, new tricks

Filipa Lara Lopes, Carlos Galvan-Ampudia, Benoit Landrein

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Filipa Lara Lopes, Carlos Galvan-Ampudia, Benoit Landrein. WUSCHEL in the shoot apical meristem: old player, new tricks. Journal of Experimental Botany, 2021, 72 (5), pp.1527-1535. 10.1093/jxb/eraa572. hal-03299266

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1 WUSCHEL in the shoot apical meristem: old player, new tricks

2 Filipa Lopes¹, Carlos Galvan-Ampudia²*, Benoit Landrein²*

3 ¹ Plant Stress Signaling, Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.

4 ² Laboratoire Reproduction et Développement des Plantes, Université de Lyon, Ecole Normale Supérieure de Lyon, UCB
5 Lyon 1, CNRS, INRAE, Lyon Cedex, France.

6 * Correspondance : benoit.landrein@ens-lyon.fr, carlos.galvan-ampudia@ens-lyon.fr

7 Abstract:

8 The maintenance of the stem cell niche in the shoot apical meristem, the structure that generates all
9 of the aerial organs of the plant, relies on a canonical feedback loop between WUSCHEL (WUS) and
10 CLV3 (CLV3). WUS is a homeodomain transcription factor expressed in the organizing center that
11 moves to the central zone to promote stem cell fate. CLAVATA3 is a peptide whose expression is
12 induced by WUS in the central zone that can move back to the organizing center to inhibit WUS
13 expression. Within the last 20 years since the initial formulation of the CLV/WUS feedback loop, the
14 mechanisms of stem cell maintenance have been intensively studied and the function of WUS has
15 been redefined. In this review, we will highlight the most recent advances in our comprehension of
16 the molecular mechanisms of WUS function, of its interaction with other transcription factors and
17 with hormonal signals and of its connection to environmental signals. Through this, we will show how
18 WUS can integrate both internal and external cues to adapt meristem function to the plant
19 environment.

20 **Keywords (alphabetically ordered):** Auxin, CLAVATA, Cytokinins, Light, Nitrate, Oxygen, Shoot apical meristem, WUSCHEL

22 Highlights: This review highlights recent advances in our comprehension of the molecular
23 mechanisms of WUSCHEL function in stem cell maintenance in *Arabidopsis* meristem and its
24 interaction with hormonal and environmental signals.

25 Introduction:

26 Plants generate organs throughout all their life thanks to the maintenance of stem cell niches
27 localized in specialized tissues referred as meristems. The shoot apical meristem (SAM) is a highly
28 organized structure, which is responsible for the generation of all of the aerial organs of the plants
29 (Barton, 2010). Meristematic cells are characterized by the expression of *SHOOTMERISTEM LESS
30 (STM)*, which encodes a homeodomain transcription factor (TF) whose activity is necessary for SAM
31 maintenance (Long *et al.*, 1996). Stem cells are located at the centre of the SAM in the central zone
32 (CZ) (Fig. 1). They are growing and dividing relatively slowly and are marked by the expression of the
33 *CLAVATA3* (*CLV3*) gene (Fletcher, 1999). The organizing centre (OC) is located below the central zone
34 at the tip of the rib meristem (RM) and is defined by the expression of the stem cell regulator
35 WUSCHEL (*WUS*) (Mayer *et al.*, 1998). Cells that are advected away from the central zone through
36 growth and division join the peripheral zone (PZ) where growth is more pronounced and where
37 organs are initiated following specific patterns of phyllotaxis (Barton, 2010). The SAM can also be
38 organized in layers (L1, L2 and L3) based on cell lineage analysis and characteristic cell division
39 orientations (Poethig, 1987) (Fig. 1).
More than 20 years ago, the maintenance of the stem cell pool in the SAM of the model plant *Arabidopsis* was proposed to be controlled by a feedback loop between WUS and CLV3 (Schoof *et al.*, 2000; Brand, 2000) (Fig. 1). WUS is a mobile homeodomain transcription factor expressed in the OC that can move to the CZ to promote stem cell fate, notably by repressing differentiation (Yadav *et al.*, 2011; Daum *et al.*, 2014). Plants lacking WUS expression are unable to maintain their stem cell niche in the SAM, which leads to termination of the meristem after the production of a very limited number of organs (Laux *et al.*, 1996). CLV3 is a small peptide whose expression is induced by WUS in the CZ but that can repress WUS expression (Brand *et al.*, 2002). Plants lacking CLV3 expression generate very large meristems producing many organs because of a lack of inhibition of WUS expression (Clark *et al.*, 1995; Fletcher, 1999). Several CLV3 receptors including CLV1, CLV2, CRN (CORYNE) co-receptors, members of the BAM (BARELY ANY MERISTEM) family as well as the recently-characterized members of the CIK (CLAVATA3 INSENSITIVE RECEPTOR KINASES) family, have been isolated (Clark *et al.*, 1993; Jeong *et al.*, 1999; DeYoung *et al.*, 2006; Müller *et al.*, 2008; Nimchuk *et al.*, 2015; Hu *et al.*, 2018). Expressed in the OC but also in different domains in the SAM, they act in concert to control WUS expression by forming a variety of homo and hetero-dimers. The binding of CLV3 to CLV1 was also shown to trigger the internalization of the receptor (Nimchuk *et al.*, 2011), a mechanism that could explain the buffering effects observed following enhancement of CLV3 expression (Müller *et al.*, 2006). As for other receptor kinases, activation of CLV1 induces a cascade of MAP KINASE (MITOGEN ACTIVATED PROTEIN) activation ultimately leading to WUS repression through mechanisms that still need to be finely dissected (Betsuyaku *et al.*, 2011). WUS also represses the expression of CLV1 through direct binding to its promoter thus adding another layer complexity to the core CLV/WUS feedback loop (Busch *et al.*, 2010). CLV signalling also affect auxin-mediated growth in flora primordia, notably in response to cold (Jones *et al.*, 2020).

Many studies have also pushed further our comprehension of the mechanisms regulating the expression of *WUS* and its function in the SAM. In this review, we will discuss recent advances aiming at characterizing the molecular mechanisms of *WUS* function, its connection to hormonal signalling and its response to environmental cues in the plant model *Arabidopsis* (for a more broader view of *WUS* function in other species see (Kitagawa and Jackson, 2019; Jha *et al.*, 2020)). *WUS* is also involved in floral organ identity and in floral meristem termination, but these functions will not be discussed here (for a review on this subject, see: (Sun and Ito, 2015)).

## Molecular mechanisms of WUS function

WUS function in the SAM relies on three distinct yet interconnected processes: its movement, its capacity to form homo and heterodimers and its binding specificity. WUS movement from OC to CZ is a central property of the CLV/WUS feedback loop but it was only confirmed 10 years after the formulation of the model (Schoof *et al.*, 2000). Using a set of translational reporter fused to various florescent proteins, Yadav and colleagues indeed showed that WUS could move from the OC to the CZ where it directly binds to the CLV3 promoter and that this movement was necessary for WUS function (Yadav *et al.*, 2011). Following this work, Daum and colleagues showed that WUS movement occurred through plasmodesmata and that specific sequences encoded within the WUS protein could promote but also restrict WUS movement in the SAM (Daum *et al.*, 2014). It is probable that the movement of WUS through plasmodesmata is regulated by specific yet uncharacterized proteins localized at plasmodesmata, similarly to what has been observed for STM (Winter *et al.*, 2007).
Like other homeodomain TF, WUS was also shown to form homodimers in *vitro* and *in vivo* (Busch *et al.*, 2010; Daum *et al.*, 2014). The mechanisms of WUS homodimerization have been recently studied in more depth (Box 1). Rodriguez and colleagues identified two distinct regions that are necessary for WUS dimerization. They also propose that the dimerization could be promoted by the binding to DNA, although it may not be strictly necessary (Busch *et al.*, 2010), and that it affects the stability of the TF (Rodriguez *et al.*, 2016). A further study from the same team showed that the homodimerization occurs in a concentration-dependent manner and that it affects binding to target genes (Perales *et al.*, 2016). As WUS movement through plasmodesmata is size-dependent (Yadav *et al.*, 2011) and that one of the sequence required for homodimerization is also necessary for mobility (Rodriguez *et al.*, 2016), the formation of dimers may reduce WUS mobility (Fuchs and Lohmann, 2020)(Box 1).

WUS has been shown to bind to DNA through three different motifs: a canonical TAAT motif for homeodomain TF, a G-box like domain and a TGAA domain (Lohmann et al., 2001; Yadav et al., 2011; Perales et al., 2016). Upon binding, WUS can act as an activator but also as a repressor thanks to the recruitment of co-repressors from the TOPLESS family (Leibfried et al., 2005; Busch et al., 2010). Interestingly, Perales and colleagues showed that the formation of homodimers could affect both the binding to DNA and the activity of WUS (Box 1). They proposed a model where WUS monomers would activate CLV3 in the CZ but where WUS dimers would repress CLV3 in the OC (Perales et al., 2016). Sloan and colleagues recently obtained the structure of fragments of WUS proteins bound to different DNA sequences which allowed them to study its binding specificity further (Sloan et al., 2020). They showed that WUS preferentially binds to TGAA sequences and that the dimerization allows cooperative and stabilized binding to specific repeated motifs.

In addition to the formation of homodimers, WUS can also form heterodimers with members of the HAIRY MERISTEM (HAM) family of transcription factors (Zhou et al., 2015, 2018) (Box 1). The triple ham1.2.3 mutant has a very intriguing phenotype where CLV3 expression increases and moves from the CZ to the OC. Given that HAM is only expressed in the L3, Zhou and colleagues proposed that the formation of heterodimers between WUS and HAM prevents the induction of CLV3 in the OC while the absence of HAM in the L1 and L2 allows the induction of CLV3 by WUS in the CZ (Zhou et al., 2018). This model is supported by two sets of computational simulations that could recapitulate both WT and mutant phenotypes (Zhou et al., 2018; Gruel et al., 2018). Very recently, Su and colleagues also showed that WUS physically interacts with STM. They also demonstrated that STM could bind to the CLV3 promoter and that this binding strengthened the one of WUS through the formation of a WUS/STM heterodimers (Su et al., 2020) (Box 1).

Taking together these studies, we could build a model where WUS homodimers or/and WUS/HAM heterodimers may inhibit CLV3 expression in the OC, while WUS/STM heterodimers would induce CLV3 expression in the CZ (Box 1). Studying the movement, DNA binding and transcriptional activity of the WUS homodimers and heterodimers should allow us to further understand how the dimerization affects WUS function in both CZ and OC.

## WUS interaction with hormone signalling

Following the identification of its targets, it was shown that WUS maintains stem cell identity by repressing differentiation, and that many of the WUS targets are involved in hormone signalling (Leibfried et al., 2005; Busch et al., 2010; Yadav et al., 2013; Ma et al., 2019) (Box 2). WUS and
124 cytokinins (CKs) have an intricate feedback loop, where WUS activates CK signalling by repressing
125 negative regulators of CK signalling of the *type-A ARR (ARABIDOPSIS RESPONSE REGULATOR)* family
126 (Leibfried *et al.*, 2005). In return, CKs promote stem cell fate by inducing WUS expression but also by
127 repressing *CLV1* expression (Gordon *et al.*, 2009; Buechel *et al.*, 2010; Nimchuk *et al.*, 2015).
128 Interestingly, it was also proposed that the positioning of the WUS domain relies on cytokinins, and
129 more specifically on the expression pattern of the cytokinin AHKs receptors (ARABIDOPSIS HISTIDINE
130 KINASEs) (Gordon *et al.*, 2009; Chickarmane *et al.*, 2012). Gruel and colleagues further supported this
131 idea through computational studies. They showed that the diffusion of two mobile signals from the
132 epidermis: one corresponding to active CKs, the other corresponding to an unknown molecule
133 restricting AHKs expression to the L3, could position the WUS domain in the OC. Interesting scaling
134 properties of the system were highlighted using this model as it was shown that WUS expression
135 domain can scale to the size and the curvature of the SAM (Gruel *et al.*, 2016) (Box 2). Identifying the
136 molecule controlling AHKs expression should allow testing the prediction of this model and
137 confirming that CK signalling indeed controls the positioning of the WUS domain in the OC.

138 In addition to cytokinins, the WUS/CLV feedback loop has also been tightly connected to auxin
139 signalling. Auxin accumulates at specific position in the PZ to induce organ emergence thanks to polar
140 transport mediated by PIN1 proteins (PIN-FORMED 1) (Reinhardt et al., 2000). Although auxin also
141 accumulates in the CZ, signalling is low in this zone which is mostly insensitive to the hormone
142 (Vernoux et al., 2011). A recent paper from Ma and colleagues proposed that the maintenance of the
143 stem cells in such a low auxin signalling state is controlled by WUS (Ma et al., 2019) (Box 2). WUS
144 notably reduces the expression of the MONOPTEROS/AUXIN RESPONSE FACTOR 5 (MP/ARF5) at the
145 CZ to lower auxin signalling. They demonstrated that WUS also directly regulates the expression of
146 other genes involved in auxin signalling and response through histone de-acetylation, thus
147 preventing cells at the CZ to differentiate. Interestingly, Galvan-Ampudia and colleagues also recently
148 showed that newly formed auxin maxima corresponding to the future primordia are formed as
149 protrusions originated from the CZ, but these only emerge in the boundaries of the CZ where the
150 temporal integration of auxin concentration allows the activation of auxin signalling (Galvan-
151 Ampudia et al., 2020) (Box 2). From these studies, we could build a model where WUS maintains
152 stem cell fate by limiting auxin signalling in the CZ through chromatin modification, restricting organ
153 emergence to the PZ where auxin signalling can occur. Interestingly, we can hypothesize that this
154 inhibition of auxin signalling by WUS may control the rate of organ emergence in the SAM as a result
155 of the way organs emerge following specific patterns of phyllotaxis in the SAM (box 2).

156 Auxin can also feedback on stem cell homeostasis thus adding another loop to the system. It has
157 indeed been shown that MP/ARF5 inhibits the expression of two negative ARRs in the CZ, which
158 could thus induce cytokinin signalling and WUS expression in the SAM (Zhao *et al.*, 2010). MP/ARF5
159 can also repress the expression of *DORNROSCHEN/ENHANCER OF SHOOT REGENERATION 1* (DRN/ESR1), a positive regulator of *CLV3* expression (Luo *et al.*, 2018)(box 2). Although
160 transcriptional repression is not through direct binding to the *CLV3* promoter, DRN/ESR1 is required
161 to maintain *CLV3* expression in the stem cells. However, *dnr/dnrl* double mutant does not
162 recapitulate *clv3* mutant, suggesting that DRN might help to fine-tune the extension of the stem cell
163 niche rather than determining it.
164

165 WUS and the transduction of environmental cues
166

In addition to internal signals, it has been recently shown that several environmental cues such as light and sugar levels, mineral nutrient availability or oxygen levels can affect meristem function through hormone signalling and the CLV/WUS loop (box 3). Light is known to modulate SAM activity through auxin and cytokinins and thus influence plant growth and development at several stages of development (Yoshida et al., 2011; Pfeiffer et al., 2016). Accordingly, Pfeiffer and colleagues demonstrated that light and metabolic signals converge towards the TOR pathway (TARGET OF RAPAMYCIN) to modulate WUS expression in the SAM of germinating seedlings. They further showed that this effect is, at least partly, dependent on the activity of two CK degrading enzymes from the CYTOKININ OXIDASE (CKX) family (Pfeiffer et al., 2016) (box 3).

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Nitrogen (N) is a major mineral nutrient for plants whose availability in the soil affects metabolism, growth, and developmental processes (Vidal et al., 2020). The perception of N in different organs and the integration of this information through long-distance signalling are key for coping with changes in N availability. Although nitrate can act as a signal itself, one of the major long-distance N signalling pathways is mediated by CK (Zhang et al., 2020) (box 3). SAM activity also responds to changes in nitrate availability in a cytokinin-dependent manner. Osugi and colleagues indeed showed that an increase in nitrate levels in the soil leads to the production of CK precursors by specific ISOPENTENYL TRANSFERASE (IPT) enzymes in the root and to their translocation via the xylem to the shoot (Osugi et al., 2017). Landrein and colleagues further showed that the generation of active cytokinin by LONELY GUY (LOG) enzymes from these precursors in the SAM induces CK signalling, and WUS expression. This activation leads to stem cell proliferation and increased meristem size and organ production rate (Landrein et al., 2018) (box 3).

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However, the effect of nitrate on meristem function may be more complex and only partly mediated by CK signalling. Nitrate is also a major source of nitric oxide (NO), a central redox signalling molecule (Fancy et al., 2017). Interestingly, keeping the balance between the different forms of reactive oxygen species (ROS), such as NO, superoxide anion ($O_2^−$) or hydrogen peroxide ($H_2O_2$), affects growth and development both in shoots and roots and is important for SAM robustness against environmental fluctuations (Foyer et al., 2018). Hence, accumulating ROS species in the SAM by mutating the AtFTSH4, an ATP-dependent mitochondrial protease that counteracts accumulation of internal oxidative stress, causes meristem termination at higher temperatures (Dolzblasz et al., 2016). Interestingly, the key enzymes regulating ROS metabolism have a distinct spatial distribution within the SAM and the different forms of ROS play distinct roles in each domain (Yadav et al., 2009; Zeng et al., 2017; Foyer et al., 2018) (box 3). For example, depleting $O_2^−$ in the CZ lead to a decrease of WUS transcript and protein levels, CLV3 expression leading to meristem termination (Zeng et al., 2017). $H_2O_2$, on the other hand, is mainly present in the PZ, where it inhibits WUS expression and promotes stem cell differentiation (Zeng et al., 2017). $H_2O_2$ production can be promoted via nitrate- or cytokinin-induced NO, through the induction of superoxide dismutase, which converts superoxide ($O_2^−$) into $H_2O_2$, pointing for a possible role of NO in the regulation of stem cell activity (Wany et al., 2018). Supporting this idea, cytokinins are required for the activation of the *CYCD3* cell cycle gene by NO, thereby promoting cell proliferation (Shen et al., 2013).

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Oxygen levels may also affect stem cell homeostasis in both plant and animals (Le Gac and Laux, 2019). Weits and colleagues showed that the SAM is a closed hypoxic niche using a microscale oxygen electrode (Weits et al., 2019) (box 3). Consistent with this, a large number of core hypoxia-

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211 induced genes are upregulated in the SAM when compared to juvenile leaves and their expression is
212 downregulated when meristems are exposed to higher oxygen concentrations (80%) (Weits et al.,
213 2019). Most importantly, increasing the levels of oxygen resulted in decreased leaf production rates,
214 supporting the importance of hypoxia for SAM activity (Weits et al., 2019). Part of this response is
215 mediated by LITTLE ZIPPER2 (ZPR2), which is degraded by the oxygen-dependent N-degron pathway
216 and is thus stabilized at low O₂ (Wenkel et al., 2007; Weits et al., 2019). Several targets of ZRP2,
217 including HD-ZIPIII transcription factors and HECATE (HEC), have been shown to be involved in stem
218 cell regulation, notably by modulating WUS expression.
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220 Recently, Wu and colleagues have shown that WUS was also involved in plant immunity, by acting as
221 a molecular barrier against virus spreading at the shoot apical meristem. Upon viral infection, WUS
222 protein is stabilized and actively represses the expression of methyltransferases necessary for
223 ribosome stability, thus reducing global protein synthesis and keeping the virus out the meristem
224 (Wu et al., 2020). How WUS protein is stabilized upon viral infection is still an open question,
225 however this work very interestingly show a new role of WUS in the protecting stem cells from
226 infections.
227

## Conclusion

228 Accumulating evidence gathered in the recent years highlighted WUSCHEL has a central regulator of
229 stem cell fate and differentiation in the SAM and as a point of convergence for both internal and
230 external signals. The recent characterization of the activity of WUS homodimers and heterodimers
231 with HAM and STM has notably been a huge step forward in our understanding of WUS function.
232 Thanks to that, we can hypothesize that WUS activity in distinct tissues (such as OC and CZ), organs
233 and stages of development might rely on the formation of specific heterodimers with other TFs,
234 including HAM and STM. The study of the interplay between the *WUS/CLV* loop and auxin and
235 cytokinins also highlighted very strong connections between stem cell maintenance and hormone
236 signalling. This work notably allowed redefining the function of WUS in controlling organogenesis
237 through the inhibition of differentiation. Finally, the recent characterization of the impact of
238 environmental signal on *WUS* expression showed that the *CLV/WUS* feedback loop is finely tuned to
239 adapt meristem function to a variety of signals, and that hormones play a central role in this process.
240 A key question that remains to be answered is how all of these signals can be integrated by both
241 hormone signalling and WUS for plants to adapt meristem maintenance and organogenesis to their
242 constantly changing local environment.
243

244
245 The work of the Landrein lab is supported by the research fund of the ENS de Lyon and by the BAP
246 department of INRAe. The work of Filipa Lara Lopes is supported by the Fundação para a Ciência e a
247 Tecnologia of Portugal (UIDB/04551/2020, PD/BD/128397/2017, LISBOA-01-0145-FEDER-028128,
248 PTDC/BIA-BID/32347/2017). The work of Galvan-Ampudia is supported by the Agence Nationale de
249 la Recherche (ANR-18-CE13-0019 2018 to Teva Vernoux).
250

251
**Fig.1 Organization of the *Arabidopsis* shoot apical meristem**

RM: Rib meristem, PZ: Peripheral zone, OC: Organizing centre, CZ: central zone. L1 to L3: Layer 1 to layer 3.

<figure><img src="image_3.png" /></figure>

**Box 1. Recent developments in our comprehension of WUS regulation of CLV3 expression**

* WUS dimerization could explain its dual functions in the OC and in the CZ

Rodriguez et al. (2016) and Perales et al. (2016) showed that WUS forms stable homodimers upon binding to DNA in a concentration-dependent, which affects its binding to the CLV3 promoter. They proposed a model where WUS dimers negatively regulate CLV3 expression in the OC while WUS monomers positively regulate CLV3 expression in the CZ (panel A).

* WUS can form heterodimers with HAM in the OC

Zhou et al. (2018) showed that WUS can physically interact with members of the HAM family of TF, which are specifically expressed in the L3 of the SAM. They proposed a model where WUS/HAM heterodimers repress CLV3 expression in the OC while WUS alone in the CZ induces CLV3 expression (panel B).

* WUS can form heterodimers with STM in the CZ

Su et al. (2020) showed that WUS can physically interact with STM and that STM binding to CLV3 promoter can enhance the stability of WUS binding to this promoter through the formation of heterodimer in the CZ (panel B).

A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>
**Box 2. Recent developments in our comprehension of the mechanisms of WUS regulation of meristem function**

* WUS modulate auxin signalling in the CZ

Ma *et al.* (2019) showed that WUS acts as a rheostat to maintain stem cells in a low auxin signalling state by modulating the expression of many genes involved in auxin signalling and response through histone acteylation.

* Auxin maxima are produced in the CZ but only emerge in the PZ

Galvan-Ampudia *et al.* (2020) showed that newly formed auxin maxima corresponding to the future primordia are formed as protrusions originated from the CZ but only emerge in the PZ, which could result from the inhibition of auxin signalling by WUS in the CZ.

* Auxin can feedback on stem cell regulation through MP/ARF5

Luo *et al.* (2018) showed that MP/ARF5 can repress the expression of DRN/ESR1, a positive regulator of *CLV3* which, thus activating *WUS* expression and stem cell fate through the inhibition of *CLV* signalling.

* A model for WUS control of organogenesis in the SAM

The emergence of new organs in the SAM results from the accumulation of auxin at specific location thanks to polar auxin transport by PIN1 proteins (Reinhardt *et al.*, 2000). This mechanism of organ positionning (phyllotaxis) has long been conceptualized by the so-called inhibitory field theory (Landrein and Vernoux, 2014). This theory states that pre-existing organs inhibit the initiation of new organs at their vicinity (by depleting auxin from the surroundings). Interestingly, theoretical work testing this theory and partly validated experimentally have shown that two key parameters could control the positionning and timing of organ emergence through inhibitory fields: the size of the inhibitory fields and the radius of the central ring on which organs are initiated (Douady and Couder, 1996; Landrein *et al.*, 2015). By combining the recent work from Ma and colleagues and from Galvan-Ampudia and colleagues, we could hypothesize that WUS may control this second parameter by inhibiting of auxin signalling. In such scenario, increasing WUS activity would inhibit auxin signalling locally but increase organogenesis globally (See below). This model, that remains to be tested, would explain the tight correlation that can be measured between WUS expression, meristem size (which is defined as the distance between the center of the SAM and the organs) and organogenesis rate (Landrein *et al.*, 2015, 2018).

<figure><img src="image_6.png" /></figure>
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**Box 3. Influence of environmental signals on stem cell homeostasis in the SAM**

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* Light and metabolic signals can modulate WUS expression in germinating seedlings

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Pfeiffer *et al.* (2016) showed that light and metabolic signals are integrated by the TOR to regulate WUS expression in the SAM of germinating seedlings. They further showed that part of this response relied on the regulation of the activity of cytokinin degrading enzymes (panel A).

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* Nitrate can modulate WUS expression through cytokinins

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Landrein *et al.* (2018) showed that the SAM can respond to quick changes in nitrate availibility in the soil thanks to long range signalling of CK precursors, that are activated in the SAM and trigger the induction of WUS expression (panel B).

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* ROS can also influence stem cell homeostasis in the SAM

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Zeng *et al.* (2017) showed that ROS-metabolizing enzymes displays specific patterns of expression in the SAM. They proposed that the balance between O2-- and H2O2 is involved in stem cell maintenance and differentiation in the SAM (panel C).

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* Oxygen levels can affect stem cell homeostasis in the SAM

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Weits *et al.* (2019) showed that the stem cell niche is under hypoxic conditions and that altering oxygen levels in the meristem can affect stem cell homeostasis, notably through the activity of HD-ZIPIII transcription factors. This effect is mediated through the degradation of ZPR2 by the N-degron pathway (panel D).

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<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

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<figure><img src="image_9.png" /></figure>

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<figure><img src="image_10.png" /></figure>

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