Toward an integrative understanding of morphogenesis: interplay between developmental biology and computational science.

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Meristem boundaries and primordia

The SAM is a highly stereotypical structure made of domains containing cells with distinct cell fates and functions which are spatially separated. The Central Zone (CZ) is made of slowly dividing cells renewing the stem cell pool; beside the CZ is the Organization Center (OC) which is important for maintaining the CZ identity. Below the CZ and OC lies the rib meristem which gives rise to the main stem and thus drives the formation of the main aerial growth axis of the plant. The Peripheral Zone (PZ) contains cells with higher division rate and is the place of organ primordia initiation. Within organ primordia, cells exhibit a higher growth rate than the surrounding cells and are the place of new growth axis formation resulting in the emergence of new lateral organs. The separation between fast growing organ primordia and the rest of the SAM is marked by a crease-shaped domain: the boundary domain (Figure 3).
The main transcription factors (TFs) sustaining the SAM organization have been intensively studied over the last decades. The CZ is defined by interplay between the WUSCHEL (WUS) mobile TF and the small peptide CLAVATA 3 (CLV3). WUS is expressed in the OC and moves to the CZ where it activates the expression of CLV3, the binding of CLV3 to a complex made of CLAVATA1-2 triggers the restriction of WUS expression to the OC. This feedback loop prevents the expansion of stem cell identity outside of the CZ (Perales et al., 2016). In the same time, SHOOT MERISTEMLESS (STM) TF that belongs to the KNOTTED-like homeobox (KNOX) gene family is expressed in the CZ where it inhibits cell differentiation through induction of cytokinin (CK) synthesis and repression of gibberellin (GA) synthesis. Local repression of STM in the PZ is associated with initiation and differentiation of organ primordia. The boundary domain is marked by the expression of TFs belonging to distinct families. JAGGED LATERAL ORGAN (JLO) and LATERAL ORGAN BOUNDARY (LOB) (two LATERAL BOUNDARY DOMAIN TFs) are thought to regulate KNOX expression and to reduce growth by downregulating brassinosteroid (BR) synthesis, respectively (Bell et al., 2012). LATERAL SUPPRESSOR (LAS) a member of GIBBERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF GAI (RGA) and SCARECROW (GRAS) TFs are expressed in the boundary and induces formation of axillary meristems (Greb et al., 2003). Another important group of TFs expressed in the boundary domains are the CUP-SHAPED COTYLEDON 1 to 3 (CUC1-3) belonging to NO APICAL MERISTEM (NAM)/ARABIDOPSIS ACTIVATOR FACTORS (ATAF)/CUC (NAC) TFs family, but since they have been of main interest in this project, their role in boundary domain formation will be discussed later in this introduction (see Figure 3 for a map of genetic actors involved in SAM patterning).
Beyond TFs, hormone distribution and signaling play a major role in SAM patterning. Among the hormones reported to have a role in SAM patterning CK, GA and BR are involved but auxin appears to be a master regulator in organ primordia initiation and I will focus on its contribution. Auxin corresponds to a class of molecules able to stimulate coleoptile or stem growth and having a chemical structure derived from tryptophan and exhibiting a short lateral chain ended by a carboxyl group. Indole Acetic Acid (IAA) the most abundant form in plants is a weak acid (pKa 4.85) and can be found as equilibrium between protonated (IAAH) and anionic (IAA-) forms depending on the pH. The protonated form can freely diffuse across the plasma membrane whereas the anionic form is unable to cross the membrane. In the apoplast, a weak proportion of IAA is protonated (IAAH) and reaches the cytosol where it dissociates. IAA can also enter the cell via auxin influx carriers belonging to the AUX1/LAX family which are amino-acid permease-like proteins acting as H+ symports. In the cytosol, almost the entire pool of IAA is anionic and can only exit via efflux carriers either from the ABC MDR transporter family or the PIN-FORMED (PIN) family. PIN transporters are often polarly distributed at the PM and their polar localization is dynamically regulated through phosphorylations/de-phosphorylations (Figure 4). The multicellular patterns of PINs enable the dynamic and directional distribution of auxin, creating maxima and minima of auxin within organ and tissue that control cellular responses, differential growth and development. The underlying mechanisms responsible for coordinating PIN1 polarity across multiple cells enabling the formation of auxin fluxes across tissues are supported by various models. The “with the flux” model postulates that PINs polarize according to the flux direction, while the “up to the gradient” model postulates that PINs polarize toward the cell with highest auxin concentration. Both models lack of proposed mechanisms to explain them, the “with the flux” model miss a flux sensing mechanism, while the “up to the gradient” model would require cells to be able to sense the auxin concentration of their neighbors (Bhatia and Heisler, 2018). Auxin distribution via Polar Auxin Transport (PAT) mediated by PIN1 is at the heart of SAM patterning events since both naphthylphthalamic acid (NPA, inhibitor of polar auxin transport) treated plant or null mutant pin1 exhibit a suppression of flower primordia initiation in PZ. This phenotype can be rescued by local application of IAA at the PZ indicating that local accumulation of IAA in the PZ is required to initiate OPs (Reinhardt et al., 2000; Reinhardt et al., 2003) (Figure 5). Conversely, imaging of auxin signaling input reporter (DII-VENUS based reporters) and auxin transcriptional output reporters (DR5 type reporters) show iterative formation of auxin responses in the PZ prior to organ primordia outgrow (Heisler et al., 2010; Brunoud et al., 2012)(Figure 5). As a consequence of differential growth between organ primordia and surrounding tissue, a circumferential pattern of tensile stress is formed around the growing organ primordia and since tensile stress has been shown to impact polarity of cells (Hamant et al., 2008), PIN1 reorients toward the initiating organ primordia. This particular orientation of PIN1 toward growing primordia contributes to deplete auxin from the rest of the SAM (Heisler et al., 2010), thus enabling the formation of an inhibitory field around the initiated organ primordia preventing any new auxin maxima from being formed. New auxin maxima and subsequent organ primordia will form sequentially at a distance from the previous organ primordia. The auxin depletion associated with organ primordia initiation contributes to decrease auxin levels in the boundary domain and thus contributes to the activation of the expression of some TFs in the boundary domain, including CUC2 (Vernoux et al., 2000).

Role of CUCs in shaping the plant body plan

CUC TFs are a subset of NAC TFs, they have been identified in the 90’s and early 2000’s in both Petunia hybrida and Arabidopsis thaliana using forward genetic screens searching for developmental defects (Souer et al., 1996; Aida et al., 1997). In petunia, the no apical meristem (nam) mutant has no apical meristem; this phenotype is also observed in the double mutants cuc1cuc2 or cuc1cuc3 of Arabidopsis thaliana (Figure 6). CUCs are expressed in numerous boundary domains in aboveground parts of the plant including cotyledon-cotyledon, SAM-organ primordia, sepal-sepal, petal-petal, stamen-stamen junctions, between carpels and between ovule primordia (Aida et al., 1999; Ishida et al., 2000; Takada et al., 2001; Vroemen et al., 2003; Goncalves et al., 2015). Except from STM and RAX that has been shown to induce CUC2 expression, rather little is known on the transcriptional regulation of CUC genes. Based on the use of pCUC2::GUS reporter, IAA treatment was reported to repress CUC2 expression; such regulation is to be correlated with the presence of auxin responsive elements within the promoter of CUC2 (Galbiati et al., 2013). The interplay between CUC2 and auxin is further supported by the fact that auxin is depleted from places where CUC2 is expressed and CUC2 has been proposed to impact PIN1 polarity, although direct evidences are lacking (Heisler et al., 2005; Bilsborough et al., 2011). In SAM, CUC3 expression has been shown to be induced by mechanical stresses whereas CUC1 is not altered in the same conditions suggesting that CUC genes are regulated through distinct pathways (Fal et al., 2016). Post-transcriptional regulation of CUC1 and CUC2 also affects their expression, their mRNAs being targeted by a microRNA. The MIR164 family comprises three members, miR164a, miR164b and miR164c that are largely functionally redundant. They differ by their expression pattern even if they exhibit overlapping patterns (Sieber et al., 2007). They negatively regulate CUC1 and CUC2 through cleavage of their transcripts thus modulating the level of their targets (Nikovics et al., 2006) (Figure 7) whereas CUC3 mRNA is not susceptible to these microRNAs.


Main growth axis and control of leaf size

How a leaf grows has been a longstanding question for scientists studying leaf development, some of them believed leaves grow from the tips (August Trecul 1853), others argue it was from the basis. Like in most scientific controversies, both views were right it just depends on the species. While Arabidopsis leaves grow following a basipetal gradient, some other angiosperm species can exhibit acropetal, bidirectional or diffuse growth gradients (Das Gupta and Nath, 2015)(Figure 9). However, the first phase of leaf growth consists in the recruitment of cells from the peripheral zone of the meristem to the leaf primordia. This phase is followed by a proliferative phase which increases both the number of cells and the whole size of the leaf primordia, then cells progressively exit the cell cycle to enter into expansion (except for cells of the stomata lineage for which this is delayed). In Arabidopsis, this transition between cell proliferation and cell expansion occurs basipetally (from the tip to the basis of the leaf). Changes in either the number of founder cells recruited into the primordia or the rate of proliferation do not usually impact the final leaf size due to the existence of compensatory mechanisms. On the other hand, changes in timing of proliferation arrest affect the final organ size (Donnelly et al., 1999; Kazama et al., 2010; Andriankaja et al., 2012; Fox et al., 2018). The progression of proliferation arrest front is controlled by a set of regulatory modules, including TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR 1 (TCPs), GROWTH REGULATING FACTORS (GRFs) and micro RNAs (Kalve et al., 2014) (Figure 10). Class I TCPs promote proliferation and cell growth (Herve et al., 2009; Kieffer et al., 2011) while class II TCPs promote cell expansion by the indirect activation of Arabidopsis Responses Regulator 16 (ARR16, a negative regulator of Cytokinin signaling pathway) (Efroni et al., 2008; Efroni et al., 2013). Class II TCPs also up-regulate miR396 that restricts the expression of GRFs proliferation promoting factors to the basal part of the leaf. The entry into cell expansion is associated with endoreplication and plants with impaired capacity to enter into endoreplication are affected in both leaf cell size and leaf size (del Pozo et al., 2006). Relative to the duration of leaf development, the longer the proliferative phase will be the more complex the leaf will be. Indeed the transition from proliferation to differentiation has been shown to be delayed in compound leaves in comparison to simple leaves. Conversely a down regulation of genes promoting the transition from proliferation to differentiation leads to a more complex leaf shape in Arabidopsis (Alvarez et al., 2016). In accordance with this view, some genetic actors involved in differentiation delay like STM and BREVIPEDICELLUS (BP) are expressed in C. hirsuta compound leaves but not in A. thaliana simple leaves (Rast-Somssich et al., 2015). Thus the regulation of proliferation and expansion impacts both leaf final size and leaf shape complexity, the latter relies on the iterative formation of new growth axis at the leaf margin; the actors and mechanisms associated with the formation of these new growth axis are developed in the following part.

New growth axis at the leaf margin

Leaves can display a range of various shapes depending on the number and orders of new growth axes formed. These new growth axes rely on differential growth and the intensity of this differential growth determines the nature of the new growth axis. To make it simpler, let’s consider a conceptual leaf growing following the proximo-distal axis, let’s now consider differential growth occurring at the margin, if the difference of growth is low, it will give rise to serration or lobe; if the difference is high, it will give rise to leaflets. For instance Arabidopsis leaves are simple with a serrated margin while in its close relative Cardamine hirsuta, differential growth is more pronounced and as a consequence, the leaves are composed of leaflets. These two leaves have one order of dissection (serrations or leaflets). Some leaves can have two or more orders of dissection, Solanum lycopersicon leaves for instance are composed of leaflets and each of these leaflets are serrated, they thus have two orders of dissection (leaflets being the first and serrations on leaflets the secondary) (Figure 11).

CUC3 expressing cells of the CUC2 expression domain exhibit reduced cell growth.

To figure out the link between expression patterns of CUCs and differential growth occurring during leaf morphogenesis, we combined live-imaging and time lapse experiments on lines carrying transcriptional reporters for CUC2 and CUC3. We first normalized mean projected signal intensities of CUC2 and CUC3 reporters then applied a threshold on these normalized signals to classify cells into three domains: CUC2 expressing cells, CUC2 and CUC3 expressing cells named CUC2 and CUC2/3 hereafter and cells expressing neither CUC2 nor CUC3 assigned as noCUC cells (Figure 2A-D). We then measured the area of CUC2 and CUC2/3 domains as well as the surfaces of cells within these domains on 29 independent leaves. These measurements show that CUC2/3 domain is more restricted to the sinuses than the CUC2 domain and is composed of smaller cells (Figure 2D, G-H).

Table of contents :

Chapter I: An introduction to plant morphogenesis from cells to shape through differential growth
Morphogenesis: shapes, cells and growth
Plant morphogenesis: a complex multiscale process
Cell cycle and cell division events:
Cell expansion
A specific view on plant morphogenesis: Heterogeneity
Differential growth shapes the above ground plant body plan
Meristem boundaries and primordia
Role of CUCs in shaping the plant body plan
Leaf (margin): a model to study differential growth
Structural organization of leaves and associated cell types
Main growth axis and control of leaf size
New growth axis at the leaf margin
Main objective of the project
Chapter II: CUC3 mediates differential growth at the leaf margin by reducing cell growth
Chapter III: Auxin responses and mapping of TIR1/AFB during leaf serration
Core auxin signaling pathway: from perception to transcriptional outputs
Structural organization of auxin signaling components:
TIR/AFBs-AUX/IAAs interaction: affinity and specificity
AUX/IAAs inhibitory action on auxin transcriptional output
ARFs oligomerization and organization of AuxREs drive the diversity of auxin responses.
Other levels of ARF regulations
Auxin signaling components involved in leaf serration
Pattern of auxin responses during leaf serration:
Pattern of TIR1, AFB2 and AFB3 during leaf serration:
Leaf phenotype of tir1/afb mutants:
Material and methods:
Chapter IV: General discussion and perspectives
Growth control: multicellular levels and cell wall properties
From mechanosensing to transcriptional activation of CUC3
Differential auxin distribution and differential growth
Toward an integrative understanding of morphogenesis: interplay between developmental biology and computational science.


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