UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA

AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMAS
MÁSTER EN BIOLOGÍA COMPUTACIONAL

DEPARTAMENTO DE BIOTECNOLOGÍA – BIOLOGÍA VEGETAL

DEPARTAMENTO BIOTECNOLOGÍA - BIOLOGÍA VEGETAL

4D Computational Analysis for the Integration of Tissue

Formation with Cell Growth and Division Dynamics during
Lateral Root Formation

TRABAJO FIN DE MÁSTER

Autor: Marina Herrero Juanco

Tutor Externo: Miguel Ángel Moreno Risueño y Laura Serrano Ron

Tutor Académico: Mary Paz González García

July, 2023
 Table of contents
Summary .................................................................................................................................................................. 1
Abbreviations ........................................................................................................................................................... 2
Introduction.............................................................................................................................................................. 3
1. Post-embryonic organogenesis .................................................................................................................... 3
2. Arabidopsis thaliana root system ................................................................................................................. 5
2.1 Use of the model species Arabidopsis thaliana to research the root system ............................................ 5
2.2 Arabidopsis root tissular organization ................................................................................................. 7
2.3 Arabidopsis primary root longitudinal zonation ................................................................................ 10
3. Lateral root development in Arabidopsis thaliana..................................................................................... 10
3.1 Lateral root formation process................................................................................................................. 10
3.2 Formative stage .................................................................................................................................. 12
3.3 Identified cell populations in the LR formative stage......................................................................... 16
Materials & Methods ............................................................................................................................................. 18
1. Light Sheet-based Fluorescence Microscopy ............................................................................................. 18
2. Preprocessing of image data ...................................................................................................................... 19
3. Lineage tracing ........................................................................................................................................... 19
4. Statistical analyses...................................................................................................................................... 20
Results .................................................................................................................................................................... 21
1. Lineage tracing of the identities from the stem-cell trajectory ................................................................. 26
2. Lineage tracing of the identities from the vascular trajectory ................................................................... 35
3. Integration of the results obtained from the different cell identities in real time .................................... 43
Discussion ............................................................................................................................................................... 45
1. The stem-cell trajectory ............................................................................................................................. 46
2. The vascular trajectory ............................................................................................................................... 48
Conclusion .............................................................................................................................................................. 51
Bibliography............................................................................................................................................................ 52
 Summary
Postembryonic organogenesis plays a crucial role in the growth and adaptation of plants
to changing environmental conditions. In Arabidopsis, the majority of the mature root
system is composed of underground lateral roots (LRs). The process of LR formation
consists of two main stages: pre-patterning and formative stages. The lateral root
primordium (LRP) progresses through several developmental stages (I-VII) and
gradually acquires the characteristic tissue organization of the primary root.
Cell division and growth have been linked to the determination of cell fate and the
shaping of organ morphology. Thereby, we wanted to address the importance of these
forces in LRP organogenesis. For this, we used Light Sheet-based Fluorescence
Microscopy (LSFM) for the generation of 4D recordings of developing LRP in several
fluorescent marker lines (one for each identity) and cell membrane markers. The main
goal of this study was to validate and deepen the knowledge about how different cell
identities and their trajectories are specified at the early phases of the LR formative stage
in Arabidopsis thaliana. This work focused on the formative stage of LR formation and
was done by studying cell lineages through MaMuT software.

Altogether, our results suggest that the cell division pattern for SCN and QC
establishment is regulated differently during LR formation, embryogenesis, and primary
root growth in Arabidopsis and that before the actual QC development there could be an
organizing like center or cells with CBF3 and weak WOX5 expression. Lastly, the
dynamic expression patterns of vascular marker genes imply that the identity of vascular
tissue is not a uniform characteristic throughout various developmental stages,
suggesting the existence of additional specification mechanism for vascular cell identity
development over time. This suggests that the initial vascular tissue identity observed
during embryonic development is transient and not maintained in postembryonic. For all
this, it was concluded that the histogenesis of the LRP is hierarchical and is organized in
at least three development trajectories.

Our study not only provides evidence of an unprecedented hierarchy in a

developmental process, but also explores and validates the cell identities previously
described, thus shedding light on the long-term question of how plants conduct
development programs to establish cell fate while growing or developing structures.
Lastly, it was concluded that the histogenesis of the LRP is hierarchical and is organized
in at least three development trajectories.

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Abbreviations
❖ CEI: Cortex/Endodermis Initials

❖ EI: Epidermis Initials

❖ FC: Founder Cell

❖ LR: Lateral Root

❖ LRC: Lateral Root Cap

❖ LRP: Lateral Root primordium

❖ LSFM: Light Sheet-based Fluorescence Microscopy

❖ OZ: Oscillation Zone

❖ PBS: Pre-Branch Site

❖ PLT: PLETHORA

❖ PR: Primary Root

❖ QC: Quiescent Center

❖ RAM: Root Apical Meristem

❖ RC: Root Clock

❖ SAM: Shoot Apical Meristem

❖ SCN: Stem Cell Niche

❖ SCR: SCARECROW

❖ scRNA-seq: Single-Cell RNA Sequencing

❖ SHR: SHORT-ROOT

❖ WOX5: WUSCHEL-RELATED HOMEOBOX 5

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 Introduction
1. Post-embryonic organogenesis

Plants complete their life cycle through postembryonic organogenesis. Plants must
produce most of their organs after germination, unlike mammals which predominantly
rely on organs developed during embryogenesis. Additionally, because plants are
sessile organisms, they must adapt to changing and adverse environmental conditions .
In this scenario, postembryonic organogenesis is essential and provides exceptional
flexibility and adaptation facilitating their survival and reproductive success in a variety
of habitats (Raps et al., 1983; Rhodes et al., 1986; Seemann et al., 1984; Bao et al.,
2014; Nibau et al., 2008).

Plants maintain their ability to reprogram cells throughout their whole existence to be
able to produce new organs. In addition, this reprogramming capacity is the foundation
of regeneration processes (Efroni et al., 2016). Interestingly, according to (Sugimoto et
al., 2010), not all plant cells appear to be reprogrammable. Meristems, which are cellular
structures in which two concurrent and opposing processes are maintained: stem cell
renewal and recruitment for differentiation, are reprogrammable allowing diverse cell
fate specification. The shoot apical meristem and the root apical meristem, which are
found in all plants, are responsible for the development of new organs in the plant's aerial
and underground portions, respectively (Heidstra and Sabatini, 2014; Perez-Garcia and
Moreno-Risueno, 2018).

Meristems contain stem cell niches (SCN). These niches are made up of stem cells
around a quiescent center (QC), a small cluster of slowly dividing cells. SCNs can be
produced post-embryonically but also during embryogenesis and sustained throughout
post-embryogenesis. The post-embryonic creation of SCN can take place during the
development of new organs, the regeneration of injured organs, the formation of a new
organ, or the development of organs from calluses. Re-specification of stem cells is a
crucial step in these processes (Heidstra and Sabatini, 2014; Efroni et al., 2016; Perez-
Garcia and Moreno-Risueno, 2018).

The development of post-embryonic organs is essential for the adaptation and

growth of plants. Plant development, growth, and reproduction are governed by
interrelated processes, facilitating plant survival in a variety of conditions.

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 The capacity of plants to modify their growth and development in response to
environmental stimuli evidence and the strong connection between post-embryonic
organogenesis and plant adaptability. Plants possess exceptional plasticity, allowing
them to alter their phenotypes in response to environmental factors. Plants are able to
modify their reproductive phase, improve survival rates, and optimize resource allocation
because of this adaptive response, known as phenotypic plasticity (Lloyd et al., 2022).
In reaction to changes in temperature, day length, and nutrient availability, for instance,
plants can modify the timing of their flowering (Amasino & Michaels, 2010). Because of
their ability to coordinate their reproductive phase with favorable conditions, plants are
better able to produce seeds and engage in pollination.

In addition to adaptability, post-embryonic organogenesis plays a crucial role in plant

production, including agricultural and horticultural practices. It is essential to comprehend
the principles underpinning plant growth and development in order to maximize crop
output, enhance crop quality, and improve cultivation methods. Scientists and farmers
can control plant development patterns, manage blooming time, and increase fruit output
thanks to their understanding of post-embryonic organogenesis (Rojas et al., 2010;
Poethig, 2009). Increased biomass, bigger fruits, or longer harvesting times are a few
productivity-related features that are the focus of genetic engineering and breeding
initiatives. Researchers may create plans to increase plant output by better
understanding the complexities of post-embryonic organogenesis. This will help to meet
the world's expanding need for food and resources (Zhou et al., 2020; Rojas et la., 2010).

To sum up, post-embryonic organogenesis and the plant life cycle are interconnected
processes crucial for plant adaptation, production, and addressing global food security.
We can thereby highlight the extreme plasticity of plant cells being able to change cell
fate to generate new organs or reconstruct a functional SCN after injury. In fact, after full
excision, the root meristem can also regrow (Sena et al., 2009). These regeneration
procedures appear to involve both embryonic and postembryonic developmental
programs since distinct tissues may be produced from non-stem cells through a re-
specification process (Efroni et al., 2016; Moreno-Risueno et al., 2015).

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 2. Arabidopsis thaliana root system

2.1 Use of the model species Arabidopsis thaliana to research the root
system

Thale cress, sometimes referred to as Arabidopsis thaliana (A. thaliana), is a small

annual plant in the Brassicaceae family and dicotyledonous angiosperm. Close relatives
of this plant species include vegetables including rapeseed, radish, and mustard as well
as commercially significant oilseed crops (Kramer, 2015).

Despite being initially reported as a model organism for genetics and molecular
biology research only became well known in the 1980s (Kramer, 2015). Its small and
straightforward genome, which has 5 pairs of chromosomes, is about 125 Mbp in size
and encodes over 33,000 genes (Kramer, 2015). With the release of “The Arabidopsis
Genome Initiative” in 2000, the scientific world gained access to the genetic data of the
plant species Arabidopsis thaliana (The Arabidopsis Genome Initiative, 2000).

Although Arabidopsis thaliana has a simple anatomical structure with a core rosette
of leaves from which a main inflorescence develops. Either the main inflorescence shoot
or the rosette can directly produce lateral branches. The small, white, and highly
coordinated blooms of Arabidopsis thaliana produce a large number of seeds that are
kept inside siliques until they are dispersed (Kramer, 2015).

This plant species has lateral roots (LR) that grow from a primary root system
(Kramer, 2015). Notably, the roots of Arabidopsis thaliana are transparent and organized
into distinct layers, typically consisting of a single layer of each specialized tissue. The
primary root exhibits continuous and directed growth, enabling the visualization of
different developmental stages along its proximodistal axis. This simplicity and
informative nature of the Arabidopsis thaliana root system make it an ideal model for
studying root development (Van Norman & Benfey, 2009).

5
 Figure 1. Illustration of a plant of Arabidopsis thaliana. Schematic representation of an
Arabidopsis thaliana plant. The primary organs are pointed out. The primary shoot and the lateral
branches, which sprout from another subbranch or straight from the rosette, are the fundamental
structural elements of the aerial portion. All branches can develop leaves, flowers, and siliques. The
primary root and lateral roots, which might come from other lateral roots or from the primary root, make
up the root system. Adapted from DataBase Center for Life Science (DBCLS), CC BY 4.0
<https://creativecommons.org/licenses/by/4.0>, via Wikimedia Commons.

Research on plants has been transformed by the use of the model plant
Arabidopsis thaliana, which has provided invaluable insights into many facets of plant
biology and development. This is because of its straightforward genome, ease of culture,
quick life cycle, and well-characterized morphological characteristics (Meinke et al.,
1998). Researchers have made tremendous progress in understanding the complex
mechanisms behind plant growth and development through the extensive use of the
model organism Arabidopsis thaliana, which has helped advance agricultural practices
and crop improvement.

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 2.2 Arabidopsis root tissular organization

A. thaliana's roots are formed by concentric layers of cells, each of which typically
correspond to a distinct tissue identity. These tissues are the epidermis, cortex,
endodermis, pericycle, and vascular cylinder, arranged from the outside in. The sagittal-
plane-organized xylem, the phloem, which is positioned perpendicular to the xylem, and
the procambium, which is dispersed between the xylem and the phloem, are components
of the vascular tissues (Figure 2 B).

Together, the cortex and the endodermis make up the ground tissue. The cortex has
the capacity of thickening in response to unfavorable environmental conditions, acting
both as a tissue for storing nutrients and as a barrier against abiotic stress (Cui, 2015;
Long et al., 2010). The casparian strip encircles the endodermis and serves as a
selective diffusion barrier for nutrients and as defense against diseases (Naseer et al.,
2012). The pericycle is very reprogrammable and for the source of callus development
in vitro because of its great cellular flexibility (Sugimoto et al., 2010; Zhang et al., 2022).
The pericycle may be considered as a critical tissue for the organogenesis and
regeneration processes occurring in the Arabidopsis root due to its flexibility (Zhang et
al., 2022; Benfey & Scheres, 2000; Perianez-Rodriguez et al., 2014; Atta et al., 2009).

The most inner layers of the root correspond to the vascular cylinder, which houses
the essential vascular tissues responsible for the transport of water, nutrients, hormones,
and signaling molecules throughout the plant. Within the vascular tissues, the functional
transport occurs through specialized tissues known as the phloem and the xylem. The
xylem primarily transports water and minerals, while the phloem is responsible for the
movement of organic molecules, such as photo-assimilates. The xylem cells form
conducting tracheary elements, which are interconnected to facilitate efficient transport.
These tracheary elements are surrounded by non-conducting parenchyma cells and
xylem fibers, providing structural support and additional functionality to the xylem tissue.
On the other hand, the phloem consists of conducting sieve elements that are connected
to companion cells, ensuring their proper maintenance and functionality. Alongside the
sieve elements, non-conducting parenchyma cells and fibers contribute to the overall
structure and support of the phloem tissue. (Bauby et al., 2007; Carlsbecker and
Helariutta, 2005; De Rybel et al., 2016)

At the distal end of the root, in the root apical meristem (RAM), the tissues are arranged
similarly to at the proximal end, except for an additional exterior tissue known as the
lateral root cap (LRC). The LRC serves as a substantial barrier that safeguards the

7
meristematic cells when the root penetrates the soil. The LRC cells continuously lose
their function and are replaced by new cells because of the friction that ongoing
development creates. In addition, the LRC affects how primary root (PR) development is
oriented in response to stimuli such gravity, water, and light (Figure 2 B) (Eapen et al.,
2005; Kumar and Iyer-Pascuzzi, 2020; Silva-Navas et al., 2016; Swarup et al., 2005).

Known as the columella, this tissue's most apical, central area is made up of
specialized cells called statocytes. (Zhang et al., 2019). As a result of an auxin regulation
process, these cells build up starch inside amyloplasts forming statoliths. The
displacement of cytosol and endoplasmic reticulum caused by the sedimentation of
statoliths is thought to be the cause of the root's gravitropic response. It also appears to
activate mechanosensitive sites inside columella cells. (Arnaud et al., 2010; Leitz et al.,
2009).

As the root expands, the SCN produces new cells. Five different initial stem cell types
are seen in the root SCN, which are arranged surrounding the infrequently dividing QC
cells (Figure 2 B). WUSCHEL-RELATED HOMEOBOX 5 (WOX5), a transcription factor,
is one highly recognized QC identity determinant. The division of QC cells is inhibited by
WOX5 activity (Forzani et al., 2014; Sarkar et al., 2007). WOX5 is a mobile protein that
spreads to the SCN for its structure and maintenance, however WOX5 expression is not
restricted to the QC cells (Kong et al., 2015), and appears in forming SCN and during
regenerative processes.

Many of the surrounding initial (stem) cells differentiate after laser ablation of QC
cells, indicating that the QC is in charge of preserving the stem cells' undifferentiated
condition or stemness (Benfey and Scheres, 2000; van den Berg et al., 1997). As a
result, initial (stem) cells are kept in an undifferentiated stage, and their division produces
the various cell types that make up the root. The initial (stem) cells include the vascular
initials, LRC and epidermis initials (LRC/EI), the cortex/endodermis initials (CEI),
pericycle initials, and columella initials (Benfey and Scheres, 2000; Fisher and Sozzani,
2016; van den Berg et al., 1995).

In addition to keeping neighboring cells in their undifferentiated condition, it has been

suggested that the QC serves as a cell reserve for root meristem repair following
damage. According to this theory, the QC divides when meristem and stem cell damage
occurs from several causes to replenish lost cells (Matosevich and Efroni, 2021). The
QC may, interestingly, regenerate from the surrounding tissues when it is deliberately
ablated (Xu et al., 2006). However, the ultimate, developed QC is only apparent in the
end stages of the regeneration process, suggesting that this re-specification of the QC

8
is a gradual process. In fact, it is possible to recognize stem cell-like division patterns
before the development of a focused QC, indicating the possibility of an intermediary
identity operating during regeneration (Efroni et al., 2016; Matosevich and Efroni, 2021).

A B

Figure 2. Tissular and developmental organization of the Arabidopsis thaliana primary root. (A)
Schematic illustration of the primary root tissular organization. (B) Longitudinal sections taken at the
differentiation and meristematic zones, respectively, in the middle and bottom root zones. According to
the legend, different cell types are colored. Adapted from Bouché, Frédéric (2017): Arabidopsis - Root
cell types. figshare. Figure. https://doi.org/10.6084/m9.figshare.4688752.v1.

9
 2.3 Arabidopsis primary root longitudinal zonation

A. thaliana roots can be divided into several developmental areas along their
longitudinal proximodistal axis. Starting from the root tip, the meristematic zone,
elongation zone, and differentiation zone can be seen in Figure 2 A.

• The meristematic zone is composed of up rapidly multiplying cells that

maintain the root's growth pace. Cells get larger as they move farther from
the SCN (Dello Ioio et al., 2007).
• The elongation zone is composed of cells that grow anisotropically in the
longitudinal direction until they accomplish the optimal size for each cell
type (Beemster and Baskin, 1998).
• The differentiation zone is composed of cells that grow into fully
functioning cell types that perform specialized roles. This includes the
creation of the casparian strip as well as the production of root hairs. Here
is where the LRs develop (Grierson et al., 2014; Lavenus et al., 2013;
Roppolo et al., 2011).
Aside from the typical root zonation, two other zones have been described:

• The transition zone is a brief transitional area that connects the apical meristem
of the meristematic zone to the elongation zone via the basal meristem (Di
Mambro et al., 2017).
• The oscillation zone (OZ) is defined by the fluctuating expression of hundreds of
genes. The OZ, which includes the basal meristem and the elongation zone, is
critical for LR development during the pre-patterning phases which will be
explained later (Moreno-Risueno et al., 2010).

3. Lateral root development in Arabidopsis thaliana

3.1 Lateral root formation process

The Arabidopsis' root system is composed of underground LRs. The LR extends from
the PR, increasing the overall surface area and mechanical strength of the root system
and allowing the plant to investigate the soil environment. Millions of higher-order root
branches can eventually grow, yielding many kilometers of root network in a small area
of soil (Dittmer, 1937). LRs begin in flowering plants and gymnosperms from a specific
cell layer in the PR called the pericycle. Specifically in Arabidopsis and most other dicots,
10
LRs are generated exclusively from pericycle cells adjacent to growing xylem tissue (the
xylem pole pericycle) (Dubrovsky & Rost, 2006; De Smet et al., 2007).

The creation of LR is a multistep development process separated into two phases

(De Smet et al., 2007; Moreno-Risueno et al., 2010; De Smet et al., 2008; Van Norman
et al., 2013):

• Pre-patterning phase: It begins in the OZ, which is located near the root tip.
This area is distinguished by the presence of the Root Clock (RC), a
molecular oscillator that generates periodic pulses in gene expression. When
some of the cells exit the OZ, the RC controls the periodic development of
pre-branching sites (PBS), which indicate places for future LR emergence.
Finally, some cells from the PBS will be specified as LR founder cells (FC).
• Formative phase: The formative phase begins with FC specification followed
by their polarization and nuclear migration, followed by numerous rounds of
carefully controlled genetic cell division, initially anticlinally and then
periclinally. Following this, many further rounds of cell division occur,
resulting in the formation of a dome-shaped primordium (LRP) that grows
through multiple stages (I-VII) and gradually acquires the typical tissue
structure of the main root. Finally, the new LR will emerge from the tissues
that surround it (Figure 3).

Arabidopsis plants exhibit a consistently spaced pattern of LR along the major

root axis under normal circumstances. As we advance shootward along the proximodistal
axis, these new organs vary from newly created LRP to fully mature LRs. LRs alternately
appear on the left and right sides of the primary root, following the primary root in a wavy
pattern. Furthermore, LRs are oriented to the outside of the bends. The chemical activity
of the RC controls the creation of this unique pattern. In addition, throughout all the
different steps, auxin signaling acts as a central regulator thanks to the versatility and
specificity of different auxin signaling modules (De Smet et al., 2007; Laskowski et al.,
2008).

To sum up, lateral root formation could be summarized in four steps (Nibau et al.,
2008):

1. Stimulation and dedifferentiation of OZ pericycle. (This is the pre-patterning

phase).
2. Cell cycle re-entry and asymmetric cell divisions leading to the early formation
of an LRP (formative phase). Stages I-IV

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 3. LRP emerges from the PR's outer layers via cell expansion (formative phase).
Stages V-VI
4. Activation of the LR meristem, which mimics PR growth (formative phase).
Stage VII.

Primary root
Figure 3 Lateral root primordia development across primary root. The primary root's tissular
organization is shown schematically while the formation of a lateral root primordia is shown through its
different stages and colored in white. Until the stages VII and VIII the primordium does not break through
the epidermis of the primary root. Adapted from Figure 1 from Yang et al. (Figure 1 from Yang et al.
(2019).

3.2 Formative stage

As mentioned previously, the formative stage begins with FC specification. These

cells begin to expand, and their nuclei begin to move towards one another. As a result,
FCs divide asymmetrically, producing two morphologically and probably functionally
distinct daughter cells. To yet, the signal that initiates the differentiation of cells from the
PBS into pluripotent FCs remains unclear (Serrano-Ron et al., 2021a; Van Norman et
al., 2013). Following the first anticlinal asymmetric divisions that produce the stage I LRP
(Figure 4 A), periclinal divisions occur to form a two-layered LRP (stage II). These first
formative divisions appear to be uniform (Figure 4 A) and strictly controlled (De Smet et
al., 2008). Cells continue to divide significantly more freely after that (von Wangenheim
et al., 2016). The LR creation process is divided into seven distinct developmental
phases (I-VII) (Malamy and Benfey, 1997). The newly created root cell types are
gradually ordered to guarantee the new organ's functioning. As a result, a preserved

12
dome-shaped primordium with tissue layers is generated (Malamy and Benfey, 1997;
Trinh et al., 2018).

The mechanical forces are crucial in the creation of an LRP. External tissues
generate mechanical opposing forces during the formation of an LRP, which play a role
in determining the LRP shape (Lucas et al., 2013a; Vermeer et al., 2014), whereas
central and lateral LRP regions emerge from non-deterministic cell division patterns (von
Wangenheim et al., 2016). In fact, computational models that are simply focused on cell
division and development may produce a layered primordium (von Wangenheim et al.,
2016). This sort of concept, however, does not explain cell type patterning and overlooks
several crucial aspects of LR creation, such as cell type patterning. Furthermore,
asymmetric divisions have been identified at specific embryonic periods in both the
central and lateral LRP areas, indicating formative identity shifts (Schutz et al., 2021).

As mentioned before meristems contain SCN, and SCN contain QC cells. When
developing a new organ, the post-embryonic generation of SCN can occur. When the
plant is developing new roots, the activity of PLETHORA (PLT) transcription factors and
GRAS-family transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR) in
combination with an auxin gradient is required to maintain the QC and SCN during post-
embryonic primary root growth (Bennett and Scheres, 2010; Perilli et al., 2012). Malamy
and Benfey (1997) previously demonstrated that the GRAS-type transcription factor SCR
is one of the first genes to display differential LRP expression. SCR is expressed
primarily in the outer layer of cells at stage II and is restricted to the endodermis and the
QC in later stages (Figure 4 A). In the PR, it interacts with SHR to regulate SCN
maintenance, ground tissue formation, and organ growth (Fisher and Sozzani, 2016).
The PLT3, PLT5, and PLT7 transcription factors are essential for the early formative
divisions of the LRP central domain, the determination of the LR meristem tissues, and
the development of an auxin gradient throughout the formative and final meristem
processes (Figure 4 A). Furthermore, PLT3, 5, and 7 activate PLT1, PLT2, and PLT4 at
later stages of development (stages II-III), which is essential for LRP cell type
specification (Du and Scheres, 2017).

Research using PLT mutants (Du and Scheres, 2017) revealed an abnormal
morphology of the LRP during the transition from stage I to stage II, as well as a loss of
the auxin response gradient. In the plt3 plt5 plt7 mutant, PLT1, 2, and 4 are not induced,
resulting in LRP severely impaired in development. Introgression of any PLT member
into the mutant primordium at stage II entirely restores layer identities and recovers
mutant meristem and tissue establishment abnormalities. A second research (Goh et al.,

13
2016) found that inhibiting SCR activity prevented periclinal divisions in the LRP
outer cell layer and disrupted the generation of the QC cells, demonstrating that SCR is
important in the specification of the new LR's QC.

14
A

Figure 4. Lateral root formation. (A) lateral root's formation process is shown schematically. Cells are
colored by cell identity. According to the legend, colored circles inside the cells illustrate gene expression.
Stages I and II show the expression of PLT 3, 5, 7, which stimulates PLT1, 2, 4 expression at stages II and
III. Later stages of tissue development depend on this hierarchy. Stage II expression of SCR contributes to
quiescent center's specification. (B) Colors are used to indicate the localization of the different cell lineages.
The illustration is based on the ability of the populations to originate from or give rise to other cell populations
(black arrows). The dashed arrows show subsequent tissue/region of the LR whose origin could come from
each cell population based on localization of biomarkers at stage IV or later. The epidermis and lateral root
cap of the LR form after stage IV of LR. Figure adapted from Mol Plant. 2021 August 02; 14(8): 1362–1378.
doi:10.1016/j.molp.2021.05.028.

15
 3.3 Identified cell populations in the LR formative stage

Considering the findings mentioned previously, it is suggested that tissue lineages

must be initiated during the early morphogenesis phase, at the early stages of LR
formation (stages II-IV), prior to the meristem formation phase (stages V-VII), when the
different tissue identities become distinguishable.

As a result, Laura Serrano Ron and a team of scientists carried out a previous study
to this project (Serrano-Ron et al., 2021b) where computational analysis of single-cell
RNA sequencing (scRNA-seq) transcriptomic data followed by in vivo validations
showed seven distinct populations in the early stages (I-IV) of LRP development, which
can be distinguished by distinctive gene expression and enriched biological functions
(Figure 4 B). To validate the anticipated cell types in vivo, specific biomarkers were
chosen, and their promoter regions were fused to proteins fluorescence reporters for
sustained expression in transgenic lines. Out of those seven cell populations, 5 of them
will be studied in this project:

• Primordial cells marked with pDR4::NLS-3Xyfp (DR4) as identity marker.

• QC transitioning cells marked with pWOX5::YFP (WOX5) as identity marker.
• Endodermis/QC transitioning stem cells marked with pCBF3::NLS-3x-
mCherry (CBF3) as identity marker.
• Vascular like II marked with pMYB108::NLS-3xYFP (MYB108) as identity
marker
• Protophloem-like (cells close to the phloem) marked with pATHMP41::NLS-
3xYFP (ATHMP41) as identity marker.
In the previous research, two cell populations Endodermis/QC-transitioning stem
cells and QC-transitioning cells were found to be enriched in stem cell-like characteristics
and predicted the establishment of the ground tissue and QC lineages, along with a new
SCN. CBF3 expression was found in endodermis-QC-transitioning stem cells during
stages II-III of LRP development. QC transitioning cells were identified by the expression
of WOX5 and PLT4 and emerged during stage IV of LRP development. The analysis of
WOX5 expression in the CBF3 mutant led to the conclusion that CBF3 was engaged in
the transition between endodermis/QC-transitioning cells and QC-transitioning cells
because its function altered WOX5 expression in conjunction with an LRP formation
phenotype. These findings identify a developmental pathway that leads to the formation
of the LRP's new stem cell niche. In addition, they discovered three LRP cell types,
vascular-like 1, vascular-like 2, and protophloem-like cells, which suggest early vascular-
like identity acquisition. MYB108 expression identified vascular-like cells at stages II-III

16
of LRP development. Interestingly, protophloem-like cells were found close to principal
root phloem tissues (also designated by ATHMP41), implying an early vasculature link
of the LRP to the primary root and an early vascular developmental trajectory
independent of stem cell specification. Finally, during late phases of LRP formation, two
LRP populations, known as primordial and flanking cells, were limited to the flanks. DR4
expression denoted primitive cells, which developed at stage I and were eventually
limited to the sides of the LRP.

The existence of developmental linkages between cell identities was suggested by

the spatiotemporal correlations between the reported biomarkers. A few further analyses
and experiments were carried out and they concluded that the LRP's ontogeny was
structured and entailed the hierarchical assembly of cell lineages. An initial cell
population of precursor cells differentiated into three developmental branches, yielding
1) a center domain of stem cells, 2) an early vascular system at the sides, and 3) a
surrounding zone with a pluripotent stem cell reservoir (Figure 4 B).

17
 Materials & Methods
1. Light Sheet-based Fluorescence Microscopy

A customized plant growth chamber coupled inside a Light Sheet-based

Fluorescence Mycroscopy (LSFM) system (Maizel et al., 2011) was employed on
4 dpi seedlings to capture LRP development in real time. 4 to 6 capillaries (100
µl micropipettes Blaubrand Cat.-N° 708744) were glued to the bottom of a Petri
dish, which was then filled with Phytagel medium (2 g/l Murashige & Skoog
(Duchefa) (Murashige and Skoog, 1962), 0.5 g/l MES (2-(NMorpholino)-ethane
sulfonic acid, Duchefa), and 8 g/l Phytagel (Sigma-Aldrich). One sterile seed was
placed over each capillary after the medium at the top of the plate had been
removed. Following an overnight stratification at 4 ºC, seeds were then placed for
vertical growth under standard growth conditions (22 ºC, long day photoperiod
(16 h light/8 h dark)).

Prior to imaging, capillaries were removed from the Petri dish, and the
medium was pushed about 1 cm away from the capillary using a small rod. The
capillary was then put into the microscope growth chamber, which was filled with
half MS medium. The cotyledons are left exposed to the air when the seedling is
placed inside the chamber. Following the correct orientation of the sample, LSFM
imaging was carried out using the program Luxendo Bruker MuViSPIM. We used
the following parameters for the detection of fluorescent markers:

• CFP: 465-490
• GFP: 500-535
• YFP: 520-555
• mCherry: 595-630 (610 LP for higher signal)
• Laser selected: 405 nm
The LRP growth was monitored for 24 to 48 hours, taking a z-stack (200 µm wide,
0.5 µm frame spacing) every 30 minutes.

This LSFM procedure was applied on seedlings with two fluorescent

markers —one for cell identity and the other for defining the cell wall—, which
were already available in the host laboratoy. As shown in Table 1, these seedlings
were all products of an F1 cross between the two parental marker lines. The 5

18
different cell identities studied in this project were described and studied
genetically on a previous project where these distinctive cell populations were
identified through scRNA-se during early LR organogenesis (Serrano-Ron et al.,
2021). In addition, the analysis of the primordial cells (whose identity marker is
DR4) population was conducted previously to this project.

Cell population Cell identity marker line x Cell wall marker line
Primordial cells pDR4::NLS-3xYFP
QC-transitioning cells pWOX5::YFP
Vascular-like 2 cells pMYB108::NLS-3xYFP pUBQ10::LTI6B-2x-Cherry
Protophloem-like cells pATHMP41::NLS-3xYFP
End/QC-transitioning cells pCBF3::NLS-3x-mCherry pUBQ10::mTq2::PIP

Table 1. Fluorescent F1 lines used for LSFM. Seedlings from the F1 of the cross between the cell
identity marker lines of the cell populations and cell wall marker lines of a different color were used for
LSFM.

2. Preprocessing of image data

The 4D datasets resulting from the LSFM underwent a drift correction process
performed with BigDataProcessor2, a Fiji plugin developed for efficient inspection
and processing of large image data (Schindelin et al., 2012; Tischer et al., 2021).
Brightness and color were also adjusted to improve visualization. Finally, the
region of interest was cropped to remove uninformative regions from the dataset.
A total of 4 recordings were analyzed, one for each cell population.

3. Lineage tracing

The cell lineage tracing analysis of the different cell populations was carried
out through the Fiji plugin MaMuT, which enables the annotation of large image
data (Wolff et al., 2018). The process consisted on labeling the individual cells
(spots), which were tagged as positive or negative for the expression of the
corresponding identity marker, and connecting them to precursor and daughter
cells. However, due to biological significance, two cell populations were labeled
differently:

• QC-transitioning cells population, marked by WOX5 expression, had three

types of cells according to the intensity of the marker’s fluorescence and
its presence: high intensity positive cells, weak intensity positive cells and
negative cells.

19
 • Protophloem-like cell population, marked by ATHMP41 expression, had
three types of cells as well. However, these cells were labeled according
to the presence of the marker and their location: positive cells inside the
LRP, positive cells outside the LRP and negative cells inside the LRP.

Cell population Number of cell Total number of Time

(marker) lineages/tracks labeled spots Frames (hours)

Primordial cells (DR4) 18 2180 42 21

QC-transitioning cells
(WOX5) 33 1984 30 15

Vascular-like 2 cells
(MYB108) 29 1888 35 17.5

Protophloem-like cells
(ATHMP41) 27 1706 33 16.5

End/QC-transitioning cells
(CBF3) 34 1910 25 12.5
Table 2. Number of total cell lineages, individual cells and frames labeled for each marker. The
video of CBF3 marker was labeled until frame 25 because the resolution was lower than the rest (cell
walls were not very visible).

Once the labeling was done, the data was exported to csv files which were
subsequently analyzed. In order to harmonize the results of all the tracked lines,
the recordings where cropped and adjusted in time so the developmental stages
would be comparable and the population of primordial cells was included in some
of the results as a control, as its lineage tracing analysis was conducted
previously to this project.

4. Statistical analyses

Through R, t-tests were performed to determine whether single pairwise

comparisons were statistically significant for each marker (p-value ≤ 0.001).

20
 Results
To investigate the link between cell division and cell fate determination a collection of 4D
datasets of LRP in various fluorescent marker lines using Light Sheet-based
Fluorescence Microscopy (LSFM) were generated. These marker lines displayed a
marker of one of the previously defined cell identities as well as a separate color marker
of the cell membrane to define cellular shape. A total of six recordings of approximately
20 hours in length were created to investigate cell identity (previously described) during
LRP formation; DR4::NLS-3xYFP, recMYB36-YFP, pMYB108::NLS-3xYFP,
pATHMP41::NLS-3xYFP, pWOX5::ER-GFP, and pCBF3::NLS-3x-mCherry. Out of these
six videos, five of them were used for this project while one did not have good resolution
and the biomarkers were not visible (MYB36) ( Figure 5). Moreover, the recording line
marked with DR4 was previously analyzed as part of Laura Serrano´s Ph.D. thesis. This
lineage tracing research revealed that cell identity (as indicated by DR4) and positional
information impact the cell division rates of LRP cells. DR4 identity was lost in the central
areas of the LRP without the need for cell division, and its recovery was an uncommon
event. The data collected from that study (Serrano-Ron et al., 2021b) has been
incorporated into the results of this project and used for future comparisons and
analyses (Figure 6).

As mentioned previously, it was discovered that the formation or a LRP implies

the sequential establishment of several cellular identities (Serrano -Ron et al., 2021b).
However, this study raised the question of how this tissue initiation couples with cell
division and growth, which are forces that have been linked to the determination of cell
fate and the shaping of organ morphology (Lucas et al., 2013a; Nakajima and Benfey,
2002; Ten Hove and Heidstra, 2008; von Wangenheim et al., 2016). To address this
question, the recordings taken previously in the host laboratory and mentiones above,
will be labeled and studied. The strategy used for the labeling and analysis of the data
consisted in dividing the primordia into longitudinal and perpendicular regions as shown
in Figure 7. The final scheme used for the analysis is shown in Figure 7G and will be
used to analyze the localization of each marker.

21
 A B C D E

Figure 5. Confocal images of the different markers; DR4 (A), CBF3 (B), WOX5 (C), MYB108 (D) and
ATHMP41(E) and cell lineage annotations made through MaMuT software. Cell lineage annotations
are represented as white circles for negative cells and colored circles for positive cells. Positive cells for
each marker have their own color: DR4 cells are turquoise, CBF3 cells are cyan blue, WOX5 cells are
two colors; green for high intensity and yellow for low intensity, MYB108 cells are yellow and ATHMP41
cells are red.

Figure 6. Total number of cells from each lateral root primordium from the different markers (DR4,
CBF3, WOX5, MYB108 and ATHMP41) over time (relative frames to developmental stage). The
total number of cells for each primordium is the sum of all the cells tagged for each video using MaMut
software. The duration of the annotations for each marker varies because of the resolution of each
marker and its consequence in the level of accuracy for annotating. CBF3 marker gave the worst
resolution for cell walls and made the annotations less accurate over time, so the annotations were done
until frame 24. The frames of this graphical representation are relative frames to the developmental
stages of the LRP because the recordings started at different times (some of them with an earlier
primordium because the marker was visible at those stages) and have different durations (frames).

22
 Figure 7. Illustration for analysis regions. (A) Simplified 3D representation of a lateral root primordium.
(B) LRP divided in three regions; “Side 1” and “Side 2” colored in white and “Middle” colored in blue. (C)
Front view of illustration B. (D) Representation of the future schematic analyses of the LRP. The blue
dashed line indicates the position of the middle region. (E) LRP divided into “Flanks” and “Center” regions
that will be analyzed. (F) Front view of the previous image where the red dashed line represents the
flanks. (G) Future and final schematic analyses of the LRP including the flanks.

Except for recMYB36-YFP, which was not visible under these conditions, the films
verified the previously documented expression patterns for the identification markers.
For example, at very early stage II LRP, all cells expressed pDR4::NLS-3xYFP, which
was quickly limited to the flanking areas and remained expressed until emergence.
pMYB108::NLS-3xYFP was evident in multiple cells along the LRP throughout stages II
and III, but its signal got weaker as the stages progressed. The activity of
pATHMP41::NLS-3xYFP could not be identified in the LRP from the beginning. In fact,
there was one positive cell that entered the LRP at stage III and then divided at early
stage IV. The nucleus of this cell changed its shape from an elongated nucleus to a round
nucleus right before incorporating into the LRP and dividing a few frames later.
pCBF3::NLS-3x-mCherry revealed increased expression in the core cells of the LRP at
stage III, however the visibility of the marker was diminished overall, most likely due to
the temporality of this cell type or bleaching produced by long-term laser treatment.
pWOX5::ER-GFP was visible from stage II and had a broad pattern of expression that
overlapped with pCBF3::NLS-3xmCherry, as predicted.

23
 Except for recMYB36-YFP, which was not visible under these conditions, the
films verified the previously documented expression patterns for the identification This
expression pattern became increasingly limited to central cells with increased marker
expression at stage IV, but the marker remained detectable only in a few central cells at
subsequent stages. This finding supports prior findings that low WOX5 expression
identified the SCN and QC-transitioning cells, but greater WOX5 expression designated
the mature QC (Kong et al., 2015; Serrano-Ron et al., 2021b). All these observations are
shown in Figure 8.

All the recordings were manually annotated for cell identity and linage tracing
using the MaMuT software (Wolff et al., 2018). As mentioned before, DR4 had been
annotated and analyzed previously to this work and will be included in the analyses and
comparisons of this project. This lineage tracing research revealed that cell identity (as
indicated by DR4) and positional information impact the cell division rates of LRP cells.
DR4 identity was lost in the central areas of the LRP without the need for cell division,
and its recovery was an uncommon event. The data collected from that study (Serrano-
Ron et al., 2021b) has been incorporated into the results of this project and used for
future comparisons and analyses.

24
 Middle plane Side plane

Middle plane Side plane

Figure 8. Confocal images of DR4 (A), CBF3 (B), WOX5 (C), MYB108 (D) and ATHMP41 (E). Stained
cell walls are shown in a gray scale. DR4 (A), CBF3 (B) and WOX5 (C) images are taken from the middle
section of the primordium where the marker is also present. However, MYB108 (D) and ATHMP41 (E)
have two images: the one on the left shows the middle region of the primordium and the one on the right
shows the cells that were positive for each marker since they were not localized in the middle region of
the primordium. The white circles represent the individual negative cells for the marker, while the cells
colored in the matching color for the marker; turquoise, cyan blue, green, yellow, and red represent the
positive cells for the marker.

25
 1. Lineage tracing of the identities from the stem-cell
trajectory

For theCBF3 marked line, the analyzed recording started at the end of stage II
and ended at stage IV (Figure 9). The LRP was initially formed by 34 cells, which were
tracked over time and gave rise to 34 cell lineages (Figure 9A). Based on expression of
pCBF3::NLS-3x-mCherry (Figure 9B), cells in these lineages were labeled as positive or
negative for CBF3 expression (Figure 9A). At first, none of the cells were positive for
CBF3. Then, at late stage III, two positive cells (lineage 1 and lineage 11) emerged. In
stage IV, an additional negative cell of lineage 11 gained CBF3 expression, although 6
hours (12 frames) later it lost CBF3 expression (Figure 9A). Notably, each cell from the
initial primordium produced a patch of cells that were essentially placed in the same
location as the mother cell; a fact that is recapitulated in all the recordings. According to
the longitudinal and perpendicular scheme of the LRP mentioned before (Figure 7), the
expression of CBF3 was restricted to cells in the central region, both in the middle and
side planes (Figure 9C).

Regarding the LRP growth over time, the number of cells tagged as positive for
CBF3 expression slightly increased between the 4th and 7th hour of the recording (end of
stage III and beginning of stage IV). On the other hand, the number of CBF3 negative
cells constantly increased and represented the overall growth of the primordium (Figure
10).

We next determined if the difference in cell division rates was due to cell identity
or location inside the LRP by examining cell division rates across LRP regions (Figure
11). As predicted, center areas contributed far more to LRP growth than flanking regions.
We discovered that CBF3 positive cells on the side planes appeared earlier and were
more abundant than CBF3 positive cells in the middle plane (both planes are in central
regions).

Finally, as cell division is considered a key factor for cell fate determination (Ten
Hove and Heidstra, 2008), we tested if cell division was influencing changes in cell
identity as assessed by CBF3 expression. We quantified the number of identity shifts
which occurred with and without cell division (Figure 12). Interestingly, none of the
identity transitions that took place during the recording was associated with cell division.

26
Figure 9. Cell lineage tracing of CBF3 marked cells. (A) Cell lineage tracing results for the
pCBF3::NLS-3x-mCherry x pUBQ10::mTq2::PIP (F1) line using the MaMuT software. Each circle
represents one cell and connections between cells indicate relationships across time (frames) in the
same lineage. According to the legend, cells are colored as negative or positive for the CBF3 marker.
(B) Quantification for CBF3 expression in cells tagged as positive and negative for CBF3 marker.
According to a t-test, the asterisk denotes significance (p-value<0.001). n ≥ 400. (C) Schematic
illustration of the tracks' locations in the recorded LRP. The numbers match the tracks listed in (A). Each
number in the left image (middle) only refers to a single cell, whereas in the right image, each number
refers to a collection of cells. According to the legend, cells and groups are colored as negative, positive
or both for the CBF3 marker.

27
 A
120
V
100

80
Number of cells

CBF3 negative
60
CBF3positive
40 Total

20

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Stages (ST) ST II ST III ST IV

B
3.5
V
3

2.5
Number of cells

1.5

0.5

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Stages (ST) ST II ST III ST IV

Figure 10. Total number of cells tagged as positive and negative for CBF3 marker in the LRP over
time (frames). (A) The total number of cells is the sum of the CBF3 negative cells and CBF3 positive
cells. (B) Total number of CBF3 positive cells until frame 30, where we can see that it decreases to 2
positive cells at frame 25. The negative cells for CBF3 marker are tagged until frame 24 because the
fluorescent marker did not give a good resolution of the cell walls which made the annotations less
accurate in future frames.

28
A
4

3
Number of cells

0
Frames 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334

Stages (ST) ST II ST III ST IV

Central regions Flanks

B
50
45
40
35
Number of cells

30
25
20
15
10
5
0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Stages (ST) ST II ST III ST IV

CBF3 negative-Center Middle CBF3 negative - Center Side 1
CBF3 negative - Center Side 2 CBF3 positive - Center Middle
CBF3 positive - Center Side 1 CBF3 positive - Center Side 2

Figure 11. Presence of the CBF3 marker in the LRP over time (frames). (A) The CBF3 positive
cells of the LRP are present in the central regions over time. (B) Total number of cells tagged as
positive and negative for CBF3 marker in the central regions of the LRP over time (frames).

29
 4

0
Negtive to positive Positive to negative

Figure 12. Total number of cell identity transitions based on the presence or absence of CBF3
expression. Both transitions happened with no cell division.

For the WOX5 marked line, the analyzed recording started at stage II and ended
at stage V (Figure 13). The LRP was initially formed by 34 cells, which were tracked over
time and gave rise to 34 cell lineages (Figure 9 A). Based on expression of pWOX5::YFP
expression (Figure 13 B), cells in these lineages were labeled as high intensity, weak
intensity and negative for WOX5 expression (Figure 13 A). At first, 6 of the cells showed
positive for WOX5 (lineages 1, 2, 5, 9, 11 and 32) and all of them were tagged as weak
intensity. Then, at the beginning of stage IV, two weak intensity cells (lineage 1 and
lineage 32) gained high intensity expression for WOX5. Between stage III and IV 6 weak
intensity cells lost their WOX5 expression (lineages 1, 2, 5, 11 and 32). Lineage 9 did
not change identity during the recording, while lineages 1 and 32 turned into mixed
lineages of high intensity and negative cells, and the rest of the lineages generated a
mix of weak intensity cells and negative cells (Figure 13 A).

According to the longitudinal and perpendicular scheme of the LRP (Figure 7),
cells showing WOX5 expression were located at the central regions of the LRP, while
the flanks only contained negative cells (Figure 13 C). WOX5 weak expression domain

30
is highly coincident with CBF3 positive. However, WOX5 high appeared later. In addition,
during the first six hours of development, the number of WOX5 weak intensity cells rose.
In the sixth hour of the recording, the weak intensity cells dropped as the high intensity
cells increased. By the end of the recording, the number of both the weak and high WOX5
intensity cells remained constant. As in the case of CBF3, the number of WOX5 negative
cells constantly increased and reflected the overall cell growth of the primordium (Figure
14).

Figure 13. Cell lineage tracing of WOX5 marked cells. (A) Cell lineage tracing results for the
pWOX5::YFP x pUBQ10::LTI6B-2x-Cherry (F1) line using the MaMuT software. Each circle represents
one cell and connections between cells indicate relationships across time (frames) in the same lineage.
According to the legend, cells are colored as negative, high or weak intensity for the WOX5 marker.
(B) Quantification for WOX5 expression in cells tagged as high intensity, weak intensity and negative
for WOX5 marker. According to a t-test, the asterisk denotes significance (p-value<0.001). n ≥ 400. (C)
Schematic illustration of the tracks' locations in the recorded LRP. The numbers match the tracks listed
in (A). Each number in the left image (middle) only refers to a single cell, whereas in the right image,
each number refers to a collection of cells. According to the legend, cells and groups are colored as
negative, high, and weak intensity, weak intensity and negative or high intensity and negative for the
WOX5 marker.

31
 105
90
Number of cells

75
60
45
30
15
0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Stages (ST) ST II ST III ST IV ST V

WOX5 negative WOX5 high intensity WOX5 weak intensity Total

Figure 14. Total number of cells tagged as high intensity, weak intensity and negative for WOX5
marker in the LRP over time (frames). (A) The total number of cells is the sum of the WOX5 negative
cells and WOX5 positive (high intensity and weak intensity cells) cells.

In order to assess the association of cell division and cell identity, we evaluated
the cell division rates of cells labeled as high intensity, weak intensity, and negative for
WOX5 as well as the identity transitions between these cell identities. The only
transitions of identity that took place during the recording were from weak intensity to
negative, which half of them occurred with cell division, and from weak to high intensity
which did none of them occurred with cell division. (Figure 15 A). The analysis of cell
division rates revealed that cells labeled as high intensity divided more than the rest. The
cells labeled as weak intensity, on the other hand, behaved similarly to negative cells
(Figure 15 B).

32
 A 5

V 4
Number of transitions

0
Negative to Negative to Weak Weak High intensity High intensity
weak high intensity intensity to intensity to to weak to negative
intensity negative high intensity intensity

B 0.05

V 0.04
divisions/number of spots)
Cell division index (cell

0.03

0.02

0.01

0
WOX5 high intensity WOX5 weak intensity WOX5 negative

Figure 15. Cell identity transitions and cell divisions of cells marked with WOX5. (A) Total number
of cell identity transitions in terms of WOX5 marker presence (high or weak) or absence. Weak intensity
WOX5 cells transitioned into other identities and did not happen with cell division. Half of the transitions
from weak to negative occurred with cell division (colored in light grey). (B) Cell division index of each
cell type calculated by dividing the total number of divisions (for that cell type) by the total number of
spots (of that cell type). High intensity WOX5 cells divide more than the rest.

We next determined if the difference in cell division rates was due to cell identity
or location inside the LRP by examining cell division rates across LRP regions (Figure
16). As expected, central regions contributed far more to LRP development than flanking
regions. WOX5 positive cells (high and low intensity) were exclusively found in the
central regions (Figure 16 A). In this regions, the weak intensity cells located in the
middle plane divided more than the ones located in sides (Figure 16 B).

33
 A 12

10
Numbner of cells

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Stages (ST) ST II ST III ST IV ST V

WOX5 weak intensity - Central Regions WOX5 high intensity - Central Regions
WOX5 weak intensity -Flanks WOX5 high intensity - Flanks

B
40

35

30
Number of cells

25

20

15

10

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Stages (ST) ST II ST III ST IV ST V

WOX5 negative - Center Middle WOX5 negative - Center Side 1
WOX5 negative - Center Side 2 WOX5 weak intensity - Center Middle
WOX5 weak intensity - Center Side 1 WOX5 weak intensity - Center Side 2
WOX5 high intensity - Center Middle WOX5 high intensity - Center Side 1
WOX5 high intensity - Center Side 2

Figure 16. Presence of the WOX5 marker in the LRP over time (frames). (A) The WOX5 high and
weak intensity cells of the LTR are present in the central regions of the primordium over time. (B) Total
number of cells tagged as positive (high and weak intensity) and negative for WOX5 marker in the
central regions of the LRP over time (frames).

34
 2. Lineage tracing of the identities from the vascular
trajectory

For the MYB108 marker line, the analyzed recording started at stage II and ended
at stage IV (Figure 17). The LRP was initially composed of 29 cells, which were tracked
over time and gave rise to 29 distinct cell lineages (Figure 17 A). Based on expression
of of pMYB108::NLS expression (Figure 17 B), cells in these lineages were labeled as
positive and negative for MYB108 expression (Figure 17 A). The expression of MYB108
was highly specific and restricted to a narrow developmental window, as it was found in
3 positive cells (lineages 27, 28 and 29) specifically in the frames ranging from stage II
to the beginning of stage III (Figure 17 A). Taking as reference the longitudinal and
perpendicular scheme of the LRP (Figure 7) the expression of MYB108 was restricted to
cells at the edge one of the side planes, mostly located in the flanking regions (lineage
27 and 29) (Figure 17 C).

We next determined if the difference in cell division rates was due to cell identity
or location inside the LRP. First, we examined the number of positive and negative cells
across time (Figure 18). As expected, the number of MYB108 negative cells constantly
increased and reflected the overall cell growth of the primordium and contributed far
more to the LRP development (the central regions specifically) (Figure 18). As mentioned
previously, the expression of MYB108 was restricted to a narrow developmental window
and lost their expression at a very specific time. This identity shift was not associated to
cell division in any of the cases (Figure 19). In addition, we analyzed positive and
negative cell populations across different LRP regions. We found that MYB108
expression was maintained in the positive cells located in the flanks for longer time than
the one located in the central region (Figure 20).

35
Figure 17. Cell lineage tracing of MYB108 marked cells. (A) Cell lineage tracing results for the
pMYB108::NLS-3xYFP x pUBQ10::LTI6B-2x-Cherry (F1) line using the MaMuT software. Each circle
represents one cell and connections between cells indicate relationships across time (frames) in the
same lineage. According to the legend, cells are colored as negative or positive for the MYB108 marker.
(B) Quantification for MYB108 expression in cells tagged as positive and negative for MYB108 marker.
According to a t-test, the asterisk denotes significance (p-value<0.001). n ≥ 400. (C) Schematic
illustration of the tracks' locations in the recorded LRP. The numbers match the tracks listed in (A). Each
number in the left image only refers to a single cell, whereas in the right image, each number refers to a
collection of cells. According to the legend, cells and groups are colored as negative or positive for the
MYB108 marker.

36
 80

70

60
Number of cells

50

40

30

20

10

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Stages (ST) ST ST III ST IV

MYB108 negative MYB108 positive Total

Figure 18. Total number of cells tagged as positive and negative for MYB108 marker in the LRP
over time (frames).

4
Number of transitions

0
Positive to negative Negative to positive

Figure 19. Total number of cell identity transitions in terms of MYB108 marker presence or
absence. All MYB108 positive cells lose their identity without cell division.

37
 A Number of cells 3

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Stages ST ST ST
(ST) II III IV

Central regions Flanks

B
25

20
Number of cells

15

10

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Stages ST ST ST
(ST) II III IV
MYB108 negative - Center Middle MYB108 negative - Center Side 1
MYB108 negative - Center Side 2 MYB108 positive - Center Middle
MYB108 positive - Center Side 1 MYB108 positive - Center Side 2

38
 C
25

20
Number of cells

15

10

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Stages (ST) ST II ST III ST IV

MYB108 negative - Flank 1 Middle MYB108 negative - Flank 1 Side 1

MYB108 negative - Flank 2 Side 1 MYB108 negative - Flank 2 Side 2

MYB108 positive - Flank 1 Middle MYB108 positive - Flank 1 Side 1

MYB108 positive - Flank 2 Side 1 MYB108 positive - Flank 2 Side 2

MYB108 negative - Flank 2 Middle MYB108 negative - Flank 1 Side 2

Figure 20. Total number of cells tagged as positive and negative for MYB108 marker in the central
(B) and flanking (C) regions of the LRP across time (frames). (A) Positive MYB108 regions over time
(frames). (B) All cells of the primordium localized in the central regions of the LRP over time (frames).
(C) All cells of LRP localized in the flanks over time (frames).

Finally, ATHMP41 marker line was analyzed. The recording started at stage II
and ended at stage IV (Figure 21). The LRP was initially composed of 27 cells, which
were tracked over time and gave rise to 27 distinct cell lineages. Based on expression
of pATHMP41::NLS expression (Figure 21 B) and the location of the cells showing
expression of the marker, cells in these lineages were labeled as positive inside the
primordium, positive outside the primordium and negative for ATHMP41 expression
(Figure 21 A). One cell was positive for ATHMP41 expression (lineage 20) during the
entire recording (Figure 21 A). Taking as reference the longitudinal and perpendicular
scheme of the LRP (Figure 7) the initial location of this cell was outside of the LRP and
at stage III it was located at the edge one of the side plane , specifically in the central
region (Figure 21 C). This expression domain is equivalent to MYB108’s but appears
later.

39
As shown in Figure 22, the number of ATHMP41 negative cells constantly increased and
reflected the overall cell growth of the primordium as it happened in the rest of the cases
(Figure 22). We next determined if the difference in cell division rates was due to cell
identity or location inside the LRP by examining cell division rates across LRP regions
(Figure 23). We found that the ATHMP41 positive cell stayed outside the primordium for
the first seven hours (frame 15), then it was located inside the LRP and a few frames
later it divided once (Figure 23).We tried to test whether cell division was contributing to
the changes in the cell identity as determined by ATHMP41 expression but since we had
1 positive cell whose expression was not lost in the recording, this question could not be
assessed with our available data.

Lastly, we analyzed the division rate for negative and positive cells for
ATHMP41 marker (Figure 24). The cell division rate for the positive cell is similar to the
negative cells, but since it is only 1 unique positive cell, it explains why contributes less
to the overall cell growth of the primordium.

40
Figure 21. Cell lineage tracing of ATHMP41 marked cells. (A) Cell lineage tracing results for the
pATHMP41::NLS-3xYFP x pUBQ10::LTI6B-2x-Cherry (F1) line using the MaMuT software. Each circle
represents one cell and connections between cells indicate relationships across time (frames) in the
same lineage. According to the legend, cells are colored as negative or positive (outside or inside of the
primordium) for the ATHMP41 marker. (B) Quantification for ATHMP41 expression in cells tagged as
positive and negative for ATHMP41 marker. According to a t-test, the asterisk denotes significance (p-
value<0.001). n ≥ 400. (C) Schematic illustration of the tracks' locations in the recorded LRP. The
numbers match the tracks listed in (A). Each number in the left image only refers to a single cell, whereas
in the right image, each number refers to a collection of cells. According to the legend, cells and groups
are colored as negative or positive for the ATHMP41 marker.

41
 70

60

50
Number of cells

40

30

20

10

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Stages (ST) ST II ST III ST IV
ATHMP41 negative ATHMP41 positive Total

Figure 22. Total number of cells tagged as positive and negative for ATHMP41 marker in the
LRP over time (frames). The total is the sum of positive and negative cells for the ATHMP41 marker.

3
Number of cells

0
Frames 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Stages (ST) ST II ST III ST IV

ATHMP41 positive outside the primordium ATHMP41 positive inside the primordium

Figure 23. Graphical representation of the ATHMP41 positive cell that incorporates into the
primordium over time (frames). The cell changes its location to inside the primordium in frame 29 and
it divides in frame 19 after its elongated nucleus changes shape into a rounder nucleus.

42
 0.03

divisions/number of spots)
Cell division index (cell
0.02

0.01

0
ATHMP41 negative ATHMP41 positive

Figure 24. Cell division rate for negative and positive cells for ATHMP41 marker. Positive
ATHMP41 cells divide more than the negative cells. The cell division rate is calculated by the
dividing the number cell divisions (for that cell type) by the number of cells (of that cell type).

3. Integration of the results obtained from the different cell

identities in real time

As the starting point of development was specific for each recording, we

represented the expression of all the marker genes over relative frames, so the results
obtained in different recordings were comparable.

Figure 25 shows an integrated vision of the stablishment of the different cell

identities in real time of development in the LRP. In this integrated model, DR4
expression (primordial cells) is the first one that stablishes and it has two growth peaks
(frames 2-3 and frame 20). Regarding the stem cells trajectory, when WOX5 weak
decreases, WOX5 high decreases (frames 8 to 12). In that fragment of time CBF3 stops
increasing and remains constant. In fact, in frame 25, CBF3 decreases and WOX5 high
increases one last time while WOX5 weak remains constant since frame 19. On the othe
hand, the first idenditity that stablishes in the vascular trajectory is MYB108 which is lost
in frame 12. A few frames later (frame 15) ATHMP41 stablishes and increases in
number. Thereby, this result represents an integrated vision in which the establishment
of the different LRP identities can be assessed in real time

43
 20

18

16

14
Number of cells

12

10

0
Relative 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
frames
to ST II
developmental
stage ST III

Relative stages (ST) ST IV

ST V

DR4 positive cells CBF3 positive cells WOX5 high intensity cells
WOX5 weak intensity cells MYB108 positive cells ATHMP41 positive cells

Figure 25. Total number of positive cells for each marker over time (frames). Each marker is colored
differently as shown in the legend. The frames of this graphical representation are relative frames to the
developmental stages of the LRP because the videos were recorded at specific times (some of them with
an earlier primordium) and have different durations (frames).

44
 Discussion

The development of the root system is an intricate post-embryonic phenomenon

crucial for the survival and adaptation of plants to the environment (Nibau et al., 2008;
Smith and De Smet, 2012). Furthermore, comprehending the molecular signals that drive
root development has shown exceptional potential in enhancing crop yield (Rogers and
Benfey, 2015), emphasizing the biotechnological significance of performing research on
the root system. In the model plant Arabidopsis thaliana, root system development
primarily relies on the generation of lateral roots. The overall process of LR formation
can be divided into: pre-patterning, involving the identification of sites from which LR will
emerge, and formation, where functional structures are newly generated from the pre-
patterned sites (Motte et al., 2019; Van Norman et al., 2013).

Despite extensive research on the formation of LR and the identification of

various molecular components and developmental processes involved (De Smet et al.,
2007; De Smet et al., 2008; Du and Scheres, 2017; Efroni et al., 2016; Matosevich and
Efroni, 2021; Moreno-Risueno et al., 2015; Moreno-Risueno et al., 2010; Perianez-
Rodriguez et al., 2021; Wachsman et al., 2020; Xuan et al., 2015; Xuan et al., 2016;
Mercadal et al., 2022; Wang D et al., 2023), there are still significant unresolved
questions. This study aims to address some of the existing gaps in understanding the
function of the genes involved in the early stages of development of a LR primordium
(Goh et al., 2016). Additionally, we aimed to characterize and understand the distinct cell
identities during the early stage of LR formation (Serraron-Ron et al., 2021b) by
analyzing 4D datasets at cellular resolution that were previously generated in the host
laboratory.

As a whole, our study has validated the physical localization of 4 cell types inside
the LRP, as well as their hierarchical, preserved temporal sequence of formation and the
functional relationships among several of them. (Serrano-Ron et al., 2021b).
Furthermore, we combined these data with cell division and lineages to make a deeper
analyses. Taken together, this data has the potential to be used for the generation of an
integrative model that combines genetic regulation with cell division and growth
dynamics.

45
 1. The stem-cell trajectory

One of the remarkable characteristics of plants is their ability to undergo continuous

growth and organ generation throughout their life cycle. This process relies on the
presence of stem cells located in the shoot apical meristem (SAM), root apical meristem
(RAM), and cambium. Within the SAM and RAM, a specialized group of cells known as
the organizing center divide at a slow pace, providing a cellular reservoir for the
surrounding stem cells (Weigel and Jürgens, 2002). These organizing centers also play
a crucial role in maintaining the stem cell identity of neighboring cells (Scheres, 2007).
However, irrespective of their specific location, many stem cells originate during
embryogenesis, emphasizing the crucial significance of their determination for post-
embryonic development.

In Arabidopsis, the embryo undergoes a highly ordered sequence of cell divisions,

during which new tissues are specified and patterned. After the longitudinal and
transverse division of the apical cell of the embryo giving rise to octant stage embryo
(separated into two tiers). All cells undergo a tangential division that separates the
protoderm (the precursor of the epidermis) from the inner tissues (the precursors of the
ground and vascular tissues). Then the globular stage is formed after a few divisions:
The outer, protodermal cells divide anticlinally, only to extend the outer layer. By contrast,
the inner cells divide longitudinally. In this context, the four basal cells differentiate into
larger outer ground tissue precursors and smaller inner vascular precursors.
Concurrently, the topmost cell of the suspensor undergoes specification as the
hypophysis and undergoes an asymmetric division. This division results in the formation
of a smaller lens-shaped cell, which serves as the precursor of the QC, and a larger
basal cell, which serves as the precursor of the distal stem cells within the root meristem
(Perilli et al., 2012; Hove et al., 2015; Bennett and Scheres, 2010).

Although extensive research has been conducted on primary root initiation,

organization, and growth, the molecular and cellular mechanisms governing the
patterning of new LRP remain poorly characterized (Tian et al., 2014). Root growth is a
consequence of the activities occurring within the RAM, which comprises a group of
proliferating cells arranged around a central SCN. The establishment of the RAM in the
PR takes place during early embryogenesis through formative cell divisions, and the
initial cell identity is maintained by the QC (Perilli et al., 2012; ten Hove et al., 2015).
Several regulatory mechanisms have been identified in Arabidopsis thaliana that controls
the establishment and maintenance of the QC in the PR. The maintenance of the QC
46
and SCN during post-embryonic primary root growth relies on the activity
of PLT transcription factors and SCR in combination with an auxin gradient (Bennett and
Scheres, 2010; Perilli et al., 2012).

In the LRP, the stem cell trajectory consists of the sequential emergence of two cell
populations. The first population within the LR primordium is distinguished by the
expression of CBF3 and it is called endodermis/QC transitioning cells due to abundant
expression of genes linked to the stem cell niche, specifically those associated with the
cortex/endodermis initial. A second population exhibits enhanced expression of WOX5
which is associated with the organizing center of the root meristem (Primc and Maizel,
2021). These two populations are situated in the central domain of the LR primordium.
Mutations in CBF3 lead to the arrest of LR development prior to the emergence of the
second population, thereby delineating a hierarchical sequence in the establishment of
the LR meristem (Primc and Maizel, 2021).

Our results validate that CBF3 marked cells are the first cells established in the LRP
region that belong to the stem cell trajectory. Notably WOX5 is also located in the very
region. Weak WOX5 expression is present from the beginning, almost concomitant with
CBF3 expression. Thus, weak WOX5 expression overlaps with CBF3, which might need
to disappear so that high WOX5 expression can be established. This suggests that
before the actual QC development there could be an organizing like center or cells with
CBF3 and weak WOX5 expression. Next, the expression pattern of WOX5 became
increasingly limited to the central cells, exhibiting higher expression levels specifically at
stage IV. Subsequently, in later stages, the marker was only detectable in a small
number of central cells. These findings support that the low expression of WOX5 defined
the SCN and the transitioning cells to the QC, while higher expression of the marker is
characteristic of the mature QC (Pi et al., 2015; Forzani et al., 2014; Sarkar et al., 2007).

In addition, the CBF3 marker seems to be restrictive for cell division since the cell
lineages expressing this identity do not divide. Thus, this identity could have associated
the absence of cell division as a characteristic feature.. On the other hand, although
WOX5 is associated with the QC, which rarely divides, cells that expressed WOX5 with
high intensity were the cells that most divided. This could be caused by a separate
mechanism of WOX5 imposing quiescence which has not been activated yet at the
beginning of stage V. This hypothesis is supported by a study (Goh et al., 2016) that
suggests that there is earlier onset of WOX5 expression during LR development
(Ditengou et al., 2008) and that QC identity is established around stage V of LRP
development. CBF3 regulates the initiation of the QC-transitioning cells and in

47
agreement with its function, its expression pattern is very similar to the one of the LRP
regulator SCR, which leads to QC specification (Goh et al., 2016). Based on the
proposed model by Laura Serrano-Ron and coworkers (Serrano-Ron et al., 2021b), SCR
would also be expressed in the endodermis/QC-transitioning stem cells, likely
contributing to initiation of the QC-transitioning cells to specify a new QC.

Notably, cells that started expressing CBF3 did not arise from an asymmetric cell
division, i.e. the identity was gained independently of cell division relaying to specification
by positional information. On the other hand, WOX5 positive cells did not originate from
an asymmetric cell division either while the marker appeared to have started expressing
after CBF3 decreased and remained constant, so the gained of the identity in these cells
could be influenced by the presence of CBF3 plus additional positional information.

During post-embryonic development, stem cells in Arabidopsis thaliana are specified

in the shoot and root meristems, which are derived from the embryonic SAM and RAM,
respectively. The SAM gives rise to the aerial parts of the plant, including leaves and
flowers, while the RAM is responsible for root growth. Similar to embryonic development,
stem cells in the post-embryonic SAM and RAM are maintained through the presence of
specific regions within the meristems. The QC in the root apical meristem is responsible
for the maintenance of stem cell populations. Overall, both embryonic and post-
embryonic development in Arabidopsis thaliana involve the specification and
maintenance of stem cells in specific meristematic regions. The genetic pathways and
signaling mechanisms involved in stem cell regulation share similarities and involve
WOX5, SCR and the PLTs , although there are differences in the specific factors and
interactions that govern stem cell behavior during each phase.

Altogether, our results suggest that the cell division pattern for SCN and QC
establishment is regulated differently during LR formation, embryogenesis and primary
root growth in Arabidopsis (Wysocka-Diller et al., 2000) and that, while SCR is not strictly
required for LR QC establishment, ‘normal’ QC-forming formative divisions related to
primary root growth and LRP outgrowth depend on SCR function (Goh at al., 2016).

2. The vascular trajectory

In Arabidopsis, all vascular tissues of the plant axis are derived from four initial
cells in the globular embryo (Scheres et al., 1994). According to Scheres et al. (1994)
and De Rybel et al. (2014), the division responsible for ground tissue formation also gives
rise to the initial vascular precursor cells in Arabidopsis thaliana embryos. Consequently,
all vascular tissues in the root originate from four provascular initial cells in the early

48
globular stage embryo. These cells undergo multiple rounds of periclinal divisions with a
specific orientation, leading to the development of a vascular bundle composed of up to
40 cells by the end of embryogenesis. The precise regulation of cell file number in the
vascular tissue during the growth of postembryonic roots remains uncertain. The
vascular bundle exhibits a diarchy pattern with a central xylem axis and two phloem
poles. While the exact specification events for these cells are largely unknown, recent
advances have improved our understanding of vascular tissue formation during
embryogenesis. Mutations in some genes during embryogenesis result in defective
divisions that generate vascular tissues and a complete failure to establish an embryonic
root (Hardtke and Berleth, 1998; Hamann et al., 2002; Smit., et al 2020a; Smit., et al
2020b).

During embryogenesis, several steps can be recognized during vascular

development: specification of vascular tissue identity, cell proliferation to generate a
bundle of cells, patterning into xylem, phloem, and cambium cell types, and finally
differentiation into functional transport cells. The regulators and effectors of all but the
first step have been dissected in some detail. The mechanisms governing the
establishment of this tissue during embryogenesis and whether there is a shared
mechanism between embryonic tissue formation and postembryonic growth are currently
unknown (Smit., et al 2020a; Smit., et al 2020b).

Vascular-like cells were marked by MYB108 expression and appeared at stage

II of LRP development located adjacent to the vasculature of the primary root and located
on the side of the LRP. Protophloem-like cells were marked by ATHMP41expression and
appeared at stage III-IV of LRP development located adjacent to the phloem poles of the
primary root, which also expressed this marker (Serrano-Ron et al., 2021b). This
suggests an early vasculature connection of the LRP to the primary root and an early
vascular developmental trajectory independent of stem cell specification. Figure 7 shows
that the decreasing of MYB108 marker overlaps with the increasing of ATHMP41 in the
same LRP area validating that these populations are in the same trajectory and that
MYB108 is the precursor/regulator of the second population. Furthermore, as mentioned
previously, MYB108 cells, the same as other precursor cell populations seem to have
been associated with a cell division restriction. Thus, MYB108 cells do not divide while
the marker is expressed.

Interestingly, the positive cell for ATHMP41 marker at stage III was outside the
primordium at earlier stages. In that fragment of time, a change in the shape of the
nucleus was observed right before the cell incorporated into the primordium. This

49
information suggests that this cell could be a primordial cell transitioning into a vascular
cell since primordial cells are thought to give rise to all the LRP precursor cell types and
display rounded nuclei.

Lastly, we do not know if cells that expressed MYB108 divided previously (as the
cells were already marked from the beginning of the 4D dataset), which means that the
identity could be gained associated to cell division or by positional information. On the
other hand, ATHMP41 positive cells did not divide previously while the marker started to
be expressed after MYB108 disappeared. Given the close connection in gene expression
and location in the LRP, it is possible that the gained ATHMP41identity in these cells
could be influenced by the presence of MYB108 plus positional information.

The dynamic expression patterns of vascular marker genes imply that the identity
of vascular tissue is not a uniform characteristic throughout various developmental
stages, suggesting the existence of additional specification mechanism for vascular cell
identity development over time. This suggests that the initial vascular tissue identity
observed during embryonic development is transient and not maintained in
postembryonic stages. Notably, recent advancements in scRNAseq have highlighted the
high divergence among different vascular cell types within postembryonic vascular cells.
In scRNAseq experiments conducted on roots, distinct clusters are formed by xylem and
phloem cells; however, vascular tissues as a whole do not form a separate cluster that
is distinct from other major tissue identities such as ground tissue and epidermis. Studies
by Ryu et al. (2019), Shulse et al. (2019), and Denyer et al. (2019) have contributed to
this observation. Thus, it is arguable whether postembryonic vascular bundle cells
possess a common identity or even require one. Instead, the presence of a transient
"general" primordial multipotent vascular identity may be necessary only during the
initiation of new vascular bundles, ensuring proper placement of the vascular bundle as
a whole and the establishment of cambial cells. Future scRNAseq investigations on
embryos and contexts such as wounded stems or graft junctions (Melnyk et al., 2018)
could provide further insights into this matter (Ryu et al., 2019; Shulse et al., 2019;
Denyer et al., 2019 Smit et al., 2020).

50
 Conclusion

In conclusion, the development of the root system in plants, particularly the

formation of lateral roots, is a complex process crucial for plant survival and adaptation.
Understanding the molecular signals and genetic regulation involved in root development
has significant implications for improving crop yield. While extensive research has been
conducted on the formation of lateral roots and the identification of various molecular
components and developmental processes, there are still significant gaps in our
understanding. This study aimed to address some of these gaps by investigating the
early stages of lateral root development and characterizing cell identities using 4D
datasets.

To sum up, the results validated the presence of distinct cell types and their
hierarchical sequence of formation within the lateral root primordium, providing insights
into the functional relationships among them. The results suggested a stem cell trajectory
within the LRP, with the expression of CBF3 marking the first cells established in the
LRP region, followed by the expression of WOX5 associated with the organizing center
of the root meristem. On the other hand, the expression patterns of MYB108 and
ATHMP41 markers suggest the existence of an early vasculature connection between
the LRP and the primary root, indicating an independent vascular developmental
trajectory from stem cell specification.

Lastly, the study highlighted the potential for integrating genetic regulation, cell
division, and growth dynamics to develop an integrative model of lateral root
development. Additionally, this project shed light on the stem cell trajectory in the root
meristem and the vascular trajectory during embryogenesis and post-embryonic growth.
The findings suggest that the establishment and maintenance of stem cells and vascular
tissues involve complex genetic pathways and signaling mechanisms that share
similarities but also exhibit distinct characteristics in different developmental stages.
Overall, this research contributes to our understanding of root development and provides
a foundation for further investigations in this field.

51
 Bibliography
Anamarija Primc, Alexis Maizel, Understanding lateral root formation, one cell at a
time, Molecular Plant, Volume 14, Issue 8, 2021, Pages 1229-1231, ISSN1674-2052.

Arnaud, C., Bonnot, C., Desnos, T., and Nussaume, L. (2010). The root cap at the
forefront. C R Biol 333, 335-343.

Atta, R., Laurens, L., Boucheron-Dubuisson, E., Guivarc'h, A., Carnero, E., Giraudat-
Pautot, V., Rech, P., & Chriqui, D. (2009). Pluripotency of Arabidopsis xylem pericycle
underlies shoot regeneration from root and hypocotyl explants grown in vitro. The
Plant journal : for cell and molecular biology, 57(4), 626–644.

Atta, R., Laurens, L., Boucheron-Dubuisson, E., Guivarc'h, A., Carnero, E., Giraudat-
Pautot, V., Rech, P., & Chriqui, D. (2009). Pluripotency of Arabidopsis xylem pericycle
underlies shoot regeneration from root and hypocotyl explants grown in vitro. The
Plant journal : for cell and molecular biology, 57(4), 626–644.

Bao, Y., Aggarwal, P., Robbins, N.E., 2nd, Sturrock, C.J., Thompson, M.C., Tan, H.Q.,
Tham, C., Duan, L., Rodriguez, P.L., Vernoux, T., et al. (2014). Plant roots use a
patterning mechanism to position lateral root branches toward available water. Proc
Natl Acad Sci U S A 111, 9319-9324.

Bargmann, B.O., Vanneste, S., Krouk, G., Nawy, T., Efroni, I., Shani, E., Choe, G.,
Friml, J., Bergmann, D.C., Estelle, M., et al. (2013). A map of cell type-specific auxin
responses. MolSyst Biol 9, 688.

Bauby, H., Divol, F., Truernit, E., Grandjean, O., and Palauqui, J.C. (2007).
Protophloem differentiation in early Arabidopsis thaliana development. Plant Cell
Physiol 48, 97-109.

Beemster, G.T., and Baskin, T.I. (1998). Analysis of cell division and elongation
underlying the developmental acceleration of root growth in Arabidopsis thaliana.
Plant Physiol 116, 1515-1526.

Benfey, P.N., and Scheres, B. (2000). Root development. Current Biology 10, R813-
R815.biology.

Bennett, T., & Scheres, B. (2010). Root development-two meristems for the price of
one?. Current topics in developmental biology, 91, 67–102.

52
Birnbaum, K.D., and Moreno-Risueno, M.A. (2021b). Reconstruction of lateral root
formation through single-cell RNA sequencing reveals order of tissue initiation. Mol
Plant 14, 1362-1378.

Carlsbecker, A., and Helariutta, Y. (2005). Phloem and xylem specification: pieces of
the puzzle emerge. Curr Opin Plant Biol 8, 512-517.

Colette A. ten Hove, Kuan-Ju Lu, Dolf Weijers; Building a plant: cell fate specification
in the early Arabidopsis embryo. Development 1 February 2015; 142 (3): 420–430.

Cui, H. (2015). Cortex proliferation in the root is a protective mechanism against

abiotic stress. Plant Signal Behav 10, e1011949.

David Rhodes and others, Metabolic Changes Associated with Adaptation of Plant
Cells to Water Stress , Plant Physiology, Volume 82, Issue 4, December 1986, Pages
890–903.

De Rybel, B., Adibi, M., Breda, A. S., Wendrich, J. R., Smit, M. E., Novák, O.,
Yamaguchi, N., Yoshida, S., Van Isterdael, G., Palovaara, J., Nijsse, B., Boekschoten,
M. V., Hooiveld, G., Beeckman, T., Wagner, D., Ljung, K., Fleck, C., & Weijers, D.
(2014). Plant development. Integration of growth and patterning during vascular tissue
formation in Arabidopsis. Science (New York, N.Y.), 345(6197), 1255215.

De Rybel, B., Mahonen, A.P., Helariutta, Y., and Weijers, D. (2016). Plant vascular
development: from early specification to differentiation. Nat Rev Mol Cell Biol 17, 30-
40.

De Smet, I., Tetsumura, T., De Rybel, B., Frei dit Frey, N., Laplaze, L., Casimiro, I.,
Swarup, R., Naudts, M., Vanneste, S., Audenaert, D., et al. (2007). Auxin-dependent
regulation of lateral root positioning in the basal meristem of Arabidopsis.
Development 134, 681-690.

De Smet, I., Vassileva, V., De Rybel, B., Levesque, M.P., Grunewald, W., Van
Damme, D., Van Noorden, G., Naudts, M., Van Isterdael, G., De Clercq, R., et al.
(2008). Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis
root. Science 322, 594-597.

Dello Ioio, R., Linhares, F.S., Scacchi, E., Casamitjana-Martinez, E., Heidstra, R.,
Costantino, Denyer, T., Ma, X., Klesen, S., Scacchi, E., Nieselt, K., and Timmermans,
M.C.P. (2019). Spatiotemporal Developmental Trajectories in the Arabidopsis Root
Revealed Using HighThroughput Single-Cell RNA Sequencing. Dev Cell 48, 840-852
e845.

53
Di Mambro, R., De Ruvo, M., Pacifici, E., Salvi, E., Sozzani, R., Benfey, P.N., Busch,
W., Novak, O., Ljung, K., Di Paola, L., et al. (2017). Auxin minimum triggers the
developmental switch from cell division to cell differentiation in the Arabidopsis root.
Proc Natl Acad Sci U S A 114, E7641-E7649.

Ditengou, F. A., Teale, W. D., Kochersperger, P., Flittner, K. A., Kneuper, I., van der
Graaff, E., Nziengui, H., Pinosa, F., Li, X., Nitschke, R., Laux, T., & Palme, K. (2008).
Mechanical induction of lateral root initiation in Arabidopsis thaliana. Proceedings of
the National Academy of Sciences of the United States of America, 105(48), 18818–
18823.

Dittmer, H. J. (1959). A Method to Determine the Length of Individual Roots. Bulletin

of the Torrey Botanical Club, 86(1), 59–61.

Du, Y., and Scheres, B. (2017). PLETHORA transcription factors orchestrate de novo
organ patterning during Arabidopsis lateral root outgrowth. Proc Natl Acad Sci U S A
114, 11709-11714.

Dubrovsky JG, Gambetta GA, Hernández-Barrera A, Shishkova S, González I.

Lateral root initiation in Arabidopsis: developmental window, spatial patterning,
density and predictability. Ann Bot. 2006 May;97(5):903-15.

Eapen, D., Barroso, M.L., Ponce, G., Campos, M.E., and Cassab, G.I. (2005).
Hydrotropism: root growth responses to water. Trends Plant Sci 10, 44-50.

Efroni, I., Mello, A., Nawy, T., Ip, P.L., Rahni, R., DelRose, N., Powers, A., Satija, R.,
and Birnbaum, K.D. (2016). Root Regeneration Triggers an Embryo-like Sequence
Guided by Hormonal Interactions. Cell 165, 1721-1733.

Fisher, A.P., and Sozzani, R. (2016). Uncovering the networks involved in stem cell
maintenance and asymmetric cell division in the Arabidopsis root. Curr Opin Plant
Biol 29, 38-43.

Forzani, C., Aichinger, E., Sornay, E., Willemsen, V., Laux, T., Dewitte, W., & Murray,
J. A. (2014). WOX5 suppresses CYCLIN D activity to establish quiescence at the
center of the root stem cell niche. Current biology : CB, 24(16), 1939–1944.

Garcia, P., Gude, I., et al. (2021a). An auxin-regulable oscillatory circuit drives the
root clock in Arabidopsis. Science advances 7.

Goh, T., Toyokura, K., Wells, D.M., Swarup, K., Yamamoto, M., Mimura, T., Weijers,
D., Fukaki, H., Laplaze, L., Bennett, M.J., et al. (2016). Quiescent center initiation in

54
the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription
factor. Development 143, 3363-3371.

Grierson, C., Nielsen, E., Ketelaarc, T., and Schiefelbein, J. (2014). Root hairs.
Arabidopsis Book 12, e0172.

Hardtke CS, Berleth T. The Arabidopsis gene MONOPTEROS encodes a

transcription factor mediating embryo axis formation and vascular development.
EMBO J. 1998 Mar 2;17(5):1405-11.

Heidstra, R., and Sabatini, S. (2014). Plant and animal stem cells: similar yet different.
Nat Rev Mol Cell Biol 15, 301-312.

Jeffrey R. Seemann and others, Photosynthetic Response and Adaptation to High

Temperature in Desert Plants : A Comparison of Gas Exchange and Fluorescence
Methods for Studies of Thermal Tolerance, Plant Physiology, Volume 75, Issue 2,
June 1984, Pages 364–368.

Josep Mercadal, Isabel Betegón-Putze, Nadja Bosch, Ana I. Caño-Delgado, Marta

Ibañes; BRAVO self-confined expression through WOX5 in the Arabidopsis root
stem-cell niche. Development 1 August 2022; 149 (15): dev200510

Kong, X., Lu, S., Tian, H., and Ding, Z. (2015). WOX5 is Shining in the Root Stem
Cell Niche. Trends Plant Sci 20, 601-603.

Kramer, E. M. (2015). Arabidopsis thaliana as a model organism in plant science:

advantages and limitations. Current Opinion in Plant Biology, 24, 1-6.

Kumar, N., and Iyer-Pascuzzi, A.S. (2020). Shedding the Last Layer: Mechanisms of
Root Cap Cell Release. Plants (Basel) 9.

Laskowski, M., Grieneisen, V.A., Hofhuis, H., Hove, C.A., Hogeweg, P., Maree, A.F.,
and Scheres, B. (2008). Root system architecture from coupling cell shape to auxin
transport. PLoS biology 6, e307.

Lavenus, J., Goh, T., Roberts, I., Guyomarc'h, S., Lucas, M., De Smet, I., Fukaki, H.,
Beeckman, T., Bennett, M., and Laplaze, L. (2013). Lateral root development in
Arabidopsis: fifty shades of auxin. Trends Plant Sci 18, 450-458.

Leitz, G., Kang, B.H., Schoenwaelder, M.E., and Staehelin, L.A. (2009). Statolith
sedimentation kinetics and force transduction to the cortical endoplasmic reticulum in
gravity-sensing Arabidopsis columella cells. Plant Cell 21, 843-860.

55
Lloyd, J.P.B., Lister, R. Epigenome plasticity in plants. Nat Rev Genet 23, 55–68
(2022). https://doi.org/10.1038/s41576-021-00407-y.

Long, T. A., Tsukagoshi, H., Busch, W., Lahner, B., Salt, D. E., & Benfey, P. N. (2010).
The bHLH transcription factor POPEYE regulates response to iron deficiency in
Arabidopsis roots. The Plant cell, 22(7), 2219–2236.

Maizel, A., von Wangenheim, D., Federici, F., Haseloff, J., and Stelzer, E.H. (2011).
Highresolution live imaging of plant growth in near physiological bright conditions
using light sheet fluorescence microscopy. Plant J 68, 377-385.

Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral
roots of Arabidopsis thaliana. Development 124, 33-44.

Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral
roots of Arabidopsis thaliana. Development 124, 33-44.

Margot E. Smit and others, A PXY-Mediated Transcriptional Network Integrates

Signaling Mechanisms to Control Vascular Development in Arabidopsis, The Plant
Cell, Volume 32, Issue 2, (2020b), Pages 319–335,

Margot E. Smit, Cristina I. Llavata-Peris, Mark Roosjen, Henriette van Beijnum, Daria
Novikova, Victor Levitsky, Iris Sevilem, Pawel Roszak, Daniel Slane, Gerd
Jürgens, Victoria Mironova, Siobhan M. Brady, Dolf Weijers; Specification and
regulation of vascular tissue identity in the Arabidopsis embryo. Development 15
(2020a); 147 (8): dev186130.

Matosevich, R., and Efroni, I. (2021). The quiescent center and root regeneration. J
Exp Bot 72, 6739-6745.

Meinke, D. W., Cherry, J. M., Dean, C., Rounsley, S. D., & Koornneef, M. (1998).
Arabidopsis thaliana: a model plant for genome analysis. Science (New York,
N.Y.), 282(5389), 662–682.

Melnyk, C.W., Gabel, A., Hardcastle, T.J., Robinson, S., Miyashima, S., Grosse, I.,
and Meyerowitz, E.M. (2018). Transcriptome dynamics at Arabidopsis graft junctions
reveal an intertissue recognition mechanism that activates vascular regeneration.
Proc Natl Acad Sci U S A 115, E2447-E2456.

Moreno-Risueno, M.A., Sozzani, R., Yardimci, G.G., Petricka, J.J., Vernoux, T.,
Blilou, I., Alonso, J., Winter, C.M., Ohler, U., Scheres, B., et al. (2015). Transcriptional
control of tissue formation throughout root development. Science 350, 426-430.

56
Moreno-Risueno, M.A., Van Norman, J.M., Moreno, A., Zhang, J., Ahnert, S.E., and
Benfey, P.N. (2010). Oscillating gene expression determines competence for periodic
Arabidopsis root branching. Science 329, 1306-1311.

Motte, H., Vanneste, S., & Beeckman, T. (2019). Molecular and Environmental
Regulation of Root Development. Annual review of plant biology, 70, 465–488.

Naseer, S., Lee, Y., Lapierre, C., Franke, R., Nawrath, C., and Geldner, N. (2012).
Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without
suberin. Proc Natl Acad Sci U S A 109, 10101-10106.

Nibau, C., Gibbs, D.J., and Coates, J.C. (2008). Branching out in new directions: the
control of root architecture by lateral root formation. New Phytol 179, 595-614

P., and Sabatini, S. (2007). Cytokinins determine Arabidopsis root-meristem size by

controlling cell differentiation. Curr Biol 17, 678-682.

Perez-Garcia, P., and Moreno-Risueno, M.A. (2018). Stem cells and plant
regeneration. Dev Biol 442, 3-12.

Perianez-Rodriguez, J., Manzano, C., and Moreno-Risueno, M.A. (2014). Post-

embryonic organogenesis and plant regeneration from tissues: two sides of the same
coin? Front Plant Sci 5, 219.

Perianez-Rodriguez, J., Rodriguez, M., Marconi, M., Bustillo-Avendano, E.,

Wachsman, G., Sanchez-Corrionero, A., De Gernier, H., Cabrera, J., Perez

Perilli, S., Di Mambro, R., & Sabatini, S. (2012). Growth and development of the root
apical meristem. Current opinion in plant biology, 15(1), 17–23.

Pi, L., Aichinger, E., van der Graaff, E., Llavata-Peris, C. I., Weijers, D., Hennig, L.,
Groot, E., & Laux, T. (2015). Organizer-Derived WOX5 Signal Maintains Root
Columella Stem Cells through Chromatin-Mediated Repression of CDF4 Expression.
Developmental cell, 33(5), 576–588.

Poethig, R. S. (2009). Small RNAs and developmental timing in plants. Current

opinion in genetics & development, 19(4), 374-378.

Richard M. Amasino , Scott D. Michaels, The Timing of Flowering, Plant Physiology,

Volume 154, Issue 2, October 2010, Pages 516–520.

Rogers, E. D., & Benfey, P. N. (2015). Regulation of plant root system architecture:
implications for crop advancement. Current opinion in biotechnology, 32, 93–98.

57
Rojas, C. A., Hemerly, A. S., & Ferreira, P. C. (2010). Genetically modified crops for
biomass increase. Genes and strategies. GM crops, 1(3), 137–142.

Roppolo, D., De Rybel, B., Denervaud Tendon, V., Pfister, A., Alassimone, J.,
Vermeer, J.E., Yamazaki, M., Stierhof, Y.D., Beeckman, T., and Geldner, N. (2011).
A novel protein family mediates Casparian strip formation in the endodermis. Nature
473, 380-383.

Ryu, K.H., Huang, L., Kang, H.M., and Schiefelbein, J. (2019). Single-Cell RNA
Sequencing Resolves Molecular Relationships Among Individual Plant Cells. Plant
Physiol 179, 1444-1456.

Sarkar, A. K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, K.,
Scheres, B., Heidstra, R., & Laux, T. (2007). Conserved factors regulate signalling in
Arabidopsis thaliana shoot and root stem cell organizers. Nature, 446(7137), 811–
814.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T.,
Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source
platform for biological-image analysis. Nat Methods 9, 676-682.

Schutz, L.M., Louveaux, M., Vilches Barro, A., Bouziri, S., Cerrone, L., Wolny, A.,
Kreshuk, A., Hamprecht, F.A., and Maizel, A. (2021). Integration of Cell Growth and
Asymmetric Division during Lateral Root Initiation in Arabidopsis thaliana. Plant Cell
Physiol 62, 1269-1279.

Sena, G., Wang, X., Liu, H.Y., Hofhuis, H., and Birnbaum, K.D. (2009). Organ
regeneration does not require a functional stem cell niche in plants. Nature 457, 1150-
1153.

Serrano-Ron, L., Cabrera, J., Perez-Garcia, P., and Moreno-Risueno, M.A.

(2021a).Unraveling Root Development Through Single-Cell Omics and
Reconstruction of Gene Regulatory Networks. Front Plant Sci 12, 661361.

Serrano-Ron, L., Perez-Garcia, P., Sanchez-Corrionero, A., Gude, I., Cabrera, J., Ip,
P.L.,

Serrano-Ron, L., Perez-Garcia, P., Sanchez-Corrionero, A., Gude, I., Cabrera, J., Ip,
P.L., Birnbaum, K.D., and Moreno-Risueno, M.A. (2021b). Reconstruction of lateral
root formation through single-cell RNA sequencing reveals order of tissue initiation.
Mol Plant 14, 1362-1378.

58
Shirley Raps and others, Adaptation of the Cyanobacterium Microcystis
aeruginosa to Light Intensity , Plant Physiology, Volume 72, Issue 3, July 1983, Pages
829–832.

Shulse, C.N., Cole, B.J., Ciobanu, D., Lin, J., Yoshinaga, Y., Gouran, M., Turco, G.M.,
Zhu, Y., O'Malley, R.C., Brady, S.M., et al. (2019). High-Throughput Single-Cell
Transcriptome Profiling of Plant Cell Types. Cell Rep 27, 2241-2247 e2244.

Silva-Navas, J., Moreno-Risueno, M.A., Manzano, C., Tellez-Robledo, B., Navarro-

Neila, S., Carrasco, V., Pollmann, S., Gallego, F.J., and Del Pozo, J.C. (2016).
Flavonols Mediate Root Phototropism and Growth through Regulation of Proliferation-
to-Differentiation Transition. Plant Cell 28, 1372-1387.

Smith S, De Smet I. Root system architecture: insights from Arabidopsis and cereal
crops. Philos Trans R Soc Lond B Biol Sci. 2012 Jun 5;367(1595):1441-52.

Sugimoto, K., Jiao, Y., and Meyerowitz, E.M. (2010). Arabidopsis regeneration from
multiple tissues occurs via a root development pathway. Dev Cell 18, 463-471.

Swarup, R., Kramer, E.M., Perry, P., Knox, K., Leyser, H.M., Haseloff, J., Beemster,
G.T., Bhalerao, R., and Bennett, M.J. (2005). Root gravitropism requires lateral root
cap and epidermal cells for transport and response to a mobile auxin signal. Nat Cell
Biol 7, 1057-1065.

The Arabidopsis Genome Initiative. (2000). Analysis of the genome sequence of the
flowering plant Arabidopsis thaliana. Nature, 408(6814), 796-815.

Thorsten Hamann, Eva Benkova, Isabel Bäurle, Marika Kientz, and Gerd Jürgens
(2002). The Arabidopsis BODENLOS gene encodes an auxin response protein
inhibiting MONOPTEROS-mediated embryo patterning

Tian, H., Jia, Y., Niu, T. et al. The key players of the primary root growth and
development also function in lateral roots in Arabidopsis .

Tischer, C., Ravindran, A., Reither, S., Chiaruttini, N., Pepperkok, R., and Norlin,
N.J.B. (2021). BigDataProcessor2: A free and open-source Fiji plugin for inspection
and processing of TB sized image data. 37, 3079-3081.

Trinh, J., Zeldenrust, F. M. J., Huang, J., Kasten, M., Schaake, S., Petkovic, S.,
Madoev, H., Grünewald, A., Almuammar, S., König, I. R., Lill, C. M., Lohmann, K.,
Klein, C., & Marras, C. (2018). Genotype-phenotype relations for the Parkinson's
disease genes SNCA, LRRK2, VPS35: MDSGene systematic review. Movement
disorders : official journal of the Movement Disorder Society, 33(12), 1857–1870.
59
van den Berg, C., Willemsen, V., Hage, W., Weisbeek, P., and Scheres, B. (1994).
Cell fate in the Arabidopsis root meristem determined by directional signalling. Nature
378, 62-65.

Van Norman JM, Xuan W, Beeckman T, Benfey PN. To branch or not to branch: the
role of pre-patterning in lateral root formation. Development. 2013 Nov;140(21):4301-
10.

Van Norman, J. M., & Benfey, P. N. (2009). Arabidopsis thaliana as a model organism
in systems biology. Wiley interdisciplinary reviews. Systems biology and
medicine, 1(3), 372–379.

Van Norman, J.M., Xuan, W., Beeckman, T., and Benfey, P.N. (2013). To branch or
not to branch: the role of pre-patterning in lateral root formation. Development 140,
4301-4310.

von Wangenheim, D., Fangerau, J., Schmitz, A., Smith, R.S., Leitte, H., Stelzer, E.H.,
and Maizel, A. (2016). Rules and Self-Organizing Properties of Post-embryonic Plant
Organ Cell Division Patterns. Curr Biol 26, 439-449.

Wachsman, G., Zhang, J., Moreno-Risueno, M.A., Anderson, C.T., and Benfey, P.N.
(2020). Cell wall remodeling and vesicle trafficking mediate the root clock in
Arabidopsis. Science 370, 819-823.

Wang D, Ma X, Hao Z, Long X, Shi J, Chen J. Overexpression of Liriodenron WOX5

in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology.
International Journal of Molecular Sciences. 2023; 24(2):906.

Wolff, C., Tinevez, J.Y., Pietzsch, T., Stamataki, E., Harich, B., Guignard, L.,
Preibisch, S., Shorte, S., Keller, P.J., Tomancak, P., et al. (2018). Multi-view light-
sheet imaging and tracking with the MaMuT software reveals the cell lineage of a
direct developing arthropod limb. Elife 7.

Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., & Benfey, P. N.
(2000). Molecular analysis of SCARECROW function reveals a radial patterning
mechanism common to root and shoot. Development (Cambridge, England), 127(3),
595–603.

Xu, J., Hofhuis, H., Heidstra, R., Sauer, M., Friml, J., and Scheres, B. (2006). A
molecular framework for plant regeneration. Science 311, 385-388.

Xuan, W., Audenaert, D., Parizot, B., Moller, B.K., Njo, M.F., De Rybel, B., De Rop,
G., Van Isterdael, G., Mahonen, A.P., Vanneste, S., et al. (2015). Root Cap-Derived
60
Auxin Prepatterns the Longitudinal Axis of the Arabidopsis Root. Current biology : CB
25, 1381-1388

Xuan, W., Band, L.R., Kumpf, R.P., Van Damme, D., Parizot, B., De Rop, G.,
Opdenacker, D., Moller, B.K., Skorzinski, N., Njo, M.F., et al. (2016). Cyclic
programmed cell death stimulates hormone signaling and root development in
Arabidopsis. Science 351, 384-387.

Zhang, Y., He, P., Ma, X., Yang, Z., Pang, C., Yu, J., Wang, G., Friml, J., and Xiao,
G. (2019). Auxin-mediated statolith production for root gravitropism. New Phytol 224,
761-774.

Zhang, Y., Umeda, M., & Kakimoto, T. (2022). Pericycle cell division competence
underlies various developmental programs. Plant biotechnology (Tokyo,
Japan), 39(1), 29–36.

Zhou, J., Li, D., Wang, G., Wang, F., Kunjal, M., Joldersma, D., & Liu, Z. (2020).
Application and future perspective of CRISPR/Cas9 genome editing in fruit crops.
Journal of integrative plant biology, 62(3), 269–286.

61