INTERNATIONAL UNIVERSITY

BIOTECHNOLOGY DEPARTMENT
PLANT PHYSIOLOGY

Cell division and cell expansion

in the growth of plant

Instructor: Tran Van Minh (PhD)

Course ID: BTBT17IU21
Name: Ho Thanh Mai
Student ID: BTBTIU17127

Ho Chi Minh City, April 3, 2019.

 CONTENTS

I. Introduction………………………………………………..….………….….02
II. The plane and symmetry of cell division………………………….………..02
III. Cell expansion and growth…………………………………………….……04
IV. The importance of cortical microtubules in plant growth………….…….06
V. Regulation of cell division and expansion by sugar and auxin signaling.
1. Auxin and cell division.
1.1. Basic molecular mechanisms of the cell cycle…….………...…..08
1.2. Auxin action on cell cycle……………...…………………………08
1.3. Auxin signaling pathways controlling cell cycle………………..09
2. Auxin and cell expansion/elongation
2.1. From cell wall loosening to expansion……….………………….09
2.2. ABP1 and cell expansion……………..…………………………..10
VI. Discussion…………………………………………………………………….11
VII. References……………………………………………………...……...……..12

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I. INTRODUCTION:
Plant growth and development results from a combination of three processes at the
cellular level: cell division, cell expansion, and cell differentiation. Cell division in
meristems, by increasing cell number, increases the potential for growth, but it is cell
expansion that accounts for the actual increase in plant mass. If the two processes
become uncoupled, cells would either become progressively larger if the doubling
time for cell mass was shorter than the cycle time, or progressively smaller if the mass
doubling time was longer than the cycle time. Despite numerous elegant experiments,
the details of how cell growth is regulated and coupled to cell division remain poorly
understood. Over the last decade, genetic analyses and genome-wide gene expression
profiling studies have significantly advanced our understanding of the signaling
pathways regulating cell proliferation and expansion.

II. THE PLANE AND SYMMETRY OF CELL DEVISION.

The plane (direction) of cell division is an important determinant of plant form. Cells
proliferate mainly via symmetric division, whereas asymmetric divisions are
associated with initiation of new developmental patterns and cell types.
Division planes in both symmetrically and asymmetrically dividing cells are
established through the action of a cortical preprophase band (PPB) of cytoskeletal
filaments, which is disassembled upon transition to metaphase, leaving behind a
cortical division site (CDS) to which the cytokinetic phragmoplast is later guided to
position the cell plate.
Asymmetric cell division is a fundamental mechanism that generates cell diversity
while maintaining self-renewing stem cell populations in multicellular organisms.
Both intrinsic and extrinsic mechanisms underpin symmetry breaking and differential
daughter cell fate determination in animals and plants.
When a single cell poised to undergo mitosis. If the planes of division of its
descendants are parallel to the plane of the first cell division, a single file of cells will
be produced. At the other extreme, if the planes of cell division of the descendant
cells are randomly aligned, an unorganized clump of cells will result. The plane of
cell division is immensely important in determining form, but so is the symmetry of
cell division. Asymmetrical cell division, in which one daughter cell receives more
cytoplasm than the other during mitosis, is fairly common in plant cells and usually
signals a key event in development. (Figure 3)

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Figure 3: The plane and symmetry of cell division influence development of form.

A Preprophase Band of microtubules can

orient itself one of two ways around the
nucleus and control the plane of division.
This process occurs during late
interphase. The microtubules hold the
nucleus in place during spindle formation
so the spindles are directed to their
respective positions to produce the
determined division pattern. (Figure 4)

Figure 4: The preprophase band and the

plane of cell division.
Asymmetric cell division (ACD) allows for the production of diverse cell types, while
still maintaining stem cell populations for additional growth. An asymmetrically
dividing cell must polarize molecular components such as mRNA, proteins, cell-
surface markers, or organelles unequally to create distinct cellular domains. Once
cellular asymmetry is achieved, coordination of the division plane with the
segregation of these components is needed so that the resulting daughter cells are not
equal.

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Asymmetric cell division generates cellular diversity, contributing to cell patterning
and stem cell maintenance during plant development. Daughter cells of different
sizes, shapes, and/or fates are formed when a cell divides in an asymmetric manner.

III. CELL EXPANSION AND GROWTH.

Plant cells grow rapidly and “cheaply” by intake and storage of water in vacuoles
AKA “Water Weight”. Plant cells expand primarily along plant’s main axis. Cellulose
microfibrils in cell wall restrict direction of cell elongation.
The uptake of water is very important and this produces the cell’s expansion. Growing
plant cells also produce additional organic material in their cytoplasm, but the uptake
of water typically accounts for about 90% of a plant cell’s expansion. Most of this
water is packaged in the large central vacuole, which forms by the coalescence of
numerous smaller vacuoles as a cell grows. The water goes to the central vacuole
which grows due to the coalescing of many small vacuoles. They do not expand
equally in all directions; usually it is along the main axis which is controlled by the
cellulose in the cell wall.
Cell expansion contributes to plant form. Orientation of cell growth is in the plane
perpendicular to the orientation of the cellulose microfibrils in the wall. Enzymes
weaken cross-links in the wall, and allow it to expand as water diffuses into vacuole
by osmosis.
Growing plant cells expand mainly
through water uptake. In a growing
cell, enzymes weaken cross-links in
the cell wall, allowing it to expand
as water flows in by osmosis. The
orientation of cell growth is mainly
in the plane perpendicular to the
orientation of cellulose microfibrils
in the wall. The microfibrils are
embedded in a matrix of other
(noncellulose) polysaccharides,
some of which form the cross-links
visible in the micrograph (TEM).
Loosening of the wall occurs when
hydrogen ions secreted by the cell activate cell wall enzymes that break the cross-
links between polymers in the wall. This reduces restraint on the turgid cell, which
can take up more water and expand. Small vacuoles, which accumulate most of this
water, coalesce and form the cell’s central vacuole.

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Cellulose microfibrils are synthesized at the cell surface by complexes of enzymes
that can move in the plane of the plasma membrane. According to one hypothesis,
microtubules form "banks" that confine the movement of the enzymes to channels of
specified direction. Each enzyme complex advances along one of these channels as
the microfibril it extends becomes locked in place by cross-linking to other
microfibrils. (Figure 5)

Figure 5: A hypothetical mechanism for how microtubules orient cellulose

microfibrils.
A root is made up of a number of cell files. It is the sum of the extension of the
individual cells in each file that constitutes root extension. As cells in adjacent files
cannot move across each other, all files must extend at the same rate. Cell
expansion occurs predominantly in the longitudinal direction, although some radial
expansion may take place close to the apex. This increases the root's diameter.
The growth rate of roots is reduced under adverse soil conditions such as low water
potential or excess salinity. This response is often associated with a shortening of the
growth-zone and a reduction in the maximum relative rate of extension. Extension
rate in the apical region of the growth-zone is usually less affected. The biochemical
basis of wall loosening and tightening in extending roots is not fully resolved. The
orientation of cellulose microfibrils in the wall is believed to control the anisotropy of
cell growth, but there is little evidence to suggest that it regulates extension rate along
the growth-zone.
In a growing root, cell division and expansion must be coordinated. However, cell
division can be inhibited (e.g., by using mitotic inhibitors such as colchicine) without
initially affecting expansion, demonstrating that these processes can be partly
uncoupled. Understanding the molecular co-regulation of cell division and expansion
in roots promises to be one of the key areas of future research.

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IV. THE IMPORTANCE OF CORTICAL MICROTUBULES IN PLANT
GROWTH
Mutants made of Arabidopsis, called Fass mutant which have confirmed the
importance of cortical microtubules in both cell division and expansion. The
microtubular arrangement in these mutants is abnormal. The preprophase band do not
form in an organized fashion which affects the arrangement of the cellulose
microfibrils in the cell wall. The poor arrangement of the cellulose microfibrils affects
the elongation of the cell causing it to elongate in all direction equally and to divide
every which way.
Fass mutants are unusually
squat and seem to align their
division planes randomly. They
lack the ordered cell files and
layers normally present. Fass
mutants develop into tiny adult
plants with all their organs
compressed longitudinally. The
squat body of the fass mutant
results from cell division and
cell elongation being randomly
oriented instead of orienting in
the direction of the normal plant
axis. (Figure 6)

F
igure 6: The fass mutant of Arabidopsis
confirms the importance of cortical
microtubules to plant growth.

V. REGULATION OF CELL DIVISION AND EXPANSION BY SUGAR

AND AUXIN SIGNALING
1. Auxin and cell division.
Mitotic cell division encompasses the sequence of events giving rise to the formation
of two daughter cells. The cell cycle is divided into four phases:

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- The replication of DNA characterizes the S phase (synthesis) whereas segregation
of the duplicated chromosomes and physical separation of the two daughter cells
(cytokinesis) take place in mitosis or M phase. Two essential gap phases separate
the S and M phases.
- The G1 phase, between mitosis and the entry into S phase, and the G2 phase,
between replication and mitosis, monitor whether the previous phase has been
fully and accurately achieved before execution of the next one.
- Cell growth occurs mainly within these two gap phases. The G1/S and G2/M
transitions are two critical regulatory steps of the cell cycle sometimes referred as
cell-cycle restriction—or checkpoints.
The plant cell cycle shares this highly ordered process with all eukaryotes and
basic molecular mechanisms are mainly conserved.

The two phytohormones auxin and cytokinin were shown to play important roles in
the induction of cell division and control of cell-cycle progression. Auxin is necessary
but not sufficient to stimulate cell division in cultured cells or plant tissues because
the presence of cytokinin is also required. Cells cultured in the absence of exogenous
cytokinin, such as BY2 tobacco cells, produce it at least at critical phases of cell
division while they are strictly dependent on exogenous auxin.

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The specific roles of auxin at the various phases of the cell cycle in dividing cell
suspension cultures has been hampered by the fact that auxin is required at the initial
step of cell-cycle entry and thus its effects cannot easily be dissociated from this
initial and critical step. The mechanisms by which auxin affects the cell-cycle
machinery are still far from being elucidated but recent data suggest that auxin acts on
multiple targets and affects transcriptional regulation as well as protein turnover of
core cell-cycle regulators.
1.1. Basic Molecular Mechanisms of the Cell Cycle.
The completion of the Arabidopsis and rice genomes has permitted the identification
of the majority of the core cell-cycle genes in plants. Recent advances have
established that plant cell-cycle progression is controlled by an evolutionarily
conserved molecular mechanism involving distinct combinations of cyclin-dependent
kinase (CDK) and cyclin complexes which phosphorylate a number of substrates at
the G1/S and/or G2/M transitions. Several key features are, however, unique to plant
cells.
Within a CDK-cyclin complex, the cyclin plays a role in the selective interaction with
substrate proteins whereas the CDK is the catalytic subunit responsible for the
recognition of the serine or the threonine target motif in the substrate proteins.
1.2. Auxin Action on Cell Cycle.
Importantly, auxin was shown to induce the expression of CDKA;1, encoding the
CDKA implicated throughout the cell cycle (Hemerly et al. 1993; Ferreira et al. 1994;
Doerner and Celenza 2000). In tobacco cell suspension culture, auxin was reported to
play an important role in the assembly of active CDKA-associated complexes
(Harashima et al. 2007). Activation of the kinase activity may however require
cytokinin for promoting the phosphorylation of the CDKA kinase as reported in
Nicotiana plumbaginifolia cells (Zhang et al. 1996).
Auxin also affects posttranscriptional regulation of cell-cycle components. Auxin
increases E2FB protein stability (Magyar et al. 2005). Authors showed that elevated
E2FB/DPA support cell proliferation in the absence of auxin whereas control BY2
cells in tobacco.
In addition to effecting core cell-cycle regulators, auxin has been reported to regulate
the expression of other genes or the turnover of proteins that are necessary for cell
proliferation. For example, auxin was reported to stabilize a potato homolog of a
human EBP1 (ErbB3 epidermal growth factor receptor binding protein) expressed
transiently in Arabidopsis cells (Horvath et al. 2006).
In plants, EBP1 seems to be required for expression of cell-cycle genes belonging to
distinct phases such as CYCD3;1 (G1 and G1/S), ribonucleotide reductase 2 (RNR2,
S-phase) or CDKB1;1 (G2 to M phase) in a dose- and auxin-dependent manner.
Modulation of EBP1 expression in Arabidopsissupports a role in cell proliferation of

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meristematic cells whereas it promotes expansion in nondividing cells (Horvath et al.
2006).
1.3. Auxin Signaling Pathways Controlling Cell Cycle.
Two auxin pathways with different levels of importance seem to be involved. One
involves the auxin binding protein, ABP1, and the other the already mentioned
Aux/IAA/SCFTIR1/AFB pathway.
ABP1 is critical for auxin regulation of the cell cycle. ABP1 was isolated through its
capacity to bind auxin and, based on its involvement in the activation of ion fluxes at
the plasma membrane in response to auxin, was characterized as an auxin receptor.
Inactivation of the protein in synchronized cells revealed that the G2/M transition was
also impaired. Local inactivation of ABP1 at the I1 position of the shoot apical
meristem of tobacco plants results in rapid arrest of cell division with changes in the
orientation of the cell division plate in cells which were progressing into the cell cycle
at the time of inactivation.
2. Auxin and cell expansion/elongation.
Cell expansion is an increase in cell size accompanying the process of plant growth.
This increase in size usually results from the combination of two processes: The
increase in cell ploidy level by endoreplication (successive rounds of DNA replication
with no mitosis), and the complex process of cell expansion, which is driven by
internal turgor pressure and restricted by the ability of cell walls to extend.
Plant cell expansion requires uptake of water, which is then stored in vacuoles, and
irreversible extension of the cell wall, which includes wall loosening (short time
frame) and deposition of new wall material (long time frame). Auxin is one of the
major stimuli affecting these mechanisms but it is essential to keep in mind that cell
expansion is also under the control of many other stimuli, such as blue light and most
of the other phytohormones. Auxin-dependent cell expansion follows a dose-response
curve in which high concentrations are inhibitory.
2.1. From Cell Wall Loosening to Expansion.
Plant cells are surrounded by a complex and dynamic wall, which plays a critical role
during development in establishing cell size and cell shape. The structure of the
primary cell wall is formed by a network of crystalline cellulose microfibrils
interlinked with hemicelluloses (mainly xyloglucan or arabinoxylan in seed plants).
This network is embedded in a matrix of pectins and also contains a small amount of
cell-wall proteins.
Rapid enlargement requires wall loosening, that involves modification of the
molecular interactions within the cell-wall network (but not of its composition),
resulting in relaxation of wall tension. Expansins have been identified as major wall-
loosening agents. These cell wall proteins are activated by acidification and, in a

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range of pH between 4.5 and 6, are thought to disrupt noncovalent binding between
cellulose and hemicelluloses.
Auxin was shown to induce rapid cell elongation in stem, coleoptile, or hypocotyl
segments within minutes after auxin treatment. This rapid effect is believed to result
from the activation of a proton pump ATPase at the plasma membrane, inducing
extrusion of H+, extracellular acidification, activation of expansins, and subsequent
wall loosening.

2.2. ABP1 and Cell Expansion.

Various lines of evidence, mainly relying on electrophysiological experiments and use
of a whole series of ABP1 molecular tools (specific monoclonal and polyclonal
antibodies, carboxy-terminal synthetic peptides, recombinant wild-type and mutated
proteins) support the hypothesis that ABP1 is involved in the initial perception of
auxin at the outer face of the plasma membrane and induces the activation of
H+ATPase and K+ inward channels.
In addition ABP1 was shown to mediate the auxin-induced enlargement (or swelling)
of protoplasts of maize coleoptiles and Arabidopsis hypocotyls. Analysis of the
swelling response of protoplasts of pea stem epidermal cells revealed a biphasic dose
response curve to IAA, one phase being dependent on ABP1 but not the other,
suggesting that at least in these epidermal cells another pathway is involved.
Finally, functional inactivation of ABP1, by use of conditional antisense or
conditional immunorepression strategies, confirmed the critical role of the protein in
the control of cell expansion in leaf tissues even in cells with increased ploidy (Braun
et al. 2008). Altogether, these data show that ABP1 is essential for auxin-dependent
cell expansion during leaf growth.

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VI. DISCUSSION.
Much progress has been made in the identification of components taking part in the
control of cell division and cell expansion. The final number, size, and shape of plant
organs largely rely on the coordination of these cellular responses and auxin plays a
central role in these processes. Much has yet to be learned about the complex network
of regulation activated by auxin and how the auxin signal is differentially transduced
in a variety of downstream responses depending on the cell, the organ, or the
environment. Elucidating the auxin signaling pathways involved and how the two
types of auxin receptors, TIR1/AFB and ABP1, cooperate to control cellular
responses to auxin are critical challenges for the coming years.
Plant tissues have two ways to regulate their growth, on the one hand by coordinating
cell expansion and on the other hand by directing the orientation of cell division
planes. Both pathways seem to be regulated by mechanical stresses within the tissue.
Cell expansion is asymmetric, reduced in the direction of maximal stress due to a
mechanical feedback on cell wall strengthening mediated by cortical microtubules.
The orientation of cell division planes often agrees with the direction of the cell’s
shortest midplane (Besson and Dumais, 2011) or the direction of maximal stress
(Hamant et al., 2008). As mechanical stresses change and adapt during the growth of
a tissue, these feedbacks from mechanical stresses on the tissue mechanics itself
provide an autonomous way to impact not only the final tissue morphology but also
the tissue growth dynamics.
It has been shown that the mechanical feedback on cell expansion can provide a mean
to reduce or amplify growth heterogeneities in tissues (Uyttewaal et al., 2012). Here,
we addressed how the orientation of cell divisions impacts a tissue’s growth
dynamics. There are two noisy rules of cell division namely random orientation of cell
division planes and random orientations that divide the cell in exactly two halves.
Further, there are three deterministic cell division rules: division perpendicular to the
previous cell division plane, along the maximum principal second moment of area
(roughly corresponding to the shortest new wall) and along the direction of maximum
principal stress. Random cell divisions release less stress within the tissue, create
larger, and more asymmetric cells with higher stress variability and growth variability
than those cell divisions following the direction of maximum principal second
moment of area or stress. In contrast, when cell shape or stress prescribe the cell
division plane, cell shape is more isotropic and stress variability is the least among all
rules – possibly indicating the sensitivity of growth regulation in those cases.
While the case of random cell divisions might appear a bit extreme in the context of
development, the choice of the cell division mode might nevertheless be regulated,
even when restricted to the rules that depend on an interaction with the tissue. There
the orientation of new cell walls agrees with the direction of maximum principal
stress (Hamant et al., 2008). The analysis presented in this work shows that both cell
divisions along the shortest midplane and along the axis of maximal stress give rise to
equivalent tissue geometry, topology, and tissue growth dynamics. We have,

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however, restricted ourselves to the case of isotropically growing tissue as present
only at the tip of the shoot apical meristem. So the conclusion is that isotropic growth
cannot provide the data to discern between both rules. A distinction is in our opinion
only possible in a tissue that grows anisotropically. In practice one could investigate
mutants in which the orientation of the new wall is abnormal, such as ton1 (Traas et
al., 1995) or pok1;pok2 (Müller et al., 2006; see also Müller, 2012 for a review) and
compare their shoot apex tissue statistics and most importantly growth dynamics in a
similar setup as in Uyttewaal et al. (2012).
In summary, the orientation of cell division does not only contribute to tissue
geometry and topology but also to the dynamics of tissue growth. Cell division planes
that are oriented along the cell’s shortest midplane or the direction of stress actively
contribute to the tissue growth dynamics by releasing stress and thus reducing stress
variability and growth variability within a tissue. While geometric and topological
tissue characteristics are limited to discern between different modes of cell division,
measuring quantities such as growth variability provides handles to distinguish cell
division modes and beyond learn about the regulation of growth via the orientation of
cell division.

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