GEB 203

Plant Developmental Biology

Md Faruk Hasan
Dept. of Genetic Engineering and Biotechnology
University of Rajshahi

27/08/2020
 What is pattern formation in early embryogenesis ?

Pattern formation is the basic directions for development of specific

structures in specific locations of embryo.
In developmental biology, pattern formation refers to the generation of
complex organizations of cell fates in space and time during embryogenesis.

Pattern formation is controlled by genes and phytohormones. The role of

genes in pattern formation is an aspect of morphogenesis, the creation of
diverse anatomies from similar genes, now being explored in the science of
evolutionary developmental biology.

Fate of plant cells in embryogenesis

Two types of hypotheses explain the fate of plant cells in embryogenesis.
1. Lineage-based mechanisms propose that cell fate is determined early in
development and passed on to daughter cells.
2. Position-based mechanisms propose that cell fate is determined by final
position.
Plant cell fate is established late in development and depends on cell position.
 Mechanisms of pattern formation in plants embryogenesis
Embryogenesis starts with a single cell, the zygote, and ends with a mature embryo in which
all cells have acquired a specific fate. The organized division and specification of cells during
embryogenesis is called pattern formation. The processes that generate all of these different
cell types from the zygote remain largely unknown. The highly organized cell divisions
observed during embryogenesis in Arabidopsis do not seem to be critically important for axis
formation and cell fate specification, as embryos mutant for the FASS gene have a completely
altered pattern of cell division but still contain all cell types that build the basic body plan,
and show a relatively normal axis.
What mechanisms are involved in pattern formation?
Conceptually, when focusing on those patterning steps that involve asymmetric cell divisions,
two mechanisms of pattern formation can be envisioned.
Firstly, a cell with intrinsic polarity divides to generate two different daughter cells. Given the
strong polarity of the egg cell and the hypophysis, this scenario could well apply to the
division of these cells. Alternatively, initially identical daughter cells acquire different
identities after division. This type of pattern formation requires cell–cell communication to
ensure that the position, rather than lineage, of a cell determines its fate.
Secondly, the mechanism could also work over longer distances, to pattern fields of cells. In
this context, an ever-increasing body of evidence shows that the plant hormone auxin is
required for pattern formation. Importantly, all processes required for auxin activity
biosynthesis and transport, auxin perception by its receptor, and auxin response are each
required for pattern formation. The patterning steps that require auxin activity, focusing on
recent findings that illuminate the mechanism of auxin action.
Different genes, Cell lineages, PIN protein localization, and auxin response maxima during
Arabidopsis embryogenesis. Arabidopsis embryos follow a regular and stereotypical cell
division pattern. Lineages are indicated by thin lines between individual stages. PIN protein
localization at membranes is marked with PIN1, PIN4, and PIN7 lines and DR5 reporter.

After division of the zygote, the one-cell embryo (same for two-cell embryos) expresses PIN7
in the basal daughter cell (bc), with the protein on the side of the apical cell (ac), which
expresses the DR5 reporter. After two more cell-division rounds, all proembryo (pe) cells
express PIN1 without apparent polarity and show DR5 reporter activity. Basal suspensor (sus)
cells express PIN7, which is polarly localized on the proembryo side. At the globular stage,
basal PIN1 polarity is established in the central lower cells of the proembryo, whereas PIN1
localizes apically in outer protoderm (pd) cells. At the same time, PIN7 polarity reverses in
suspensor cells and PIN4 is activated in the uppermost suspensor cell. This cell now expresses
the DR5 reporter and is specified as hypophysis (hyp). During the transition stage, PIN1
polarity at the flanks of apical embryo half converges in adjacent cells accompanied by the
appearance of new DR5 maxima. These sites mark the initiation of the cotyledons.
At the torpedo stage, primordia for each of the seedling organs can be distinguished. Discrete
regions of the embryo give rise to the root apical meristem (RAM), the hypocotyl (hypo), the
cotyledons (cot), and the shoot apical meristem (SAM).
 Role of Auxin in pattern formation in embryogenesis
Auxin controls several cell specification and pattern formation processes in the early embryo.
In postembryonic auxin-dependent growth and patterning, auxin has been proposed to
accumulate in concentration gradients, with cells converting different threshold
concentrations to distinct responses. The other conceptual extreme of auxin activity would be
that of a trigger that would elicit a predefined response above a certain threshold. Multiple
distinct responses are specified by the perceived auxin concentration. It appears that
alterations in auxin concentrations cannot bypass the predefined cell fate in the embryo. For
example, when auxin levels are ubiquitously increased through expression of a biosynthesis
gene and inhibition of transport, DR5 is activated throughout the embryo. However, this is
associated with fusion of cotyledons, but no ectopic specification of pattern elements.
Likewise, removal of auxin causes a failure to specify the root or cotyledons, but no cell-fate
transformations. A prepattern determines the developmental potential that can be triggered by
auxin, in which the concentration of auxin could determine response amplitude. Inappropriate
accumulation of auxin in cotyledon tips either through expression of dominant-negative Rab5
or apolar PIN1 is sufficient to convert these cells to root identity. The size of the Aux/ IAA
and ARF families, as well as the presence of six TIR1/AFB receptors would allow enormous
combinatorial complexity to equip each cell with a unique response machinery to enable
unique cellular auxin responses. Systematic analysis of Aux/IAA and ARF functions in the
embryo, as well as identification of target genes and processes of auxin in the embryo will
provide critical insight into the prominent role of auxin as an embryo patterning molecule.
 Auxin response during embryogenesis in Arabidopsis

Figure: Cell lineages, PIN protein localization, and auxin response maxima during embryogenesis. (A)
After division of the zygote, the one-cell embryo expresses PIN7 in the basal daughter cell (bc), with the
protein on the side of the apical cell (ac), which expresses the DR5 reporter. After two more cell-division
rounds, all proembryo (pe) cells express PIN1 without apparent polarity and show DR5 reporter activity.
Basal suspensor (sus) cells express PIN7, which is polarly localized on the proembryo side. At the globular
stage, basal PIN1 polarity is established in the central lower cells of the proembryo, whereas PIN1 localizes
apically in outer protoderm (pd) cells. At the same time, PIN7 polarity reverses in suspensor cells and PIN4
is activated in the uppermost suspensor cell. This cell now expresses the DR5 reporter and is specified as
hypophysis (hyp). During the transition stage, PIN1 polarity at the flanks of apical embryo half converges in
adjacent cells accompanied by the appearance of new DR5 maxima. These sites mark the initiation of the
cotyledons. (B) At the torpedo stage, primordia for each of the seedling organs can be distinguished.
Discrete regions of the embryo give rise to the root apical meristem (RAM, green), the hypocotyl (hypo,
blue), the cotyledons (cot, yellow), and the shoot apical meristem (SAM, red).
Distinctive embryo phenotypes of auxin mutants
Characteristic defects of auxin-related mutants at three different stages of embryo development.

Fig.: The figures show examples of defects (red) that can occur at each developmental stage. (A)
Transverse instead of longitudinal division of the apical cell at the one-cell stage. (B) At the globular
stage, two phenotypes can be distinguished. The hypophysis divides aberrantly (left) resulting in a
rootless seedling. In addition, vascular divisions may be incorrect. (C) At the heart stage, embryo
cotyledon formation and/or separation are impaired, resulting in cotyledon fusion, an aberrant
cotyledon number, or complete absence of cotyledons. Other auxin-related mutants have both
cotyledon and root meristem defects. The root meristem defects in such mutants are the result of
aberrant hypophysis division except for the plt1 plt2 plt3 plt4 mutant in which the hypophysis
descendants divide abnormally.
 Effects of genes in early embryonic pattern formation
and morphogenesis
Arabidopsis and other crucifers display a regular pattern of cell division in
early embryogenesis. This regularity enabled primordia of seedling
structures to be recognised from early embryogenesis and facilitated the
identification of precursors for the meristems of the root and the shoot.
Moreover, the mutant seedling phenotypes of two genes involved in pattern
formation in the Arabidopsis embryo, GNOM and MONOPTEROS, were
shown to correlate topographically with abnormal patterns of cell division in
the early embryo. These results supported the view that oriented cell
divisions are associated with pattern formation in the Arabidopsis embryo.
The fs phenotype, however, suggests that oriented cell divisions are not
instrumental for pattern formation: although the primordia of seedling
structures cannot be recognized by morphological criteria at the heart stage,
the body pattern of the seedling is essentially normal.
Thus, fs embryos mimic the situation found in plant species such as cotton:
the pattern of cell division in the embryo is not regular and yet the seedling
has the same body pattern as in crucifers.
It is therefore appropriate to view the regularity of cell division in the
Arabidopsis embryo as a special case: cells at specific positions respond to
pattern formation by changing cell shape or by undergoing oriented cell division.
So, morphogenesis would be the consequence of pattern formation. The fs
phenotype indicates that pattern formation is not dependent on morphogenesis.
The additive phenotype of the fass gnom double mutant suggests that the two
processes are independent; pattern formation and morphogenesis are normally
integrated to produce the wild-type seedling.
In order to identify factors influencing pattern formation in the Arabidopsis
embryo. Although the division of the zygote produces a variably oriented cell
wall and subsequent divisions of both its daughter cells can be abnormal, cell
polarity is not affected since the apical and the basal cell differ in staining
intensity as they do in wild-type.
The fs seedling itself demonstrates that polarity is maintained throughout
development since it represents a polarised structure. Thus, cell polarity as set up
in the zygote might be essential for cells to acquire information about their
relative positions which may underlie pattern formation. In some plant species,
cell fate is then presaged by very regular patterns of cell division and cell
arrangements, if the FS gene is functional.
In essence, the fs phenotype suggests that pattern formation and morphogenesis
are two distinct processes in establishing the body organisation of the seedling.
List of genes related to embryonic pattern formation

GENES SYMBOLS
PEPTIDYLPROLYL CIS/TRANS PIN1
ISOMERASE, NIMA-INTERACTING 1/
PIN FORMED1
SHOOT MERISTEMLESS STM
WUSCHEL WUS
CLAVATA CLV
HOBBIT HBT
MONOPTEROS MP
GURKE, GK
GNOM GN
FACKEL FK
PASTICCINO PAS
FASS FS
REGULATORY PARTICLE NUMBERS RPN
 Effects of mutant genes in early embryonic pattern
formation and morphogenesis
1. Mature embryo development: Role of pin1-1

PINFORMED1 (PIN1) has profound effects on cotyledon development. During

embryogenesis, the pin1 mutation affects cotyledon positioning, number, growth
and separation, resulting in disruption of bilateral symmetry. The cotyledon defects
in pin1 were already apparent in torpedo stage embryos, where cotyledons with
unequal sizes were initiated at asymmetrical positions. These observations indicate
that pin1 mutations disrupt bilateral symmetry and cotyledon separation from early
embryogenesis onwards.
2. Shoot formation: STM mutation effects

Wild type shoot apex stm mutant

Shoot Meristemless (stm)

phenotype

Fig.: STM mutation wild-type prevents cell differentiation in Peripheral Zone

STM gene is required for SAM formation during embryogenesis and is

expressed by only a specific set of cells within the embryo apex. In the
mutants stm organs originating from the shoot meristems are fused, which
indicates a role of STM in restricting cells and organ formation.
3. Shoot Development: Effect of WUS gene phenotype

Wild Type Wild type SAM

wus mutant
wus SAM

The interaction between the WUS and CLV pathways establishes a negative
feedback loop between the stem cell and the meristematic regions. Indeed, WUS
expression is sufficient to induce meristem cell identity, and expression of CLV
genes represses meristem maintenance and WUS activity. The apical region of
the globular embryo is partitioned into two domains: a peripheral domain of
STM-expressing cells and a central domain.
4. Shoot Development: Effect of CLV gene phenotype

Normal SAM
CLV localization in
Central Zone of SAM

1. CLV localization in Central Zone of SAM

clavata SAM
2. Mutant phenotype: Huge apical meristems
 CLV wild type restricts stem cell accumulation

The cell differentiation is controlled by the genes CLAVATA (CLV). The CLV
genes are required to limit the meristem size. CLV and STM genes are
known to play opposite or competitive roles in the regulation of meristem
activity.
 5. Shoot- Root Development: Effect of HBT and MP genes

Normal cell division create Abnormal cell division create Has a basal peg not a root
shoot and root stub and normal shoot

HOBBIT: Mutations in HOBBIT (HBT) gene affect the development of the basal,
but not the central region, resulting in the lack of a functional root meristem, and
therefore root formation. hbt seedlings initiate but do not develop adventitious
roots from their hypocotyls because they are unable to form new root meristem.
MONOPTEROS: Mutations in the MP gene strongly perturb embryo
development, affecting the establishment of the embryo axis. In addition, mp
mutants also display defects in cotyledon positioning and these cotyledons are
frequently fused.
 6. Shoot- Root Development: Effect of GK, FK, MP and GN genes

Normal cell division

create shoot and root

Abnormal cell division create Has a basal peg not a root

stub and normal shoot
-Gk mutations cause apical defects in seedlings and also reduced the efficiency of
seed germination and seedling growth
-fk embryo mutants don't exhibit the characteristic heart shape and mutant embryo
appears smaller and wider than wild-type embryos. The radial organization of the
vasculature is also abnormal in fk, and tissue layers are disorganized
-mp mutant is devoid of hypocotyls, root and root meristem
-gn mutant shows loss of apical basal polarity and disorganized growth
 7. Seedling Development: Effect of FS gene
Fig.: Seedling phenotype of
the strong allele fassR226-32
(right) showing extreme
compression of all body
elements; wild-type on the left.
FASS mutations affect cell elongation and
orientation of cell walls resulting in a
seedling that is strongly compressed in
the apical-basal axis and enlarged in the
hypocotyl. Mutations in FASS (FS) gene
produce disordered patterns of cell
division. The fass mutants show inability
to form microtubule preprophase bands,
which predict the site of future cell wall
deposition in plant cells. However, the
full range of appropriate cell types is
present and complete in the fass mutants,
albeit disordered and seedlings are
produced.
8. Growth of leaves and roots: Effect of RPn10-1

WT rpn10-1 Embryos of rpn10-1

mutants stop their
growth at the globular
stage with defects in
the formation of the
embryonic root, the
protoderm, and
procambium.

Fig.: The rpn10-1 mutant, leads to growth

inhibition and the accumulation of
polyubiquitinated target proteins.