Lecture 8 in Developmental Biology of Plants

DEVELOPMENTAL
BIOLOGY
Lecture 8

INTRODUCTION
1. Higher plants continue to generate new organs and tissues throughout their
postembryonic life.
2. This process is achieved through the activity of specific domains formed during
embryogenesis: the shoot and root apical meristems.
3. Shoot and root meristems generate all the progeny cells required for tissue and organ
differentiation, and add permanently new organs to the growing plant body.
4. Progress has been made in understanding shoot and root morphogenesis through the
isolation of genes from developmental mutants, and the establishment of their temporal
and spatial patterns of expression using molecular biology techniques.
5. This understanding of developmental genes and how they are controlled by hormones
and other components of culture media can improve the monitoring of in vitro
manipulation and aid in investigating processes of adventitious regeneration in tissue
culture.

VEGETATIVE SHOOT MORPHOGENESIS
Presentation title 3
Structural organization of the vegetative shoot apical meristem
1. The future shoot apical meristem (SAM) becomes commonly recognizable at early stages
during the establishment of the basic body of the embryo.
2. The future SAM remains usually quiescent until germination, but in some species, it
becomes active and organized before germination.
3. During the vegetative phase, the SAM varies in shape, size, and meristematic activity
among different species.
4. The shape and size of the shoot apex may change during development and during a
plastochron, which is the time between the formation of successive leaves.
5. Regardless of the species, some common features characterize the organization of
functional SAMs of Angiosperms during the vegetative phase.

Molecular basis of SAM identity and organization
1. SAM maintenance and organization genes encode transcription factors or receptor/signal molecules.
2. Loss-of-function mutations in the STM gene cause no SAM and produce seedlings without leaves,
implying that cotyledons and leaves are not fully homologous organs.
3. The STM gene belongs to the KNOX family and marks the early initiation of SAM during
embryogenesis, occurring in all types of SAM during development.
4. The KN1 gene is a good marker of the undetermined state of the SAM cells, while the RS1 gene is
normally expressed in the SAM.
5. Mutations in WUS and ZLL genes affect the initiation of true leaves but not of cotyledons.
6. WUS-expressing cells act as an organizing center, conferring stem-cell identity to the overlying
initial cells, while ZLL maintains the stem cells of the shoot meristem in an undifferentiated state and
mediates signaling from the vascular initial cells to the embryo apex.

Molecular basis of SAM identity and organization
1. Loss-of-function clavata1 mutation leads to an increase in shoot and floral SAM size, causing stem fasciation and generation of
flowers with extra organs.
2. The CLV1 gene encodes a receptor serine/threonine kinase and is expressed in the central region of SAM corresponding to the
rib zone. CLV3 is expressed throughout SAM development, predominantly in the L1 and L2 tunica layers of the region
corresponding to the central zone.
3. CLV3 signal informs central zone cells of their proper rate of cell division or rate at which they should exit the central zone
for differentiation. CLV pathway functions as a negative feedback loop from the central stem cells to the organizing center.
4. The signal is the small protein CLV3 which is secreted by the stem cells, moves intercellularly and activates a receptor that
contains the CLV1 and CLV2 proteins to repress WUS.
5. The mgoun1 and 2 mutants also display an enlarged vegetative SAM with occasional fasciation and accumulation of cells in
the peripheral zone. The corresponding MGOUN genes would affect the partitioning of peripheral cells into leaf or floral organ
primordia.
6. Fasciations are the result of the fusion of two or several growth points, resulting in linear, circular, or radiate fasciations.
Inhibition of auxin transport can also result in fused leaf organs, similar to what occurs in the cuc mutants, which are altered in
organ separation.

Phyllotaxy and patterns of leaf development
1. Phyllotaxy, or the positioning of leaves on a stem, is determined by a combination
of genetic, environmental, and developmental factors.
2. Leaf primordia arise in regular geometric arrays, which could be due to a self-
organized growth process.
3. The signal for leaf initiation may spread acropetally from underlying tissues
towards the point where a leaf is going to rise.
4. Wall-loosening protein expansin and auxin are potential factors involved in leaf
initiation.
5. Many mutations that affect shoot apical meristem (SAM) size also affect phyllotaxy.
6. Auxin transporters and cytokinins may play a role in controlling phyllotaxy.

1. Leaf initiation involves an increased rate of proliferation of cells that are recruited from the SAM
peripheral zone.
2. KNOX family genes are downregulated at sites where leaf initiation is in preparation and can be
considered as negative markers of leaf determination.
3. CUC genes need to be appropriately expressed in the presumptive boundary cells to prevent the
formation of fused organs, but the mechanism by which these boundaries are set in relation to the
predicted auxin maxima that define sites of leaf initiation is unknown.
4. Early in leaf initiation, ANT 1 and FIL genes start to be expressed in the leaf founder cells and
appear as positive markers.
5. Close interactions between the SAM and its flanking regions seem to control the acquisition of leaf
determination by the lateral cells. This process is probably highly disturbed in some cases observed
in vitro where leaf initiation seems to occur independently of the presence of organized SAMs.

1. Leaves are determined organs that grow to a specific size and shape
2. Leaves display bilateral symmetry and dorsiventrality
3. Factors intrinsic and extrinsic to the leaf primordium contribute to the
specification of cells as abaxial or adaxial
4. Genes like YABBY, KAN1, and PHAN are key determinants of abaxial and adaxial
identity
5. Different rates of expansion and cell division influence final leaf shape, which can
be simple or complex
6. Interactions between the SAM and developing leaves are required for proper leaf
development

Development of axillary and adventitious meristems
1. During post-embryonic development, the shoot apical meristem (SAM) produces repeated units
called phytomers.
2. Each phytomer consists of a leaf, one or more axillary buds, and a stem portion comprising a node
and an internode.
3. Axillary buds are secondary shoot meristems located at the junction between the stem and the leaf
base and responsible for lateral branching.
4. Axillary meristems can originate either at the same time as the leaf primordia or later from already
differentiated tissues.
5. The control of subsequent axillary bud growth is by the main shoot apex, with cytokinin as a key
factor in promoting bud growth whereas auxin may have an inhibitory effect.
6. Increased cytokinin production mediated by gene transfer may reduce apical dominance but failed
to induce axillary bud formation in some mutants.

Development of axillary and adventitious meristems
1. Adventitious buds can arise spontaneously in different parts of plants.
2. Dedifferentiation can be induced in cells, leading to a return to a proliferative, meristematic
condition.
3. The formation of adventitious buds on leaves can be experimentally induced by the overexpression
of homeobox genes, such as KN1 and KNAT1, or cytokinin biosynthetic genes.
4. There may be reciprocal links between cytokinins and homeobox genes.
5. Different genes, such as CUC1 and CUC2, promote shoot regeneration from calluses in the
presence of cytokinins.
6. Various genes that promote adventitious shoot formation have been identified in Arabidopsis, such
as CKI1 and ARR2.

REPRODUCTIVE MORPHOGENESIS
• 1. In plants, there is no germ line, and germ cell differentiation is delayed until late in
development.
• 2. The transition to flowering in plants occurs in shoot apical meristems, which are
reprogrammed to make inflorescence or floral organs upon receiving appropriate signals.
• 3. The change in determination of the SAM cells during flowering can be split into several
successive steps, including induction, evocation, and formation of inflorescence and
flowers.
• 4. Flower organs gain characteristic forms and functions for pollination and fertilization
to occur.
• 5. SAM reprogramming towards flowering in plants is likely accompanied by the
acquisition of new properties by the differentiated organs, as shown by tissue culture
experiments.

Structural rearrangements of the SAM during the reproductive phase
1. The size and growth rate of the shoot apical meristem (SAM) increase during the transition from making
leaves to making flowers.
2. This transition is accompanied by the loss of the cytophysiological zonation of the SAM and the activation of
cells of the central zone.
3. The modified SAM can generate either a unique flower or an inflorescence meristem that will give rise to
several flower meristems.
4. The L1 layer contributes to the epidermis, stigma, part of the transmitting tract, and integument of the ovules,
while the L2 and L3 layers contribute to the parenchyma and other internal tissues to different degrees.
5. Movement of transcription factors between cells is a generalized phenomenon in planta, and much work
remains to be done to detect such movements in systems grown in vitro and to demonstrate their involvement
in regeneration processes under the influence of growth substances.
6. Under inductive conditions, the switch to the flowering stage is marked by a prefloral phase followed by a
reproductive phase during which flower primordia organize and diversification occurs depending on the
species. Under non-inductive conditions, the SAM continues to produce leaves but acquires a new type of
structure, defining the "intermediate phase."

Control of flowering time
1. Adult SAMs (shoot apical meristems) require environmental and internal signals for competent
flowering depending on the species.
2. Arabidopsis, a model plant, has been studied to identify key genes controlling flowering time
through genetic analysis, including the photoperiod, vernalization, autonomous, and gibberellin
pathways.
3. The photoperiod pathway involves CONSTANS (CO) and FT genes that respond to long-day
conditions and delay flowering when mutated.
4. The vernalization pathway is controlled by FLOWERING LOCUS C (FLC) and FRIGIDA (FRI)
genes and is activated by exposure to cold winter conditions.
5. Epigenetic effects, such as chromatin modeling and miRNA regulation, also play a role in
controlling genes involved in flowering time.

Determination of floral and inflorescence meristem identity
1. LFY/FLO genes play a role in promoting floral transition and in later stages.
2. Inflorescence identity is controlled by two sets of genes, with TFL promoting
inflorescence identity and AP1 specifying floral meristem identity.
3. AP1 functions downstream of LFY, which is required for the proper expression of
floral organ identity genes during the late stage of the reproductive phase.

Determination of floral organ identity
1. Floral organs form in concentric rings, called whorls, and the order of development is sepals, petals,
stamens, and carpels.
2. Homeotic genes known as MADS box genes are responsible for specifying organ identities in the
flowers, and there are three main classes of homeotic genes involved in flower organ identity: A, B, and
C.
3. The ABC model predicts that the overlapping action of these genes would specify organ identity, and
LFY plays a pivotal role in regulating floral organ identity as it regulates the expression of A, B, and C
class genes.
4. A new class of floral identity MADS box genes (SEPALATA) was recently described and in the revised
model, SEP genes are referred to as E class while a D class specifying ovule identity completes the model.
5. Gymnosperms lack the A-function gene, which is likely the reason why these plants do not have petals
and sepals, and further insights are still necessary to study genes that have no obvious counterparts
among species.

ZYGOTIC EMBRYOGENESIS
Pattern formation from the zygote to the mature embryo
1. Zygotic embryos develop within the embryo sac from fertilized ovules in a pattern.
2. Monocotyledonous and dicotyledonous seedlings differ in appearance due to the number
of cotyledons.
3. Double fertilization generates diploid zygote and triploid endosperm.
4. Embryogenesis can be divided into three phases: morphogenesis, embryo maturation, and
desiccation.
5. The initial zygote of Arabidopsis reaches 20,000 cells in nine days, growing into a mature
embryo of 500 μm in diameter.
6. During the third phase, embryos accumulate storage macromolecules, enter metabolic
quiescence, and can withstand desiccation.

Genes controlling embryogenesis
1. Embryogenesis in higher plants requires coordination of genetic programs and communication between
different parts of the developing seed.
2. A precise order of events ensures the correct positioning of embryonic organs, such as the shoot and root
meristems, the cotyledons, and the hypocotyls.
3. A large number of embryonic mutants have been identified in Arabidopsis and maize, including mutants
altered in their pattern formation, meristem establishment, and maturation programs.
4. The expression of homeodomain-containing transcription factors, such as WUSCHEL (WUS), partitions plant
embryos early into regions marked by these factors, similar to animal embryos.
5. Various genes have been identified as involved in apico-basal and radial patterning, including MONOPTEROS
(MP), FACKEL (FK), GURKE (GK), GNOM (GN), HOBBIT (HBT), BODENLOS (BDL), SCARECROW
(SCR), and KNOLLE (KN).
6. The protoderm expresses the Arabidopsis thaliana meristem layer 1 (AtML1) and LIPID TRANSFER
PROTEIN (LTP) genes, which are tissue-specifically expressed and play important roles in embryonic
development.

Phytohormones and embryo formation
1. Phytohormones such as auxin and cytokinins play a role in seed development.
2. Abscisic acid is involved in seed maturation.
3. Auxin is involved in apical-basal patterning of the embryo and cotyledon
separation.
4. Mutations in genes such as MONOPTEROS and GNOM affect embryo axis
formation and vascular development, which are auxin response factors.
5. CUC1 and CUC2 genes are involved in organ separation, but their control by
auxin is not yet known.
6. Cytokinins may act together with auxin in embryogenesis, affecting cell division
and differentiation in the vascular cylinder.

Asexual embryogenesis in planta
1. Some species have the ability to form embryos that don't originate from fertilized
egg cells.
2. Somatic embryogenesis, the process of forming embryos from non-germ cells,
occurs naturally in some plants, such as Malaxis.
3. Genes involved in zygotic embryogenesis have been little studied for their role in
somatic embryogenesis.

ROOT MORPHOGENESIS
1. A root pole is differentiated during embryogenesis with a future root apical
meristem.
2. Shoot and root organs from many species may regenerate meristems of either type
in planta and in tissue culture, but there is no reliable report of direct interconversion
of these meristems during normal development.
3. The emergence of roots has constituted one of the important and determining
parameters for the growth of plants on land.
4. Gene cassettes are providing some similar developmental functions in shoot and
root.
5. A range of processes appear to be only root-specific.

ROOT MORPHOGENESIS
Structural organization of the root apical meristem
1. Angiosperm roots have a stereotypical anatomy with cylindrical layers of tissue types
enclosing a central vascular cylinder.
2. The root tip consists of a cap and the root proper, both composed of columns of cells, with
the cap protecting the root tip as it pushes through the soil.
3. The root apical meristem (RAM) mediates longitudinal growth of the root and elaborates a
root cap made up of cells that continuously slough off as the root grows.
4. In species with closed RAM types, the cell files that make up the cylindrical layers converge
into the root apex, while in species with open RAM types, the cell files do not converge but
appear to constrict and extend into the root cap.
5. The RAM displays zonation, with the quiescent center controlling stem cell differentiation,
and the proliferating zones giving rise to the cortex, central cylinder, and cap. Cells beyond
the RAM elongate and differentiate.

ROOT MORPHOGENESIS
Genes involved in primary root identity and patterning
1. Root development starts with root specification.
2. HBT, BDL, and AXR6 genes have a critical role in root formation during embryogenesis.
3. Mutations in these genes result in seedlings that lack a primary root but can form roots post-
embryonically.
4. RML1 gene mutation leads to glutathione depletion and stops cell division in roots but not shoots.
5. RCH1 gene encodes a receptor kinase, which is RAM specific.
6. CLE19 and CLE40 genes are expressed in the RAM.
7. WOX5 gene is expressed in the quiescent center of Arabidopsis root.
8. MP protein is similar to AUXIN RESPONSE FACTOR 1 and mediates responses to auxin.
9. PIN auxin efflux protein regulates root growth and patterning.
10.The diameter and height of the RAM are regulated by auxin and hormones.

ROOT MORPHOGENESIS
Lateral and adventitious root formation
1. Lateral and adventitious rhizogenesis occur postembryonically from roots or
stems.
2. Understanding these processes could lead to better manipulation of root initiation
and architecture.
3. Auxin has been considered a key root-inducing factor, but its connections with
genes involved in root formation are not yet clear.
4. The auxin requirement for inducing lateral roots from non-excised roots is higher
than from excised roots.
5. Nitrate and inorganic phosphate nutrition play a major role in controlling the
development of the root system.

ROOT MORPHOGENESIS
Lateral and adventitious root formation
6. Embryonic root and lateral root formation share common factors, but lateral root
formation is distinct, as shown by mutant analysis.
7. Auxin is required for the early steps of cell dedifferentiation and root initiation,
but later inhibits root growth.
8. Lateral root primordia commonly originate endogenously some distance behind
the main root apices from dedifferentiation of the parent root pericycle.
9. Vascular strands are important in determining the sites of primordium initiation
through the allocation of trophic, hormonal, and possibly other signaling factors.
10. The pericycle has been proposed to be a mono-layered extended meristem, and
histological origins of adventitious root meristems are more diverse.

SECONDARY MERISTEMS AND RADIAL GROWTH
In most dicotyledons and in gymmosperms, expansion growth of stems and roots
occurs through the establishment and the functioning of 2 to 4 internal meristematic
layers dividing periclinally. These are called secondary meristems or cambium.
Cambia were formerly divided into vascular cambium giving rise to secondary xylem
and phloem, and cork cambium that produces cork. The cork cambium is now
usually named phellogen, and the term cambium is preferably used for vascular
cambium.

Origin and function of the vascular cambium
1. The vascular cambium allows the development of tree-like forms and ensures the
perennial life of trees through the regular renewal of secondary xylem and phloem.
2. The origin of cambial layers is different in stems and in roots.
3. The cambium forms a complete ring of dividing cell layers and gives rise to
cylinders of secondary tissues.
4. In roots, the vascular cambium is initiated by divisions of the procambial cells that
remained undifferentiated between the primary xylem and the primary phloem.
5. The cambium produces secondary phloem and xylem by periclinal divisions and
increases in circumference by anticlinal divisions.

6. The cambium presents alternate periods of meristematic activity and arrested
growth, giving rise to successive rings on the wood.
7. Auxin is considered as the main phytohormone involved in the regulation of
cambial activity.
8. Cytokinin is also essential for promoting the division of procambial cells.
9. A regulation by miRNA of the PHAVOLUTA gene has been shown to be involved
in vascular cambium cell determination.
10. Active cambial cells have inherent cell cycling capacity, explaining their ability to
form rapidly a callus both following wounding and in tissue culture without
dedifferentiation processes.

THE CELL CYCLE
Cell proliferation, polyploidy, and development
1. Plant development involves various morphogenetic processes that require cell
division activity, both in planta and in tissue culture.
2. Cells are induced to divide at the onset of any type of adventitious organ
formation, and cell cycle reactivation is necessary for callusing and regenerating
processes in tissue culture.
3. The eukaryotic cell division cycle is divided into four phases: G1, S, G2, and M.
4. Sucrose added to excised pea root tips rapidly stimulates the entry of G1 cells into
S and G2 into M.
5. The duration of S and M phases is rather constant for a given species under
standard conditions, while the G1 phase's duration varies.

THE CELL CYCLE
Cell proliferation, polyploidy, and development
6. Quantitative data show that the cell cycle duration is always shorter in the peripheral zone
(PZ) than the central zone (CZ) of the vegetative shoot apical meristem of various species.
7. Cell size is a component of developmental programs, and a critical cell size is required to
commit the cells to enter the S phase for a new round of division.
8. During temporary arrests of meristematic activity, cells stop the cell cycle in a prolonged
G1 (G0) phase.
9. In non-cycling differentiated parts of plants, endoreduplication can occur, leading to
supernumerary rounds of DNA synthesis.
10. Endoreduplication occurs in seed or plant tissues with high metabolic activity and might
provide a mechanism whereby cells increase the availability of DNA template and thus
increase the level of gene expression.

THE CELL CYCLE
Molecular control of the cell cycle
1. The plant cell cycle is regulated by cyclin-dependent kinases (CDKs) that bind to positive
regulators called cyclins.
2. The controlling factors of the plant cell cycle are highly conserved and similar in
mammals and plants.
3. CDKs are serine/threonine kinases that control progression of the cell cycle in all
eukaryotes.
4. CDKs are regulated by association with cyclin regulatory submits and by
phosphorylation/dephosphorylation events.
5. CDKs can be separated into two main classes: the PSTAIRE class and the non-PSTAIRE
class.

THE CELL CYCLE
Molecular control of the cell cycle
6. In Arabidopsis, the CDC2a gene (CDK A) is expressed during both in G1-to-S and in G2-
to-M transitions, while CDC2b (CDK B) gene is more associated with the progression
through G2.
7. More recently, three other CDK classes (C, D and E) have been identified to which has
been added the distantly related CDK activating kinase CAK1, renamed CDK F1.
8. The number of cyclins defined in Arabidopsis has expanded to 49 distributed in 8 classes
and 23 subgroups.
9. The expression of the main cyclin genes has been studied, giving information on how their
transcripts accumulate during the various phases of the cell cycle.
10. The genes encoding CYCD cyclins are induced at a specific time during cell cycle re-entry
but generally remain expressed at a constant level in actively dividing cells.

THE CELL CYCLE
Hormonal control of the cell cycle
1. Auxin and cytokinin are essential for cell division.
2. Their exact roles in this process are not yet fully understood.
3. Auxin is involved in the initiation of DNA synthesis and cell enlargement.
4. Cytokinins are involved in the process of mitosis.
5. A combination of auxin and cytokinin is required to reinitiate cell proliferation in tissue
culture.
6. Other growth regulators such as gibberellic acid and abscisic acid may also influence the
mitotic cycle.

Lecture 8 in Developmental Biology of Plants

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