The document discusses the genetic and molecular mechanisms underlying the flowering process in plants, emphasizing the roles of floral meristem identity and floral organ identity genes in determining flower structure. It describes the classical ABC model of floral organ identity, where the interaction of classes of genes specifies the development of sepals, petals, stamens, and carpels. The document also highlights the involvement of microRNAs and MADS-box genes in regulating floral development and transitions between vegetative and reproductive phases.

1. DR. VIBHA KHANNA
Asso. Prof. (Botany)
SPC GOVERNMENT COLLEGE
AJMER (Rajasthan)

2. PLANT PHYSIOLOGY
THE FLOWERING PROCESS
PRESENTATION 5:
Floral Development:
Genetic And Molecular Analysis

3. Flowering Process
Vegetative
Meristem
• Undergoes floral induction on genetic level
Inflorescence
Meristem
• Produce small leaves before it next produces floral meristem.
Floral
Meristem
• Under goes a series of developmental changes (Floral development),
on genetic level, that eventually give rise to the four basic structures of
the flower --- sepals, petals, stamens and carpels.
• Each of these structures is derived sequentially from a whorl that
develops from the floral meristem.
Whorl 1 to 4
• 1: First to appear, and it develops into the sepal
• 2. Develops into petals
• 3: Define the stamen (male reproductive organs) and
• 4. Forms carpel (female reproductive organs).

4. Flowering Process
• From a genetic perspecitive, two phenotypic
changes that control vegetative and floral growth
are programmed in the plant.
• The first genetic change involves the switch from
the vegetative to the floral state (floral induction).
• The second genetic event follows the
commitment of the plant to form flowers (floral
development).
• A series of genes are sequentially turned on and
off to obtain its final unique identity.

5. Genes That Regulate Floral Development
• Two major categories :
– Floral meristem identity genes : Activity of these
genes, in the immature primordia formed at the
flanks of the shoot apical meristem (inflorescence
meristem), results in its transition to form the
floral meristem.
– Floral organ identity genes: Floral organ identity
genes directly control floral organ identity.
• {Five key genes initially were identified in Arabidopsis
that specify floral organ identity. APETALA1 [AP1],
APETALA2 [AP2], APETALA3 [AP3], PISTILLATA [PI] and
AGAMOUS [AG].}

6. Floral meristem identity genes
• The CENTRORADIALUS (CEN) gene in snapdragons and the
Arabidopsis homologue of CEN (TERMINAL FLOWER 1 or TFL1) is
expressed during the vegetative phase of development, and has the
additional function of delaying the inflorescence development, i.e., the
reproductive transition. They suppress the terminal flower formation.
• The gene that suppresses terminal flower formation, basically,
suppresses expression of genes, which specifies floral meristem
identity.
• The next step in the reproductive process is the specification of floral
meristems—those meristems that will actually produce flowers .
• In Snapdragons FLORICAULA (FLO), specifies floral meristem identity.
• In Arabidopsis, LEAFY (LFY), APETALA 1 (AP1), and CAULIFLOWER (CAL)
are floral meristem identity genes .
• LFY is the homologue of FLO in snapdragons, and its upregulation
during development is key to the transition to reproductive
development .
• Expression of these genes is necessary for the transition from an
inflorescence meristem to a floral meristem.

7. Floral meristem identity genes
• LEAFY is the first gene expressed.
• The LEAFY mRNA first appears in the flanks of the inflorescence meristem,
the exact location from which the floral meristem will develop. Its
expression rises through stages 1 and 2 (floral meristem identification
stage), and disappears in stages 3 and 4 (first expression of floral organ
identity genes).
• LEAFY has two functions ---
– It is necessary for the conversion of the inflorescence meristem to a floral
meristem and
– It define the petals and stamens.
• APETALA1, another gene involved in the commitment to flowering, also
plays a role in sepal and petal development.
• As LEAFY, APETALA1 is expressed in cells at the margin of the inflorescence
meristem during stage 1 and 2.
• Because whorls 1 and 2 give rise to sepals and petals, respectively, it is not
surprising that APETALA1continues to be expressed in these whorls during
stages 3 and 4, while its expression disapppers from whorls 3 and 4 during
these stages.

8. Floral organ identity genes
• Floral organ identity genes
– Floral organ identity genes directly control floral organ
identity.
– Five key genes initially were identified in Arabidopsis that
specify floral organ identity. APETALA1 [AP1], APETALA2
[AP2], APETALA3 [AP3], PISTILLATA [PI] and AGAMOUS
[AG].
• These genes are homeotic genes.
• All homeotic genes that have been identified so far, in
plants, encode transcription factors.
• Most plant homeotic genes belong to a class of related
sequences known as MADS box genes.

9. The ‘Classical’ ABC Model
• The ABC model partially explains the determination of
floral organ identity
• The ABC model was discovered by Elliot Meyerowitz,
Enrico Coen in 1991.
• According to this model, the five floral organ identity
genes, described above, fall into three classes ABC
defining three different kinds of activities.

10. The ‘Classical’ ABC Model: Class A
• Class A activity , encoded by AP1 and AP2 , controls organ identity
in the first and second whorls.
• Loss of class A activity results in the formation of carpels instead of
sepals in the first whorl and of stamens instead of petals in the
second whorl.
• Loss of class A activity results in the spread of class C activity
throughout the meristem.
• ovulata is an A function gene of snapdragon similar to APETALA2.

11. The ‘Classical’ ABC Model: Class B
• Class B activity , encoded by AP3 and PI, controls organ
determination in the second and third whorls.
• Loss of class B activity results in the formation of sepals instead of
petals in the second whorl, and of carpels instead of stamens in the
third whorl.
• Loss of class B activity results in the expression of only class A and
class C activities.
• deficiens and globosa are snapdragon genes that have homologous
functions to the Arabidopsis B function genes.

12. The ‘Classical’ ABC Model: Class C
• Class C activity , encoded by AG, controls events in the third and fourth
whorls.
• Loss of class C activity results in the formation of petals instead of stamens
in the third whorl and of sepal instead of carpel in the fourth whorl.
• Loss of class C activity results in the expression of only class A activity
throughout the floral meristem.
• In the absence of class C activity, the fourth whorl (normally a carpel) is
replaced by a new flower.
• A snapdragon gene similar to AGAMOUS is pleniflora.

13. The Quartet Model: ABCE Model
• The Quartet model first discover by Theissen 2001.
• According to the Quartet model, floral organ identity is
regulated by tetrameric complexes of the ABCE
proteins.
• The ABCE Model was formulated based on genetic
experiments in Arabidopsis and Antirrhinum.
• Arabidopsis class E genes are required for the activities
of the A,B and C genes.
• In Sepallata1-4 mutants, all of the floral organs
resemble vegetative leaves, suggesting that SEP genes
are required for floral meristem identity.
• ABCE Model for floral organ determination in which
SEPs act as Class E genes required for floral organ
identity.

14. The ABCE model for flower patterning
• According to the `classic' ABC model of flower
patterning, the combinatorial activity of three
genetic functions, A, B and C, are sufficient to
specify the four floral organs.
• According to the ABC model,
– `A' on its own specifies sepal fate in the outermost
whorl,
– `A' and `B' together determine petals in the second
whorl.
– The stamens in the third whorl are specified by the
combinatorial activities of `B' and `C', whereas
– `C' alone is responsible for the formation of carpels in
the centre of the flower.
• In Arabidopsis,
– A-class activity is conferred by APETALA1 (AP1)
and AP2, the latter of which is a target of miR172.
– AP3 and PISTILLATA (PI) have B-class function, and
– C-class function in the centre of the emerging flower
is conferred by AGAMOUS (AG).
• More recently, the `ABC' model has been
expanded to include a fourth, `E', function.
– The E-class genes SEPALLATA1 (SEP1), SEP2,
SEP3 and SEP4 play a crucial role as co-regulators in
all four whorls, and underpin the leafy nature of all
floral organs.

15. Class D Genes Are Required For Ovule Formation.
• Class D activities were first discovered in petunia.
• Silencing, MADS box genes known to be involved in floral
development in petunia, FLORAL- BINDING
PROTEIN7/11[FBP7/11] , resulted in the growth of styles
and stigmas in the locations normally occupied by ovules.

16. Regulation of Phase Transition by microRNA
• A model of the sequential
action of miR156, miR172
and their respective targets
in regulating phase
transitions in A. thaliana.
• Abbreviations: AG,
AGAMOUS; AP1, APETALA1;
AP2, APETALA2; FUL,
FRUITFULL; LFY, LEAFY;
miR156, mature miRNA156;
miR172, mature miRNA172;
SEP3, SEPALLATA3; SOC1,
SUPRESSOR OF
OVEREXPRESSION OF
CONSTANS 1; SPL,
SQUAMOSA PROMOTER
BINDING PROTEIN-LIKE.

17. miR172/AP2 regulate transition to
flowering
• The different pathways that regulate flowering can be categorised as:
– pathways that actively promote flowering, such as the photoperiod,
light quality, gibberellic acid and ambient temperature pathways, or
– pathways that enable flowering by removing floral repressors from the
system.
• SPL genes and miR172 are the`enabling factors', as their activity
ultimately leads to the shutting down of the AP2-like floral repressors
that regulate `meristem competence’.
• miR156, which prevents precocious flowering until plants have
reached a permissive age, is also an `enabling factor'.
• However, the miR156-targeted SPL genes
– regulate the expression of miR172 and thus the AP2-like floral
repressors,
– SPL proteins have also directly bind to and promote the expression of
floral integrator genes, such as SOC1 and floral meristem identity genes
such as LFY, FUL and AP1.
• AP2, besides its role as a floral repressor also functions in floral
patterning.

18. Regulation of flower development by
miR172/AP2
• Besides its role in regulating phase transitions, miR172 and
its targets appear to also play a role in floral patterning.
• AP2 expression is actually restricted to the outer two
whorls, while the expression of miR172 is restricted to the
centre of young floral primordia.
• AP2 and miR172 are engaged in a negative-feedback loop
that presumably helps to establish a sharp boundary
between the inner and the outer whorls of the emerging
flower.
• In this scenario, AP2 not only directly binds to AG and
prevents its transcription in the outer two whorls, but it is
also at the same time cleared from the centre of the floral
primordium by the activity of miR172.

19. ↑ in miR172 Induces Transition to Flowering
• Increased levels of miR172 result in the
downregulation of AP2-like
transcription factors that normally
repress flowering.
• Release from this repression, in
combination with the flower-promoting
actions of SPL3, SPL4 and SPL5, makes
the plant competent to flower and the
transition to flowering can occur.
• In addition to its role as a floral
repressor, AP2 contributes to the
patterning of the emerging flower.
• Both AP2 and miR172 participate in
establishing a sharp boundary between
the vegetative outer organs (sepals,
petals) and the inner whorls of
reproductive organs (stamen, carpels).

20. MADS-box
• The MADS box is a conserved sequence motif.
• The genes which contain this motif are called the MADS
box gene family.
• In plants, MADS box genes are involved in controlling all
major aspects of development, including male and female
gametophyte development, embryo and seed
development, as well as root, flower and fruit
development.
• The MADS box gene family got its name later as an
Acronym referring to the four founding members.
– MCM1 from the budding yeast , Saccharomyces cerevisiae.
– AGAMOUS from the thale cress Arabidopsis thaliana.
– DEFICIENS from the snapdragon Antirrhinum majus.
– SRF from the human Homo sapiens.

21. MADS-box genes
• The MADS box encodes the DNA binding MADS domain.
• MADS domain proteins are generally transcription factors.
• Each of these genes contains a 56-amino acid sequence that is
necessary for the protein to bind to DNA.
• This sequence is located near the amino terminal end of the
protein, and is called the MADS-box.
• Plant MADS-box genes contain a second conserved sequence called
the K-domain because its sequences is similar to the coiled coil
domain of keratin. This region is primarily involved in the protein-
protein interactions.

22. Activity of MADS-box Genes
• The MADS box gene transcription factors form tetramers that
bind to CC[A/T]6GG sequences, the so called CArG-box, in the
regulatory regions of their target genes.
• When the tetramers bind two different CArG-boxes on the same
target gene, the boxes are brought into close proximity, causing
DNA bending.

23. Activity of MADS-box Genes
• The Model is based on the observation that MADS box genes
dimerize, and two dimers can come together, forming a tetramer.
• These tetramers are hypothesized to bind CArG-boxes on target
genes and modify their expression.

24. Activity of MADS-box Genes
• Although all MADS box proteins can form higher order
complexes, not all of these are able to bind DNA.
• For example, Class B factors [AP3 and PI] bind DNA only
as heterodimers, whereas both homodimers and heterodimers
of Classes A, C and E can bind DNA.
• According to the Model, tetramers composed of different
homodimers and heterodimers of MADS domain proteins
can exert combinatorial control over floral organ identity.

25. An Overview of Flower Development
• Floral meristem identity genes initiate a cascade of gene
expression that turns on region-specifying (cadastral) genes,
which further specify pattern by initiating transcription
of floral organ identity genes .
• SUPERMAN (SUP) is an example of a cadastral gene
in Arabidopsis that plays a role in specifying boundaries for
organ identity gene expression.
• The patterning of the flower is explained by the `classical'
ABC model, according to which the combinatorial interaction
of three classes of homeotic functions (A, B and C), provides
the positional information that determines the fate of the
emerging floral organs
• The ABC genes code for transcription factors that initiate a
cascade of events leading to the actual production of floral
parts.

26. An Overview of Flower Development
• In brief,
– in the outermost whorl of the Arabidopsis flower, sepals are specified by
the activity of AP1 and AP2 (A class).
– In the second whorl, the activity of A-class proteins overlaps with those
of the B-class proteins PISTILLATA and APETALA3 to establish petals.
– Similarly, overlapping activity of the B-class proteins with that of the C-
class protein AGAMOUS (AG) induces stamen fate in the third whorl.
– Finally, in the innermost whorl, AG controls the formation of carpels.
– In addition to the ABC genes, class D genes, specifically regulate ovule
development.
– More recently, four highly redundant genes, SEPALLATA1 (SEP1), SEP2,
SEP3 and SEP4, that are required for the pattering of all four whorls of
the emerging flower have been identified and have been added to the
ABC model as E-class function genes.
• An important feature of the ABC model is that A-class and C-class
function are mutually exclusive.
• Genes capable of producing B are normally inhibited in whorl 4 by
cadastral genes like SUPERMAN.

27. The Molecular Expression of Floral Organ Genes
• The genetic model predicts that the A organ identity genes will be expressed
in the tissues from which sepals and petals are derived.
Although APETALA2 is classified as an A function gene, mutants of this genes
also affect stamen and carpel development. This gene is shown to be
expressed in all four whorls. The expression of the other A
gene, APETALA1 appears to be restricted to whorls 1 and 2.
• The genetic model has also suggested that C organ identity genes are
negatively regulated by the expression of A genes. This would lead to a
hypothesis stating that the expression of C genes such as AGAMOUS would
not appear in cells giving rise A function organs. Early expression of
the AGAMOUS genes is restricted to whorls 3 and 4; later in development,
the expression of AGAMOUS is restricted to specific cell types.
– In stamens, the gene is not found in any cells that give rise to the pollen, nor is it
expressed in the pollen grain itself.
– And in the carpel cells, AGAMOUS is only epxpressed in the outer cells of the
ovule.
• B gene function genes have been suggested to control petal and stamen
function (whorl 2 and 3, respectively). Both APETALA3 and PISTILLATA are
found to be expressed in the appropriate whorls. PISTILLATA though is also
found to be expressed in whorl 4 that gives rise to carpels.

28. Glossary
• Cadastral genes: Genes that restrict the action of other genes to
specific regions of the organism.
• Homeobox genes are a large family of genes involved in directing
the body structure formation.
• Homeotic genes: Group of genes that control the pattern of body
formation. They encode proteins which are the transcription
factors that direct cells to form various parts of the body. (A
homeotic protein can activate one gene but repress another,
producing effects that are complementary and necessary for the
ordered development of an organism.)
• Hox genes: the developmental control genes responsible for
directing the body plan along the AP axis (anterior–posterior axis)
and the proteins it codes for are transcription factors.
• miRNAs (microRNAs) are short non-coding RNAs that regulate gene
expression post-transcriptionally. They generally bind to the 3'-UTR
(untranslated region) of their target mRNAs and repress protein
production by destabilizing the mRNA and translational silencing.