Stamens
Carpels
25
Flowering and Fruit Development
Flowering and fruit development have long held the
interest of developmental biologists and physiologists
because they represent a dramatic change in the pattern
of shoot development and have significant economic
implications. The switch of the shoot apical meri-
stem from vegetative to floral organs and the subse-
quent development of fruit is a critical step in the
developmental history of a plant and must be regulated
precisely in order to ensure reproductive success. In
the previous chapter it was shown that synchronization
of flowering time with an environmental cue such as
photoperiod is not a simple event. It is possible only
through the interactions of many genetic and biochem-
ical pathways: pathways involved in signal perception
and transduction; pathways involved in the regulation
of the circadian clock; and pathways involved in the
development of the floral primordia.
Over the years, flowering in a number of species,
including maize (Zea mays), petunia (Petunia sps.), snap-
dragon (Antirrhinum sps.), tobacco (Nicotiana tobacum),
and annual ryegrass (Lolium temulentum), has been the
subject of molecular and genetic studies. More recently,
focus has shifted to the model plant Arabidopsis, where
a number of genes that influence flowering have been
identified and a model proposed to account for the
genetic specification of floral organ initiation. In many
cases, especially winter cereals and biennials, a period
of low temperature can significantly alter the flowering
433
Petals
Sepals
response. The low-temperature treatment, called ver-
nalization, influences flowering time under long days.
Finally, flowering is followed by the development of a
specialized organ, the fruit, which ensure the proper
environment for seed maturation and dispersal of the
mature seed.
In this chapter we will examine
• the molecular genetic control of flower initiation
and development in the shoot apical meristem,
• the phenomenon of vernalization and its relation-
ship to other flowering time pathways, and
• the basic principles of fruit set and fruit develop-
ment.
25.1 FLOWER INITIATION AND
DEVELOPMENT INVOLVES
THE SEQUENTIAL ACTION
OF THREE SETS OF GENES
As noted in the previous chapter, flowering in many
plants is influenced by environmental factors such as
photoperiod and temperature and involves the synthesis
of a mobile floral stimulus in the leaves. Other plants do
not require external inputs and the signal is generated
in the leaves when the plant simply reaches a minimum
434 Chapter 25 / Flowering and Fruit Development

stage of development. In any case, flower development
is initiated when that signal arrives at the shoot apical
meristem. During the vegetative state, the shoot apical
meristem is programmed to produce leaf primordial.
When the floral signal arrives from the leaf, the meri-
stem acquires floral identity and secondary inflorescence
meristems, or floral primordia, arise in the axils of the
uppermost leaf primordia.
The use of genetic mutants that alter flowering-time
and the initiation of floral primordia has been a pow-
erful tool for identifying many of the genes involved
and dissecting the various pathways that lead to flower-
ing (Table 25.1). As a general rule, mutants that cause
early flowering indicate a wildtype gene that normally
represses flowering while mutants that cause late flow-
ering point to a wildtype gene that normally promotes
flowering.
Extensive research with Arabidopsis has identified
three sequential stages to the flowering process, each
with its own set of genes. The first set of genes com-
prises the flowering-time genes. Flowering-time genes
determine when the plant initiates flowering, either in
response to the appropriate environmental signal or
by monitoring the developmental state of the plant.
Most mutations of the flowering-time genes cause the
plants to flower later than normal, although a few will
cause flowering to advance. One role of flowering-time
genes is to activate the expression of floral-identity
genes. Floral-identity genes commit undifferentiated
primordia to the production of floral rather than veg-
etative structures. Mutations in floral-identity genes
cause primordia that would normally develop as flowers
to produce structures with vegetative characteristics.
The floral-identity genes in turn activate a set of
organ-identity genes that serve to control the sub-
sequent development of floral organs such as sepals,
petals, stamens, and carpels. Expression of floral- and
organ-identity genes, however, is not strictly linear.
Some identity genes overlap in both the time of their
expression and their function. As we shall see later, muta-
tions in the organ-identity genes may cause abnormal
development of any or all of the floral organs.
25.1.1 FLOWERING-TIME GENES
INFLUENCE THE DURATION
OF VEGETATIVE GROWTH
Flowering-time genes provide the connection between
florigen, or the floral induction signal, and the transi-
tion to the production of floral organs. Flowering-time
mutants may therefore interfere with the production of
the signal in the leaf (including the timing mechanism, or
circadian clock), translocation of the signal to the apex,
or its activity in the apex. Most flowering-time mutants
identified thus far cause plants to flower later than nor-
mal, indicating that the mutants interfere with pathways
that normally promote flowering. Note that flowering
time, as it is used here, refers to a developmental time

TABLE 25.1 Some principal genes involved in flowering.

Gene Name Gene product Pathway/function

AG AGAMOUS (Unknown) Organ identity

AP1 APETALA 1 (Unknown) Floral/organ identity
AP2 APETALA 2 (Unknown) Organ identity
AP3 APETALA 3 (Unknown) Organ Identity
CCA1 CIRCADIAN CLOCK ASSOCIATED 1 Transcription factor Circadian clock
CO CONSTANS Transcription factor Long-day pathway
ELF3 EARLY FLOWERING 3 Novel protein Circadian clock
ELF4 EARLY FLOWERING 4 Novel protein Circadian clock
FCA FCA RNA-binding protein Autonomous pathway
FLC FLOWERING LOCUS C Transcription factor Floral repressor
FT FLOWERING LOCUS T Lipid-binding protein Floral promoter
GA1 GIBBERELLIC ACID 1 (Unknown) Gibberellic acid pathway
LD LUMIDEPENDENS Nuclear protein Autonomous pathway
LFY LEAFY Transcription factor Floral identity gene
TOC1 TIMING OF CAB 1 Transcription factor Circadian clock
VRN1 VERNALIZATION 1 DNA-binding protein Vernalization pathway
VRN2 VERNALIZATION 2 Repressor protein Vernalization pathway
VRN3 VERNALIZATION 3 Equivalent to FT Vernalization/LD pathway
 25.1 Flower Initiation and Development Involves the Sequential Action of Three Sets of Genes
rather than chronological time (e.g., days to flower-
ing). For example, Arabidopsis is a facultative long-day
plant with a critical photoperiod of 8 to 10 hours.
The vegetative Arabidopsis plant grows as a rosette and
flowering is preceded by stem elongation. Thus, under
long days Arabidopsis flowers with 4 to 7 leaves in the
rosette (about 3 weeks) but under short days flowering
is delayed until 20 leaves have formed (7 to 10 weeks).
Note that flowering is not related to elapsed time, but
to the number of rosette leaves produced before the
flowering stem appears. Late-flowering mutants simply
extend vegetative growth and increase the number of
leaves in the rosette before the stem elongates and the
flowers develop.
It is interesting to note that, although a large num-
ber of late-flowering mutants have been described, no
single Arabidopsis mutant that remains vegetative indef-
initely has yet been identified. This fits with the general
assumption that there are multiple pathways controlling
flowering time with a certain amount of built-in redun-
dancy. Redundancy provides that inactivation of genes in
one pathway is at least partially compensated for by other
genes or complementary pathways. In Arabidopsis, at
least five separate, but interacting, pathways for control-
ling flowering time have been identified (Figure 25.1).
Several flowering-time mutants, including fca, ld,
and fve, flower later than wildtype plants under both
LD and SD conditions but remain sensitive to ver-
nalization. Because flowering in the mutants is equally
affected under both LD and SD conditions, the cor-
responding wildtype genes are thought to be active
in an autonomous pathway that monitors develop-
mental stage and initiates flowering in response to
Long-day
pathway
PHYA
CRY2
GI
Low-temperature Autonomous
pathway pathway
CO
FLC FCA
FT
LD
FVE
Vegetative Flower
LFY
primordia primordia
ELF3
GA
pathway
PHY B
Repressor
pathway
FIGURE 25.1 Five separate genetic pathways control
flowering time in Arabidopsis. All pathways appear to
converge on LEAFY (LFY ), a floral-identity gene in the
shoot apical meristem that mediates the switch from a
vegetative meristem to a floral meristem.
435
internal developmental signals. Such a signal is com-
monly reflected in a minimum leaf number that must
be achieved before flowering can proceed. Flower-
ing of a second group of mutants, including constans
(CO) and gigantea (GI), is delayed under LD condi-
tions, but not under SD conditions. These mutants also
show no response to low-temperature treatments, or
vernalization, which is normally linked to a LD flow-
ering response and not a SD response. As described in
Chapter 24, the CO gene is believed to be a central
component in the photoperiodic or long-day pathway
and is responsible for promoting the mobile floral stim-
ulus FT. Both CO and GI have been cloned and studied
in some detail. It appears that GI operates before CO
in the same pathway and that floral promotion under
long days depends on the amount of CO protein and,
subsequently, FT protein that is produced. The action
of FT at the shoot apical meristem is at least in part
mediated by a transcription factor FD. At this point it
appears that the combination of FT and FD proteins
is responsible for initiating flowering under long days
by activating floral identity genes in the shoot apical
meristem.
A single mutant identified as ga1, which flowers
late under LD conditions and does not flower at all
under SD conditions, is thought to represent a sepa-
rate pathway mediated by gibberellin, the gibberellic
acid or GA pathway. Finally, there is a small group of
genes that appear to act primarily as floral repressors.
This conclusion is based on the observation that their
loss-of-function mutants [e.g., elf3 (early flowering 3) and
phytochrome B (phyB)] cause early flowering. Some, such
as the phyB mutants, retain a response to photoperiod
while others, such as elf3, do not. The loss of photope-
riod response in elf3 appears due to disruption of the
circadian rhythm component of photoperiodic timing.
Other early-flowering mutants, such as emf1 and emf2
(embryonic flower 1-2), flower almost immediately fol-
lowing germination, without forming any rosette leaves.
Instead, the plants form reproductive structures on the
surface of their cotyledons. Because wildtype plants
eventually flower, it appears that the repressor activity
of EMF genes must be intended to prevent precocious
flowering. The activity of repressor genes must decline
during development or at some point be turned off,
eventually allowing one or more of the promotory path-
ways to take precedence. In addition to the above four
genetic pathways, a possible fifth pathway that mediates
low-temperature effects on flowering has been suggested
by recent studies.
One of the challenges that remain is to sort out to
what extent known flowering-time genes are involved in
the production, transmission, and perception of the
floral induction signal. In this regard, an interest-
ing late-flowering mutation has recently been isolated
from maize (Zea mays). Flowering in maize follows
436 Chapter 25 / Flowering and Fruit Development
an autonomous pathway and wildtype plants normally
flower after producing a fixed number of leaves. Maize
plants carrying the mutant id1 (indeterminate 1), how-
ever, continue to produce only leaves long after the
wildtype plant has produced ears and tassels. When they
eventually do flower, the floral structures are aberrant.
However, unlike many of the Arabidopsis flowering-time
genes, which are expressed in the shoot apical meristem
and activate floral-identity genes, id1 is expressed only in
the young leaves. This pattern suggests that the function
of ID1 may be more closely related to the synthesis of
the floral stimulus (or repression of floral inhibitors) in
the leaves.
25.1.2 FLORAL-IDENTITY GENES
AND ORGAN-IDENTITY
GENES OVERLAP IN TIME
AND FUNCTION
While the principal effect of flowering-time mutants
is on the duration of vegetative development, muta-
tions in the floral-identity genes disrupt the transition
of the undifferentiated primordia to floral meristems.
At least four floral-identity genes have been isolated
from Arabidopsis: LEAFY (LFY ), APETALA1 (AP1),
APETELA 2 (AP2), and CAULIFLOWER (CAL). Floral-
identity genes are expressed in the apical meristem prior
to the formation of floral organs but their expression
is up-regulated rapidly following floral induction by
long days or application of gibberellin. However, their
individual roles have been difficult to study because
of extensive redundancy (i.e., a loss of function due
to one mutation is readily compensated by one of the
other wildtype genes). The LEAFY gene appears to
play a key role in floral meristem identity. This can
be demonstrated by placing the gene under the control
of a strong gene promoter (designated 35S) from the
cauliflower mosaic virus. Transgenic plants that con-
tain the 35S::LEAFY combination (called a construct)
bypass the requirement for a floral induction signal
and express the gene constitutively. In such plants, a
shortened primary shoot terminates early in clusters of
flowers and all secondary shoots produce flowers from
the rosette. By contrast, the lfy mutant produces more
inflorescence branches than a wildtype plant but the
‘‘flowers’’ consist of green, leaf-like organs. Moreover,
constitutive expression of the flowering-time gene CO
leads to a rapid activation of LEAFY in wildtype plants.
LEAFY appears to have a central role in the flowering
process. It is probably the principal target of the mobile
flowering-time mobile stimulus FT when it arrives in
the meristem. LEAFY , in turn, activates organ identity
genes such as APETALA1 (AP1).
The Arabidopsis flower is rather typical among
advanced flowering plants, consisting of four distinct
whorls of floral organs (Figure 25.2).The outermost
Stamens
Carpels
Petals
Sepals
FIGURE 25.2 (A) The Arabidopsis flower consists of four
distinct whorls. The outermost whorl (whorl 1) consists
of four sepals, which are green and leaf-like. The next
whorl (whorl 2) consists of four yellow petals. The third
whorl contains six stamens, and the innermost whorl
(whorl 4) contains two fused carpels at the base of the
pistil.
whorl (whorl 1) consists of four sepals, which are green
and leaf-like. The next whorl (whorl 2) consists of four
yellow petals. The third whorl (whorl 3) contains six
stamens, or male reproductive organs, and the inner-
most whorl (whorl 4) contains two fused carpels at the
base of the female reproductive structure, the pistil.
Mutations in combination with studies of temporal and
spatial expression patterns have identified five genes
that are involved in the determination of organ iden-
tity: APETALA1 (AP1), APETALA2 (AP2), APETALA3
(AP3), PISTILATA (PI), and AGAMOUS (AG). Note
that AP1 and AP2 have both been previously identi-
fied as floral identity genes as well. Mutations in the
organ-identity genes generally result in the modifica-
tion, displacement, or total absence of floral organs. In
addition, mutations in any one of these genes generally
influence the development of two adjacent floral organs.
The influence of organ-identity genes on the devel-
opment of the Arabidopsis flower can best be understood
by viewing the floral meristem as three overlapping
developmental fields or fields of gene activity; desig-
nated A, B, and C. Field A includes the sepals and petals
(whorls 1 and 2), field B includes the petals and stamens
(whorls 2 and 3), and field C includes the stamens and
central carpels (whorls 3 and 4). This view is referred
to as the ABC model for floral organ specification, in
which a particular gene or pair of genes is associated
with each developmental field, but controls the identity
of two adjacent whorls of organs (Figure 25.3). Accord-
ing to this model, expression of AP1 alone specifies
sepals; AP1 in combination with AP3 and PI specifies
petals; AP3 and PI in combination with AG specify sta-
mens; and AG alone specifies the carpels. Expression of
AP2 is apparently required throughout the meristem, in
part to suppress, in combination with a sixth, unknown
gene (X ), the expression of AG in those whorls that are
destined to become sepals and petals.
 25.2 Temperature Can Alter the Flowering Response to Photoperiod 437

1 2 3 4 meristem would be unable to form either petals or

Whorls Sepals Petals Stamens
A filled instead with sepal-like and carpel-like structures.
Developmental B Similarly, in the ag mutant, stamens and carpels are not
fields
C formed and all four whorls are filled with sepals or petals.
APETALA3/ PISTILLATA
Genes APETALA1 AGAMOUS
APETALA2
FIGURE 25.3 The ABC model for floral organ specifi-
cation in Arabidopsis. (B) The floral meristem is visu-
alized as being controlled by three developmental fields,
identified as A, B, and C. Field A is involved in the speci-
fication of whorls 1 and 2. Field B is involved in the speci-
fication of whorls 2 and 3. Field C is involved in the
specification of whorls 3 and 4. Field A alone specifies
sepals, fields A and B together specify petals, fields B
and C together specify stamens, and field C alone speci-
fies carpels. Each field is associated with a specific gene
or gene pair. APETALA1 is expressed only in field A,
APETALA3 and PISTILLATA are expressed in field B,
and AGAMOUS is expressed only in field C. AGAMOUS
also represses (T-bar) the expression of APETALA1 in
field C. APETALA2 is expressed throughout the meri-
stem and, in conjunction with an unknown gene (X),
represses the expression of AGAMOUS in field A. (After
J. D. Bewley et al., 2000.)
The ABC model can be used to either predict
or interpret what will happen to organ development
in loss-of-function mutants for each of these genes
(Figure 25.4). For example, in the ap3 (or pi) mutant,
developmental field B would be inoperative and the
Carpels stamens. Not only is this the case, but the whorls that
would normally be filled with petals and stamens are
No doubt this model will continue to be refined as new
genes are discovered and the various genes and their
products are subjected to further molecular analysis.
Although it is becoming more evident which genes
are active in controlling which aspects of floral initiation
and development, their biochemical and physiologi-
cal function is not yet clear. However, most of the
gene products that have been analyzed thus far share
certain characteristics, called motifs, which are typi-
cal of transcription factors. Transcription factors are
DNA-binding proteins that enable RNA polymerases
to recognize promoters and begin transcription of the
gene in eukaryotes. It thus appears that the principal
function of most flowering-time, floral-identity, and
organ-identity genes is to regulate other genes that may
then direct synthesis of the components that actually
make up the individual organs.
25.2 TEMPERATURE CAN ALTER
THE FLOWERING RESPONSE
TO PHOTOPERIOD
There are many examples of interactions between tem-
perature and photoperiod, particularly with respect to
flowering behavior (see Salisbury, 1963, for an exten-
sive listing). In most cases the interaction results in
relatively subtle changes in the length of the critical

Genotype
Observed
Whorls Interpretation FIGURE 25.4 The ABC model helps to
Phenotype
interpret floral-identity in loss-of-function
mutants. In wildtype plants, all three devel-
4. Carpels AP3 opmental fields are active and produce the
Wildtype 3. Stamens AP1 AG normal complement of sepals (Se), petals (Pe),
2. Petals Se Pe St Ca stamens (St), and carpels (Ca). The apetala3
1. Sepals mutant represents a loss of B function. This
leaves AP1 to be expressed alone in whorl
2, replacing the normal petals with sepaloid
structures. Also, AG is expressed alone in
4. Carpels
ap3
whorl 3, producing carpeloid structures in
3. Carpeloid stamens
(apetala3) AP1 AG place of normal stamens. The agamous mutant
2. Sepaloid petals
Se Pe Ca Ca represents a loss of C function, leaving AP1
1. Sepals
Loss of B function to be expressed in all four whorls. The result
is petaloid structures in whorl 3 and sepaloid
4. Sepaloid carpels
structures in whorl 4.
3. Petaloid stamens AP3
ag
(agamous) 2. Petals AP1
Se Pe Pe Se
1. Sepals
Loss of C function
438 Chapter 25 / Flowering and Fruit Development
photoperiod or a tendency toward daylength neutral-
ity or an inability to flower altogether at high or low
temperature extremes. There are other plants, however,
for which flowering is either quantitatively or qualita-
tively dependent on exposure to low temperature. This
phenomenon is known as vernalization. Vernalization
is a means of preventing precocious reproductive devel-
opment late in the growing season, ensuring instead
that seed production does not begin until the beginning
of the next growing season so that the seed will have
sufficient time to reach maturity.
Vernalization refers specifically to the promotion
of flowering by a period of low-temperature and should
not be confused with other miscellaneous effects of
low-temperature on plant development. The term itself
is a translation of the Russian yarovizatsya; both words
combining the root for spring (Russian, yarov; Latin,
ver) with a suffix meaning ‘‘to make’’ or ‘‘become.’’
Coined by the Russian T. D. Lysenko in the 1920s, ver-
nalization reflects the ability of a cold treatment to make
a winter cereal mimic the behavior of a spring cereal
with respect to its flowering behavior. The response
had actually been observed many years earlier by agri-
culturalists, but didn’t receive critical attention of the
scientific community until J. G. Gassner showed in 1918
that the cold requirement of winter cereals could be
satisfied during seed germination. For his part, Lysenko
received considerable notoriety for his conviction that
the effect was an inheritable conversion of the winter
strain to a spring strain. His position— a form of the
thoroughly discredited Lamarkian doctrine of inheri-
tance of acquired characteristics—was adopted as Soviet
dogma in biology and remained so until the 1950s.
The adoption of Lysenko’s views as official dogma
had a significant impact on Soviet biology and placed
agriculture in the USSR at a severe disadvantage for
decades.1
25.2.1 VERNALIZATION OCCURS MOST
COMMONLY IN WINTER
ANNUALS AND BIENNIALS
Typical winter annuals are the so-called ‘‘winter’’ cere-
als (wheat, barley, rye). ‘‘Spring’’ cereals are normally
daylength insensitive. They are planted in the spring
and come to flower and produce grain before the end of
the growing season. Winter strains, however, if planted
in the spring would normally fail to flower or pro-
duce mature grain within the span of a normal growing
1 the crown, including the apical meristem, remains
The story of vernalization is a classic example of what can
happen when science becomes enmeshed in political
ideology. For the interested student, this unfortunate episode
in the history of science has been artfully documented by
D. Joravsky in his book The Lysenko Affair (Chicago:
University of Chicago Press, 1970).
season. Winter cereals are instead planted in the fall.
They germinate and over-winter as small seedlings,
resume growth in the spring, and are harvested usually
about midsummer. The over-wintering cold treatment,
or vernalization, renders the plants sensitive to long
days.
One of the most thorough studies of vernaliza-
tion and photoperiodism was carried out on the Petkus
cultivar of rye (Secale cereale) by F. G. Gregory and
O. N. Purvis, beginning in the 1930s. There are two
strains of Petkus rye: a spring strain and a winter strain.
The spring strain of Petkus rye is a facultative long-day
plant. Under short days, floral initiation does not occur
until after about 22 leaves have been produced, typi-
cally requiring a season of about 4.5 months. Under
the appropriate long-day regime, however, flowering in
the spring strain is initiated after as few as seven leaves
have been produced, requiring only about two months.
When sown in the spring, the winter strain is insensi-
tive to photoperiod. The winter strain flowers equally
slowly—requiring four to five months— regardless of
daylength.
If seeds of the winter strain are sown in the fall,
however, the germinated seedlings are subjected to
an over wintering low-temperature treatment. When
they resume growth in the spring, winter strain plants
respond as long-day plants in the same way as the spring
strain. The effect of the over wintering cold treatment
can also be achieved by vernalizing the seed, that is, by
holding the germinated seed near 1◦ C for several weeks.
Note that the low-temperature treatment, at least in the
case of winter annuals, does not alone promote early
flower initiation. Rather, the effect of vernalization is to
render the seedling sensitive to photoperiod.
Another example of vernalization is seen in bien-
nial plants. Biennials are monocarpic plants that normally
flower (and die) in the second season, again follow-
ing an over-wintering cold treatment. Typical bien-
nials include many varieties of sugar- and table-beet
(Beta vulgaris), cabbages and related plants (Brassica
sp.), carrots (Daucus carota) and other members of
the family Umbellifereae, foxglove (Digitalis purpurea),
and some strains of black henbane (Hyoscyamus niger).
Biennials share with the winter annuals the property
that subjecting the growing plant to a cold treat-
ment stimulates a subsequent photoperiodic flowering
response.
Biennials typically grow as a rosette, charac-
terized by shortened internodes, in the first season
(Figure 25.5). Over winter, the leaves die back but
protected. New growth the following spring is char-
acterized by extensive stem elongation, called bolting,
followed by flowering. The cold requirement in
biennials is qualitative (i.e., absolute). In the absence of
a cold treatment many biennials can be maintained in
 25.2 Temperature Can Alter the Flowering Response to Photoperiod
FIGURE 25.5 Vernalization and stem elongation in cab-
bage. Left: Cabbage plants were vernalized for six weeks
at 5◦ C before being returned to the greenhouse. Center:
Plants were sprayed weekly with a solution containing 5
× 10−4 M gibberellic acid. Right: Control plants grown
at normal greenhouse temperature remain in a rosette
habit. Except for the vernalization treatment, all plants
were maintained in the greenhouse under a long-day
(16-hour) photoperiod.
the nonflowering rosette habit indefinitely. As a rule,
vernalized plants, whether winter annuals or biennials,
tend to respond as long-day flowering plants, although
some biennials are daylength-indifferent following
vernalization. One exception to the rule is the perennial
Chrysanthemum morifolium, a SDP. Some varieties of
Chrysanthemum require vernalization before responding
as a quantitative SDP. As a perennial, Chrysanthemum
normally requires vernalization on an annual basis.
Many other plants such as pea (Pisum sativum) and
spinach (Spinacea oleracea) can be induced to flower
earlier with a cold treatment but it is not an absolute
requirement.
25.2.2 THE EFFECTIVE TEMPERATURE
FOR VERNALIZATION IS
VARIABLE
The range of temperatures effective in vernalization
varies widely depending on the species and duration of
exposure. In Petkus rye, the effective range is −5◦ C
to +15◦ C, with a broad optimum between +1◦ C and
+7◦ C. Within these limits, vernalization is proportional
to the duration of treatment. Flowering advances sharply
after as little as one to two weeks’ treatment at 1◦ C to
2◦ C and is maximally effective after about seven weeks
at that temperature (Figure 25.6). Within the effective
range, the temperature optimum is generally higher
for shorter treatment periods. Presumably, a longer
exposure to lower temperatures within the effective
439
120
100
Days to flowering
80
60
40
20
40
10 20 30 40 50
Duration of cold treatment (days)
FIGURE 25.6 Vernalization in Petkus rye (Secale cereale).
Seeds were germinated in moist sand at 1◦ C for the
time indicated. Cold treatments were scheduled so that
all seeds were returned to the greenhouse at the same
time. The number of days to flowering progressively
decreased with increasing length of the cold treat-
ment. (From Purvis, O. N., F. G. Gregory. 1937. Annals
of Botany, N.S. 1:569–591. Copyright, The Annals of
Botany Company.)
range is required because the metabolic reactions leading
to the vernalized state progress more slowly.
Like flowering, the vernalized state is more or less
permanent in most species, giving rise to the con-
cept of an induced state. For example, vernalized
Hyoscyamus, a LDP, can be held under short days
for up to 10 months before losing the capacity to
respond to long-day treatment. On the other hand,
all cold-requiring plants that have been studied are
capable of being devernalized—vernalization can be
reversed if followed immediately by a high-temperature
treatment. Flowering in vernalized winter wheat, for
example, can be fully nullified if the seedlings are held
near 30◦ C for three to five days. For most plants, then,
there is a ‘‘neutral’’ temperature where neither vernal-
ization nor devernalization occurs. For Petkus rye the
neutral temperature is about 15◦ C. Vernalized seeds of
Petkus rye can also be devernalized by drying them for
several weeks or by maintaining the seeds under anaer-
obic conditions for a period of time following the cold
treatment.
440 Chapter 25 / Flowering and Fruit Development
25.2.3 THE VERNALIZATION
TREATMENT IS PERCEIVED
BY THE SHOOT APEX
A vernalization treatment is effective only on actively
growing plants. Cold treatment of dry seeds will not
suffice. Thus winter cereals may be vernalized as soon
as the embryo has imbibed water and the germination
process has been initiated. Other plants, in particular
the biennials, must reach a certain minimum size before
they can be vernalized. Hyoscyamus (black henbane), for
example, is not sensitive before 10 days of age and does
not reach maximum sensitivity until 30 days of age. In
either case, the cold treatment appears to be effective
only in the meristematic zones of the shoot apex. This
can be shown by localized cooling treatments or ver-
nalization of moistened embryos. Early studies showed
that even the cultured apex of isolated rye embryos
was susceptible to vernalization. Thus the induced state
established in a relatively few meristematic cells can be
maintained throughout the development of the plant.
Most biennials, however, cannot be induced as seeds.
In these plants it is the over-wintering stem apex that
perceives the stimulus, although there are some reports
suggesting that leaves and even isolated roots may be
susceptible in some cases.
25.2.4 THE VERNALIZED STATE
IS TRANSMISSIBLE
Experiments with isolated embryos have shown that ver-
nalization treatments are effective only when the embryo
is supplied with carbohydrate and oxygen is present,
indicating that it is an energy-dependent metabolic pro-
cess. Still, the nature of the induced state has eluded
researchers for many years. To the extent that the
meristem itself is the site of perception, any necessity
for a transmissible hormone appears to be ruled out.
A cold-induced, permanent change in the physiological
or genetic state of the meristematic cells (referred to
as ‘‘mitotic memory’’) would be self-propagating, that
is, it could be passed on to daughter cells through cell
division. There is some support for this argument. In
plants such as Petkus rye and Chrysanthemum, only tissue
produced in a direct cell line from the induced meristem
is vernalized. If the cold treatment is localized to a single
apex, it will flower, but all the buds that did not receive
the cold treatment will remain vegetative.
In other experiments, especially with Hyoscyamus,
transmission of the vernalized state across a graft union
has been demonstrated. A list of successful experiments
has been tabulated by A. Lang in his 1965 review (see
Further Reading). These experiments are comparable
to the transmission of florigen’’ across a graft union
(Chapter 24) and result in flowering in nonvernal-
ized receptor plants. If a vernalized Hyoscyamus plant is
grafted to a nonvernalized plant, both will flower under
long days. Transmission requires a successful (i.e., liv-
ing) graft union and appears to be coordinated with the
flow of photoassimilate between the donor and receptor.
Experiments such as those described above led
G. Melchers to propose the existence of a transmissi-
ble vernalization stimulus called vernalin. Like florigen,
vernalin has resisted all attempts at isolation and remains
a hypothetical substance. Unfortunately, the vernalin
story is to some extent clouded by interpretation. The
grafting experiments all require vernalization followed
by long days. They do not clearly distinguish between
the transmission of ‘‘vernalin’’ and the possibility that
the nonvernalized partner is responding instead to the
floral stimulus itself (e.g., FT), which would be trans-
mitted from the vernalized donor under long days.
Adding to the complexity of vernalization is the
apparent involvement of gibberellins in the response to
low temperature (see Figure 25.5). This was dramatically
demonstrated by A. Lang in 1957 when he showed that
repeated application of 10 µg of GA3 to the apex would
stimulate flowering in nonvernalized biennial strains of
Hyoscyamus and several other biennials maintained under
short days. No such promotion occurred in Xanthium
and other short-day plants treated with gibberellin under
noninductive long days. Subsequently it has been shown
that gibberellin levels tend to increase in response to
low-temperature treatments in several cold-requiring
species.
The role of gibberellins is not clear, although in
noninduced plants very high concentrations of the gib-
berellin precursor ent-kaurenoic acid accumulate in the
shoot apex. This suggests that the cold treatment is
required to complete the synthesis of gibberellins in
these plants.
25.2.5 GIBBERELLIN AND
VERNALIZATION OPERATE
THROUGH INDEPENDENT
GENETIC PATHWAYS
Results such as those described in the previous section
have raised the question: Are vernalin and gibberellin
equivalent? The answer, on both physiological and
genetic grounds, is no. It is true that gibberellin appears
to substitute for the cold requirement of some vernal-
izable plants and for the long-day requirement in some
long-day plants, or, in the case of vernalization, both.
But virtually every situation in which gibberellin has suc-
cessfully substituted for low temperature or long days
in promoting flowering involves bolting, or the rapid
elongation of stems from the rosette vegetative state.
Far less success has been achieved with gibberellins
in caulescent long-day plants—those whose stems are
already elongated in the vegetative state. Moreover,
the developmental pattern in responsive plants differs
 25.2 Temperature Can Alter the Flowering Response to Photoperiod
significantly depending on whether stem elongation is
stimulated by low temperature or gibberellin treatment.
Following low-temperature treatment, flower buds are
evident at the time stem elongation begins. Following
gibberellin treatment, on the other hand, the stem first
elongates to produce a vegetative shoot. Flower buds do
not appear until later. These results suggest independent
pathways for vernalization and gibberellins.
Recent genetic studies of flowering have confirmed
that gibberellin and vernalization operate via separate
genetic pathways (see Figure 25.1). When a triple
mutant was constructed containing mutant alleles
(co-2, fca-1, ga1-3) that impair each of the long-day,
autonomous, and GA-dependent pathways, the mutant
plants failed to flower under either long days or short
days in controlled environment rooms. After 90 to 100
rosette leaves had been produced without flowering, the
plants were then transferred to a long-day greenhouse.
After six months, the majority of the mutant plants
had died without ever flowering. However, if the triple
mutant seedlings were first vernalized at 5◦ C for 7
weeks, all the plants flowered after approximately 50
leaves had been produced. The most straightforward
interpretation of these results would be that (1) the
triple mutant has an absolute requirement for vernaliza-
tion and (2) vernalization promotes flowering through
yet another genetic pathway that is separate from the
GA-dependent, long-day, and autonomous pathways.
Both the autonomous pathway and vernalization
reduce the expression of the gene FLOWERING LOCUS
C (FLC). The product of this gene is a transcription
factor that represses flowering. However, when the
wildtype FLC gene is absent, control by the autonomous
pathway is also eliminated but the effect of vernalization
is not. Thus it appears that the autonomous pathway
acts solely through controlling FLC expression, but
vernalization is able to promote flowering through two
pathways: either through suppressing FLC expression or
through some yet-to-be-discovered FLC-independent
mechanism.
25.2.6 THREEE GENES DETERMINE
THE VERNALIZATION
REQUIREMENT IN CEREALS
This brings us to the question of whether vernalin, like
florigen, is a hormone. Or a better question to ask might
be: what is the molecular basis for vernalization? It has
actually been known for more than 30 years, primarily
through plant breeding experiments, that three genes
(VERNALIZATION 1, 2, and 3, or VRN1, VRN2, and
VRN3) have a major role in determining the vernaliza-
tion requirements in cereal grains. These genes have
now been isolated and characterized.
The VRN1 gene encodes a transcription factor and
is induced by a vernalization treatment. Furthermore,
441
there is a quantitative relationship between the amount
of VRN1 expressed in a vernalized plant and the amount
by which flowering time is reduced under long days.
VRN2 represses flowering under long days by block-
ing the expression of the floral stimulus FT. On the
other hand, VRN2 is itself repressed by VRN1. Vari-
eties of winter cereals that lack a functional copy of
VRN2 respond normally to long days without requiring
a prior cold treatment. VRN3 is the cereal equivalent
(called an ortholog) of FLOWERING LOCUS T (FT) in
Arabidopsis.
A model to illustrate how these three genes inter-
act to control flowering in winter wheat and barley
is presented in Figure 25.7. Prior to receiving a cold
treatment, the winter cereals are unable to respond to
long days because FT (or VRN3) expression is repressed
by the presence of VRN2. When the seeds are sown
in late summer or early autumn, the shoot apex devel-
ops vegetatively until winter, when VRN1 expression
is promoted. In the spring, growth is renewed; the
low-temperature-induced expression of VRN1 remains
high; and VRN1 suppresses any further expression of
Arabidopsis Winter cereals
Low Long days Low
temperature (PPDI) temperature
VRN1
VRN2
CO
FLC FT VRN2 VRN1
(VRN3)
VRN1
Flowering
FIGURE 25.7 A model comparing the regulation of flow-
ering by vernalization in winter cereals and Arabidop-
sis. Long days (mediated by PHOTOPERIOD 1 (PPD1)
in cereals) are sensed by the CONSTANS gene (CO)
which activates FLOWERING LOCUS T (FT) expres-
sion. VRN2 prevents floral induction before winter by
repressing FT expression. A low-temperature vernaliza-
tion treatment induces expression of VRN1, which
represses VRN2 and allows expression of FT under long
days. How the low temperature induces VRN1 expres-
sion isn’t known. In cereals, VRN1 also acts as a floral
meristem identity gene. In Arabidopsis, FT expression
is repressed by FLOWERING LOCUS C (FLC). Flow-
ering proceeds under long days following vernalization
because the low temperature represses FLC expression.
(Based on Trevaskis et al. 2007. Trends in Plant Science.)