Seed germination,

seedling growth
photomorphogenesis
BIOL 304
11/07/2008-11/10/2008

Life cycle of a flowering seed plant

2n
2n
2n

1n

2n

2n

Single
fertilized
egg

Embryogenesis &
seed development

Seed
Apical
-basal
axis
radial axis

germination and
seedling growth
Separable in time and
space
3

How long can seeds remain dormant?

1879: 20 bottles of seeds buried
--dug up at 5-10 year intervals and tested
--after 10 years, most still viable
--by 2000, 2 species still alive

William James Beal

lotus seeds carbon dated as 1500-3000 years old have

germinated
ability to remain dormant:
--species specific
--can be very long
4

Seed dispersal
Animal dispersal:

seeds are inside fleshy fruits that can be eaten by

animals.

Seeds with barbs or other structures that can be

tangled in animal fur or feathers.

Seeds can be collected and buried by animals

(squirrels with acorns)

Seed dispersal
Wind dispersal: some seeds have wings or other hairlike or feather-like structures.

ABA promotes seed dormancy but

inhibits seed germination
ABA treatment: maintain dormancy, inhibit
germination (inhibit radicle elongation)
Mutants for ABA biosynthesis or signaling:
precocious germination or late germination.

Seed dispersal
Floating in water: aquatic plants and
plants that live near water have
seeds that can float.
other ways: "shoot" seeds out of pods

Seed germination and seedling growth

1. Renew the growth and development of the embryo
2. Initiate postembryonic growth and development
3. The basic structure of a seedling is pre-determined
during embryogenesis.

Biochemical analysis: hormone requirement

10

ABA
ABA: appear at the late stages of embryo
development (embryo maturation).
stimulate accumulation of seed storage
proteins
promote desiccation tolerance
promotes seed dormancy
prevent precocious seed germination
How would you test that ABA promotes seed dormancy
but inhibits seed germination?
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ABApromotesseeddormancybutinhibitsseedgermination
ABAtreatment:maintaindormancy,inhibitgermination(inhibit
radicalelongation)
MutantsforABAbiosynthesisorsignaling:precocious
germinationorlategermination

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At the molecular level, ABA regulates

expression of over 1300 genes.

Genes activated by ABA:

Seed storage genes

desiccation
dormancy genes

Genes repressed by ABA:

Germination promoting genes
(i.e. a-amylase)

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Biochemical analysis: hormone requirement

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GA promotes germination
GA levels increase during early seed
germination.
exogenous GA breaks seed dormancy
GA biosynthetic mutants (ga1, 2, 3) cannot
germinate unless supplied with exogenous GA.
Mutants involved in GA signaling (gid1) fail to
germinate correctly and cannot be rescued by
exogenous GA.
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GA breaks seed dormancy by

mobilizing reserves
1. GA moves from the embryo
to the aleurone.
2. GA stimulates the synthesis
of -amylase and protease.
3. Protease activates -amylase
which form an active enzyme
with -amylase to digest
starch to glucose.
4. Glucose is mobilized to the
growing embryo during seed
germination.

16

ABA and GA antagonize each other

ABA represses expression of many
of the same genes that are
activated by GA, and vice versa
high ABA/GA ratio promotes
dormancy and opposes germination
low ABA/GA ratio opposes
dormancy and promotes
germination

17

 -amylase: a critical enzyme involved in reserve

mobilization during seed germination

GA GA+
ABA

RNA analysis

ABA

Protein analysis

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Additional factors
that control
germination

Ethylene opposes ABA action, promotes germination

BR slightly stimulates germination.
Sugars can promote or oppose germination, depending
on concentration
Seed coat inhibits germination (mechanical or
chemical resistance)
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There are also environmental factors

several conditions are monitored: water, light, and temp.
all seeds require imbibition--rehydration
some seeds require light (mostly small seeds), others
are inhibited from germinating by light, others are
indifferent to it (most crop plants)
most seeds require a prolonged exposure to cold
(4C for up to 4 weeks) for the maximal germination
efficiency

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Seedling growth &

photomorphogenesis

Germination

Seedling
growth
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Light is essential for a plant life cycle

Energy source for photosynthesis
Environmental cue for plant growth and
development:
Seed germination
Seedling growth (photomorphogenesis)
Flowering
Direction of growth (phototropism)
(auxin)
Plant movement
Stress responses
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Photomorphogenesis vs. Skotomorphogenesis:

Photomorphogenesis:
Light grown seedlings display:
- Arrested hypocotyls
- Unfolding and expansion of
the cotyledons
- Greening cotyledons.
Skotomorphogenesis:
Dark-grown seedlings become
etiolated, characterized by
elongated hypocotyls, closed
cotyledons and apical hooks.
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Photoreceptors
Phytochromes: detect red (R) and far red (FR) light and
affect many aspects of plant development
Cryptochromes: detect blue light and UV-A light;
important for seedling development and flowering
Phototropin: detect blue light and UV-A light; important
for phototropism
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Unidentified receptor: detects UV-B light

The discovery of phytochrome

The Borthwick and Hendricks experiment:
1950s: In dark, lettuce seeds were irradiated
with red light (1 min) or far-red light (3 min).
Germination rate was scored 48 hours after.
Red light = ~620-680 nm
Far-red light ~700-800 nm

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Irradiations
R
R,Fr
R,Fr,R
R,Fr,R,Fr
R,Fr,R,Fr,R
R,Fr,R,Fr,R,Fr

Germination%
88%
22%
84%
18%
72%
22%

Red light (1 flash) stimulated seed germination,

Far-red (3) negated the stimulating effect of red light.
The stimulation and negation relationship was
photoreversible.
HYPOTHESIS: the exsistence of a photoreversible light
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absorbing pigment (phytochrome)

Photoreversible phytochrome

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Phytochromes are chromoproteins

Two parts: protein & chromophore (light-absorbing moiety)

apoprotein

Chromophore
(covalently attach
to the protein via a
cysteine residue)

28

Absorption spectra of purified phytochromes:

Pr = Red light (620-680nm)
Pfr = far-red light (700-800 nm)
Phytochromes also absorb blue light.

29

apoprotein

Chromophore

Photoconversion involves a rotation (a cis-trans

isomerization) of the double bond between the
C and D rings.

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Two forms of a phytochrome

Pr: inactive form of

phytochrome, activated
by absorption of farred light (620-680 nm)

Conformational
changes

Pfr: active form of

phytochrome,
inactivated by
absorption of red light
(700-800 nm)

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Phytochrome activity is regulated at multiple levels

Transcriptional regulation: Pfr inhibits the transcription
of phytochrome genes. Red light inhibits Phy genes
expression but far red light can reverse the
inhibition. (negative feedback inhibition)

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Regulation of phytochrome activity

Protein stability: The Pfr form is less stable than the Pr
form, so over time the Pfr/Pr ratio falls in the
absence of any light stimulus. Phytochromes are
degraded by the ubiquitin/proteosome system.

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Regulation of phytochrome activity

Protein translocation

Translocation from
the cytoplasm into
the nucleus

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Regulation of phytochrome activity

Arabidopsis possesses 5 phytochromes.
All share the same chromophore but the
apoproteins are encoded by five different
genes: phyA, B, C, D, and E
Have slightly different amino acid sequences
Protein abundance and stability are variable.
overlapping but different functions
35

Distinct function of phyA and phyB

In far-red light, the phyA mutants fail to
germinate.
In red light, the phyB mutants fail to
germinate.

36

Distinct function of phyA and phyB in

regulating hypocotyl elongation
In light:
The hypocotyl of the
phyA mutant is shorter
than the phyB mutant
The phyAphyB double
mutants are even more
elongated

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Effects of PHYA and PHYB

PHYB

PHYA

PHYA

PHYB

Hypocotyl length
Hypocotyl length

PHYA
Hypocotyl length
PHYB
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Effects of PHYA and PHYB on

hypocotyl length are additive
PHYA
Hypocotyl length
PHYB
Suggesting: they regulate two independent pathways
that converge on repression of the same genes
regulating cell elongation.

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Phytochromes
act by
regulating gene
expression

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Phytochrome under natural conditions

Grown in dark:

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Phytochrome under natural conditions

End-of-day signals:
Twilight at the end of day has more far red light
than red light.

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End-of-day signals:

A. Helianthus
B. Phaseolus
C. Pharbitis
White: white light
Blue: far-red light
Gray: far-red, red

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Phytochrome under natural conditions

Canopy shading:
The chlorophyll in
Leaves absorbs well
red and blue light but
is rather transparent
to far-red light.

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Responses to blue and UV-A light

Receptor: the cryptochrome 1 and 2 proteins.

de-etiolating (inhibition of hypocotyl elongation and
stimulation of cotyledon expansion), the opening and
closure of stomata, flowering time, and the setting
of endogenous clocks
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The blue light receptor: cryptochromes

Cryptochrome 1 and 2: the apoprotein is a flavoprotein
and binds to two chromophores.

CRY1

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Blue light and phototropism

Phototropism: the differential growth of plants
towards a light source
Photoreceptor for phototropism: phototropin, a
flavoprotein with two chromophores.
Spectrum: the blue and UV-A region.

Phototropin

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Cryptochromes and phototropin

phototropin mutants (nph): lack of phototropism, normal
suppression of hypocotyl elongation in blue light
cryptochrome mutants (cry): normal phototropism, lack of
normal suppression of hypocotyl elongation in blue light

What about double mutants?

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Dissecting light signaling pathway

?
Mutant analysis
Photomorphogenesis
vs.Skotomorphogenesis
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Non photomorphogenic mutants

Mutants lack of
photomorphogenesis
under certain light
conditions:
Photoreceptor
transcription factor
(hy5, laf1, hfr1, hyh)

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Constitutively Photomorphogenic (COP) mutants

In dark: the plant develops
expanded cotyledon,
shortened hypocotyl, and
accumulate anthocyanin
(instead of chlorophyll)

51

Among the 10 cop mutants identified:

COP1 encodes an E3 ubiquitin
ligase
COP10 encodes an E2 ubiquitin
ligase variant
the other 8 encode members of a
complex known as the COP9
signalosome (CSN). some CSN
components resemble components
of the 26S proteasome

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Light signaling pathway

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