GIBBERELLINS: REGULATORS OF

PLANT HEIGHT AND SEED

GERMINATION
By:
Khanifah 4401414065
Erna Setyana 4401414075
Indira Anggraini S. 4401414105
Addina Nur L. 4411414030
• The second grup of plant hormones to be
characterized was the giberellin (GAs)
• Giberellin also play important roles in a variety
of other physiological phenomena,such as seed
germination,transtition to flowering and pollen
development
• The identification of a GA receptor in rice in 2005 has phe
crystal structures of the Paved the way for recent findings that
include the GA receptors for both rice and Arabidopsis sp.
• The bindings of bioactive GA to its receptor initiates a chain of
events that leads eventually to the responses observed at the
whole plant level
 Giberellins : Their Discovery and
Chemical Structure
• Plant pathologist found that these symptom in rice were
caused by apathogenic fungus,Gibberella fujikuroi, that
had infected plants.Culturing the fungus in laboratory
and analyzing the culture filtrate enable japanese
scientists in 1930s to obtain impure crystals with plant
growth-promoting activity.They named this mixture of
compounds gibberellin A
Gibberellic acid was first purified from
Gibberella culture filtrate
• Japanese scientics separated and
characterized three different gibberellins from
the original gibberellin A sample,naming them
gibberelin A1(GA1),gibberelin
A2(GA2),gibberellin 3 (GA3)
• Specatucar response were obtained in stem
elongation of dwarf and rosette
plans,parmaize and manyrosset pants
 All gibberellin are based on and ent
gibberallane skeleton
• The GAe s are diterpenoids that are formed
from 4 isoprenoid unit each consisting of five
carbons
• They possess a tetracyclyc (four-ringed) ent
gibberelane skeleton (containing only 19
carbon atoms because carrbon 20 is missing)
 Effect of gibberellins on growth and
development
• Many of these properties of GAs have been
exploited in agriculture for decades,and
manipulation of the GA content of crop plants
affect shoot size,fruit set dan fruit growth
Gibberelin promote seed germination
• During germination,GAs induced the synthesis
of hydrolytic enzyme such as amylases and
proteases in cereal grains.
• The enzyme degrade the stored food reserves
accumulated in the endosperm or embryo as
seed matured
• This degradation of carbohydrates and storage
proteins provide nourisment and energy to
support the seedling growth
 Gibberellin can stimulate stem and
root growth
• Applied GAs may not have dramatic effect on stem elongation
in plants that already tall since bioactive GA may not to be
limiting in some tall plants
• Gibberellin are also important for root growth. Extreme dwarf
mutans of pea and arabidopsis,in which GA biosynthesis is
blocked ,have shorter roots than wild type plants,and GA
application to the shoot enhances both shoot and root
elongation
 Gibberellin regulate the transtition
from juvenile to adult phases
• In many conifers,the juvenile phase,which may
last up to 20 years can be shortened by
treatment with GA 3 or with mixture of GA4
and GA7 and much younger plants can be
induced to enter the reproductive cone
producing phase precociously
 Gibberellin influence floral initiation
and sex determination
• GA can subtitute for the long day requirement
for flowering in many plants,especially rosette
species. Interaction of photoperiod and GA in
flowering is complex
• The nature effect of GA on sex determination
can vary with species, however it’s also
influenced by enviromental factor such as
photoperiod and nutritional status
 Gibberelin promote pollen
development and tube growth
• Gibberellin deficient dwarf mutans have
impaired anther development andpollen
formation and both these defects ,which
leads to male sterility can be reserved by
treatment with bioactive GA
• In addition,reducing the level of bioactive GA
in arabidopsis by overexpressing a GA
deactivating enzyme severely inhibitss pollen
tube growth
 Gibberelin promote fruits set and
parthenocarpy
• Fruits can be stimulated to enlarge by
treatment with GA3
• This treatment also promotes growth of the
pedicel(fruit-bearing stalk) and consequently
reduce fungal infection that can be
problematic in the compact cluster of
untreated grapes.
 Gibberelin promotes early seed
development
• Treatment with GA will not restore normally
can be attributed to reduced level of bioactive
GA in very young seeds
• The effect of GA deficiency on seed arbotion
can give a constitutive GA response
 Commercial uses of gibberelline
• The major commercial uses of GA are to
promote the growth of fruit crops,to stimulate
the barley malting process in the beer brewing
industry and to increase sugar yield in
sugarcane
 Biosynthesis and Deactivation of
Gibberellins
• Gibberellins constitute a large family of
tetracyclic diterpene acids synthesized via a
terpenoid pathway
• Knowledge of GA biosynthesis and
deactivation is important asit contributes to
our understanding of GA homeostasis
 Gibberellins are synthesized via
terpenoids pathway
• Terpenoids are compound made up of five
carbon isoprenoid building blocks.the GA are
diterpenoids are formed from four such as
isoprenoid units
• The GA biosynthetic pathway can be divided
into 3 stages each residing in a different
cellular compartment plastid,ER,or cytosol
 Some enzyme in the GA pathway are
highly regulated
• Work withbiosynthetic mutans of
arabidopsis,pea and maize facilitated the
cloning of gene for many of the enzyme in GA
biosynthesis and deactivation.
• 3 enzyme in 3 stage of pathway these are GA
20-oxidase,GA3-oxidase and GA 2-oxidase
 Gibberellin regulates its own
metabolism
• Depression of biosynthesis is achieved
through down regulation (inhibition of
expression )of some of the GA20ox and GA3ox
genes encoding the formation of bioactive GA
.Thiseffect of GA on its ow biosynthesis is
termed negative feedback regulation
• The ability GA to promote the expression of
genes involved in its own deactivation is
termed positive feedback regulation
 GA biosynthesis occurs at multiple
plant organs and cellular sites
• All molecules in GA biosynthesis leading from GGPP to
a C19-GA can be demonstrated in cell-free systems from
seeds or seed-parts, providing definitive evidence that
developing seeds are sites of GA biosynthesis
• The first committed enzyme in GA biosynthesis is
expressed in immature seed, shoot apices, root tips,
and anthers of wild-type Arabidopsis plants
• Compared to the cellular locations of GA 3-Oksidase
and the products of genes known to be up-regulated
by GA4, some cells did not make GA4 but could respond
to it.
Environmental condition can influence
GA biosynthesis
• Gibberellins play an important role in
mediating the effects of environmental.
• Lights and temperature can have profound on
GA metabolism and GA responses
• The environment can alter the metabolism of,
or response to, other hormones in addition to
GAs
• GA1 is the major bioactive GA for stem growth in
Pea and malze
• LE and le have are two alleles of gene that
regulates tallness in Pea. If GA20 is applied to the
le mutant of pea, it is not bioactive, whereas GA1
is bioactive and rescues the mutant phenotype
(plant grow tall)
• LE gene encode an enzyme that 3-hydroxylates
GA20 to produce GA1
• Less GA1 is produced in plants homozygous for
the recessive allele than in wild-type plants
 continued

• The height of pea plants is directly correlated

with the amount of endogenous GA1
• In maize: A less abundant GA, GA4, has higher
affinity for the GA receptor than GA1 when
tested in vitro, and GA4 may have an
important role in reproductive growth of rice
• In Arabidopsis: applied GA1 has less biological
activity than GA4.GA4 is assumed to be the
main biologically active GA
• The identity of the bioactive GAs in crop plants,
together with the characterization of key enzymes in
GA biosynthesis and deactivation, has enabled genetic
engineers to alter the levels of bioactive GA in crops,
and thus affect plant height.
• The conserved or the GA20ox, GA3ox, and GA2ox
enzymes between species means that genes encoding
these enzymes in one species can be introduced and
expressed in another species
• In some other agricultural crops the desire affect is
often to decrease growth
 Dwarf mutants often show other
phenotype defects
• The severely dwarfed ga1, ga2, and ga3
mutants of Arabidopsis have dormant seeds
that cannot germinate unless treated with GA
• GA-deficient mutants would have short stems
and reduced seed growth
• Homozygous mutations in three genes
encoding enzyme in the GA pathway all give
dwarf plants and normal seed development
because of gene redundancy
• The targets for auxin regulation –that is, which
genes are up- or down- regulated– are different
in different species, ad even in different organs
or tissues of the same species.

Auxins can regulate GA biosynthesis

GIBBERELLIN SIGNALING: SIGNIFICANCE
OF RESPONSE MUTANTS
• The main classes of mutants can be distinguished:
– A mutation that renders a positive regulators of GA
signaling nonfunctional give rise to a dwarf phenotype
– A mutation that renders a negative regulators of GA
signaling nonfunctional give rise to a tall phenotype
– A third class of mutants, in which a negative
regulators is made constitutively active, also give GA
nonresponsive dwarf plants
 GID1 encodes a soluble GA reseptor
• The characterization of a recessive dwarf mutant
of rice, the wild-type allele codes for globular
protein, GID1, which is a GA receptor
• To be identified as a receptor, a protein must
fulfill the following criteria:
– The binding of the ligansdto the protein must be
specific
– Ligand binding should be saturable
– Ligand binding should be of high affinity
– Ligand binding should be rapid and reversible
• Single nucleotide substitution in gid1-1 and gid1-2 and
a deletion in gid1-3 all produced GA-insensitive dwarf
plants and prevented the mutants proteins from
binding radiolabeled GA4
• The top part of a bioactive GA molecule is hydrophobic
and interacts with the N-terminal helical extension
switch region of G1D1 receptor protein
• Once the lid has closed, residues on the outer surface
of the lid of G1D1 are able to interact with specific
residues on the amino-terminal end of GAI, which is a
DELLA protein.
• Bioactive GA is an allosteric activator of G1D1, allowing
a conformational change of shape in G1D1 that
facilitates its binding to a DELLA protein.
• The GA-G1D1 complex appears to induce a coil-to-helix
conformational change on the DELLA domain
DELLA-domain protein are negative regulators
of GA response
• DELLA-domain protein are a subclass of the GRAS
family of transcriptional regulators.
• All GRAS protein have homology at the C-terminal
(GRAS) domain.
• Those involved in GA responses also have a
domain at the N-terminal end in which the first
five amino acid are aspartic acid (D), glutamic
acid (E), leucine (L), leucine (L), and alanine (A),
hence the notation “DELLA domain”.
• From extensive studies in cereal and
Arabidopsis it was concluded that DELLA
protein are negative regulators of GA
response.
• When they are degraded by proteolysis a GA
response can occur.
Mutation of negative regulators of GA may
produce slender of dwarf phenotypes
• Mutations that prevent negative regulators of GA
response from acting as negative regulators will
produce tall plant.
• This is exactly what we see in “slender” mutants
of rice and barley.
• There are five DELLA genes in Arabidopsis that
encode five DELLA proteins: GA-INSENSITIVE
(GAI), REPRESSOR or ga1-3 (RGA), RGL1, RGA2,
and RGL3, all of which are negative regulators of
GA response.
• Both RGA and GAI encode negative regulators
of GA response, but that different mutations
in theses negative regulators of GA signaling
could produce opposite phenotypes, either
slender (tall) or dwarf, depending on where
within the genes the mutations occurred.
• If a mutation was in C-terminal GARS domain,
the mutant plant was often taller than
expected.
• If the mutation was in N-terminal part of the
protein that contained the DELLA domain, the
mutant protein was an irreversibles repressor,
thus giving rice to the insensitive dwarf
phenotype of gai-1.
Gibberellins signal the degradation of negative
regulators of GA response
• DELLA-domain are nuclear-localized negative
regulatore of GA action that must be
degraded in order for GA response to occur.
• The presence bioactive GA the nuclear-
localized RGA is degraded , whereas it is not
degraded when GA levels are very low.
F-box proteins target DELLA domain proteins
for degradation
• For a protein (such as DELLA protein) to be
degraded by the 26S proteasome, it must be
“tagged” for degradation by the attachment of
a string of ubiquitin residues.
• This tagging occurs when F-box component
recruit proteins into the SCF complex, leading
to the addition of many ubiquitin molecules to
the protein and its subsequent degradation by
the 26S proteasome.
Negative regulators with DELLA domain have
agricultural importance
• In wheat the semidominant Reduced height
mutations, and in maize the d8 mutation give
rise to GA-insensitive dwarf plant.
• The dissemination of wheat cultivars
expressing Rht mutations, with reduces GA
response ang shortened stem, was pivotal to
success of the Green Revolution.
Gibberellin Responses: Early Targets of
DELLA Proteins
• As you know from the earlier part of this
chapter, Gas can affect many aspects of plant
growth and development rangking from seed
germination to flower and fruit development.
DELLA proteins can activate or suppress gene
expression
• Several of the immediate targets of DELLA
proteins are gene encoding GA biosynthesis
enzymes or the GA reseptor, implying strong
homeostatic regulation to maintain GA level
and GA response within physiological limits.
• Other immediate targets of DELLA proteins
are genes encoding transcription factors or
transcriptional regulators.
• DELLA proteins interact with Phytochrome-
interacting factors (PIFs).
• When DELLA protein binds to a PIF, it prevents the
PIF from activating gene transcription, thus the
target genes of a PIF are (inderectly) down-regulated
by DELLA proteins.
Gibberellin Responses: The Cereal
Aleurone Layer
• Cereal grains have three part: the embryo, the
endosperm, and the fused testa-pericarp (seed
coat-fruit wall).
• Living cells of the aleurone layer, which surrounds
the endosperm, synthesize and release hydrolitic
enzymes into the endosperm during germination.
• The isolated aleurone layer, consisting of a
homogeneous population of target cell for GA,
provides a unique opportunity to study the
molecular aspects of GA action in the absence of
nonresponding cell types.
GA synthesized in the embryo

• The secretion of starch-degrading enzymes by

barley aleurone layers depends on the
presence of the embryo.
• The significance of the GA effect became clear
when it was shown that the embryo
synthesizes and releases GAs into the
endosperm during germination.
Aleurone cell may have two types of GA receptors

• Given the great diversity of GA responses, the

existence of multiple receptors may not be too
surprising, though at the present time there is
no difinitive identification of plasma
membrane-localized GA recepetor.
• Within the aleurone cells there sre both Ca2+ -
independent and Ca2+ - dependent GA
signaling pathways.
Gibberellin enhance the transcription og -
amylase mRNA
• GA3 – stimulated -amylase production was
shown to be blocked by inhibitors of
transcription and translation.
• Isotope-labeling studies demonstrated that be
stimulation of -amylase activity by bioactive
GA involved de novo synthesis of the enzyme
from amino acid, rather than activation of
preexisting anzyme.
• Cereal grains can be cut in two, and half-grains
that lack the embryo (the source of bioactive GA
in intact grains) make a good experimental
system for studying the action of applied GA.
• the upregulation of genes encoding several -
amylase isoforms in rice embryoless half-grains
that have been treated for 8 hours with GA3 .
• In these half-grains the only living cells, and the
only living cells in which GA signaling occurs, are
in the aleurone layer.
• The purification of -amylase mRNA, which is
produced in relatrively large amounts in aleurone cells,
enabled the isolation of genomic clones containing
both the structural gene for -amylase and its
upstream promoter sequences.
• Sequences conferring GA responsiveness, termed GA
response element (GAREs).
• Identical GARes have been found in all cereal -
amylase promoters so far examined, and their
presence has been shown to be necessary and
sufficient for the induction of -amylase gene
transcription by GA.
GAMYB is a positive regulator of α-amylase
transcription
The hypothesis that GAMYB turns on α -amylase gene
expression is supported by the following findings :
• Synthesis of GAMYB mRNA begins to increase as
early as 1 hour after GA treatment, preceding the
increase in a-amylase mRNA by several hour
• A mutation in the GARE the prevents MYB binding
also prevents a-amylase expression
• In the absence of GA , constitutive expression of
GAMYB can induce the same responses that GA
Cycloheximide, an inhibitor of translation, has
no effect on the production of GAMYB mRNA,
indicating that protein synthesis is not required
for GAMYB expression. GAMYB can therefore be
defined as a primary or early response gene. in
contrast, similar experiments show that the a-
amylase gene is a secondary or late response
gene.
 DELLA-domain proteins are rapidly
degraded
• It is known that within 5 minutes of GA aplication
to barley aleuron cells there is an effect on the
DELLA protein, SLN1.
• As a consequence of DELLA degradation, and via
some intermediary steps that have not yet been
defined, the expression of GAMYB is up-
regulated.
• Finally, the GAMYB proteins bind to a highly
conserved GARE in the promoter of the gene for
a-amylase, activating its transcription.
 Gibberellin Responses: Anther
Development and Male Fertility
• The GAMYB transcription factor activates α-
amylase gene expression during the GA
response of cereal aleurone layers, leading to
strach degradation in germinating grain.
• The effect of bioactive GA on pollen
development is also mediated by GAMYB
GAMYB regulates male fertility
• loss of function mutants of GAMYB in rice
have revealed the role of this transcription
factor as a positive regulator of floral organ
development.
• In some flowers, even on the same plant,
phenoptypic abnormalities are more
pronounced, including grossly malformed
pistils in the most severe cases
In wild type plants, bioactive GA is involved in regulating
programmed cell death of tapetal cell. in addiction, GA
regulates the biosynthesis of sporopollenim components
that provide a thought outer coat for pollen grains. there
are several lines of evidence
• GA up-regulates the expression of genes that encode
two enzymes in lipid metabolism during pollen
development. The genes, CYP703A3 and KAR, have
GAMYB binding sites by mutation prevents their
expression
• Reporter line studies showed that GAMYB-GUS (a
translational fusion to measure GAMYB protein level)
and CYP703A3: GUS (a promoter: GUS fusion to measure
the transcription of CYP703A3) are co-expressed in wild
type anthers, but CYP703A3:GUS is not expressed in the
gamyb mutant, in which there is no GAMYB protein
These and other findings provide compelling
evidence that in rice the formation of functional
pollen grains requires GA-induced expression of
GAMYB, which turns on transcription of genes
involved in the biosynthesis of pollen wall
components of its signal transduction pathway,
rice is male sterile.
Events downstream of GAMYB in rice
aleurone and anthers are quite different
• a comparison of GAMYB-induced gene
expression as determined by microarray
analyses of embrioless half grains and anthers
of rice have revealed some very surprising
results: although GAMYB regulatesmany
genes in both aleurone layers and in anthers,
the GAMYB-regulated genes in the two
systems are quite different.
MicroRNA regulate MYBs after transcription in
anthers but not aleurone
MicroRNAs are small gene-encoded RNAs containing
about 20 to 25 nucleotides that regulate the expression
of other genes postrattranscriptionally. MiRNAs bind to
ribonuclease to spesific mRNA molecules. the mRNAs
that hybridize to these mRNA guides are quickly
digested expression of the target genes. MicroRNAs can
alert GAMYB expression in rice anthers, but not in
aleurone. this shows another difference between GA-
induced, GAMYB-regulated gene expression in the
aleurone and in anthers
Giberellin responses: Stem Growth
Rapid stem elongation occurs in rice in response to
flash flooding, when the water level rises very
quickly. however this tends to deplete carbohydrate
reserves in the plants, and decreases their survival
after the flooding subsides. interestingly,
submergence-tolerant lines of rice, which elongate
less when underwater, have enhanced survival rates
after flooding. this sub mergence-tolerance is
directly linked to the presistence of DELLA proteins,
which reduces GA sensitivity
Giberellin stimulate cell elongation and
cell division
Giberellin stimulate both cell elongation and cell
division, as evidenced by increase in cell length
and cell number, in response to aplications of
bioactive Gas. Certain plants or plant organs
associated with GA-induced growth display
greater cell number than their respective control
plants.
• Internodes of tall peas have more cells than those
of dwarf peas, and the cells are longer
• Mitosis increases marked in the rib or ground
meristem of rosette long-day plants growing in
short days, after treatment with bioactive GA
• The dramatic stimulation of internode elongation
in deep-water rice when treated with GA, is due
in part to increased cell division activity in the
intercalary meristem found in spesific monocots
Giberellin has no effect on the osmotic parameters
but has consistenly been observed to cause
increase in both the mechanical extensibility of cell
walls and the stress relaxatio of the walls of living
cells. An analysis of pea genotypes differing in GA1
content or sensitivity showed that GA decreases the
wall yield threshold( the minimum force that will
cause wall extension). Thus, both GA and auxin
seem to exert their effects by modifying cell wall
properties
In the case of auxin, cell wall loosening appears
to be mediated, in part, by cell wall acidification.
However, thois does not appear to be
mechanism of GA action, since GA-stimulated
increase in proton extrusion has not been
demonstrated. On the other hand, GA is never
present in tissues in the complete absemnce of
auxin-induced wall acidification
 Gas regulate the transcription of cell
cycle kinases
• In submergence- indunced cell, GA activites
the transition from G1 to S phase, leading to
an increase in mitotic activity. the stimulation
of cell division results from a GA-indunced
expression of the genes for several cyclin-
dependent protein kinase (CDKs). the
transcription of the genes- first, those
regulating the transition from G2 to M phase-
is induced in the intercalary meristem by GA
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