Gibberellins GAs

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GibberellinsGAs
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Gibberellins (GAs)
a class of plant hormones affect several
important plant processes eg., seed
germination stem elongation flowering male
sterility
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Gibberellins
1926 Japanese scientist Gibberella
fujikuroi gibberellin A (terpenoid cpd) 1954,
1955 US and UK scientists 1958 GA1 in higher
plant GAx 1987 synthesis/metabolism
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Gibberellins
1991 84 GAs 1995 89 GAs 64 plants, 12
fungi 13 both 1996 more than 100 /
136 1997 genes being cloned
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Gibberellic acid (GA3)
End metabolic product in fungi Plant GA20
GA5 GA3 Commercial High activity Slow
degradation Similar to GA1 additional double
bond
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Gibberellins
GA4 GA7 nonpolar, slowly diffuse GA9
GA12 precursor GA29 GA34 deactivated
form Different tissues Different forms of GA
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Gibberellins
fungi algae bacteria moss
fern gymnosperm angiosperm
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Gibberellins
growing, differentiated tissues young,
developing, expanding leaves developing
seeds/fruit
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Gibberellins
elongated internode/petiole shoot/stem apex root
cap/tip xylem sap
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Synthesis and Metabolism Mevalonic acid
pathway in cytosol Non mevalonic acid
pathway in plastid
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Mevalonic acid pathway
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In higher plants from GA12 aldehyde Early
13-hydroxylation pathway (GA1) Non
13-hydroxylation pathway (GA4) with
GA20oxidase genes pathway shifted GA4
increased / GA1 decreased
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GA12 aldehyde precursor of GA derivatives by
oxidation (C20) and hydroxylation (C13 C3 C2)
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Vegetative tissue conserved synthetic
pathway 13-OH pathway to GA20 (C19-GA) then
3b-OH to GA1 except arabidopsis and
cucumber non 13-OH pathway to GA4 Reproductive
tissue/seed various pathways different forms
of GA
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From mevalonic acid (6C) GGPP (20C-linear
cpd) ent kaurene (1st specific cpd) GA12
aldehyde (first GA) GAx
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Gibberellins
Isoprene (5C) as basic unit ent-Gibberellane
skeleton tetracyclic diterpenoid cpd
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2 main types C20-GA and C19-GA GA derivatives
by modification of 4 rings C20 oxidation
CH3 CH2OH CHO COOH Hydroxylation at C2 C3
and C13 number, position stoichiometry
Loss of C20 (C20 to C19 GA)
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GA inactivation
2b-OH GA20 GA29 GA1 GA8 C20 oxidation
to COOH
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GA inactivation
Conjugation by glucose Glycosylation inactiv
e, storage and transport Glucose via COOH GA
glycoside Glucose via OH GA glycosyl ether
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GA synthesis mutants Pea na mutant
dwarf ent-kaurene GA12 aldehyde Pea le
mutant dwarf exogenous GA1 tall exogenous
GA20 no response cloned Le gene 3b
hydroxylase GA20 GA1
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Considering 2 loci na Le normal ent-kaurene Na
le normal GA20 Grafting 1. na Le scion Na
le stock tall 2. Na le scion Na Le
stock dwarf
Conclusion?
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GA mechanism in elongation
Unlike auxin (acidification) Increase wall
extensibility Decrease minimum force for wall
extension
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GA mechanism in elongation
By (may) decrease Ca concentration in the
wall increase Ca uptake into the cell reduce
crosslinking of lignin-related cpd (via
peroxidase)
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GA mechanism in germination Activate
transcription of a amylase gene In scutellum
and aleurone
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GA detection and assay Bioassay Easy but not
specific Fractionation Plant
response Lettuce hypocotyls elongation Microdrop
/dwarf rice a amylase production
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GC-MS Solvent extraction Chromatography
(polarity) GC (boiling point) MS
(mass) Identification and quantification High
sensitivity and more specific
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GA inhibitors
Inhibit ent-kaurene synthesis AMO1618 Cycocel
Inhibit ent-kaurene oxidation Paclobutrazol Un
iconazol Ancymidol Tetcyclasis Inhibit later
steps by dioxygenases Bx-1112 LAB1988999
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Hormone Responses Perception receptor Signal
transduction second messenger (cAMP, cGMP) G
protein Ca-Calmodulin enzyme transcription
factor
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At last step Gene expression Specific region
in promoter cis element DNA-binding protein
transcription factor
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GA studies
Exogenous GA / GA inhibitor GA mutant Gene
identification / Gene cloning Gene expression /
Transformation
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GA synthesis
Enzyme gene product of multigene family Each
gene with specific pattern of expression AtGA20ox
1 shoot growth AtGA20ox2 inflorescence
development AtGA20ox3 early seedling development
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At later steps of synthetic pathway
Genes controlled by GA, light and daylength GA
inhibit transcription of GA20oxidase (GA19 to
GA20) inhibit 3b hydroxylase promote 2b
hydroxylase
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Light promote conversion of GA1 to inactive
GA8 reducing shoot elongation
Negative feedback reduce production of active
GA20 and GA1
Daylength (LD) floral initiation activates
GA20oxidase activity GA53 to GA44 GA19 to GA
20
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Pea, Pisum sativum In de-etiolated pea seedling,
exposed to red, blue, far red, all reduce GA1
level
Lettuce Lactuca sativa seed germination Red
light activates LsGA3ox1 expression GA1
increase Far-red light inhibits
LsGA3ox1 Auxin promote GA1 production inhibit
deactivation steps to GA29 and GA8
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GA synthetic mutants
Arabidopsis seed germination assay 5
complementation groups (56 lines) ga1 ga2 ga3
ga4 and ga5 all recessive, dwarf, and male
sterile ga1 and ga2 reversed by ent-kaurene ga3
reversed by ent-kaurenal
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Genes
GA1 kaurene synthase (ent-CDP synthase) GA3
Cyt P450-dependent monooxygenase GA4 3b
hydroxylase GA5 GA20oxidase
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Pea (sln) decrease 2b hydroxylase
activity increase active GA tall plant with
light green leaves
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Signal transduction mutants Stature
mutants Decreased response to GA Increased
response to GA
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Decreased signaling mutants
Dwarf Complete phenocopy of GA-deficient
mutants No response to exogenous GA
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Decreased signaling mutants
Partially / fully dominant Arabidopsis
gai Maize D8 D9 Wheat Rht1 Rht2
Rht3 Negative regulators
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Arabidopsis gai mutant
Dwarf Higher level of active GA and
GA20oxidase Semidominant
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Arabidopsis gai mutant
gai1-1 51 bp inframe deletion loss of 17 amino
acid constitutive repressor
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Arabidopsis gai mutant
intragenic suppressor of gai loss of function
allele WT phenotype
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Maize D8 mutant Dwarf Higher level of active
GA 6 dominant alleles with different severity
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Wheat Rht mutant
8 dominant alleles with different severity Dwarf
prevent lodging Wheat N fertilizer increase
yield increase height Norin10 dwarf line 2
mutated loci Rht1 or Rht-B1b (chrs 4B) Rht2
or Rht-D1b (chrs 4D)
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All genes cloned deduced amino acid
sequence GAI / Rht / d8 homologs Conserved
domains I and II in N terminal gai
mutant deletion in domain I D8 / Rht mutation
in domain I and/or II N terminal essential for
GA response
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Increased signal transduction mutants
Similar to WT GA Tall by elongated
internodes Arabidopsis spy rga Barley sln
spy Rice slr Tomato pro Pea la
crys Recessive / Negative regulators
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Increased signal transduction mutants
Arabidopsis rga Identified by suppression
analysis of ga1-3 New mutant taller ga1-3 lt
ga1-3 lt WT new locus repressor of ga1-3
(rga)
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Increased signal transduction mutants
rga recessive (deletion mutation) increase stem
elongation reverse ga1-3 delayed flowering
time no effect on GA biosynthesis RGA negative
regulator Gene 82 homology to GAI especially
in N region
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Original gai mutant gain of function Loss of
function allele of GAI ? Phenotype
normal Increase paclobutrazol resistance Low GA
normal height
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At least two components in Arabidopsis GA
signaling pathway
GAI and RGA homopolymeric Serine / Threonine
residue leucine heptad for protein-protein
interaction putative nuclear localizing signal
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Barley sln
slender mutant recessive long internodes and
narrow leaves male sterile increase a-amylase
w/o GA low endogenous GA resistant to GA
synthesis inhibitors
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negative regulator sln x dwarf mutant sln
phenotype SLN GAI/RGA homolog Dominant allele
of SLN mutant Mutation in N terminal Dwarf
barley
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Rice slr
slender rice recessive phenocopy of barley sln 1
bp deletion in NLS domain (nuclear localization
signal )
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Rice slr
frame shift mutation stop codon truncated
protein SLR gene SLN homolog Modified SLR
17 aa deletion in DELLA domain Transformation
dwarf rice
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GA signal component Dicot / Monocot GAI RGA
Rht d8 SLN SLR Putative transcription
repressor
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Arabidopsis spy
spindly mutant, recessive paclobutrazol-resistant
long hypocotyls light green leaves early
flowering spy ga1-2 spy phenotypes spy
gai spy phenotypes
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Arabidopsis spy
SPY gene product O-GlcNAc transferase Signaling
molecule Involved in protein-protein
interaction Negative regulator
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Before responses Expression of GA-regulated
genes Protein-DNA interaction Transcription
factor cis elements
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Transcription factor GAMyb
Barley HvGAMyb Bind specific sequence
in promoter of a-amylase gene Increase gene
expression Overexpression of HvGAMyb gene GA
treatment
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Arabidopsis GAMyb-like genes AtMyb33 AtMyb65
AtMyb101 Functional homologs of barley
GAMyb Transform barley aleurone with
AtMyb33 Activate a-amylase production
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Arabidopsis facultative LD plants Transfer
plants from SD to LD 11x increase of GA1 3x
increase of GA4 increase AtMyb33 expression
in shoot apex shoot apex transition to
flowering
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Potential target for AtMyb LFY promoter LEAFY
meristem-identity gene Evidence AtMyb
binding to a specific 8-bp sequence in LFY
promoter
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cis elements specific regions in
promoter transcription factor binding
site identified by deletion or site specific
mutagenesis gene expression after promoter
modification
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Conserved sequences among GA-regulated genes
- amylase box TATCCAT - GARE TAACAA/GA -
Pyrimidine box C/TCTTTTAC/T
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GA and a-amylase production Perception at
membrane receptors Increase intracellular
Ca Decrease intracellular pH Increase
CaM Increase cGMP Increase GAMyb
transcription Increase a-amylase activity Some
protein phosphorylation
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