Hormone
Responses
Perception: receptor
Signal transduction: second messenger (cAMP, cGMP)
G protein
Ca-Calmodulin
enzyme
transcription factor
At last step: gene expression
Specific region in promoter (cis element)
DNA-binding protein (transcription factor)
GA studies
Exogenous GA
GA inhibitor
GA mutant
Gene identification/cloning/expression/transformation
GA synthesis
Enzyme: gene product of multigene family
Each gene with specific pattern of expression
AtGA20ox1: shoot growth
AtGA20ox2: inflorescence development
AtGA20ox3: early seedling development
At later steps of synthetic pathway
Genes controlled by GA, light and daylength
GA: - transcription of GA20oxidase (GA19 to GA20)
- 3 hydroxylase
+ 2 hydroxylase
Red light: activates LsGA3ox1 expression
Far-red light: inhibits LsGA3ox1
Daylength (LD): activates GA20oxidase activity
GA53
to GA44
GA19
to GA20
GA synthetic mutants
Arabidopsis:
genes:
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
GA1
GA3
GA4
GA5
kaurene synthase (ent-CDP synthase)
Cyt P450-dependent monooxygenase
3 hydroxylase
GA20oxidase
Pea: decrease 2 hydroxylase activity
increase active GA
tall plant with light green leaves
Signal transduction mutants
Stature mutants
Decreased or increased response to GA
Decreased signal transduction mutants
Dwarf
Complete phenocopy of GA-deficient mutants
No response to exogenous GA
Partially/fully dominant
Arabidopsis gai
Wheat Rht1 Rht2 Rht3
Maize D8 D9
Negative regulators
Arabidopsis gai mutant
Dwarf
Higher level of active GA and GA20oxidase
Semidominant
gai1-1
51 bp inframe deletion
loss of 17 amino acid
constitutive repressor
intragenic suppressor of gai
loss of function allele
WT phenotype
Maize D8 mutant
Dwarf
Higher level of active GA
6 dominant alleles with different severity
Wheat Rht mutant
8 dominant alleles with different severity
Dwarf: prevent lodging
wheat + N fertilizer = inc yield (also inc height)
Norin10: dwarf line
2 mutated loci: Rht-B1b (chrs 4B)
Rht-D1b (chrs 4D)
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*
Increased signal transduction mutants
Similar to WT + GA
Tall by elongated internodes
Arabidopsis
Barley
Rice
Tomato
Pea
spy rga
sln spy
slr
pro
la crys
Recessive
Negative regulators
Arabidopsis rga
Identified by suppression analysis of ga1-3
New mutant: taller
ga1-3 < ga1-3* < WT
new locus: repressor of ga1-3 (rga)
rga: recessive (deletion mutation)
inc. stem elongation
reverse ga1-3 delayed flowering time
no effect on GA biosynthesis
RGA: negative regulator
Gene: 82% homology to GAI (esp. in N region)
Original gai mutant: gain of function
Loss of function allele of GAI ?
Phenotype: normal
Inc. paclobutrazol resistance
Low GA = normal height
Barley sln
slender mutant, recessive
long internodes, narrow leaves
male sterile, inc. -amylase w/o GA
low endogenous GA
resistant to GA synthesis inhibitors
negative regulator
sln x dwarf mutant = sln phenotype
SLN = GAI/RGA homolog
Dominant allele of SLN mutant
Mutation in N terminal
Dwarf barley
Rice slr
slender rice
recessive
phenocopy of barley sln
1 bp deletion in nu. localization signal (NLS) domain
frame shift mutation, stop codon, truncated protein
SLR gene = SLN homolog
Modified SLR: 17 aa deletion in DELLA domain
Transformation: dwarf rice
GA signal component
Dicot / Monocot
GAI RGA Rht d8 SLN SLR
Putative transcription repressor
Arabidopsis spy
spindly mutant, recessive
paclobutrazol-resistant
long hypocotyls, light green leaves
early flowering
spy ga1-2 = spy phenotypes
spy gai = spy phenotypes
SPY gene product: O-GlcNAc transferase
Signaling molecule
Involved in protein-protein interaction
Negative regulator
Before responses
Expression of GA-regulated genes:
Protein-DNA interaction
Transcription factor and cis elements
Transcription factor: GAMyb
Barley: HvGAMyb
Bind specific sequence in promoter of -amylase gene
Increase gene expression
Overexpression of HvGAMyb gene = GA treatment
Arabidopsis: GAMyb-like genes
AtMyb33 AtMyb65 AtMyb101
Functional homologs of barley GAMyb
Transform barley aleurone with AtMyb33
Activate -amylase production
Arabidopsis: facultative LD plants
Transfer plants from SD to LD
11x inc. of GA1
3x inc. of GA4
inc. AtMyb33 expression in shoot apex
shoot apex transition to flowering
Potential target for AtMyb: LFY promoter
LEAFY: meristem-identity gene
Evidence AtMyb binding
to a specific 8-bp sequence in LFY promoter
cis elements
specific regions in promoter
transcription factor binding site
identified by deletion or site specific mutagenesis:
gene expression after promoter modification
conserved sequences among GA-regulated genes
- amylase box: TATCCAT
- GARE:
TAACAA/GA
- Pyrimidine box:
C/TCTTTTAC/T
GA and -amylase production
Perception at membrane receptors
Inc. intracellular Ca
Dec. intracellular pH
Inc. [CaM]
Inc. cGMP
Inc. GAMyb transcription
Inc. -amylase activity
Some protein phosphorylation