4. Gibberellins (GAs)
a class of plant hormones
affect several important plant processes
eg., seed germination
stem elongation
flowering
male sterility
5.
6. 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
7. Gibberellins
1991 84 GAs
1995 89 GAs
64 plants, 12 fungi
13 both
1996 more than 100 / 136
1997 genes being cloned
8. Gibberellic acid (GA3)
End metabolic product in fungi
Plant GA20 GA5 GA3
Commercial
High activity
Slow degradation
Similar to GA1
additional double bond
9. Gibberellins
GA4 GA7 nonpolar, slowly diffuse
GA9 GA12 precursor
GA29 GA34 deactivated form
Different tissues
Different forms of GA
17. 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
19. 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
20.
21. From mevalonic acid (6C)
GGPP (20C-linear cpd)
ent kaurene (1st specific cpd)
GA12 aldehyde (first GA)
GAx
25. 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)
28. GA inactivation
* Conjugation by glucose
Glycosylation:
inactive, storage and transport
Glucose via COOH: GA glycoside
Glucose via OH: GA glycosyl ether
29. GA synthesis mutants
Pea na mutant: dwarf
ent-kaurene GA12 aldehyde
Pea le mutant: dwarf
exogenous GA1 tall
exogenous GA20no response
cloned Le gene: 3b hydroxylase
GA20 GA1
30. 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?
31. GA mechanism in elongation
Unlike auxin (acidification)
Increase wall extensibility
Decrease minimum force
for wall extension
32. 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)
33. GA mechanism in germination
Activate transcription of
a amylase gene
In scutellum and aleurone
34. GA detection and assay
Bioassay
Easy but not specific
Fractionation
Plant response
Lettuce hypocotyls elongation
Microdrop/dwarf rice
a amylase production
35. GC-MS
Solvent extraction
Chromatography (polarity)
GC (boiling point)
MS (mass)
Identification and quantification
High sensitivity and more specific
36. GA inhibitors
Inhibit ent-kaurene synthesis
AMO1618
Cycocel
Inhibit ent-kaurene oxidation
Paclobutrazol Uniconazol
Ancymidol Tetcyclasis
Inhibit later steps by dioxygenases
Bx-1112
LAB1988999
37. Hormone Responses
Perception: receptor
Signal transduction:
second messenger (cAMP, cGMP)
G protein
Ca-Calmodulin
enzyme
transcription factor
38. At last step
Gene expression
Specific region in promoter
cis element
DNA-binding protein
transcription factor
39. GA studies
Exogenous GA / GA inhibitor
GA mutant
Gene identification / Gene cloning
Gene expression / Transformation
40. 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
41. 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
42. Negative Light: feedback: Daylength promote reduce conversion (LD): production floral of init GA1 ioaft iaocntive to inactive GA20 GA8
and GA1
activates GA20oxidase activity
reducing shoot elongation
GA53 to GA44
GA19 to GA20
43. 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
44. 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
51. Arabidopsis gai mutant
gai1-1
51 bp inframe deletion
loss of 17 amino acid
constitutive repressor
52. Arabidopsis gai mutant
intragenic suppressor of gai
loss of function allele
WT phenotype
53. Maize D8 mutant
Dwarf
Higher level of active GA
6 dominant alleles
with different severity
54. 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)
55. 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*
56. Increased signal transduction mutants
Similar to WT + GA
Tall by elongated internodes
Arabidopsisspy rga
Barley sln spy
Rice slr
Tomato pro
Pea la crys
Recessive / Negative regulators
57. Increased signal transduction mutants
Arabidopsis rga
Identified by suppression analysis of ga1-3
New mutant: taller
ga1-3 < ga1-3* < WT
new locus: repressor of ga1-3 (rga)
58. 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
59. Original gai mutant: gain of function
Loss of function allele of GAI ?
Phenotype: normal
Increase paclobutrazol resistance
Low GA = normal height
60. 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
61. 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
62. negative regulator
sln x dwarf mutant = sln phenotype
SLN= GAI/RGA homolog
Dominant allele of SLN mutant
Mutation in N terminal
Dwarf barley
63. Rice slr
slender rice
recessive
phenocopy of barley sln
1 bp deletion in NLS domain
(nuclear localization signal )
64. Rice slr
frame shift mutation
stop codon
truncated protein
SLR gene = SLN homolog
Modified SLR:
17 aa deletion in DELLA domain
Transformation: dwarf rice
65. GA signal component
Dicot / Monocot
GAI RGA Rht d8 SLN SLR
Putative transcription repressor
66. Arabidopsis spy
spindly mutant, recessive
paclobutrazol-resistant
long hypocotyls
light green leaves
early flowering
spy ga1-2 = spy phenotypes
spy gai = spy phenotypes
68. Before responses
Expression of GA-regulated genes:
Protein-DNA interaction
Transcription factor
cis elements
69. Transcription factor: GAMyb
Barley: HvGAMyb
Bind specific sequence in
promoter of a-amylase gene
Increase gene expression
Overexpression of HvGAMyb gene
= GA treatment
70. Arabidopsis: GAMyb-like genes
AtMyb33 AtMyb65 AtMyb101
Functional homologs of barley GAMyb
Transform barley aleurone with AtMyb33
Activate a-amylase production
71. 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
72. Potential target for AtMyb
LFY promoter
LEAFY: meristem-identity gene
Evidence AtMyb binding
to a specific 8-bp sequence
in LFY promoter
73. cis elements
specific regions in promoter
transcription factor binding site
identified by deletion or
site specific mutagenesis:
gene expression after promoter modification
75. 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
