gybbrellin and its role in plant

1. 1. Gibberellins GAs
2. 2. a class of plant hormones affect several important plant processes eg., seed germination
stem elongation flowering male sterility Gibberellins (GAs)
3. 3. 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
4. 4. Gibberellins 1991 84 GAs 1995 89 GAs 64 plants, 12 fungi 13 both 1996 more than 100 /
136 1997 genes being cloned
5. 5. Gibberellic acid (GA3) End metabolic product in fungi Plant GA20 GA5 GA3 Commercial
High activity Slow degradation Similar to GA1 additional double bond
6. 6. Gibberellins GA4 GA7 nonpolar, slowly diffuse GA9 GA12 precursor GA29 GA34
deactivated form Different tissues Different forms of GA
7. 7. fungi algae bacteria moss fern gymnosperm angiosperm Gibberellins
8. 8. growing, differentiated tissues young, developing, expanding leaves developing seeds/fruit
Gibberellins
9. 9. elongated internode/petiole shoot/stem apex root cap/tip xylem sap Gibberellins
10. 10. Synthesis and Metabolism Mevalonic acid pathway in cytosol Non mevalonic acid
pathway in plastid
11. 11. Mevalonic acid pathway
12. 12. In higher plants from GA12 aldehyde Early 13-hydroxylation pathway (GA1) Non 13hydroxylation pathway (GA4) with GA20oxidase genes: pathway shifted GA4 increased /
GA1 decreased
13. 13. GA12 aldehyde: precursor of GA derivatives by oxidation (C20) and hydroxylation (C13
C3 C2)
14. 14. Vegetative tissue: conserved synthetic pathway 13-OH pathway to GA20 (C19-GA) then
3-OH to GA1 except: arabidopsis and cucumber non 13-OH pathway to GA4 Reproductive
tissue/seed: various pathways different forms of GA
15. 15. From mevalonic acid (6C) GGPP (20C-linear cpd) ent kaurene (1st specific cpd) GA12
aldehyde (first GA) GAx
16. 16. Isoprene (5C) as basic unit ent-Gibberellane skeleton tetracyclic diterpenoid cpd
Gibberellins
17. 17. 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)
18. 18. * 2-OH: GA20 GA29 GA1 GA8 * C20 oxidation to COOH GA inactivation
19. 19. * Conjugation by glucose Glycosylation: inactive, storage and transport Glucose via
COOH: GA glycoside Glucose via OH: GA glycosyl ether GA inactivation
20. 20. 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:3 hydroxylase
GA20 GA1

21. 21. 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?
22. 22. Unlike auxin (acidification) Increase wall extensibility Decrease minimum force for wall
extension GA mechanism in elongation
23. 23. By (may) decrease Ca concentration in the wall increase Ca uptake into the cell reduce
crosslinking of lignin-related cpd (via peroxidase) GA mechanism in elongation
24. 24. GA mechanism in germination Activate transcription of amylase gene In scutellum and
aleurone
25. 25. GA detection and assay Bioassay Easy but not specific Fractionation Plant response
Lettuce hypocotyls elongation Microdrop/dwarf rice amylase production
26. 26. GC-MS Solvent extraction Chromatography (polarity) GC (boiling point) MS (mass)
Identification and quantification High sensitivity and more specific
27. 27. Inhibit ent-kaurene synthesis AMO1618 Cycocel Inhibit ent-kaurene oxidation
Paclobutrazol Uniconazol Ancymidol Tetcyclasis Inhibit later steps by dioxygenases Bx-1112
LAB1988999 GA inhibitors
28. 28. Hormone Responses Perception: receptor Signal transduction: second messenger
(cAMP, cGMP) G protein Ca-Calmodulin enzyme transcription factor
29. 29. At last step Gene expression Specific region in promoter cis element DNA-binding protein
transcription factor
30. 30. Exogenous GA / GA inhibitor GA mutant Gene identification / Gene cloning Gene
expression / Transformation GA studies
31. 31. Enzyme: gene product of multigene family Each gene with specific pattern of expression
AtGA20ox1: shoot growth AtGA20ox2: inflorescence development AtGA20ox3: early seedling
development GA synthesis
32. 32. Genes controlled by GA, light and daylength GA: inhibit transcription of GA20oxidase
(GA19 to GA20) inhibit 3 hydroxylase promote 2 hydroxylase At later steps of synthetic
pathway
33. 33. Light: promote conversion of GA1 to inactive GA8 reducing shoot elongation Negative
feedback: reduce production of active GA20 and GA1Daylength (LD): floral initiation
activates GA20oxidase activity GA53 to GA44 GA19 to GA20
34. 34. 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 Pea, Pisum sativum In de-etiolated pea seedling, exposed to red,
blue, far red, all reduce GA1 level
35. 35. 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 GA synthetic mutants
36. 36. GA1 kaurene synthase (ent-CDP synthase) GA3 Cyt P450-dependent monooxygenase
GA4 3 hydroxylase GA5 GA20oxidase Genes
37. 37. Pea (sln) decrease 2 hydroxylase activity increase active GA tall plant with light green
leaves
38. 38. Signal transduction mutants Stature mutants Decreased response to GA Increased
response to GA

39. 39. Dwarf Complete phenocopy of GA-deficient mutants No response to exogenous GA

Decreased signaling mutants
40. 40. Partially / fully dominant Arabidopsis gai Maize D8 D9 Wheat Rht1 Rht2 Rht3 Negative
regulators Decreased signaling mutants
41. 41. Dwarf Higher level of active GA and GA20oxidase Semidominant Arabidopsis gai mutant
42. 42. gai1-1 51 bp inframe deletion loss of 17 amino acid constitutive repressor Arabidopsis gai
mutant
43. 43. Arabidopsis gai mutant intragenic suppressor of gai loss of function allele WT phenotype
44. 44. Maize D8 mutant Dwarf Higher level of active GA 6 dominant alleles with different
severity
45. 45. 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) Wheat Rht mutant
46. 46. 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*
47. 47. Similar to WT + GA Tall by elongated internodes Arabidopsis spy rga Barley sln spy Rice
slr Tomato pro Pea la crys Recessive / Negative regulators Increased signal transduction
mutants
48. 48. Arabidopsis rga Identified by suppression analysis of ga1-3 New mutant: taller ga1-3 <
ga1-3* < WT new locus: repressor of ga1-3 (rga) Increased signal transduction mutants
49. 49. 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 Increased signal transduction mutants
50. 50. Original gai mutant: gain of function Loss of function allele of GAI ? Phenotype: normal
Increase paclobutrazol resistance Low GA = normal height
51. 51. 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
52. 52. slender mutant recessive long internodes and narrow leaves male sterile increase amylase w/o GA low endogenous GA resistant to GA synthesis inhibitors Barley sln
53. 53. negative regulator sln x dwarf mutant = sln phenotype SLN = GAI/RGA homolog
Dominant allele of SLN mutant Mutation in N terminal Dwarf barley
54. 54. slender rice recessive phenocopy of barley sln 1 bp deletion in NLS domain (nuclear
localization signal ) Rice slr
55. 55. frame shift mutation stop codon truncated protein SLR gene = SLN homolog Modified
SLR: 17 aa deletion in DELLA domain Transformation: dwarf rice Rice slr
56. 56. GA signal component Dicot / Monocot GAI RGA Rht d8 SLN SLR Putative transcription
repressor
57. 57. spindly mutant, recessive paclobutrazol-resistant long hypocotyls light green leaves early
flowering spy ga1-2 = spy phenotypes spy gai = spy phenotypes Arabidopsis spy
58. 58. SPY gene product: O-GlcNAc transferase Signaling molecule Involved in protein-protein
interaction Negative regulator Arabidopsis spy

59. 59. Before responses Expression of GA-regulated genes: Protein-DNA interaction

Transcription factor cis elements
60. 60. Barley: HvGAMyb Bind specific sequence in promoter of -amylase gene Increase gene
expression Overexpression of HvGAMyb gene = GA treatment Transcription factor: GAMyb
61. 61. Arabidopsis: GAMyb-like genes AtMyb33 AtMyb65 AtMyb101 Functional homologs of
barley GAMyb Transform barley aleurone with AtMyb33 Activate -amylase production
62. 62. 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
63. 63. Potential target for AtMyb LFY promoter LEAFY: meristem-identity gene Evidence AtMyb
binding to a specific 8-bp sequence in LFY promoter
64. 64. cis elements specific regions in promoter transcription factor binding site identified by
deletion or site specific mutagenesis: gene expression after promoter modification
65. 65. - amylase box: TATCCAT - GARE: TAACAA/GA - Pyrimidine box: C/TCTTTTAC/T
Conserved sequences among GA-regulated genes
66. 66. GA and -amylase production Perception at membrane receptors Increase intracellular
Ca Decrease intracellular pH Increase [CaM] Increase cGMP Increase GAMyb transcription
Increase -amylase activity Some protein phosphorylation