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Gibberellins
GAs
a class of plant hormones
affect several important plant processes
eg., seed germination
stem elongation
flowering
male st...
Gibberellins
1926 Japanese scientist
Gibberella fujikuroi
gibberellin A (terpenoid cpd)
1954, 1955 US and UK scientists
19...
Gibberellins
1991 84 GAs
1995 89 GAs
64 plants, 12 fungi
13 both
1996 more than 100 / 136
1997 genes being cloned
Gibberellic acid (GA3)
End metabolic product in fungi
Plant GA20 GA5 GA3
Commercial
High activity
Slow degradation
Similar...
Gibberellins
GA4 GA7 nonpolar, slowly diffuse
GA9 GA12 precursor
GA29 GA34 deactivated form
Different tissues
Different fo...
fungi algae
bacteria
moss fern
gymnosperm
angiosperm
Gibberellins
growing, differentiated tissues
young, developing, expanding leaves
developing seeds/fruit
Gibberellins
elongated internode/petiole
shoot/stem apex
root cap/tip
xylem sap
Gibberellins
Synthesis and Metabolism
Mevalonic acid pathway
in cytosol
Non mevalonic acid pathway
in plastid
Mevalonic acid pathway
In higher plants
from GA12 aldehyde
Early 13-hydroxylation pathway
(GA1)
Non 13-hydroxylation pathway
(GA4)
with GA20oxida...
GA12 aldehyde: precursor of GA derivatives
by oxidation (C20) and hydroxylation (C13 C3 C2)
Vegetative tissue: conserved synthetic pathway
13-OH pathway to GA20 (C19-GA)
then 3β-OH to GA1
except: arabidopsis and cu...
From mevalonic acid (6C)
GGPP (20C-linear cpd)
ent kaurene (1st specific cpd)
GA12 aldehyde (first GA)
GAx
Isoprene (5C) as basic unit
ent-Gibberellane skeleton
tetracyclic diterpenoid cpd
Gibberellins
2 main types:
C20-GA and C19-GA
GA derivatives by modification of 4 rings
* C20 oxidation: CH3 CH2OH CHO COOH
* Hydroxylat...
* 2β-OH: GA20 GA29
GA1 GA8
* C20 oxidation to COOH
GA inactivation
* Conjugation by glucose
Glycosylation:
inactive, storage and transport
Glucose via COOH: GA glycoside
Glucose via OH: GA ...
GA synthesis mutants
Pea na mutant: dwarf
ent-kaurene GA12 aldehyde
Pea le mutant: dwarf
exogenous GA1 tall
exogenous GA20...
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 L...
Unlike auxin (acidification)
Increase wall extensibility
Decrease minimum force
for wall extension
GA mechanism in elongat...
By (may)
decrease Ca concentration in the wall
increase Ca uptake into the cell
reduce crosslinking of lignin-related cpd
...
GA mechanism in germination
Activate transcription of
α amylase gene
In scutellum and aleurone
GA detection and assay
Bioassay
Easy but not specific
Fractionation
Plant response
Lettuce hypocotyls elongation
Microdrop...
GC-MS
Solvent extraction
Chromatography (polarity)
GC (boiling point)
MS (mass)
Identification and quantification
High sen...
Inhibit ent-kaurene synthesis
AMO1618
Cycocel
Inhibit ent-kaurene oxidation
Paclobutrazol Uniconazol
Ancymidol Tetcyclasis...
Hormone Responses
Perception: receptor
Signal transduction:
second messenger (cAMP, cGMP)
G protein
Ca-Calmodulin
enzyme
t...
At last step
Gene expression
Specific region in promoter
cis element
DNA-binding protein
transcription factor
Exogenous GA / GA inhibitor
GA mutant
Gene identification / Gene cloning
Gene expression / Transformation
GA studies
Enzyme: gene product of multigene family
Each gene with specific pattern of expression
AtGA20ox1: shoot growth
AtGA20ox2: ...
Genes controlled by GA, light and daylength
GA: inhibit transcription of GA20oxidase
(GA19 to GA20)
inhibit 3β hydroxylase...
Light: promote conversion of GA1 to inactive GA8
reducing shoot elongation
Negative feedback: reduce production of active ...
Lettuce: Lactuca sativa seed germination
Red light: activates LsGA3ox1 expression
GA1 increase
Far-red light: inhibits LsG...
Arabidopsis:
seed germination assay
5 complementation groups (56 lines)
ga1 ga2 ga3 ga4 and ga5
all recessive, dwarf, and ...
GA1 kaurene synthase (ent-CDP synthase)
GA3 Cyt P450-dependent monooxygenase
GA4 3β hydroxylase
GA5 GA20oxidase
Genes
Pea (sln)
decrease 2β hydroxylase activity
increase active GA
tall plant with light green leaves
Signal transduction mutants
Stature mutants
Decreased response to GA
Increased response to GA
Dwarf
Complete phenocopy of
GA-deficient mutants
No response to exogenous GA
Decreased signaling mutants
Partially / fully dominant
Arabidopsis gai
Maize D8 D9
Wheat Rht1 Rht2 Rht3
Negative regulators
Decreased signaling mutants
Dwarf
Higher level of active GA
and GA20oxidase
Semidominant
Arabidopsis gai mutant
gai1-1
51 bp inframe deletion
loss of 17 amino acid
constitutive repressor
Arabidopsis gai mutant
Arabidopsis gai mutant
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
8 dominant alleles with different severity
Dwarf: prevent lodging
Wheat + N fertilizer: increase yield
increase height
Nor...
All genes cloned:
deduced amino acid sequence
GAI / Rht / d8 homologs
Conserved domains I and II in N terminal
gai mutant:...
Similar to WT + GA
Tall by elongated internodes
Arabidopsis spy rga
Barley sln spy
Rice slr
Tomato pro
Pea la crys
Recessi...
Arabidopsis rga
Identified by suppression analysis of ga1-3
New mutant: taller
ga1-3 < ga1-3* < WT
new locus: repressor of...
rga: recessive (deletion mutation)
increase stem elongation
reverse ga1-3 delayed flowering time
no effect on GA biosynthe...
Original gai mutant: gain of function
Loss of function allele of GAI ?
Phenotype: normal
Increase paclobutrazol resistance...
At least two components in
Arabidopsis GA signaling pathway
GAI and RGA
homopolymeric Serine / Threonine residue
leucine h...
slender mutant
recessive
long internodes and narrow leaves
male sterile
increase α-amylase w/o GA
low endogenous GA
resist...
negative regulator
sln x dwarf mutant = sln phenotype
SLN = GAI/RGA homolog
Dominant allele of SLN mutant
Mutation in N te...
slender rice
recessive
phenocopy of barley sln
1 bp deletion in NLS domain
(nuclear localization signal )
Rice slr
frame shift mutation
stop codon
truncated protein
SLR gene = SLN homolog
Modified SLR:
17 aa deletion in DELLA domain
Tran...
GA signal component
Dicot / Monocot
GAI RGA Rht d8 SLN SLR
Putative transcription repressor
spindly mutant, recessive
paclobutrazol-resistant
long hypocotyls
light green leaves
early flowering
spy ga1-2 = spy pheno...
SPY gene product:
O-GlcNAc transferase
Signaling molecule
Involved in protein-protein interaction
Negative regulator
Arabi...
Before responses
Expression of GA-regulated genes:
Protein-DNA interaction
Transcription factor
cis elements
Barley: HvGAMyb
Bind specific sequence in
promoter of α-amylase gene
Increase gene expression
Overexpression of HvGAMyb ge...
Arabidopsis: GAMyb-like genes
AtMyb33 AtMyb65 AtMyb101
Functional homologs of barley GAMyb
Transform barley aleurone with ...
Arabidopsis: facultative LD plants
Transfer plants from SD to LD
11x increase of GA1
3x increase of GA4
increase AtMyb33 e...
Potential target for AtMyb
LFY promoter
LEAFY: meristem-identity gene
Evidence AtMyb binding
to a specific 8-bp sequence
i...
cis elements
specific regions in promoter
transcription factor binding site
identified by deletion or
site specific mutage...
- amylase box: TATCCAT
- GARE: TAACAA/GA
- Pyrimidine box: C/TCTTTTAC/T
Conserved sequences among
GA-regulated genes
GA and α-amylase production
Perception at membrane receptors
Increase intracellular Ca
Decrease intracellular pH
Increase ...
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
gybbrellin and its role in plant
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gybbrellin and its role in plant

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deepak yadav --gybbrellin

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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 13-hydroxylation 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 3β-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. * 2β-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:3β 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 α 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 α 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 3β hydroxylase promote 2β 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 3β hydroxylase GA5 GA20oxidase Genes
  37. 37. Pea (sln) decrease 2β 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 α-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 α-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 α-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 α-amylase production Perception at membrane receptors Increase intracellular Ca Decrease intracellular pH Increase [CaM] Increase cGMP Increase GAMyb transcription Increase α-amylase activity Some protein phosphorylation

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