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Gibberellins GAs. a class of plant hormones affect several important plant processes eg., seed germination stem elongation flowering male sterility Gibberellins.

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Presentation on theme: "Gibberellins GAs. a class of plant hormones affect several important plant processes eg., seed germination stem elongation flowering male sterility Gibberellins."— Presentation transcript:

1 Gibberellins GAs

2 a class of plant hormones affect several important plant processes eg., seed germination stem elongation flowering male sterility Gibberellins (GAs)

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4 Gibberellins 1926 Japanese scientist Gibberella fujikuroi gibberellin A (terpenoid cpd) 1954, 1955US and UK scientists 1958GA1 in higher plant GAx 1987synthesis/metabolism

5 Gibberellins GAs GAs 64 plants, 12 fungi 13 both 1996more than 100 / genes being cloned

6 Gibberellic acid (GA3) End metabolic product in fungi Plant GA20 GA5GA3 Commercial High activity Slow degradation Similar to GA1 additional double bond

7 Gibberellins GA4 GA7nonpolar, slowly diffuse GA9 GA12precursor GA29 GA34deactivated form Different tissues Different forms of GA

8 fungi algae bacteria moss fern gymnosperm angiosperm Gibberellins

9 growing, differentiated tissues young, developing, expanding leaves developing seeds/fruit Gibberellins

10 elongated internode/petiole shoot/stem apex root cap/tip xylem sap Gibberellins

11 Synthesis and Metabolism Mevalonic acid pathway in cytosol Non mevalonic acid pathway in plastid

12 Mevalonic acid pathway

13 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

14 GA12 aldehyde: precursor of GA derivatives by oxidation (C20) and hydroxylation (C13 C3 C2)

15 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

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17 Frommevalonic acid (6C) GGPP (20C-linear cpd) ent kaurene (1st specific cpd) GA12 aldehyde (first GA) GAx

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20 Isoprene (5C) as basic unit ent-Gibberellane skeleton tetracyclic diterpenoid cpd Gibberellins

21 2 main types: C20-GA and C19-GA GA derivatives by modification of 4 rings * C20 oxidation: CH 3 CH 2 OH CHO COOH * Hydroxylation at C2 C3 and C13: number, position stoichiometry * Loss of C20 (C20 to C19 GA)

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23 * 2  -OH:GA20GA29 GA1GA8 * C20 oxidation to COOH GA inactivation

24 * Conjugation by glucose Glycosylation: inactive, storage and transport Glucose via COOH: GA glycoside Glucose via OH: GA glycosyl ether GA inactivation

25 GA synthesis mutants Pea na mutant: dwarf ent-kaureneGA12 aldehyde Pea le mutant: dwarf exogenous GA1tall exogenous GA20no response cloned Le gene:3  hydroxylase GA20 GA1

26 Considering 2 loci na Lenormal ent-kaurene Na lenormal GA20 Grafting 1. na Lescion Na lestocktall 2.Na lescion Na Le stockdwarf Conclusion?

27 Unlike auxin (acidification) Increase wall extensibility Decrease minimum force for wall extension GA mechanism in elongation

28 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

29 GA mechanism in germination Activate transcription of  amylase gene In scutellum and aleurone

30 GA detection and assay Bioassay Easy but not specific Fractionation Plant response Lettuce hypocotyls elongation Microdrop/dwarf rice  amylase production

31 GC-MS Solvent extraction Chromatography (polarity) GC (boiling point) MS (mass) Identification and quantification High sensitivity and more specific

32 Inhibit ent-kaurene synthesis AMO1618 Cycocel Inhibit ent-kaurene oxidation PaclobutrazolUniconazol AncymidolTetcyclasis Inhibit later steps by dioxygenases Bx-1112 LAB GA inhibitors

33 HormoneResponses Perception: receptor Signal transduction: second messenger (cAMP, cGMP) G protein Ca-Calmodulin enzyme transcription factor

34 At last step Gene expression Specific region in promoter cis element DNA-binding protein transcription factor

35 Exogenous GA / GA inhibitor GA mutant Gene identification / Gene cloning Gene expression / Transformation GA studies

36 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

37 Genes controlled by GA, light and daylength GA: inhibit transcription of GA20oxidase (GA 19 to GA 20 ) inhibit 3  hydroxylase promote 2  hydroxylase At later steps of synthetic pathway

38 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 GA 53 toGA 44 GA 19 toGA 20

39 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

40 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

41 GA1 kaurene synthase (ent-CDP synthase) GA3 Cyt P450-dependent monooxygenase GA4 3  hydroxylase GA5 GA20oxidase Genes

42 Pea (sln) decrease 2  hydroxylase activity increase active GA tall plant with light green leaves

43 Signal transduction mutants Stature mutants Decreased response to GA Increased response to GA

44 Dwarf Complete phenocopy of GA-deficient mutants No response to exogenous GA Decreased signaling mutants

45 Partially / fully dominant Arabidopsis gai Maize D8 D9 Wheat Rht1 Rht2 Rht3 Negative regulators Decreased signaling mutants

46 Dwarf Higher level of active GA and GA20oxidase Semidominant Arabidopsis gai mutant

47 gai bp inframe deletion loss of 17 amino acid constitutive repressor Arabidopsis gai mutant

48 intragenic suppressor of gai loss of function allele WT phenotype

49 Maize D8 mutant Dwarf Higher level of active GA 6 dominant alleles with different severity

50 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

51 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*

52 Similar to WT + GA Tall by elongated internodes Arabidopsisspy rga Barleysln spy Riceslr Tomatopro Peala cry s Recessive / Negative regulators Increased signal transduction mutants

53 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

54 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

55 Original gai mutant: gain of function Loss of function allele of GAI ? Phenotype: normal Increase paclobutrazol resistance Low GA = normal height

56 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

57 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

58 negative regulator sln x dwarf mutant = sln phenotype SLN= GAI/RGA homolog Dominant allele of SLN mutant Mutation in N terminal Dwarf barley

59 slender rice recessive phenocopy of barley sln 1 bp deletion in NLS domain (nuclear localization signal ) Rice slr

60 frame shift mutation stop codon truncated protein SLR gene = SLN homolog Modified SLR: 17 aa deletion in DELLA domain Transformation: dwarf rice Rice slr

61 GA signal component Dicot / Monocot GAI RGA Rht d8 SLN SLR Putative transcription repressor

62 spindly mutant, recessive paclobutrazol-resistant long hypocotyls light green leaves early flowering spy ga1-2 = spy phenotypes spy gai= spy phenotypes Arabidopsis spy

63 SPY gene product: O-GlcNAc transferase Signaling molecule Involved in protein-protein interaction Negative regulator Arabidopsis spy

64 Before responses Expression of GA-regulated genes: Protein-DNA interaction Transcription factor cis elements

65 Barley: HvGAMyb Bind specific sequence in promoter of  -amylase gene Increase gene expression Overexpression of HvGAMyb gene = GA treatment Transcription factor: GAMyb

66 Arabidopsis: GAMyb-like genes AtMyb33 AtMyb65 AtMyb101 Functional homologs of barley GAMyb Transform barley aleurone with AtMyb33 Activate  -amylase production

67 Arabidopsis:facultative LD plants Transfer plants from SD to LD 11xincrease of GA 1 3x increase of GA 4 increase AtMyb33 expression in shoot apex shoot apex transition to flowering

68 Potential target for AtMyb LFY promoter LEAFY: meristem-identity gene Evidence AtMyb binding to a specific 8-bp sequence in LFY promoter

69 cis elements specific regions in promoter transcription factor binding site identified by deletion or site specific mutagenesis: gene expression after promoter modification

70 - amylase box:TATCCAT - GARE: TAACAA/GA - Pyrimidine box:C/TCTTTTAC/T Conserved sequences among GA-regulated genes

71 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

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