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Gibberellins

Gibberellins. History of discovery from a plant disease phenomenon. Prior to world war II Japanese scientists were investigating a rice disease that made plants grow very tall but prevented grain formation. They showed that a chemical secreted by the disease fungus caused the hyper-elongation.

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Gibberellins

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  1. Gibberellins • History of discovery from a plant disease phenomenon. • Prior to world war II Japanese scientists were investigating a rice disease that made plants grow very tall but prevented grain formation. They showed that a chemical secreted by the disease fungus caused the hyper-elongation. • In the 1950s this chemical was isolated in the US and the UK, and named gibberellin. • Many gibberellins differing in their biological activity were subsequently shown to exist, and they have been numbered in the order of their discovery, up to now over 100.

  2. Gibberellin A1 or GA1

  3. GA effects Causes tallness. Plant becomes tall Apply Gibberellic Acid (GA3) or GA1 Dwarf pea

  4. Any plant has several GAs. • Different GAs differ in their biological activity according to the ease with which they are metabolized to GA1. • They form a metabolic sequence. • Some inactive GAs are metabolic products of active GAs. • Biosynthetic pathway • Glyceraldehyde phosphate isoprenoid pathway • Kaurene GA12-aldehyde GA20GA1GA8

  5. Gibberellin Biosynthesis Pathway Glyceraldehyde phosphate Copalyl Pyrophosphate Geranylgeranyl Pyrophosphate GA12 GA12-aldehyde ent-Kaurene GA53 GA44 GA19 GA20 GA29 GA1 GA8

  6. Tallness is specifically attributable to GA1. • Dwarf plants synthesize only small amounts of GA1. • nana (very dwarf) plants have a block in GA synthesis prior to GA12-aldehyde. • Many dwarfs have a block between GA20 and GA1 (GA1 is 3b-OH GA20) (le allele in peas).

  7. Gregor Mendel Mendel’s dwarf pea Mendel’s tall pea

  8. Mendel with his Augustinian Colleagues in Brünn

  9. Mendel with a Plant, Enlarged from the Augustinian Group Photograph

  10. Gibberellins and Tallness in Peas • 1956 - Gibberellin-like substances found in pea shoots by bioassay (Radley). • No relationship between the total gibberellin content and tallness was found to exist. • 1982 - Gibberellins in pea shoots identified by gas chromatography / mass spectrometry (Davies et al.). • 1984 - Demonstrated that tallness in peas is regulated by one particular biologically-active gibberellin, namely GA1 (Ingram et al.).

  11. Stem Length and Level of GA1 in Peas with Different Le Alleles le-1 Le le-2

  12. GA 3-hydroxylase GA1 GA20 Conversion of GA20 to GA1 by GA 3-hydroxylase, which adds a hydroxyl group (OH) to carbon 3 of GA20

  13. Gene for GA 3-hydroxylase isolated from pea by probing pea cDNA library with gene from Arabidopsis dwarf mutant suspected of being a mutant in this enzyme.

  14. Parents F13 offspring kb 1.2 - 1.0 - 0.2 - T D D T D T D T T T D T T D T T - Tall, D - Dwarf Cosegregation of GA 3-hydroxylase gene 1.2kb RFLP fragment with tall (Le) / dwarf (le-1) phenotype in an F13 segregating population Phenotype ratio:3Tall: 1 dwarf; Genotype ratio 1TT : 2Tt : 1tt RFLP ratio: 1 x (1.2kb) : 2 x (1.2 + 1.0 + 0.2kb) : 1 x (1.0 + 0.2kb) (Lester et al., 1997)

  15. Parents F13 offspring kb 1.2 - 1.0 - 0.2 - T D D T D T D T T T D T T D T T - Tall, D - Dwarf Cosegregation of GA 3-hydroxylase gene 1.2kb RFLP fragment with tall (Le) / dwarf (le-1) phenotype in an F13 segregating population This demonstrates that the identified gene is indeed Mendel’s gene regulating tallness (Lester et al., 1997)

  16. Pea Gibberellin 3-hydroxylase deduced amino acid sequences In Dwarf Pea alanine threonine

  17. Le gene: 1666 Bases 2 Exons (1- 488; 1033-1666) separated by an Intron Allele Molecular Basis Level of GA 3- hydroxylase le-1 G A at base 685; alanine threonine at AA 229 1/20Le Close to iron-binding motif of other 2ODDs

  18. Activity of Le and le-1 recombinant GA 3-hydroxylases assayed by HPLC Wild-type - Tall (Le) GA1 GA20 Radioactivity le-1 Dwarf mutant HPLC Retention time

  19. Mass Spectrum of 13C GA1 Produced from 13C GA20 by E. coli Transformed with Le cDNA

  20. Gibberellin Biosynthesis Pathway Glyceraldehyde phosphate Copalyl Pyrophosphate Geranylgeranyl Pyrophosphate GA53 GA12 GA12-aldehyde ent-Kaurene GA44 GA19 GA20 GA29 Le/le GA 3-hydroxylase GA1 GA8

  21. Gibberellin Biosynthesis Pathway ls Glyceraldehyde phosphate Copalyl Pyrophosphate Geranylgeranyl Pyrophosphate na GA53 GA12 GA12-aldehyde ent-Kaurene GA20ox sln GA44 GA19 GA20 GA29 GA2ox GA20ox GA20ox le GA3ox GA1 GA8 GA2ox

  22. GA20ox gene of Arabidopsis is expressed most strongly in the shoot apex and in young developing leaves. The false-colour image of light emitted by transgenic Arabidopsis plants containing the firefly luciferase (LUC) coding sequence coupled to the AtGA20ox promoter. (From P.Hedden IACR/BBSRC Ann rep 2000)

  23. On left:Wheat plants transformed with GA2ox, the gene for the enzyme that converts GA1 into inactive GA8. Non-transformed on right. Now we await lawn grasses so transformed!!

  24. Other GA effects • Bolting of LD plants plus flowering in some cases. GA1 naturally increases in long days producing bolting Bolting in Spinach (Spinacea oleracea)

  25. Other GA effects • Induction of germination in dormant seeds. • Mobilization of reserves in cereal grains; used in beer manufacture. • Promotion of fruit development especially grapes and some fruits. Used commercially in grapes. • Increasing yield in sugar cane

  26. Regulation by both changing hormone levels and changes in sensitivity Rapid decline in GA1 due to degradation • Dark-grown pea seedlings transferred to light: • GA1 level drops rapidly due to metabolism of GA1; • Then increases to a higher level, similar to light grown plants, over the next 4 days. (Redrawn from Changes in Gibberellin A1 Levels and Response during De-Etiolation of Pea Seedlings; O'Neill, Ross, & Reid, 2000).

  27. Regulation by both changing hormone levels and changes in sensitivity Sensitivity to GA of pea seedlings transferred to the light rapidly falls, so that the elongation rate of plants in the light is lower than in the dark even though their GA1 content is higher. (Redrawn from Changes in Gibberellin A1 Levels and Response during De-Etiolation of Pea Seedlings; O'Neill, Ross, & Reid, 2000).

  28. Intact Intact Decap. GA1 Decap. + IAA Decap. Level, ng.g-1 15 Decap. + IAA 10 5 0

  29. IAA (from the apical bud) promotes and is required for GA1 biosynthesis in subtanding internodes (Data, slide and model by John Ross and Damian O’Neill)

  30. Mode of Action Action in reserve mobilization in cereal grains GA produced by germinating embryo causes the production of -amylase by the aleurone layer of cereal grains.

  31. -amylase then degrades the starch in the endosperm into sugars for use by the embryo.

  32. Starchy Endosperm -amylase Embryo Sugars GA Aleurone Starch Maltose (G-G) Glucose Sucrose

  33. The -amylase is shown to be newly synthesized in the presence of GA (none synthesized in its absence). • Enzyme appearance inhibited by RNA and protein synthesis inhibitors. • Specific mRNA for "-amylase isolated after GA treatment. • GA thus shown to cause the synthesis of mRNA for -amylase.

  34. An upstream sequence of the amylase gene was found to bind a protein only in the presence of GA. • This sequence indicated that the binding protein might be a specific type known from animals - myb. • GA-myb present only following GA treatment. • Shown to bind to upstream regulatory sequence on the "-amylase gene.

  35. Transcription inhibitor GAmyb gene a-amylase gene GA receptor shown to be at surface of aleurone cells.

  36. GA receptor shown to be at surface of aleurone cells. GA GA signal transduction chain Transcription inhibitor GAmyb gene a-amylase gene

  37. Transcription inhibitor GA receptor shown to be at surface of aleurone cells. GA GA signal transduction chain GAmyb gene GAmyb a-amylase gene a-amylase mRNA a-amylase

  38. Mode of Action • In stem elongation GA acts by blocking the effect of an inhibitory transcription factor. • The final action of GA in stem elongation has not been established. • It does promote cell division below the stem apex.

  39. Shown by mutants in Arabidopsis GA biosynthesis mutant ga1 Wild type • Some mutants (gai) are very dwarf and do not respond to GA – signal transduction receptor damaged. • Dwarf cereals used worldwide in the Green Revolution have this mutation. • In mutants lacking GA, a second mutation partially restores growth: • A negative regulator has been turned off. Called RGA. Gibberellin Signal Transduction in Stem Elongation

  40. GA biosynthesis mutant ga1 Action of Gibberellin in Stem Elongation • This shows that stem elongation is normally repressed and what GA does is to negate the repression. GA GAI / RGA Growth GAI/RGA: act in the absence of GA to suppress growth

  41. GA biosynthesis mutant ga1 GA acts to block the actions of GAI and RGA Action of Gibberellin in Stem Elongation • This shows that stem elongation is normally repressed and what GA does is to negate the repression. GA GAI / RGA Growth

  42. GA biosynthesis mutant ga1 Action of Gibberellin in Stem Elongation • GAI/RGA are transcription inhibitors GA GAI / RGA – negative transcription factors mRNA transcription Growth

  43. GA biosynthesis mutant ga1 GA acts to block the actions of GAI and RGA Action of Gibberellin in Stem Elongation GA GAI / RGA - transcription factors mRNA transcription Growth

  44. Concept: Plant Hormones act by negating an inhibitor of hormone type growth. (the same as a double negative in the English language is a positive) In the absence of the inhibitor mutant plants display the + hormone morphology even in the absence of the hormone.

  45. Hormone receptor Negative regulator Response Plant Hormones negate an inhibitor receptor The normal null response is a hormone type response, but it is normally repressed by a member of the signal transduction chain so that you get the minus-hormone morphology. Binding of hormone to the receptors turns off the inhibition. Negative regulator Response

  46. receptor Negative regulator Response Plant Hormones negate an inhibitor receptor The normal null response is a hormone type response, but it is normally repressed by a member of the signal transduction chain so that you get the minus-hormone morphology. A mutation inactivates the negative regulator so that the hormone- type response is seen in the absence of the hormone. Negative regulator Response

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