GibberellinsGAs

a class of plant hormones

affect several important plant processes

eg., seed germination

stem elongation

flowering

male sterility

Gibberellins (GAs)

Gibberellins

1926 Japanese scientist Gibberella fujikuroigibberellin A (terpenoid cpd)

1954, 1955 US and UK scientists1958 GA1 in higher plant

GAx1987 synthesis/metabolism

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 to GA1

additional double bond

Gibberellins

GA4 GA7 nonpolar, slowly diffuse

GA9 GA12 precursor

GA29 GA34 deactivated form

Different tissues

Different forms of GA

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 GA20oxidase genes:

pathway shifted

GA4 increased / GA1 decreased

12GA aldehyde: precursor of GA derivatives

20 13 3 2by oxidation (C ) and hydroxylation (C C C )

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

From mevalonic acid (6C)

GGPP (20C-linear cpd)

ent kaurene (1st specific cpd)

GA12 aldehyde (first GA)

GAx

Isoprene (5 C) 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

* Hydroxylation at C2 C3 and C13:

number, position

stoichiometry

* Loss of C20 (C20 to C19 GA)

* 2-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 glycosyl ether

GA inactivation

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

Considering 2 locina Le normal ent-kaureneNa le normal GA20

Grafting1. na Le scion

Na le stock tall

2. Na le scionNa Le stock dwarf

Conclusion?

Unlike auxin (acidification)

Increase wall extensibility

Decrease minimum force

for wall extension

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)

GA mechanism in elongation

GA mechanism in germination

Activate transcription of

amylase gene

In scutellum and aleurone

GA detection and assayBioassay

Easy but not specificFractionation Plant response

Lettuce hypocotyls elongationMicrodrop/dwarf rice amylase production

GC-MSSolvent extraction

Chromatography (polarity)

GC (boiling point)

MS (mass)

Identification and quantification

High sensitivity and more specific

Inhibit ent-kaurene synthesis

AMO1618

Cycocel

Inhibit ent-kaurene oxidation

PaclobutrazolUniconazol

Ancymidol Tetcyclasis

Inhibit later steps by dioxygenases

Bx-1112

LAB1988999

GA inhibitors

Hormone Responses

Perception: receptor

Signal transduction:

second messenger (cAMP, cGMP)

G protein

Ca-Calmodulin

enzyme

transcription factor

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: inflorescence development

AtGA20ox3: early seedling development

GA synthesis

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

Light : promote conversion of GA1 to inactive GA8

reducing shootelongation

N NNNNNNN NNNNNNNN 20 1: reduce production of active GA and GADaylength (LD): floral initiation

activates GA20oxidase activity

GA53 to GA44

GA19 to GA20

Lettuce: Lactuca sativa seed germination

Red light: activates LsGA3ox1 expression

GA1 increase

Far-red light: inhibits LsGA3ox1

NNNNN 1: promote GA production

29 8inhibit deactivation steps to GA and GA

N NNN Pisum sativumNN NN-NNNNNNNNN NNN NNNNNNNNN NNNNNNN NN NNNN NNNNN NNN NNN 1, all reduce GA level

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

GA1 kaurene synthase (ent-CDP synthase)

GA3 Cyt P450-dependent monooxygenase

GA4 3 hydroxylase

GA5 GA20oxidase

Genes

Pea (sln)

decrease 2 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

gai1-151 bp inframe deletionloss of 17 amino acidconstitutive 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

Norin10: dwarf line

2 mutated loci: Rht1 or Rht-B1b (chrs 4B)

Rht2 or Rht-D1b (chrs 4D)

Wheat Rht mutant

All genes cloned:deduced amino acid sequenceGAI / Rht / d8 homologsConserved domains I and II in N terminal

gai mutant:deletion in domain ID8 / Rht: mutation in domain I and/or II

*N terminal essential for GA response*

Similar to WT + GATall by elongated internodes

Arabidopsis spy rgaBarley sln spyRice slrTomato proPea la crys

Recessive / Negative regulators

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)

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

Increased signal transduction mutants

Original gai mutant: gain of function

Loss of function allele of GAI ?

Phenotype: normal

Increase paclobutrazol resistance

Low GA = normal height

At least two components in Arabidopsis GA signaling pathway

N NN NNN NN N

NNNNNN N NNNNNNNNN NNNNNNN/

NNNNNNN NNNNNN NNN NNNNNNN-NNNNNNN NNNNNNNNNNN

NNNNNNNN NNNNNNN NNNNNNNNNN NNNNNN

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

negative regulator

sln x dwarf mutant = sln phenotype

SLN = GAI/RGA homolog

Dominant allele of SLN mutant

Mutation in N terminal

Dwarf barley

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

Transformation: dwarf rice

Rice slr

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 phenotypes

spy gai= spy phenotypes

Arabidopsis spy

SPY gene product:

O-GlcNAc transferase

Signaling molecule

Involved in protein-protein interaction

Negative regulator

Arabidopsis spy

Before responses

Expression of GA-regulated genes:

Protein-DNA interaction

Transcription factor

cis elements

Barley: HvGAMyb

Bind specific sequence in

promoter of -amylase gene

Increase gene expression

Overexpression of HvGAMyb gene

= GA treatment

Transcription factor: GAMyb

Arabidopsis: GAMyb-like genes

AtMyb33 AtMyb65 AtMyb101

Functional homologs of barley GAMyb

Transform barley aleurone with AtMyb33

Activate -amylase production

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

Potential target for AtMyb

LFY promoter

LEAFY: meristem-identity gene

Evidence AtMyb binding

to a specific 8-bp sequence

in LFY promoter

cis elements

specific regions in promoter

transcription factor binding site

identified by deletion or

site specific mutagenesis:

gene expression after promoter modification

- amylase box: TATCCAT

- GARE: TAACAA/GA

- Pyrimidine box: C/TCTTTTAC/T

Conserved sequences among

GA-regulated genes

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