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The growth hormone auxin

COOH

N H

Discovery of auxin

Auxin: the growth hormone

water for 18 hours

+IAA for 18 hours

COOH

N H

The roles of auxin in plant growth and development ·Root development ·Vascular initiation and patterning ·Flower development ·Embryogenesis ·Tropism

Control + IAA

Auxin appears to be necessary and sufficient for plant organogenesis

Control

+ IAA

Auxin functions are regulated at multiple levels

1. Auxin homeostasis (biosynthesis, conjugation, and degradation) 2. Auxin polar transport 3. Auxin signal transduction

Approaches used for dissecting mechanisms of auxin actions

1. Biochemical approaches: characterization of auxin inducible genes

AUX/IAA genes Auxin Response Element (AuxREs) Plants treated with water Plants treated with auxin RNA isolation RNA isolation

The AUX/IAA genes

· Transcription is rapidly induced by auxin · Independent of protein synthesis - primary response · Induced by cycloheximide · Unstable nuclear proteins · Large gene family (29 in Arabidopsis) AUX/IAA proteins probably serve as negative regulators

Domain structures of AUX/IAA proteins

I II III IV

· degradation proteosome mediated · auxin promotes degradation · Domain II is important for stability

The Auxin Response Elements

1. Analysis of the promoter regions of auxin inducible genes 2. Identify the conserved elements: TGTCTC

1. Construct an auxin reporter 2. Identify the transcription factors binding to the AuxREs

Auxin reporter DR5

Cotyledons

DR5-GFP auxin reporter

Auxin response elements

GFP Gene

hypocotyl

Root meristem

Transcription factors bind to AuxREs

Use AuxREs Cotyledons as a bait for yeast one hybrid screen

Auxin response factors: 22 members in Arabidopsis

hypocotyl

Root meristem

Approaches used for dissecting mechanisms of auxin actions

1. Biochemical approaches: characterization of auxin inducible genes

AUX/IAA genes and ARFs (auxin response factors)

2. Genetic approaches: Identification and

characterization of mutants resistant to exogenous auxin and auxin polar transport inhibitors. axr mutants, tir mutants

Auxin resistance mutant screens

+ auxin

auxin

Two types of mutants: 1) Auxin uptake: aux1, axr4 2) Auxin response: axr1, axr2, axr3, axr5, axr6, tir1

Auxin resistance mutant screens

Recessive: aux1, axr1,, axr4, axr5, axr6, msg1, tir1 Dominant: axr2, axr3, msg2, iaa28,

Dominant axr mutants contain mutations in the domain II in Aux/IAA proteins

AR / A17 X 3 IA ar x3-1 ar x3-3

VGPV VWPR L G

axr3-1

axr3-3

Col-0

A plausible auxin signaling mechansim

1. Aux / IAA proteins are short lived and function as negative regulators 2. Mutations in Aux /IAA confer dominant auxin resistance; probably increase the half-life of Aux /IAA proteins

3. Auxin signaling may depend on the degradation of Aux / IAA proteins

Auxin signaling mechanisms

ARF Aux / IAA auxin ARF Aux / IAA ARF / ARF Removal of Aux /IAA Transcription

Predictions from the model

auxin ARF Aux / IAA ARF / ARF Removal of Aux /IAA 1. Inactivation of ARF should cause the same phenotypes as those induced by stabilized Aux/IAA 2. Inactivation of Aux/IAA should lead to auxinoverproduction phenotypes 3. Overexpression of ARF may suppress dominant Aux/IAA mutants Transcription

Arabidopsis pattern mutants

Basal mutant (mp, bdl) BDL encodes an Auxin / Indole-3-Acetic Acid protein (IAA12)

MP encodes an auxin response factor (ARF5)

How is Aux/IAA degraded?

auxin ARF Aux / IAA ARF / ARF Removal of Aux /IAA Transcription

Ubiquitin-mediated protein degradation machinery?

An auxin signal transduction pathway

Low [auxin]

High [auxin]

Previous genetic studies 1) The responses to excess exogenous auxin 2) Root elongation as a primary physiological readout 3) Known auxin mutants pin1 and pid are not auxin resistent

Polar auxin transport

Carrier mediated auxin transport

Influx: a. passive b. AUX1 permease Efflux: Polarity set up by efflux carries if they are located only at the base

PIN proteins as auxin efflux carriers

PIN proteins are polarly localized

Current hypothesis for plant organogenesis

Auxin gradients determine the formation of lateral organs

Auxin peak

Auxin functions are regulated at multiple levels

1. Auxin biosynthesis

2. Auxin polar transport 3. Auxin signal transduction

Auxin biosynthesis

1. Necessary for determining the physiological roles of auxin 2. Necessary for understanding auxin movements and dynamics 3. Provides genetic foundations for dissecting the mechanisms of auxin in plant development

Proposed tryptophan dependent auxin biosynthetic pathways

NH2 N H N N H OH N N H

Nitrilase

O COOH

COOH O NH2 N H N H

H N H N H OH

O

Trp

iaaM

O NH2 N H

Hydrolase

Auxin overproduction yucca1-D mutant

WT

yucca1-D

WT

yucca1-D Zhao et al. (2001) Science

YUCCA encodes a flavin-monooxygenase involved in auxin synthesis

O

OH NH2

NH2

HN OH

N OH C N

O OH N H

N H

N H

YUCCA

N H

N H

N H

Nitrilase

Trp

IOX

Auxin

Zhao et al. (2001) Science

Overexpression of YUCCA genes causes auxin over-production

Flower defects in yuc1yuc4 double mutants

Disruption of shoot-root axis by yuc1yuc4yuc10yuc11

yuc1 yuc4 yuc10 yuc11 WT

Can exogenous auxin rescue the yucca mutant phenotypes?

1. Auxin transport 2. Auxin gradient 3. The right dosage

Production of auxin in situ by the bacterial auxin biosynthesis gene iaaM

COOH NH2 N H N H

iaaM

O NH2

iaaH

OH N H

O

When expressed in plants, iaaM converts tryptophan to indole-3acetamide, which is hydrolyzed by non-specific hydrolases in plants

Complementation of yuc mutants by expressing the iaaM gene under the control of a YUCCA promoter

A YUC promoter

iaaM gene

Transform yuc mutants

Can the iaaM gene rescue yuc phenotypes?

The yuc1yuc4 double mutant is rescued by the iaaM gene under the YUC1 promoter

Proposed IAA biosynthetic pathway in plants

Sugawara S. et.al. PNAS 2009;106:5430-5435

©2009 by National Academy of Sciences

YUC genes have very restricted expression domains

YUC4 in situ

YUC4 in situ

Previous genetic studies 1) The responses to excess exogenous auxin 2) Root elongation as a primary physiological readout 3) Known auxin mutants pin1 and pid are not auxin resistent

Genetic screens for yuc1yuc4 enhancers

yuc1yuc4

The npy1 (naked pins in yuc ) mutant is an yuc1yuc4 enhancer

wt

yuc1yuc4

npy1yuc1yuc4

npy1yuc1yuc4

Analogous mechanisms between phototropic response and auxin-regulated organogenesis

Blue light Developmental YUCCA signals auxin NPH3 PID NPY1

PHOT1

Auxin transport (PIN3)? ARF7 / NPH4?

Auxin transport (PIN1)? ARF5 / MP?

Phototropic responses

Organogenesis

Formation of pins is a hallmark for defects in auxin pathways

wt

pin1

pid

mp

Pin-like maize mutants

wt

ba1

spi1

Bif1

bif2

BARREN STALK1 (BA1) encodes a bHLH transcription factor SPARSE INFLORESCECNE1 (SPI1) encodes a YUC-like auxin biosynthesis enzyme BARREN INFLORESCENCE2 (BIF2) encodes a protein kinase involved in auxin transport and signaling. One of its targets for phosphorylation is BA1.

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