Part II: Phytohormones and Tropisms

- Form and function of multicellular organisms depend on efficient communication among cells, tissues and organs - Morphogenesis depends on chemical signals: Julius von Sachs (1832-1897) Hormone: from Greek horman - "to set in motion" chemical messenger that mediates intercellular (between cells) communication interacts with specific cellular proteins called receptors present in cell in very low concentrations control plant development and responses to stress: Growth hormones Stress hormones Auxin Salicylic acid Gibberellins Jasmonic acid Cytokinins Oligosaccharines Ethylene Abscisic acid Brassinosteroids (plant steroid hormone) Oligosaccharines

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Lecture 7: Auxin I

Biosynthesis Metabolism Transport

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The auxin concept A historical perspective

From experiments on coleoptile phototropism Darwin concluded that a growth stimulus is produced in coleoptile tip and transmitted to growth zone.

Growth stimulus passes through gelatin but not through water-impermeable barriers

Coleoptile = sheath/protective organ of the youngest leaves in grasses

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The auxin concept A historical perspective

The growth stimulus is of chemical nature.

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The auxin concept A historical perspective

The growth-promoting substance can diffuse into a gelatin block.

Coleoptile-bending assay

IAA = Auxin

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Auxin stimulates the elongation of oat coleoptile sections

primary leaves

coleoptile

Incubation in water for 18h

Incubation in auxin for 18h

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auxin from the greek word auxien, meaning to increase or to grow

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What is auxin?
Indole

Structures of three natural auxins

All plants

Peas

Mustard and corn

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Synthetic auxins

Often used as herbicides

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Auxin biosynthesis
Young leaf primordium GUS stain; location of auxin synthesis Synthesis in meristems, young leaves, developing fruits and seeds - GUS (-glucuronidase) fused to promoter containing an auxin response element - transform Arabidopsis with construct (Ti plasmid) using Agrobacterium - Visualize production/distribution of auxin by GUS staining (expression of GUS reporter gene) Auxin is produced by a cluster of cells located at sites where hydathodes will develop Hydathodes = grand-like modifications of ground and vascular tissues for release of liquid water (guttation) Aloni et al. 2003 Planta 216: 841
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Gradient of diluted GUS activity Differentiating vascular strand, sink for auxin

Wherever free auxin is produced, GUS expression (blue staining) occurs

GUS reporter gene

LB 35S Promoter AuxinResponseElement GUS RB

Agrobacterium
T-DNA

T-DNA

T-DNA

Vector

Plant transformation

Nucleus

Plant Cell

Look for GUS activity

Look for blue staining in tissue

X-Gluc (5-bromo-4-chloro-3indolyl-beta-D-glucuronic acid)

Substrate for GUS

BIOL 754 Fall 10 08

Auxin biosynthesis
Young leaf primordium GUS stain; location of auxin synthesis Synthesis in meristems, young leaves, developing fruits and seeds - GUS (-glucuronidase) fused to promoter containing an auxin response element - transform Arabidopsis with construct (Ti plasmid) using Agrobacterium - Visualize production/distribution of auxin by GUS staining (expression of GUS reporter gene) Auxin is produced by a cluster of cells located at sites where hydathodes will develop Hydathodes = grand-like modifications of ground and vascular tissues for release of liquid water (guttation) Aloni et al. 2003 Planta 216: 841
BIOL 754 Fall 11 08

Gradient of diluted GUS activity Differentiating vascular strand, sink for auxin

Wherever free auxin is produced, GUS expression (blue staining) occurs

Tryptophan biosynthetic pathway

Arabidopsis mutants trp2 and trp3 are blocked in the last two steps of tryptophan biosynthesis; IAN accumulates up to 11fold in mutants vs. wt (Radwanski et al. 1996).

In tomato, IPA synthesized independently of tryptophan (Nonhebel et al. 1993). Depending on species either IAN or IPA may serve as the intermediate between either indole-3-glycerol phosphate or indole, respectively, and IAA (Arabidopsis can do both).
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Web Topic 19.4

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orange pericarp (orp) mutant of maize

- orp has mutations in both loci encoding tryptophan synthase. - Pericarps surrounding each mutant kernel (orange) accumulate glucosides of anthranile acid and indole, which is responsible for the orange color

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Multiple pathways exist for the biosynthesis of auxin

Brassicaceae Poaceae Musaceae
Most common

Pathogenic bacteria
Pseudomonas savastonoi Agrobacterium tumefaciens

Tomato has both IPA and TAM pathway

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Most auxin in plants is in a covalently bound form

- Free IAA is biologically active form - Majority of auxin found in covalently bound form (hormonally inactive) - Low-molecular-weight conjugated auxins (Fig.) - High-molecular-weight conjugates: IAA-glucan (7-50 glucose units per IAA), IAA-glucoseproteins found in cereal seeds
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Auxin is degraded by multiple pathways

Enzymatic (oxidative) breakdown

IAA may be oxidized nonenzymatically in vitro in highintensity light (this does not occur in vivo)

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Factors regulating the steady-state level of auxin

Web Topic 19.5

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Auxin concentrations in different regions of the shoot

IAA in different regions of WT tobacco Auxin concentration in leaves

Tobacco transformant expressing TRP monoxygenase and IAM hydrolase from Agrobacterium tumefaciens to produce IAA throughout the plant.
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Subcellular localization of auxin

Distribution of IAA in cell is regulated by pH _ - IAA does not cross membranes unaided - IAAH diffuses across membranes - Auxin accumulates in more alkaline compartments Cytosol
2/3 of IAA IAA is metabolized by conjugation or by nondecarboxylative catabolism

Chloroplast
1/3 of IAA conjugation and nondecarboxylation do not occur in chloroplast

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Auxin transport
Method for measuring polar auxin transport
Polar transport requires energy and is gravity independent Donor
(A) (B)

Receiver

(B)

Donor

Auxin moves from apical to basal end (basipetally); Unidirectional transport = polar transport

(A)

Receiver
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A chemiosmotic model to explain polar auxin transport

Mitchell, 1961: Chemiosmotic Theory: chemical potential across the membrane can provide energy for ATP synthesis Proton motive force (PMF) or p = E + pH
E = Transmembrane electric potential pH = pH difference across membrane

Auxin influx driven by PMF Auxin efflux driven by E

Influx = entry/uptake of auxin into cells Efflux = loss of auxin from cells

ATP synthase, ATPase

For recap see Chapter 7 21

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A chemiosmotic model to explain polar auxin transport

1. IAA enters cell either passively (IAAH) or actively (IAA-)

(AUX1)

2. Cell wall is maintained at acidic pH by H+-ATPase

3. IAA- predominates in cytosol

Polarity of auxin transport is governed by efflux step rather then the influx step.

4. IAA anions exit cell via auxin anion efflux carriers (PIN proteins) concentrated at the basal end of cell

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