Hoe -Han Goh†, Nic Mullin‡, Alexis Peaucelle¤, Andreas Backhaus†, Jamie Hobbs‡ and Andrew Fleming†

†Dept. Animal and Plant Sciences, University Sheffield, UK, ‡Dept. Physics and Astronomy, University of Sheffield, UK, ¤INRA Versailles, Versailles, France

Introduction

Hypotheses

Aims:

References

The use of AFM for in vivo quantification of plant cell wall extensibility

Plant development relies largely on cell expansion. Cell wall looseningis a pre-requisite for cell expansion in which expansins, a conservedfamily of non-enzymatic proteins, play a major role1,2. Ectopicexpression of expansin at meristem and leaf flank has been used toinduce leaf initiation and aberrant leaf shape3,4. However, to whatextent this reflects an in vivo function for expansins remains unclear.Indeed, the role of cell wall mechanics in controlling morphogenesisremains debatable.

Atomic force microscopy can be used to characterise the

mechanical properties of cell wall during early leaf growth in

tobacco and Arabidopsis

• Differences in cell wall extensibility can be measured and mapped using

AFM with great resolution showing spatial heterogeneity of extensibility at

cellular and tissue levels.

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1. The cell wall protein expansins function in differential growth during leaf development

2.The differential regulation of expansin expression leads to organ-level coordination of leaf size and shape

alterations of plant cell wall properties modulate leaf growth

ModelPlant tissue can be modelled as growing network of viscoelastic springs (cell walls) with strain force generated by internal turgor pressure to explore general cellular behaviour during leaf development.

Cell wall is more extensible at leaf abaxial side (ab)

1. Functional analysis of expansins involve in leaf morphogenesis

2. In vivo quantification of expansin activity

3.Utilisation of a leaf growth model based on viscoelastic nature of plant cell wall

in testing various hypotheses regarding mechanical force and morphogenesis

• To determine the role of expansins in leaf morphogenesis

• To determine the role of plant cell wall in controlling leaf growth and form

Project outline:

1Cosgrove, D. J. 2000 Loosening of plant cell walls by expansins. Nature 407, 321-326.2Sampedro, J. & Cosgrove, D. J. 2005 The expansin superfamily. Genome Biology 6, 242-250.3Fleming, A. J., McQueen-Mason, S., Mandel, T. & Kuhlemeier, C. 1997 Induction of leaf primordia by the cell wall protein expansin. Science 276, 1415-1418.4Pien, S., Wyrzykowska, J., McQueen-Mason, S., Smart, C. & Fleming, A. 2001 Local expression of expansin induces the entire process of leaf development and modifies leaf shape. Proceedings of the National Academy of Sciences of the United States of America 98, 11812-118175Donnelly, P. M., Bonetta, D., Tsukaya, H., Dengler, R. E. & Dengler, N. G. 1999 Cell cycling and cell enlargement in developing leaves of Arabidopsis. Developmental Biology 215, 407-419.

Future plan:

Leaf staging for expansin expression study

• Identifying expansins involve in leaf development from expression studies

• Testing the function of expansin through inducible amiRNA silencing approach and

viscoelastic leaf growth model

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m)

Day after sowing

Petiole

Lamina

Width

Length

±1SE

5mm

5mm

Cantilever

deflection, s

Cantilever spring

constant, k

Force, F F = ks

Schematic diagram of the vertical tip movement during the approach and retract phases of a force spectroscopy measurement

Approach Retract

Jump to contact AdhesionRepulsive contact

Separation Separation

Mechanisms of atomic force microscopy (AFM)

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100µm

200µm0.5mm

1mm

Tobacco primodia

Measurements on leaf number 6 of Arabidopsis thaliana Col-0 show that leaf development can be categorised into three phases: cell division predominant stage, cell expansion stage and mature stage.

Aniline blue staining

of callose which

formed during cell

division indicating

planes of cell division

*Occurrences of cell division are indicated by GUS staining

Scale bars = 100µm

Cantilever deflection

Quadrant photodiode

laser detector

Laser emission

Sample

Piezo

elementZ

Z

Type I

Type II

Electrical

current (V)

, Z

(hysteresis caused by viscous behaviour of the leaf surface)

Topography of mapped surface Force measurement

Height

Force

Height

Force

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Day 16