pH and expansin action on single suspension-cultured tomato (Lycopersicon esculentum) cells

The aim of this study was to measure key material properties of the cell walls of single suspension-cultured plant cells and relate these to cell-wall biochemistry. To this end, micromanipulation was used to compress single tomato cells between two flat surfaces until they ruptured, and force-deformation data were obtained. In addition to measuring the bursting force, we also determined the elastic (Young’s) modulus of the cell walls by matching low strain (≤20% deformation) experimental data with a cell compression model, assuming linear elastic cell walls. The walls were most elastic at pH 4.5, the pH optimum for expansin activity, with an elastic modulus of 2.0 ± 0.1 GPa. Following the addition of exogenous expansins, cell walls became more elastic at all pH values. Western blot analysis of proteins from walls of cultured cells revealed the presence of expansin epitopes, suggesting that the inherent pH dependence of elasticity and other compression phenomena is related to the presence of endogenous expansin proteins and their wall-loosening ability. Although strict application of the linear-elastic model could not be applied to large deformations—for example, up to cell bursting—because of irreversible behaviour, the deviation of the data from the model was generally small enough to allow estimation of the strain in the cell wall at failure. This strain was greater at pH 4.5 and when expansins were added to the suspension. The changes in elasticity are consistent with suggestions about the mode of expansin action. The estimated strains at failure are compatible with data on the failure of Acetobacter-derived cellulose–xyloglucan composites and proposed mechanisms of such failure. Through the measurement of cell-wall material properties using micromanipulation, it may be possible to understand more fully how cell-wall composition, structure and biochemistry lead to cell mechanical behaviour.

[1]  J. Verbelen,et al.  Xyloglucan endotransglucosylase activity loosens a plant cell wall. , 2007, Annals of botany.

[2]  S. McQueen-Mason,et al.  Two endogenous proteins that induce cell wall extension in plants. , 1992, The Plant cell.

[3]  Andrew J. Fleming,et al.  Induction of Leaf Primordia by the Cell Wall Protein Expansin , 1997 .

[4]  J. Passioura.  The physical chemistry of the primary cell wall: implications for the control of expansion rate , 1994 .

[5]  R. Cleland,et al.  The Acid Growth Theory of auxin-induced cell elongation is alive and well. , 1992, Plant physiology.

[6]  K. Nishitani,et al.  Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. , 1992, The Journal of biological chemistry.

[7]  A. D. Tomos,et al.  THE PRESSURE PROBE: A Versatile Tool in Plant Cell Physiology. , 1999, Annual review of plant physiology and plant molecular biology.

[8]  L Wang,et al.  Modelling the mechanical properties of single suspension-cultured tomato cells. , 2004, Annals of botany.

[9]  D J Cosgrove,et al.  Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[10]  John O. Outwater,et al.  Ball Tonometry: A Rapid, Nondestructive Method for Measuring Cell Turgor Pressure in Thin-Walled Plant Cells , 2000, Journal of Plant Growth Regulation.

[11]  D. Cosgrove,et al.  Expansins in growing tomato leaves. , 1995, The Plant journal : for cell and molecular biology.

[12]  J. Lockhart.  An analysis of irreversible plant cell elongation. , 1965, Journal of theoretical biology.

[13]  M. Gidley,et al.  Probing expansin action using cellulose/hemicellulose composites. , 2000, The Plant journal : for cell and molecular biology.

[14]  D. Cosgrove,et al.  Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco. , 1998, Plant physiology.

[15]  D. Cosgrove,et al.  An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls , 1993, Planta.

[16]  A MICRO‐PENETRATION TECHNIQUE FOR MECHANICAL TESTING OF PLANT CELL WALLS , 1996 .

[17]  D J Cosgrove,et al.  Expansive growth of plant cell walls. , 2000, Plant physiology and biochemistry : PPB.

[18]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[19]  D. Cosgrove.  Relaxation in a high-stress environment: the molecular bases of extensible cell walls and cell enlargement. , 1997, The Plant cell.

[20]  J. Rose,et al.  Characterization of a new xyloglucan endotransglucosylase/hydrolase (XTH) from ripening tomato fruit and implications for the diverse modes of enzymic action. , 2006, The Plant journal : for cell and molecular biology.

[21]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[22]  A. Bennett,et al.  Detection of expansin proteins and activity during tomato fruit ontogeny. , 2000, Plant physiology.

[23]  L. D. Talbott,et al.  Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure. , 1992, Plant physiology.

[24]  D J Cosgrove,et al.  How Do Plant Cell Walls Extend? , 1993, Plant physiology.

[25]  S. Rochange,et al.  Expression of a heterologous expansin in transgenic tomato plants , 2000, Planta.

[26]  D J Cosgrove,et al.  Enzymes and other agents that enhance cell wall extensibility. , 1999, Annual review of plant physiology and plant molecular biology.

[27]  H. Kende,et al.  Expansins in Deepwater Rice Internodes , 1997, Plant physiology.

[28]  Jenny Blewett,et al.  A micromanipulation method to measure the mechanical properties of single tomato suspension cells , 2004, Biotechnology Letters.

[29]  S. McQueen-Mason,et al.  Expansin Mode of Action on Cell Walls (Analysis of Wall Hydrolysis, Stress Relaxation, and Binding) , 1995, Plant physiology.

[30]  D. Cosgrove.  Cell wall loosening by expansins. , 1998, Plant physiology.

[31]  N. Carpita,et al.  Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. , 1993, The Plant journal : for cell and molecular biology.