The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.

While callose is a well-known permeability barrier and leak sealant in plant cells, it is largely unknown whether this cell wall polymer can also serve as a load-bearing structure. Since callose occurs in exceptionally large amounts in pollen, we assessed its role for resisting tension and compression stress in this cell. The effect of callose digestion in Solanum chacoense and Lilium orientalis pollen grains demonstrated that, depending on the species, this cell wall polymer represents a major stress-bearing structure at the aperture area of germinating grains. In the pollen tube, it is involved in cell wall resistance to circumferential tension stress, and despite its absence at the growing apex, callose is indirectly involved in the establishment of tension stress resistance in this area. To investigate whether or not callose is able to provide mechanical resistance against compression stress, we subjected pollen tubes to local deformation by microindentation. The data revealed that lowering the amount of callose resulted in reduced cellular stiffness and increased viscoelasticity, thus indicating clearly that callose is able to resist compression stress. Whether this function is relevant for pollen tube mechanics, however, is unclear, as stiffened growth medium caused a decrease in callose deposition. Together, our data provide clear evidence for the capacity of cell wall callose to resist tension and compression stress, thus demonstrating that this amorphous cell wall substance can have a mechanical role in growing plant cells.b-1,3-Glucan (callose) is one of the most dynamic components of the plant cell wall. It is known to be synthesized and deposited at the outer surface of the plasma membrane by callose synthases that are localized in the membrane (Carpita and Gibeaut, 1993). The synthesis of this amorphous polymer is an important part of plant cell responses to pathogen attacks as well as physical and chemical stresses (Currier, 1957;Esau and Cronshaw, 1967;Coffey, 1976;Delmer and Amor, 1995). While injured plant cells use the polymer as a leak sealant, in certain plant cell types callose is produced during normal development (Esau, 1948;Currier, 1957;Heslop-Harrison, 1964;Scott et al., 1967;Morrison and O'Brien, 1976;Waterkeyn, 1981;Stone and Clarke, 1992). From the location of these callose deposits, it has been concluded that the polymer acts as a permeability barrier, as in pollen mother cell walls (Heslop-Harrison, 1964) and muskmelon endosperm envelopes (Yim and Bradford, 1998); as a matrix for deposition of other cell wall materials, as in developing cell plates and sieve-plate pores; and as a sealing or plugging material at the plasma membrane of pit fields, plasmodesmata, and sieve-plate pores (Eschrich, 1975). Despite the widespread occurrence of callose, its functions other than as a leak-sealing cement or permeability barrier are not well understood (Stone and Clarke, 1992). One of the few suggestions of callose playing a role in cell wall mechanics was made based on circumstantial evidence...

Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP 3 ) levels. Moreover, the utilization of diacylglycerol kinase and InsP 3 -gated calcium release receptor inhibitors suggested that InsP 3 or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt-and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses.

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