The mode of action of cell wall-degrading enzymes and their interference with Nod factor signalling in Medicago sativa root hairs.

Medicago sativa L. (alfalfa) root hairs respond to Nod factors [NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion fluxes. Protoplasts prepared from these cells using the cell wall-digesting enzymes pectolyase and cellulase do not, or to a rather small extent, respond to Nod factors. In an effort to understand this activity loss we analyzed the mode of action of both enzymes with respect to their effects on the root hairs as well as their interference with the Nod factor response. (i) In the presence of the enzymes, Nod factor at saturating concentrations neither depolarized the plasma membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes, Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s, pectolyase depolarized the plasma membrane, alkalized the external space, acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii) Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike pectolyase, the cellulase response was only weakly refractory to a second addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects similar to those of untreated enzymes. We conclude that the effects shown do not originate from enzymatic activity, but from interactions of the proteins with cell wall or plasma membrane constituents. It is further concluded that these enzymes (pectolyase more so than cellulase) trigger defense-related signal pathways, which makes protoplasts prepared with such enzymes unsuitable for studies of symbiotic or defense-related signalling.

Single-cell measurements of the contributions of cytosolic Na(+) and K(+) to salt tolerance.

Ion concentrations in the roots of two barley (Hordeum vulgare) varieties that differed in NaCl tolerance were compared after exposure to NaCl. Triple-barreled H(+)-, K(+)-, and Na(+)-selective microelectrodes were used to measure cytosolic activities of the three ions after 5 and 8 d of NaCl stress. In both varieties of barley, it was only possible to record successfully from root cortical cells because the epidermal cells appeared to be damaged. The data show that from the 1st d of full NaCl stress, there were differences in the way in which the two varieties responded. At 5 d, the tolerant variety maintained a 10-fold lower cytosolic Na(+) than the more sensitive variety, although by 8 d the two varieties were not significantly different. At this time, the more tolerant variety was better at maintaining root cytosolic K(+) in the high-NaCl background than was the more sensitive variety. In contrast to earlier work on K(+)-starved barley (Walker et al., 1996), there was no acidification of the cytosol associated with the decreased cytosolic K(+) activity during NaCl stress. These single-cell measurements of cytosolic and vacuolar ion activities allow calculation of thermodynamic gradients that can be used to reveal (or predict) the type of active transporters at both the plasma membrane and tonoplast.