Nanoinfusion: An integrating tool to study elicitor perception and signal transduction in intact leaves

Abstract

To study elicitor effects in intact leaves of Hordeum vulgare cv. Ingrid, chitin fragments were delivered to substomatal cavities with a micropipette. Responses were monitored by a calibrated reference microelectrode and a pH-sensitive microelectrode, simply positioned below neighbouring open stomata in the air-filled space of the substomatal cavities. Flooding of a leaf spot of approx. 600 x 300 μm with physiological aqueous solutions caused an immediate transient polarization of the extracellular solution in the order of -25 ± 12 mV. Immediately after the pipette solution was consumed, the extracellular solution was again polarized, still before the cavities dried out. In dry cavities, the extracellular equilibrium potential was -34 ± 10 mV. On flooding, the extracellular pH rose to 5.7 ± 0.3 after approx. 12 ± 7 min and returned to a stable level of 5.2 ± 0.3 after 30 min. Sequential infusion on the same leaf spot, first with elicitor-free solution, then with the same solution containing 25 μM N-acetyl-chitooctaose, into the still-flooded cavities yielded an elicitor-specific pH maximum between 6 and 7 approx. 10 min after flooding. A pronounced pH maximum > 7 occurred between 40 and 240 min after flooding in wild-type plants. The use of elicitor nanoinfusion for the integrated development of resistance inducers in cereals, wine and poplar is discussed.