Localization of (+)-Catechin in Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.