In planta localization of stilbenes and (+)-catechin within Norway spruce phloem: induced responses after inoculation with blue-stain fungus

The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutively produced or inducibly expressed, i.e., activated as a response to insect or pathogen attack. Many defense traits exist in both constitutive and inducible form. For example, axial phloem parenchyma cells are critical in conifer bark defense. These cells regularly form in Pinaceae phloem. Earlier research has shown that the axial phloem parenchyma is the main site of phenolic accumulation in spruce bark, including stilbene and (+)-catechin compounds. Earlier studies have also indicated that both stilbenes and catechins play a role in bark defense against pathogens. However, temporal and spatial changes in localization and accumulation patterns of stilbenes and catechins across the tissues and cells of phloem are poorly studied after wounding and fungal attack.In this study, we artificially induced bark defenses in Norway spruce (Picea abies (L.) Karst.) saplings by wounding and fungal inoculation with blue-stain fungus Endoconidiophora polonica. The changes in the distribution and accumulation patterns of stilbenes, (+)-catechins and resin acids were analysed across freeze-fixed Norway spruce phloem, representing the condition of living tissues (i.e. in planta) by using cryo-time-of-flight secondary-ion mass spectrometry (cryo-TOF-SIMS). Quantitative patterns of compounds across phloem were analysed by using gas chromatography-mass spectrometry (GC-MS). TOF-SIMS analysis revealed that at the cellular level, stilbenes and (+)-catechins were localized in the axial phloem parenchyma, and their accumulation significantly increased towards the inoculation site. Also in the bulk tissue of phloem analysed by GC-MS, the (+)-catechin content significantly increased after one week of inoculation, and continued to linearly rise during the course of 23 days of experiment. The catechin content was higher closer to the inoculation site (up to 5 mm) as compared to the further site (from 5 to 10 mm from the inoculation site; p<0.1). Fungal treatment had also a significant effect on stilbene content, namely that of piceid and astringin. In all treatments, stilbene content first decreased but then increased after three weeks from the onset of the experiment. However, the relative change in stilbene content was marginal as compared to that of (+)-catechin content. TOF-SIMS analysis showed that abietic acid (representative of resin acids) was localized at the outer layers of cortex, and its spread across the tissues and cells was highly variably but not overlapping with the location of axial parenchyma.The results shed new light on the temporal and spatial strategies of defense compound accumulation within spruce phloem as studied at the cellular and tissue level. The findings indicate that stilbenes offer a more constitutively based defense whereas (+)-catechin content merely increases after fungal challenge in the axial phloem parenchyma.