Ozone and Wounding Stresses Differently Alter the Temporal Variation in Formylated Phloroglucinols in Eucalyptus globulus Leaves

Abstract

Formylated phloroglucinol compounds (FPCs) are a class of plant specialized metabolite present in the Myrtaceae family, especially in the genus Eucalyptus. FPCs are widely investigated due to their herbivore deterrence properties and various bioactivities of pharmaceutical relevance. Despite the increasing number of studies elucidating new FPCs structures and bioactivity, little is known about the role of those compounds in planta, and the effects of environmental stresses on FPC concentration. Ozone (O₃) and wounding are key stress factors regularly confronted by plants. In this study, we investigated how O₃, wounding, and their combination affected individual and total FPC foliar concentration of the economically important species Eucalyptus globulus. Six individual FPCs, including five macrocarpals and one sideroxylonal, showed different response patterns to the single and combined stresses. Total macrocarpals only increased under single O₃ treatment, whereas total sideroxylonals only increased in response to wounding treatment, suggesting different physiological roles played by the two groups of FPCs predominantly existing in E. globulus foliage. Total FPCs increased significantly under individual wounding and O₃ treatments but not under the combined treatment. A principal component analysis indicated that all different treatments had unique FPC fingerprints. Total phenolic contents increased in all O₃ and wounding treatments, and a marginally positive correlation was found between total FPCs and total phenolic contents. We suggest that, depending on the concentration and composition, FPCs play multiple physiological roles in planta, including serving as antioxidants to scavenge the reactive oxygen species brought about by O₃ and wounding stresses.

Keywords: Eucalyptus globulus; formylated phloroglucinol compounds (FPCs); macrocarpal; ozone; sideroxylonal; wounding.

Figure 1

Structural formulas of six different FPCs identified in E. globulus leaves.

Figure 2

Representative UHPLC-DAD-qToF-MS/MS extracted ion chromatograms showing FPCs detected in E. globulus leaves. The FPCs detected in E. globulus leaves are divided into four groups according to m/z values [M−H]⁻ of 489.2857 (a), 471.2752 (b), 485.2544 (c), and 499.1609 (d). Based on co-elution with authentic standards, known FPCs are indicated by arrows with letters. See FPC identification in Material and Methods and the peak details in Table 1. Abbreviations: A, macrocarpal A; D, macrocarpal D; J, macrocarpal J; L, macrocarpal L; N, macrocarpal N; and Sid A, sideroxylonal A.

Figure 3

Temporal changes in individual FPCs (means ± SE, n = 3) in E. globulus leaves subjected to separate and combined ozone (O₃₎ and wounding treatments. Linear mixed models were applied to analyze the fixed effects of different treatments (wounding, O₃, and O₃ + wounding) and recovery times on the temporal changes in FPCs concentrations in E. globulus leaves. The summary of the linear mixed model is shown in Table 2.

Figure 4

Principal component analysis (PCA) of FPCs as affected by wounding and O₃ treatments in E. globulus leaves—loading (a) and score plot (b). Individual FPCs concentrations across the recovery period were used in this analysis. The O + W in (b) corresponds to the combined O₃ and wounding treatment. Abbreviations: A, macrocarpal A; D, macrocarpal D; J, macrocarpal J; L, macrocarpal L; N, macrocarpal N; and Sid A, sideroxylonal A.

Figure 5

Time-dependent changes (means ± SE, n = 3) in the concentration of total macrocarpals (a) and total sideroxylonals (b) in E. globulus leaves subjected to separate and combined wounding and O₃ treatments. Details of the statistical summary are shown in Table 3. Total macrocarpals are expressed as mg g⁻¹ DM macrocarpal A equivalents, and total sideroxylonals are expressed as mg g⁻¹ DM sideroxylonal A equivalents.

Figure 6

Temporal changes (mean ± SE, n = 3) in the concentration of total FPCs (a), total phenolic compounds (b), and their correlation (c) in E. globulus leaves subjected to separate and combined O₃ and wounding treatments. The data selected for non-linear correlation analysis are the mean of total FPCs and total phenolics concentrations at each recovery time point from different treatments and controls. Total phenolic contents (TPC) are expressed as mg g⁻¹ DM gallic acid equivalents. Details of the statistical summary are shown in Table 3.