The antioxidant system in Olea europaea to enhanced UV-B radiation also depends on flavonoids and secoiridoids

Highlights

• Moderate and high UV-B doses activated similar antioxidant protective mechanisms.

• High UV-B dose promote higher activation of the antioxidant enzymes and AsA/GSH.

• Flavonoids, secoiridoids and HCAds respond similarly to 6.5 and 12.4 kJ m⁻²d⁻¹ UV-B

• UV-B treatments reduce some protective compounds level, such as oleuropein.

Abstract

The Mediterranean crop Olea europaea is often exposed to high UV-B irradiation conditions. To understand how this species modulates its enzymatic and non-enzymatic antioxidant system under high UV-B radiation, young O. europaea plants (cultivar “Galega Vulgar”) were exposed, for five days, to UV-B radiation (6.5 kJ m⁻² d⁻¹ and 12.4 kJ m⁻² d⁻¹). Our data indicate that UV-doses slightly differ in the modulation of the antioxidant protective mechanisms. Particularly, superoxide dismutase (SOD), guaiacol peroxidase (GPox) and catalase (CAT) activities increased contributing to H₂O₂ homeostasis, being more solicited by higher UV-B doses. Also, glutathione reductase (Gr) activity, ascorbate (AsA) and reduced glutathione (GSH) pools increased particularly under the highest dose, suggesting a higher mobilization of the antioxidant system in this dose. The leaf metabolites’ profile of this cultivar was analysed by UHPLC-MS. Interestingly, high levels of verbascoside were found, followed by oleuropein and luteolin-7-O-glucoside. Both UV-B treatments affected mostly less abundant flavonoids (decreasing 4ʹ-methoxy luteolin and 4ʹ or 3ʹ-methoxy luteolin glucoside) and hydroxycinnamic acid derivatives (HCAds, increasing β-hydroxyverbascoside). These changes show not only different mobilization with the UV-intensity, but also reinforce for the first time the protective roles of these minor compounds against UV-B, as reactive oxygen species (ROS) scavengers and UV-B shields, in complement with other antioxidant systems (e.g. AsA/GSH cycle), particularly for high UV-B doses. Secoiridoids also standout in the response to both UV-B doses, with decreases of oleuropein and increases 2ʹʹ-methoxyoleuropein. Being oleuropein an abundant compound, data suggest that secoiridoids play a more important role than flavonoids and HCAds, in O. europaea protection against UV-B, possibly by acting as signalling molecules and ROS scavengers. This is the first report on the influence of UV-B radiation on the secoiridoid oleuropein, and provides a novel insight to the role of this compound in the O. europaea antioxidant defence mechanisms.

Introduction

UV-B radiation (280–315 nm) is a major high-energy component of natural sunlight, playing a crucial role in terrestrial ecosystems, particularly in the Mediterranean region often exposed to values around or above 7 kJ m⁻² d⁻¹ (Correia et al., 2012). The levels of UV-B radiation reaching Mediterranean ecosystems also depend on environmental factors such as ozone layer, clouds or air pollution (Verdaguer et al., 2012), and values as high as ~12 kJ m⁻² d⁻¹ have been reported (e.g. Forster et al., 2011).

UV-B radiation is perceived by different sensitive proteins including the ultraviolet-B receptor (UVR8) initiating the plant's response by inducing a switch in the development signalling cascades, and by regulating secondary metabolites pathways (Huché-Thélier et al., 2016). However, UV-B in excess becomes a stressor raising plant's reactive oxygen species (ROS) (Rácz et al., 2018). Plants response to UV-B radiation depends on dosage, time, species/genotype and environmental factors (Escobar et al., 2017), and is mediated by a complex network of interactions combining physical and biochemical protection/repair mechanisms. Such mechanisms are well evidenced in the morpho-physiology of several Mediterranean native species (e.g. Verdaguer et al., 2012; Bernal et al., 2013; Dias et al., 2018).

UV-B radiation is a major source of ROS, like O₂^•- and H₂O₂, against which plants activate sophisticated antioxidant defence systems. The enzymatic system includes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (Gr), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX), which stimulation by enhanced UV-B exposure has already been widely demonstrated (e.g. Martínez-Lüscher et al., 2015; Escobar et al., 2017; Mariz-Ponte et al., 2018; Rácz et al., 2018). The non-enzymatic system includes multiple families of metabolites (e.g. ascorbate, glutathione, flavonoids, phenolic acids and carotenoids). Some reports have addressed the role of ascorbate (AsA) and reduced glutathione (GSH) in the maintenance of homeostasis under high UV-B radiation (Yao et al., 2015). For example, AsA plays a crucial role in the protection of membranes and influences plant protective pathways involving e.g. anthocyanins, violaxanthin de-epoxidase activity, and non-photochemical energy dissipation (Gill and Tuteja, 2010). GSH is a redox buffer (scavenges ¹O₂, H₂O₂ and OH^•) being also involved in the regulation of gene expression and of cell division (Gill and Tuteja, 2010; Silva et al., 2013).

Polyphenols are among the largest and more complex family of antioxidants. Metabolomic approaches have contributed to unveil the profiles of protective polyphenols in plants, including against excessive UV-B, and to clarify how UVR8 photoreceptor mediates secondary metabolism (e.g. Wargent et al., 2015; Huché-Thélier et al., 2016; Escobar et al., 2017). Phenylpropanoids, such as flavonoids (e.g. quercetin and luteolin) and hydroxycinnamic acids (e.g. caffeic, ferulic and sinapic acids) accumulated mainly in the leaf epidermal cells, screening UV-B radiation that can reach photosynthetic leaf tissues (Neugart et al., 2014; Escobar et al., 2017). These and other secondary metabolites can also act as antioxidants through the scavenging of ROS, such as O₂^•-, OH^• and ¹O₂ (Escobar et al., 2017). Some studies suggest that hydroxycinnamic acids are predominantly involved in UV-B screening (e.g. p-coumaric, ferulic and caffeic acid with ε_max in the 310–325 nm), whereas flavonoids, especially o-dihydroxy B-ring substituted flavonoids (e.g. quercetin 3-O and luteolin 7-O-glycosides), counteracting the generation of ROS (Bernal et al., 2013). Also, compared to monohydroxy B-ring-substituted flavonoids (e.g. apigenin 7-O and kaempferol 3-O-glycosides), o-dihydroxy B-ring-substituted flavonoids have a greater antioxidant capacity (Brunetti et al., 2013). The ROS scavenging properties of these compounds rely on the hydroxyl group of the B-ring structure that donate a hydrogen and an electron to a radical, stabilizing them and generating a relative stable flavonoid phenoxyl radical (Mierziak et al., 2014). The formed molecule may react with another radical forming a stable quinone structure (Sarian et al., 2017). Some reports pinpoint that o-dihydroxy B-ring-substituted flavonoids can be found in several cell compartments near the centres of ROS generation, or be transported from their sites of biosynthesis to these compartments, such as mesophyll chloroplast (where they have a role as scavengers ¹O₂), nucleus (where may inhibit ROS-generation making complexes with Fe and Cu ions), and vacuoles (where they serve as co-substrates for peroxidases to reduce H₂O₂ escape from the chloroplast) (Agati et al., 2012; Brunetti et al., 2013). Moreover, flavonoids can also prevent oxygen radical formation by inhibiting the activity of the enzymes involved in their generation (Mierziak et al., 2014).

Another group of polyphenols, the secoiridoids, is much less studied in part because they are exclusive to the Oleaceae family (e.g. Talhaoui et al., 2015). This family of polyphenols plays, together with flavonoids, a crucial defensive role to different stressful conditions in species of major importance in the Mediterranean landscape like O. europaea (Rodrigues et al., 2015). For instance, the synthesis of the secoiridoid oleuropein increases in response to drought, salinity, pathogen infection and herbivorous suggesting some involvement in plant stress defence mechanisms (Daane and Johnson, 2010; Petridis et al., 2012a, 2012b). Moreover, whilst the plant mechanisms tailoring secoiridoids response to UV-B are not well known, oleuropein is a major compound in O. europaea organs, contributing to the sensory-value of its fruit and oils, and its antioxidative/bioactivity role is being largely studied in medicine (e.g. Rodrigues et al., 2015).

Olea europaea L. (Oleaceae) is one of the most socio-economically important cultivated fruit tree in the Mediterranean region, and is an example of well adapted species to the high-irradiance and semi-arid conditions of this region. Little is known about the performance and defence mechanisms underlying O. europaea tolerance to high UV-B radiation, and available studies report the increase of leaf cuticle thickness (Liakoura et al., 1999), the up-regulation of the antioxidant enzymes system (Koubouris et al., 2015), the accumulation of lipophilic compounds (fatty acid, alkanes and terpenes) (Dias et al., 2018) and some flavones (luteolin, quercetin and apigenin derivatives) (Liakopoulos et al., 2006). Interestingly, being oleuropein a major compound of O. europaea leaves, together with luteolin and verbascoside (Rodrigues et al., 2015), it remains unknown the role of oleuropein in this species’ defence system against UV-B. Moreover, the abundance and ratio of this compound depend on the environmental conditions, on the cultivar and organ. For example, in some cultivars (e.g. “Koroneiki”, “Megaritiki” and “Kalamon”), oleuropein was identified as the major component (Talhaoui et al., 2015).

In the present work we hypothesized that O. europaea tolerance to enhanced UV-B radiation relies on an equilibrated response of non-enzymatic antioxidants [by adjusting secoiridoid (e.g. oleuropein), phenylpropanoid (e.g. luteolin and verbascoside derivatives) and ascorbate/reduced glutathione metabolite levels] combined with an up-regulation of the antioxidant enzyme system. For that, an ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) – based phenolic metabolite profiling, combined with oxidative stress and antioxidant system analyses, were conducted in O. europaea plants exposed to moderate and high UV-B doses in order to: a) identify which enzymatic/non-enzymatic defence mechanisms are activated by each UV-B dose; b) assess the phenolic-metabolome profiles in response to increased UV-B radiation; and c) identify relevant UV-responsive phenolic compounds that may be related with O. europaea defence strategies.