Nanostructured TiO2-induced photocatalytic stress enhances the antioxidant capacity and phenolic content in the leaves of Vitis vinifera on a genotype-dependent manner
Graphical abstract
Introduction
Engineered nanomaterials (ENMs) have received a huge attention and come to the forefront of research due to their special features and wide range of potential applications [[1], [2], [3]]. In agriculture, the exploitation of ENMs has grown exponentially over the past few years. The effects of ENMs on plants are intensively studied in relation to germination, growth and development [4,5]. A large number of scientific publications and patents have already appeared relating to carbon-based ENMs, titanium dioxide nanoparticles (TiO2 NPs), silver, silica, alumina and clay nanostructures [[6], [7], [8]]. TiO2 is a widely used n-type semiconductor metal oxide possessing unique optical and electrical properties [9]. In nature, it has four polymorphs such as anatase, rutile, brookite and TiO2(B). Recently, the toxicity of these nanoparticles in the environment is studied intensively [10]. The phytotoxicity of TiO2 NPs is strongly debated in the literature, which is probably due to the different model plants and experimental conditions applied. In addition, the effects of photoreactivity of TiO2 NPs on plants generally are not taken into consideration.
It exhibits appealing photocatalytic activity [11,12], as the irradiation of TiO2 NPs by light with appropriate energy triggers charge separation of the photogenerated exciton, and the resulting free electrons and holes can be exploited in various redox reactions at the surface of the particles. These reactions can lead to the formation of different reactive oxygen species (ROS), which are efficient weapon against pathogenic microorganisms [13], and can also be exploited in the degradation of hazardous organic compounds [[14], [15], [16]].
ROS, moreover, have crucial roles in plants [17]. Depending on cellular concentrations, which are determined by production yields and detoxifying capacities, high amounts of ROS may promote oxidative damage but low amounts may act as signal molecules for upregulating antioxidant defence. Different abiotic stresses – such as cold, heat, drought, salt, nutrient deficiency, UV, toxic metals etc. – boost ROS formation above normal level [18,19]. Plants protect themselves against ROS by both enzymatic and non-enzymatic antioxidants [20]. Plants also make use of ROS as signalling molecules for regulating various physiological responses including development and stress tolerance [17,21,22]. Natural factors, such as biotic or abiotic stressors may result in oxidative cell damage when enhanced ROS production is not counterbalanced by increased antioxidant defence. On the other hand, some ROS, especially hydrogen peroxide [23] and singlet oxygen [24] are also regarded as promoters of antioxidant responses as signal molecules.
The non-enzymatic defence system of grapevine (Vitis vinifera L.) consists of various antioxidants including phenolic compounds, one of the most important groups [25,26]. The major phenolic components of grapevine leaves, caftaric acid and glycosylated quercetins, were shown to be efficient hydrogen peroxide and singlet oxygen scavengers [27], contributing to the observed high total antioxidant capacities in the Pinot noir cultivar [28]. These secondary metabolites are able to scavenge ROS inhibiting the lipid peroxidation, and preventing protein and DNA damages in plants [29,30]. In grapevine the total phenolic content is a sensitive factor for both biotic and abiotic stresses. For example, production of polyphenols was shown to significantly increase in esca diseased grapevine leaves compared to the healthy ones [31]. Content of trans-caffeoyltartaric acid, trans-coumaroyl-tartaric acid, quercetin- and kaempferol derivatives changed similarly to total phenolics production. Drought stress can influence differently the phenolic levels in plants depending the duration and severity of stress. While short-term drought boosted the flavonoid levels in leaves [32,33], the long-term water deficient condition resulted in the depletion of total phenolics content both in grapevine leaves and roots [34]. The level of three phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid (all acids were found in ester-bound form) decreased significantly under the drought stress [34]. Cold stress also resulted in a decrease of the phenolics levels leading to a lower radical scavenging capacity in the grapevine leaves [35]. Acclimative responses of grapevine leaves to stress conditions, on the other hand, rely on increased phenolic contents. UV exclusion was shown to decrease the p-caffeoyl-tartaric acid content of cv. Graciano grapevine leaves under field conditions [36], and the correlation between physiological performance and quercetin-3-O-glucoside and kaempferol-3-O-glucoside in leaves of UV-B stressed vines have been observed in Pinot noir and Riesling cultivars [37]. Also, the more chill-tolerant Maerchal foch grapevine cultivar was characterized by a higher content of phenolic compounds in parallel with better radical-scavenging capacity than the sensitive Kismis lucistyj variant [35].
On the basis of this established link between oxidative stress responses and grapevine leaf phenolics, the present study explored the potential of TiO2 NPs to enhance leaf antioxidant capacity via exogenous, solar UV-mediated ROS production.
In our previous work, photosynthetic response of Vitis vinifera cv. Cabernet sauvignon treated with TiO2 NPs were investigated [38]. We found that TiO2 NPs decreased the photosynthetic rate, while intercellular CO2 level and stomatal conductance increased, suggesting a non-stomatal limitation of the photosynthesis. A slight modification in the flavonol profile was also revealed. This work stimulated us to further investigate the non-enzymatic system of grapevine. We extended the experiments to five grapevine varieties during the blooming phenophase period and under the relatively high UV radiation of June (see data in supplementary material). In addition to verifying the ROS generating potential of Degussa P25 nanoparticles in vitro, we examined the effects of the exogenous ROS generated by these nanoparticles on grapevine leaves. The present study is focused on alterations in phenolic profiles and leaf antioxidant capacities in response to Degussa P25 treatment.
Section snippets
Chemicals and reagents
Acetonitrile and methanol (LiChrosolv® Reag. Ph Eur, Merck, Germany) were gradient grade for liquid chromatography. Formic acid (Alfa Aeasar, 97%) was purchased from Molar Chemicals Ltd., Hungary. Reference substances of quercetin 3-O-rutinoside, quercetin 3-O-glucoside, quercetin 3-O-glucuronide, kaempferol 3-O-glucoside, kaempferol 3-O-glucuronide were obtained from Extrasynthese (Genay, France). Dimethyl sulfoxide, DMSO, (≥99.9%, Sigma-Aldrich), 2,4,6-tris(2-pyridyl)-s-triazine, TPTZ, (98%,
Structural features of TiO2 NPs and EPR investigation of their ROS production in vitro
Foliar exposure of five different grapevines was carried out by using Degussa P25 TiO2 which is one of the most active commercial photocatalysts, therefore it has been frequently used as reference TiO2 in heterogeneous photocatalysis [49]. It is composed of a mixture of anatase and rutile crystalline phases as revealed by our XRD pattern presented in Fig. 1a.
It was recently reported that this dual crystal phase is mainly comprised of individual anatase and rutile nanoparticles, together with
Conclusions
Aqueous dispersion of P25 TiO2 NPs was used for the foliar exposure of five grapevine cultivars in field condition, in order to study their stress responses. The presence of superoxide and hydroxyl radicals as well as singlet oxygen were confirmed by in vitro experiments proposing that foliar application of TiO2 NPs in field condition can cause strong photocatalytic stress. These photocatalytically produced ROS altered significantly the total phenolics, antioxidants and nutrients content of
Acknowledgments
This paper was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences, and by the ÚNKP-18-4 New National Excellence Program of the Ministry of Human Capacities. The work was also supported by Hungarian National Research, Development and Innovation Office (OTKA 124331) and by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the 20765-3/2018/FEKUTSTRAT ‘Innovation for sustainable and
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