Profile of Essential Oils From the Leaves of Annona Grafted

Mechanical damage, during grafting, results in the formation of reactive oxygen species, which are neutralized by the enzymatic and non-enzymatic antioxidant systems which may influence the essential oil composition of grafts and rootstocks because of the formation of oxygenated terpenes, substances with higher reactivity against pathogens. Various studies have demonstrated the biological activity of Annonaceae and the important pharmacological potential of the substances produced by the genus Annona. Thus, we studied the essential oils of leaves collected from the graft (Annona × atemoya) and lateral budding from the rootstock (Annona emarginata), in the same individual, in order to characterize chemical profile. The extraction was carried out by hydrodistillation and separation, quantification and identification of the substances were performed by gas chromatography coupled to a mass spectrometer. Chemical profiles of essential oils were evaluated by principal component analysis (PCA). Annona × atemoya and Annona emarginata presented chemical profiles of essential oil with specific substances. Some substances are common in oils of both species. Germacrene D is predominant in Annona × atemoya and α and β-selinene, β-elemene and spathulenol occurred in Annona emarginata. Our results suggest that the profile of such essential oil may be another indicator for the success of the combination of these two species.


Introduction
The Annonaceae family has the important genus Annona, mainly because of its edible fruits.Several studies have demonstrated in Annona biological activity of substances, which have important pharmacological potential (Boyom et al., 2011;Fontes et al., 2013; D. M. S. Ocampo & R. C. Ocampo, 2006;Siqueira et al., 2011).
Essential oils are substances produced by specialized metabolism and are characterized by the odor they produce.Essential oils are predominantly made up of terpenes (monoterpenes and sesquiterpene (Bakkali, Averbeck, Averbeck, & Idaomar, 2008).
The grafting process results in transferring molecules, such as mRNAs, between the rootstock and scion, which may interfere in the synthesis of other molecules involved in the primary (Baron et al., 2019;Baron, Bravo, Maia, Pina, & Ferreira, 2016;Kanehira et al., 2010) and specialized metabolisms, as essential oils.
The great susceptibility of atemoya to pathogens is controlled by means of grafting on A. emarginata, whose resistance (Baron et al., 2019) may be related, at least in part, to biosynthesized substances in the specialized metabolism, such as terpenes, which are present in essential oils.Thus, we studied the essential oils of leaves collected from the graft (Annona × atemoya) and lateral budding of the rootstock (Annona emarginata), in the same individual, in order to characterize the chemical profile.

Plant Material
Annona × atemoya plants were grafted onto Annona emarginata (Schltdl.)H. Rainer variety of terra-fria in commercial crop.Although productivity was guaranteed, among other factors, by the removal of lateral budding of the rootstock, in this study the leaves of these shoots were collected, which allowed the study of the essential oil of the graft and rootstock in the same individual under the same conditions.
The plant material was collected on Paraizinho farm in the city of Pardinho, São Paulo, Brazil (23°5′3″ S, 48°22′38″ W; 895 m above sea level).The collection was conducted during late spring between 9:30 AM and 10:00 AM.Subsequently, the leaves were dried at 40 °C to a constant dry weight.

Oil Extraction and Analysis
After drying, 80 g dry masses of the leaves of the two species were hydro-distillized for 2 h to have essential oils extracted in a Clevenger-type apparatus.Essential oils were separated from the aqueous phase using the solvent dichloromethane (0.5 mL, Merck ® ).The extracted oils were stored in amber glass vials at -20 °C prior to the analysis of their chemical composition (Campos, Baron, Marques, Ferreira, & Boaro, 2014).
The compounds were identified by comparing the obtained mass spectra with the database system GC-MS (Nist.62 lib.), literature (Adams, 2007), and retention index (RI).In order to obtain RI of the substances, a mixture of n-alkanes (C 9 -C 24 ; Sigma Aldrich ® 99%) was employed and analyzed under the same operating conditions of samples, and the equation by Van den Dool & Kratz (Van Den Dool & Kratz 1963) were used.

Statistical Analysis
Principal component analysis (PCA) was carried out with the substances presented in the essential oil using XLSTAT program (2017).

Results and Discussion
Substances classified as monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes were identified in essential oils of both species, all of them from terpenes class (Table 1).
Sesquiterpenes were predominant in essential oils, while sesquiterpene hydrocarbons and oxygenated sesquiterpenes constituted by 90.1% and 72.9% of essential oils of atemoya and A. emarginata, respectively.
Monoterpenes are highly affected by temperature and luminous intensity, and thus, can be volatilized (Bakkali et al., 2008;Wang, Owen, Li, & Peñuelas, 2007).The percentage difference of sesquiterpenes observed in both species, which was higher in atemoya, may be due to the crown of atemoya and was more exposed to light and temperature.Atemoya was grafted on A. emarginata, whose collected lateral budding (thief branch) was more protected from light and temperature.
The multivariate analysis revealed that the rootstock species (A.emarginata) and graft (Annona × atemoya) presented oils with major substances, characterizing two different groups of chemical profiles.A. emarginata was mainly discriminated by α and β-selinene, β-elemene and spathulenol, totalizing 41.75% and Annona × atemoya by germacrene D (42.82%) (Figure 1).Biotic factors have the potential to alter the chemical composition and production of essential oils in which the molecules are formed by the interaction between plants and environment (Dudareva, Klempien, Muhlemann, & Kaplan, 2013;Holopainen & Gershenzon, 2010).
Moreover, a high percentage of oxygenated terpenes in essential oils of atemoya (12.19%) and A. emarginata (20.14%) was identified, which may indicate stress, because the comparison between the chemical profiles of essential oils from the ungrafted rootstock A. emarginata, grown in a greenhouse, demonstrated the absence of oxygenated terpenes (Campos et al., 2014).
Thus, the mechanical damage resulting from grafting (Suzuki & Mittler, 2012) may have been the cause of stress, leading to the formation of reactive oxygen species (ROS) (Suzuki & Mittler, 2012).The increased production of oxygenated terpene fractions may have contributed to ROS neutralization by non-enzymatic antioxidant system (Gill & Tuteja, 2010).Terpene oxygenates are molecules with high reactivity against pathogens (Oda, Fujinuma, Inoue, & Ohashi, 2011), which may explain their high percentage in A. emarginata, which is used as the rootstock for atemoya.Moreover, the pathogen resistance in A. emarginata may originate from the union of the species with atemoya because terpene oxygenates were observed after grafting and have not been observed in non-grafted plants (Campos et al., 2014).Furthermore, the union of plants at different ages can be an additional reason for the increased ROS production.
After grafting, the evaluation of the essential oil composition of the leaves of atemoya variety Thompson identified 28 substances, of which germacrene D was the most abundant (42.8%).In the essential oils extracted from A. emarginata, 36 substances were identified, with β-selinene (12.6%) and α-selinene (12.2%) forming the major components (Table 1).Germacrene D is a sesquiterpene hydrocarbon that is related to ecological roles in the interaction of plants with their predators and pollinators (Prosser et al., 2004), functions as an attraction agent, and facilitates the location of plants for food and ovipositor.Sesquiterpenes, similar to pheromones, bind to the receptors located on the antennae of insects, as demonstrated in Heliothis virescens (Mozuraitis, Stranden, Ramirez, Borg-Karlson, & Mustaparta, 2002;Skiri, Galizia, & Mustaparta, 2004;Stranden et al., 2003).
These substances demonstrated biological activity, which may be related to insect-plant interactions, including the attraction of insects and protection or defense of plants against pathogens, and operated as antioxidant non-enzymatic substances against oxidative stress.Table 1.Chemical composition (%) of the essential oils of atemoya (Annona × atemoya) and Annona emarginata