Metabolic response induced by abiotic elicitors in Anacardium othonianum seedlings

Abstract Applying elicitors in plants growing under natural conditions change the metabolic content of volatile compounds extracted from their tissues. Here, we have investigated how abiotic elicitors influence the volatile composition of the ethyl acetate extract of Anacardium othonianum seedlings propagated in vitro and ex-vitro cultures. Abiotic factors such as salicylic acid, silver nitrate, light quality, and cultivation mode affected the composition of volatile compounds in ethyl acetate extract of A. othonianum leaves seedlings. GC/MS analysis demonstrated that α-tocopherol was the main metabolite in cultures added with chemical elicitors, reaching 22.48 ± 4.06% in the case of 30 µM salicylic acid. Higher concentrations of salicylic acid decreased the amount of α-tocopherol. The addition of AgNO3 had a linear effect on α-tocopherol. Thus, the production of this metabolite indicates that salicylic acid and AgNO3 elicitors positively affect α-tocopherol production at specific concentrations helping to understand how plant culture can be better manipulated. Graphical Abstract


Introduction
Elicitation is a strategy that uses physical or chemical agents to induce plants to synthesize phytoalexins and other bioactive metabolites.this strategy has been used to extend plant resistance, increasing the number of beneficial secondary metabolites (Kim et al. 2007;Saini et al. 2014).Elicitors can be classified as biotic or abiotic, depending on their nature (narayani and Srivastava, 2017).the phytochemical profile of plants can be influenced by abiotic factors such as ultraviolet radiation, light quality, cultivation mode, and treatment with elicitors during plant growth and development (Saini et al. 2014;matsuura et al. 2018;artés-Hernández et al. 2022;Espinosa-Leal et al. 2022).
Anacardium othonianum Rizz., is a native Brazilian tree known as the Cerrado cashew tree. A. othonianum is essential in the economy of many Brazilian families because fruits and cashew nuts are used as a food product (Bessa et al. 2013a;Curado et al. 2016;pereira et al. 2021).Furthermore, A. othonianum parts are used in Brazilian folk medicine to treat various diseases, including diabetes and rheumatism.However, A. othonianum seedlings grow slowly, which can hinder the production of this plant and restrict its commercialization.Several authors have studied the growth aspects of A. othonianum concerning the composition of the growth medium of the in vitro culture.these studies aimed to understand which factors influence the growth of A. othonianum and its metabolic composition (Bessa et al. 2013b;Gazolla et al. 2017;Bessa et al. 2019;Rosa et al. 2020;Faria et al. 2021;artés-Hernández et al. 2022).
Secondary metabolites represent an interface between the plants and the environment where they live.the metabolic composition may indicate various aspects of plant development given that the synthesis of these metabolites is often affected by surrounding conditions.thus, the metabolic composition of plants can be used to understand how biotic and abiotic factors influence plant growth (artés-Hernández et al. 2022).thus, to our knowledge, there are no reports on how elicitors impact the composition of volatiles in the ethyl acetate extract of A. othonianum seedlings propagated in vitro and ex-vitro cultures.therefore, in this study, we aimed to verify how abiotic factors such as light quality, salicylic acid, silver nitrate, and cultivation mode influence the composition of volatile compounds in the ethyl acetate extract of leaves of A. othonianum seedlings propagated in vitro cultures.

GC/MS analysis
tables S1, S2, and S3 list the main volatile chemical compounds (relative areas higher than 5%) in the extracts obtained from the leaves of a. othonianum seedlings treated with different light colors or chemical elicitors as determined by GC/mS analyses.

In vitro and ex-vitro culture
the volatile extracts of in vitro and ex-vitro A. othonianum seedling leaves were different.the ex-vitro A. othonianum extract contained a larger number of compounds than the in vitro A. othonianum extract.We characterized the volatiles in the ex-vitro and in vitro A. othonianum extracts and compared between them (table S1).
the phytosterols β-sitosterol, stigmasterol, and campesterol have been associated with plant defense responses against biotic and abiotic stress types.these compounds have beneficial effects on human health.For example, they reduce cholesterol and protect against cardiovascular diseases.therefore, biotechnological strategies for increasing phytosterol production in plants are desirable (miras-moreno et al. 2016).
differences between the chemical profile of the in vitro and ex vitro A. othonianum extracts showed that the tissue culture technique interfered with the original plant metabolism (Castellar et al. 2014).In vitro and ex-vitro metabolic modulation have been studied and can be used to understand how to manipulate plant culture (narayani and Srivastava 2017; matsuura et al. 2018).

Influence of light
in a previous study with A. othonianum, we observed that light color did not influence the average length of the aerial parts or the average number of expanded leaves.on the other hand, red and yellow light increased the weight of fresh and dry leaves by 1.08-and 1.05-fold, respectively, as compared to the control (white light) (Gazolla et al. 2017).
table S2 shows that light quality influenced the volatile chemical composition of A. othonianum.among the ten identified compounds, only the diterpene 16-kaurene and the phytosterol β-sitosterol emerged during treatment with any of the light colors.Blue light elicited the highest β-sitosterol content (18.53 ± 1.55%).We only detected linoleic acid (4.64 ± 0.45%) in seedlings exposed to green light, whilst α-tocopherol (10.77 ± 2.71%) was detected in seedlings exposed to all color light.α-tocopherol has been suggested to protect plants against photooxidation.the photosynthetic process produces large amounts of free radicals, so the biological activity of α-tocopherol is a consequence of its antioxidant properties (Gobbo-neto and Lopes 2007;Cardoso et al. 2019;isah 2019).Cultivation under red light is known to produce α-tocopherol and can be used to improve seedling health and to obtain better cultures.
Here, we found that treatment with light negatively affected the biosynthesis of the alcohol dodecanol and the hydrocarbon 3-heptyl-hexadecane.Light irradiation affects several aspects of plants, including cell growth and the biosynthesis of metabolites (de Carvalho et al. 2020).therefore, the relationship between light wavelength and the composition of volatiles in a plant must be known.this study provides the basis for molecular biology, which aims to elucidate how metabolite production is regulated under the influence of light at different wavelengths, thereby helping to identify factors that can influence the chemical defenses of the plant species under abiotic stress, such as light quality.Studies have shown that light intensity and type can influence the concentration or composition of fixed and volatile secondary metabolites, such as terpenoids (Batista et al. 2018).

Influence of salicylic acid and AgNO 3 elicitors
We used salicylic acid and agno 3 as abiotic elicitors during the experiments with A. othoninum because these chemical elicitors interact by different mechanisms of action.Salicylic acid is essential for inducing systemic acquired resistance (SaR) and plays an important role in plant responses to abiotic stress.agno 3 can inhibit ethylene biosynthesis (Sudha andRavishankar 2002, Giri andZaheer 2016).
our results showed that adding salicylic acid or agno 3 to the culture medium significantly affected the volatile composition compared to the control (table S3).α-tocopherol was the main metabolite in cultures added with a chemical elicitor, reaching 22.48 ± 4.06% in the case of 30 µm salicylic acid.Higher salicylic acid concentration decreased the amount of α-tocopherol (7.03 ± 0.65%), indicating that a higher concentration of this acid negatively affected the A. othonianum immune response.on the other hand, agno 3 addition had a linear effect on α-tocopherol (21.29 ± 13.88% after the addition of 30 µm agno 3 and 36.48 ± 2.32% after the addition of 60 µm agno 3 ).
plants treated with 30 µm salicylic acid contained the highest β-sitosterol and 3-heptyl-hexadecane levels.according to miras-moreno et al. ( 2016), the joint action of elicitors is a strategy that is commonly used to enhance the biosynthesis of phytosterols.this fact has been observed for the association of agno 3 (10 μm) and methyl jasmonate (100 μm), which enhanced phytosterol biosynthesis in Lemna paucicostata plants in vitro.moderate doses of salicylic acid and agno 3 can increase the concentration of antioxidant and anti-inflammatory compounds, inducing stress resistance. in contrast, higher salicylic acid concentrations decrease the stress response and antioxidant activity (munné-Bosch and penuelas 2003;namdeo 2007;de Carvalho et al. 2020).
Concerning the biomass (table S4) and growth parameters (table S5), A. othonianum seedlings elicited with salicylic acid or agno 3 had higher fresh weight, dry weight, and shoot length values and number of leaves than the control, regardless of the chemical elicitor type or concentration.Salicylic acid at 30 µm favored higher fresh (1.37-fold) and dry weight (1.76-fold) compared to the control.Likewise, salicylic acid at 30 µm increased the shot length and the number of leaves by 1.11-and 1.44-fold, respectively, compared to the control.
When we compared all the treatments (ex-vitro, in vitro, light quality, and chemical elicitors), we concluded that the most promising strategy to improve phytosterol production is in vitro cultivation under blue light to obtain higher β-sitosterol content. in turn, red and yellow lights not only provide higher dry and fresh weights, but they also increase the production of the diterpene 16-kaurene.Regarding the chemical elicitors, salicylic acid and agno 3 are beneficial in terms of biomass (fresh and dry weight) and growth parameters (shoot length and number of leaves).However, salicylic acid favors high β-sitosterol and 3-heptyl-hexadecane levels, regardless of the concentration.Finally, agno 3 increases α-tocopherol production linearly.
the in vitro and ex-vitro elicitation technique is essential for understanding metabolic modulation in seedlings.the results have been suggesting that this method could be better designed to increase the metabolic diversity induced in vitro and ex-vitro, aiming at an increase in the plant's immune response and knowledge about its metabolic modulation with the treatment of different elicitors.

Conclusion
the results obtained in this study suggest that red light, the cultivation mode (in vitro and ex-vitro), salicylic acid, and agno 3 can be used to improve the resistance of cashew (A.othonianum) seedlings, which the change in the composition of volatile compounds can confirm.the results help to understand metabolic modulation and how to plant culture can be better manipulated.