Effect of jasmonic acid on major terpenes and density of glandular trichomes in Lippia graveolens kunth (Verbenaceae)

Abstract The effect of exogenous application of jasmonic acid (JA) on the concentration of main terpenes and density of glandular trichomes was investigated in the Mexican oregano, propagated from seeds from 3 localities. JA 1 mM was applied locally and to the whole plant. JA locally applied increased the number of trichomes, with a mean of 20 trichomes more with respect to the controls in plants from Tecomavaca and Zapotitlán Salinas, and significantly increased the thymol concentration by 185% systemically and 255% locally, compared to the control. JA applied to the whole plant decreased the number of trichomes and increased the concentration of caryophyllene from 0.79 to 1.7 mg g−1, and α-caryophyllene from 0.3 to 0.8 mg g−1 in plants from San Rafael with reference to water control. The results suggest a plasticity of morphologic and phytochemical responses, and a potential use of JA to improve phenolic monoterpenes and sesquiterpenes production.


Introduction
There are around 40 species known as Mexican oregano including Lippia graveolens. It grows in arid and semi-arid zones in Mexico and it is widely used as a seasoning in food and medicinally for the treatment of gastrointestinal illness, ear pain, inflammation, and as an abortifacient (Pascual et al. 2001). Essential oil content is characterized by phenolic monoterpenes including thymol and carvacrol (Dom ınguez et al. 1989). However, the essential oil content has shown great variability at both within and among populations (Hern andez et al. 2009). It has been reported that environmental factors are key players in the production of metabolites, for example light affects flavonoids (Moreno-Rodr ıguez et al. 2014), and water status impacts oil content (Silva and Dunford 2005).
Elicitation, defined as an induction of a natural mechanism of plant resistance using biotic or abiotic factors (Malekpoor et al. 2015), is one of the most utilized methods for evaluating responses due to stress in plants. Jasmonates, including jasmonic acid (JA), are used as elicitors. Exogenous application of JA induces the biosynthesis of secondary metabolites and increases the number of glandular trichomes (Boughton et al. 2005;Wasternack and Strnad 2017). However, there are no studies of the effect of elicitors in L. graveolens on the accumulation of useful compounds under controlled conditions. This study evaluated whether the application of JA on L. graveolens obtained from wild seeds collected in three different regions of Mexico, affects the density of glandular trichomes and the concentration of major monoterpenes.

Density of glandular trichomes
Peltate trichomes were identified on the adaxial and abaxial sides of leaves ( Figure S1), as has been previously reported in this species (Mart ınez-Natar en et al. 2011). JA increased the density of glandular trichomes in leaves of locally treated plants, although in some cases it was masked by a possible effect of the solvent. This potential influence in the control of acetone was not uniform or homogeneous, so it is not possible to assign a direct effect of the solvent on the variables studied. Significant differences in the density of glandular trichomes with respect to the controls (P˂ 0.05) were found in the apical region of the adaxial side of leaves from San Rafael, and in the apical and basal regions of the abaxial side on plants from Tecomavaca and Zapotitl an Salinas ( Figure S2) 48 h after treatments. However, 8 days after spraying JA on the whole plant significantly decreased the number of trichomes in comparison with controls (P 0.05) in most experimental groups (Table S2). The effect of JA was more evident in mature leaves with respect to young leaves, although the latter have higher density of glandular trichomes (especially in plants from Zapotitl an Salinas P˂0.016). Kjaer et al. (2012) obtained a similar result and discussed that leaf size increase leads to a 'dilution' effect on the number of trichomes due to the increased area of treated leaves. The effect of JA differed also between adaxial and abaxial surfaces. There is evidence of a differential response in the trichomes on the two sides of the same leaf associated with environmental conditions, for instance less number of trichomes under high air humidity (Lihavainen et al. 2017). The type of response also differs according to the morphotype of trichomes (Tozin et al. 2017) which reflects differences in function among the types of structures and leaf surfaces. Significant differences in the number of glandular trichomes were also detected between the apical and basal regions of the leaves (Tables S2 and S3). In peppermint, new trichomes formation is limited to the basal zone after leaves reached one-half of their mature size (Turner et al. 2000).

Concentration of main terpenes
JA increased thymol content in plants from Zapotitl an Salinas treated locally in comparison with controls and systemic leaves ( Figure S3), with mean concentrations of 4. 74, 1.84 and 1.78 mg g À1 , respectively. Babst et al. (2009) described more intense responses to JA in secondary metabolism genes locally than systemic. But in the same way as in this study, differences between local and systemic also reflected developmental stage of the leaves. There was a systemic effect of JA in plants from Tecomavaca with an increase in thymol concentration compared to control groups (P 0.05) but in plants from San Rafael, caryophyllenes increased compared to both controls. Malekpoor et al. (2015) documented that the application of JA decreased the concentrations of some sesquiterpenes in basil. Similarly, in vitro cultures with JA in lavender have shown an increase in monoterpene concentration and a decrease in the concentration of sesquiterpenes in comparison to control without JA (Andrys et al. 2018).
In relation to the whole plant treatment, JA increased thymol (P < 0.05) and carvacrol (P < 0.05) in mature leaves from Zapotitl an Salinas and Tecomavaca, respectively ( Figure S4) compared to control groups. Thymol and carvacrol accumulations due to JA have been reported in species of Thymus (Alavi- Samani et al. 2015). The comparison between young and mature leaves did not show significant differences in any monoterpene. For some aromatic species the progressive increase in terpenes in young leaves and stability in the accumulation in mature leaves has been reported, although the tendencies can vary between majority and minority components (Rohloff 1999;Gershenzon et al. 2000). At the molecular level, the transcripts responsible for the enzymes involved in the biosynthetic pathway of metabolites have been shown to be higher in young leaves than in mature ones (Babst et al. 2009;Pandelo et al. 2012).
The origin of seeds was a critical factor in both variables. Those differences may be associated with the environmental conditions of each particular zone. All three localities are semi-arid regions, but Zapotitl an Salinas is the driest with 25% relative humidity in May in contrast with 35% in Tecomavaca and 34% in San Rafael. This agrees with Lihavainen et al. (2017) who reported lower densities of trichomes at higher relative air humidity. On the other hand, Zapotitl an Salinas soil contains an elevated concentration of salt (Paredes-Flores et al. 2007). High concentrations of salt in the soil have been shown to alter the distribution and size of different glandular trichome morphotypes and the composition of essential oils in Mentha (Karray-Bouraoui et al. 2009). The phenotype of adaptive defence traits, besides being influenced by a genetic component, is determined by environmental conditions. PCA separated the Zapotitl an Salinas samples from the other two locations in accordance to the accumulation of o-cymene, carvacrol, and caryophyllene as well as the number of trichomes on the adaxial and abaxial sides of the apical region of the leaves ( Figure S5).

Conclusions
JA may induce the formation of new glandular trichomes locally and systemically when it is applied directly on few leaves, but the response is the opposite when the whole plant is treated. Additionally, JA affected the accumulation of major terpenes variably. There were significant increases of thymol and carvacrol in plants from Tecomavaca and Zapotitl an Salinas, and caryophyllenes in plants from San Rafael with respect to the controls. This fact points out the importance of the origin of the plant material and environmental conditions in the induction of secondary metabolites. In our study, despite the geographical proximity among areas, soil conditions and relative humidity are particularly contrasting, which may be shaping the phytochemical and morphological adaptive traits in each population. These results show that JA could be a powerful technique for managing a particular and directed compound accumulation which positively affects the production of plant compounds.