Up to the best of our knowledge, there is no study on the effect of environmental stresses on surahalala plants. However, according to previous studies23,24,25, essential oil contents and the expression profiling of their related genes along with the growth of aromatic plants are significantly affected by altering the environmental conditions. Chrysargyris et al.26 reported that water deficit is one the major environmental factors changing the synthesis and production of secondary metabolite in medicinal plants. Moreover the such stress condition can cause lower plant growth and matter production in significant amounts. On the other hand, proline has reported to be an effective free amino acid in response to environmental stresses where it can play as osmoregulatory elements in plants. In most of cases application exogenous proline led to higher tolerant in plants under water deficit stress. However, its application has sowed different effects on the contents of secondary metabolites and the expressional levels of their related genes27,28,29,30,31,32.
In order to quantify the effect of PEG application as a simulator of water deficit condition and the effect of proline in such situation, SOD activity and H2O2 content were measured. The results according to higher H2O2 content and SOD activity under higher PEG levels showed that PEG application was able to properly simulate the stress condition. In addition, the content of free proline increased in response to PEG application in surahalala plants. Though, PEG led to significant decrease in plant growth by affecting the shoot and root weights. Moreover, the results of proline application on SOD activity and H2O2, on one hand, and on fresh shott and root weights of the surahalala plants, on the other hand, indicated that higher concentration of proline than 15 µm might causes stress-like condition in this plant. The application of proline up to 15 µm showed mainly decrease in the SOD activity and the content of H2O2 along with higher shoot and root weights in comparison with the control, under all PEG levels. The content of proline, however, was continuously increased in response to higher level of exogenous application of proline. These results were verified by different advanced data mining methods in which the associations of SOD and H2O2 with root and shoot weights were highly negative. In most previous studies application of proline resulted in higher dry mater production and the tolerance of the plants33. Although, in our study was verified that higher contents of proline than a critical point might lead to negative effect on the plants. In concordance with our study, different authors showed that proline regulation application was able to decrease the negative effects of environmental stress on Trifolium repens L.30, tobacco32, Glycyrrhiza uralensis27, and chickpea28 mostly via decreasing the content of H2O2 and malondialdehyde and also increasing the activity of enzymatic antioxidants. Also, Zali and Ehsanzadeh33 and34 Showed that proline was capable of increasing the growth of fennel And Phaseolus vulgaris under water deficit conditions and nutrient deficiency.
Measuring secondary metabolites in the current study indicated that PEG application has positive effect on the contents of most essential oils. On the other hand, in all metabolite such as 1,8-cineole, Carvone, Thymol, and β-citronellol the highest PEG level (20%) did not show highest content, instead it showed lower contents in compare with 10% level of PEG. This result indicates that environmental stresses might play a role in activating some genes that are involved in both essential oils synthesis pathways and stress responsive genes. Nonetheless, the complex polyploidy levels in line with lack of genomic and transcriptomic information related to the majority of aromatic plants have limited further study even on model plants such as mint species35. In some studies23,36,37, associations between genes that are involved in ABA, jasmonic acid and gibberellin responsive pathways which are enhanced under specific stresses and the metabolite gene network have been reported.
In line with our results, Akula and Ravishankar38 and Aftab39 reported higher secondary metabolites in response to environmental stresses, but there is no study related to the stress effect on metabolite contents of surahalala. Meanwhile, there were positive association between 1,8-cineole, Carvone, Thymol, and β-citronellol contents with proline content in surahalala and they showed low Euclidian distances from one another as well as placing in similar clusters. These essential oils were increased by increasing the level of proline up to 15 µm and then decreased dramatically. The α-pinene and estragole contents showed negative response to proline application. Since 1,8-cineole, carvone, thymol, and β-citronellol levels were increased in response to 10% PEG and up to 15 µm proline in surahalala plants, it seems that low water deficit condition and moderate application of proline may a proper combination of treatment leading to increase their concentrations per dry matter unit in this plant. On the other hand, α-pinene and estragole showed negative associations with proline, but their contents were generally equal to, or in some cases higher than, the control proline treatment. Therefore, the suggested treatment combination of low water deficit level and moderate application of proline would not have significant negative impacts on these compounds.
As a class of secondary metabolites, monoterpenes are mainly identified in fungi, but different contents of them have also been reported in some medicinal plants such as eucalyptus40 pepper mint, and recently in surahalala2. These compounds showed variety functions in plants and are involved in some basic and specialized metabolisms. Some of these compounds play a role in odor synthesizing for attracting the pollinators. There are reports related to their operations in defense mechanisms against herbivores and plant pathogens by synthesizing some toxic compounds. There are some studies indicating that these compounds are involved in sinical transduction under various stresses after the starting damage to the plant cell2. In the current study, H2O2 as signaling molecule showed significant and positive associations with α-pinene and estragole based on the results of data mining which are concordance the previous studies33,41. Other reports regarding the importance of monoterpenes are related to their impacts on symbiosis relationship of their plants by accumulating in their roots and rhizomes42,43. In addition to the importance of monoterpenes on the plants, different effects of these compounds on human diseases have been reported.
In aromatic and pharmaceutical plants, the content of essential oils and terpenoid compounds are important substances with high economic values. The major contents of surahalala essential oils, according to Mozafari et al.2, are consisted of monoterpenes. 1,8-cineole is a commercially significant monoterpene compound that has pharmaceutical applications and is considered as a potential biofuel. According to the literatures, this compound has anti-inflammatory impacts and Linghu et al.44 reported that it was able to subside the inflammatory effect of human umbilical vein endothelial cells (HUVECs) disease phenotype45. Similarly, carvone is known to have anticarcinogenic properties in human tissues. Carvone is also able to synthesis attractive odors and flavors applicable in aromatherapy and food products46. In our study carvone content showed high Euclidian distances from SOD activity and H2O2 indicating revers associations between these compounds in some conditions. In line with these results, Huchelmann et al.47 described that carvone is able to hinder the production of a stress-induced compounds and metabolites in tabacum leaf. In addition to 1,8-cineole, pinene has anti-inflammatory and also antimicrobial activities. Surahalala plants under the water deficit and proline treatments showed to have thymol in their shoot which is able to be used as an effective antimicrobial compound. Having antiseptic effects caused thymol to be used as ingredient in mouthwash and toothpaste products41. Another monoterpene that was distinguished in surahalala plants is estragole which can act as an agent for attracting the pollinators, play a role in defense mechanism against pathogens and herbivores, and be applied in food products and spices as flavoring compounds48. Similarly, β-citronellol is used in perfume factories and can act in pollinators attractions along with repellent effects on some organisms mainly mosquito49.
As it verified by the results of this study and mentioned in the results of some other studies6,11,15,17, different the environmental conditions lead to change in the contents of secondary metabolites. The contents and biosynthesis of the monoterpenes are normally regulated by similar or different molecular and genomic pathways which are most commonly functioning as the connected gene networks. Altering environmental conditions or treating plants with various compounds may directly trigger or repress some genes in monoterpenes genomic pathways, or the genes that are responsible for transcribing their sequences into RNAs. The levels of changes under altered conditions could be quantified by expression rates of related genes and considering the transcriptomic patterns of the plants’ tissues. In this study, all considered monoterpenes were affected by the stress condition and proline treatments. Hence, assessing the expression rates of the genes involved in their biosynthesis can help us to find the origin of the changes in such metabolites and lead the way to future genetic engineering toward upregulate or downregulate their expression. The monoterpenes that are considered in this study are the product of enzymatic activities transforming specific precursors.
According to Chen et al.35, terpene synthase (TPS) gene family is largely involved in terpenes biosynthesis of Mentha longifolia. In their study, TPS family was divided into six subfamilies sharing a great number of mostly identical and some similar motifs. After considering these motifs in all available data bases of the aromatic plants we found over 90% similarity among these motifs indicating that these subfamilies are most likely available in surahalala with similar sequences and tasks. However, sequence analyses of complete cds of different TPSs within and between different species by Huchelmann et al.47 indicated that the similarity of these genes within each species genome is significantly higher than the similarity between genomes of different species.
Almost all of the precursors of different monoterpenes are product of similar pathways in which TPS genes act in nearly end of these pathways. In these pathways, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are synthesized at the initial points. IPP and DMAPP are direct products of two other pathways consist of mevalonate pathway (MVA) in the cytosol and phosphate pathway (MEP) in the chloroplast. Generally, IPP and DMAPP are directly transferred into geranylgeranyl pyrophosphate (GGPP), farnesyl diphosphate (FPP), and geranyl diphosphate (GPP) by catalyzing activities of prenyltransferases. After that, different TPS genes catalyze these compounds into the precursors of monoterpenes and other secondary metabolites. The produced precursors are then modified by enzymatic activities transcribed from different enzymatic genes35.
The content of 1,8-cineole in this study was higher than its content in other plants, and the proline treatments specially under water deficit condition sufficiently increased the levels of this monoterpene. Huchelmann et al.47 attributed the low level of 1,8-Cineole in nicotiana tabacum to rapid conversion of GPP precursor into other monoterpenes other than 1,8-Cineole. Therefore, proline treatment water deficit condition most likely trigger the expression of some genes involved in either higher production of 1,8-Cineole precursor or more rapid conversion of the precursor GPP into this monoterpene in surahalala plants. Other than treated surahalala plants, control plants showed slightly higher 1,8-cineole content than other plants. In study of Chen et al.50, 1,8-Cineole has shown negative effects on germination and significantly reduced its percent in Arabidopsis; consequently, the problems with direct germination of surahalala plants may be the results of higher 1,8-cineole content in this plant which led us to produce it under in vitro condition. On the other hand, one of genes involved in production of 1,8-cineole precursor is TPS27, which its relative expression rate in surahalala plant under moderate water deficit and slight concentration of proline was significantly higher than control conditions and high concentrations of PEG (20%) and proline (20 µm).
The expression rate of L3H gene was significantly increased by applying PEG and proline treatments in surahalala which its highest rate was achieved by 10 or 15 µm proline under 10% PEG. The expression rate of this gene showed positive and significant correlation with carvone content in surahalala and based on the data mining results, the Euclidian distance of this monoterpene and L3H expression rate was the significantly lower than other gene expression profiling. Carvone results from MEP pathway that produces geranyl diphosphate (GDP) and it showed negative regulatory effects on MVM pathway resulted in negative association and high distance between this monoterpene and other assessed monoterpene in this study, for example estragole, that are produced by MVM pathway. After biosynthesizing of GDP, limonene synthase (LS) enzyme converts it to limonene by separating the diphosphate group. Next, hydroxylation of limonene (C6 position) by L3H and L6H by co-factoring of NADPH transforms it into carveol and finally into carvone by dehydrogenases mechanism51. One of the reasons for increasing the content of carvone under water deficit condition in the current study may be the production of signaling molecule such O– and OH– that are capable of dehydrogenating different compounds. On the other hand, proline might get involved directly in L3H production pathway or indirectly help NADPH cofactor in carvone pathway, lead to higher expression profiling of L3H by both ways. In the study of Xie et al.49 higher gibberellin content, which is affected by stress conditions, showed positive interaction with carvone content.
The anti-inflammatory and antimicrobial of α-pinene have verified by different studies. According to Wu et al.52, α-pinene is a specific product from aromatic plants which the most of other organisms are not able to produce it. α-pinene is directly synthesized by the activity of pinene synthase (PS) enzyme from GGPP. In the current study, the expression rate of TPS2 gene was increased in response to application 10% PEG, while it decreases by application 20% PEG. This result indicates that moderate stress on surahalala plant can lead to higher conversion of GGPP into this monoterpene. The results of data mining verified these results and showed that the expression rate of TPS2 and also the content of α-pinene were positively correlated and they had low Euclidian distance from those treatments that contained 10% PEG in two-dimensional representation of canonical correlation. Under no stress and sever stress conditions expression rate of TPS2 showed no regular pattern in response to proline treatments, while its rate was the highest under moderate stress level (10%PEG) with no proline application. Therefore, proline treatment has a negative regulatory effect on the expression rate of TPS2. To the best of our knowledge, there is no study related to the effect of proline treatment on expression rate of TPS2 gene or the content of α-pinene. Wu et al.52 showed that in addition to enzymatic activity of TPS2, fusion of the pinene synthase (PS) with heterologous geranyl diphosphate synthase (GPPS), they called GPPS-PS significantly increased the content of α-pinene and β-pinene.
In the previous study by Hoseiny et al.2, thymol was detected in surahalala shoots. In the current study, the content of thymol was increased as the results of proline treatments and PEG application, however, the best combination for achieving higher thymol content was 15 µm proline level applied under 10% PEG as moderate water deficit condition. One pathway of biosynthesis of thymol is through conversion of GPP to neryl pyrophosphate (NPP) which in turn it transforms to γ-terpinene and then into p-cymene. The final product of this pathway is either carvacrol or thymol. In surahalala both of these monoterpenes were detected by gas chromatography method used in this study; though, the content of thymol was higher in almost all samples. Majdi et al.53 described TPS1 as a significant agent in thymol biosynthesis. The expression profiling of this gene (TPS1) showed similar pattern to the content of thymol in response to the treatments. The main product of TPS1 activity in metabolite biosynthesis pathway is to γ-terpinene, one of the precursors of thymol. Canonical correlation and correlation plot showed that TPS1 gene expression in addition to low two-dimensional distance and high correlation with thymol, it showed a close relationship with β-Citronellol content as well. Additionally, the relative expression of TPS1 showed significant association with 1,8-cineole content based on the results from heatmap analysis. This means that TPS1 is probably involved in other metabolite biosynthesis pathways in surahalala and it likely is not a pathway specific enzyme.
The availability of estragole metabolite in surahalala was first reported by Hoseiny et al.2. The importance of this metabolite in surahalala is related to its role as a defense compounds against different microorganisms and herbivores, in addition to pollinator attractant 48. Estragole is a subset of phenylpropenes group that also contain isoeugenol, eugenol, and transanethole (isoestragole). A part of flavoring properties of some plant species such as banana (Musa sapientum)54, melon (Cucumis melo)55, tomato (Solanum lycopersicum)56, and strawberry (Fragaria vesca)57 is resulting from the phenylpropenes group available in these fruit in their sequestered glycosides or as free volatiles forms. Estragole is normally biosynthesized via IPP pathway which its precursor are the compounds of para-hydroxy group such as coniferyl acetate41 and p-allylphenol58. O-methyltransferases (OMTs) is an important enzyme act in transforming para-hydroxy compounds to estragole by using a methyl donor (S-adenosylmethionine; SAM)48. The content of estragole in surahalala plants were significantly increased in response to higher water deficit condition and higher proline treatments. Similarly, the expression profiling of OMT genes showed positive response to these treatments. Unlike other considered metabolites in this study, the highest content of estragole in surahalala plants was obtained in highest proline level (20 µm) under highest PEG concentration (20%). The effectiveness of these treatments comes from their impact on methyl productions pathways because some methylated compounds could be used in signal transduction activity in response to stress conditions. As it was mentioned earlier, the highest level of proline in surahalala plant is probably causes nutrient stress or make water deficit severer leading to activating some networks in stress signaling pathways that take advantage of methylated compounds.
β-Citronellol is another metabolite that was detected in surahalala and its availability in this plant was also reported previously by Hoseiny et al. 2. This metabolite significantly increases the importance of using surahalala extracts in industrial products as the result of its application in insect (specially mosquito) repellents and perfumes59. β-Citronellol content and the expression rate of geraniol dehydrogenase GDH3 showed significant and positive correlation (0.73) resulting from their similar patterns in response to PEG and proline application in this study. Base on the clustering results of metabolites and the differential expressions of considered genes in this study obtained from heatmap method, GDH3 expression rate and β-Citronellol showed the closest relationship with 10 and 15 µm proline and 10% PEG treatments. GDH is one the significant enzymes act in biosynthesis of geraniol and citronellol by dehydrogenizes the GPP precursors. Moreover, GDH3 expression rate showed low Euclidian distances in two-dimensional plots of biplot and canonical correlation from TPS1 and L3H expression rates and the thymol content in surahalala.
