The effect of drought stress on the expression of key genes involved in the biosynthesis of phenylpropanoids and essential oil components in basil (Ocimum basilicum L.)
Graphical abstract
Drought stress may enhance the amount of methylchavicol content, in part, through increasing the expression of chavicol O-methyl transferase (CVOMT) gene, which is involved in the last biosynthetic step of estrogole (methylchavicol) formation.
Introduction
Plant essential oils, including general terpenoids and phenylpropanoids as major constituents, are important sources of aromatic and flavoring in food, industrial, and pharmaceutical products (Charles and Simon, 1990). Phenylpropanoids are a group of small phenolic molecules, which are key elements in many important herbs and species, including cloves, cinnamon, basil and tarragon (Tahsili et al., 2012). Ocimum basilicum, belonging to family Lamiaceae, is one of the best known genera for its medicinal properties and economically important aromatic oils (Rastogi et al., 2014). The Ocimum genus comprises annual and perennial herbs and shrubs native to the tropical and subtropical regions of Asia, Africa, and Central South America and includes 50–150 species and subspecies (Javanmardi et al., 2002, Labra et al., 2004). The glands of O. basilicum (2n = 48) are rich in phenylpropenes as well as monoterpenes and sesquiterpenes (Gang et al., 2001, Iijima et al., 2004, Werker et al., 1993). It is indigenous to Persia and India (Bilal et al., 2012) and is widely cultivated for the production of essential oils and containing high proportions of phenylpropanoid derivatives, such as eugenol, methyleugenol, chavicol, metheylchavicol, often combined with various proportions of linalool, a monoterpen, and sesquiterpenes (Tahsili et al., 2012). These compounds are synthesized and stored in peltate glands found on the surface of leaves, stems, and flowers (Iijima et al., 2004). Among the plants known for medicinal value, the plants of genus Ocimum are rich in phenolic compounds and are very useful for their therapeutic potentials (Ramesh and Satakopan, 2010). Studies indicate that O. basilicum possesses analgesic, anti-inflammatory, antimicrobial, and cardiac stimulant properties (Bilal et al., 2012). Major aroma compounds found in volatile extracts of basil exhibited varying amounts of anti-oxidative activity. Furthermore, ingestion of these aroma compounds may help to prevent in vivo oxidative damage, such as lipid peroxidation, which is associated with cancer, premature aging, atherosclerosis, and diabetes (Lee et al., 2005). Despite the wide uses and the importance of sweet basil and its essential oils, little is known about the biosynthesis and developmental regulation of the compounds responsible for the flavor quality of the fresh and dried herbs (Wang and Pichersky, 1999).
Biosynthesis of phenylpropanoid compounds is produced from the shikimate pathway and regulated by several groups of enzymatic reactions through metabolic channels (Dixon et al., 1992). All phenylpropanoids are derived from cinnamic acid, which is formed from phenylalanine by the deamination action of phenylalanine ammonia-lyase (PAL) (Fig. 1). PAL is one of the most important enzymes with a key role in regulation of phenypropanoid production in plants (Achnine et al., 2004, Iijima et al., 2004). Cinnamate 4-hydroxylase (C4H), is a member of the cytochrome P450 monooxygenase superfamily, catalyzes the hydroxylation of trans-cinnamic acid to 4-hydroxycinnamate (p-coumaric acid), and is the second key enzyme of phenylpropanoid biosynthetic pathway (Chen et al., 2007). C4H plays a pivotal role at the interface between the cytosolic phenylpropanoid pathway and membrane-localized electron-transfer reactions (Koopmann et al., 1999). The third enzyme of the general phenylpropanoid pathway is 4-coumarate: CoA ligase (4CL), which may play a central role in regulating overall flux of the hydroxycinnamic acids into subsequent biosynthetic pathways (Rastogi et al., 2013). The 4CL is positioned at the metabolic branch point that connects general phenylpropanoid metabolism with different end-product-specific pathways (Stuible and Kombrink, 2001). The last biosynthetic step involved in the formation of estrogole (methylchavicol) is catalyzed by enzyme chavicol O-methyltransferase (CVOMT) (Gang et al., 2001, Lewinsohn et al., 2000).The availability of the sequences of these genes essentially facilitates the identification of the conditions under which their expression levels are enhanced (Nasrollahi et al., 2014).
Environmental and ecological factors are known to remarkably affect the essential oil content and composition in aromatic plants (Nasrollahi et al., 2014). The production of the plant specialized metabolites and the expression level of the genes involved in their biosynthesis are strongly associated with growth conditions. Plants exposed to drought stress generally produce higher levels of specialized metabolites (Selmar and Kleinwächter, 2013). Drought stress may also change the essential oil yield and composition in plants (Khorasaninejad et al., 2011, Nowak et al., 2012, Petropoulos et al., 2008). The increase of essential oil percentage and its main constituents under drought stress have been reported in both O. basilicum and O. americanum (Khalid, 2006). Linalool and methylchavicol contents of sweet basil, as well as the percentage of total essential oil, increased under water stress (Simon et al., 1992). Successful and efficient use of deliberate drought stress can directly enhance specialized metabolite production. This enhancement can be achieved by applying special irrigation regimes that are both simple and inexpensive, but this approach requires extensive examination to optimize metabolite production (Selmar and Kleinwächter, 2013).
Because of the economic importance of essential oil production, basil is considered as a target for bioengineering. Little is known about the gene regulation of phenylpropanoids in aromatic plants. Understanding the expression pattern of the genes involved in phenylpropanoid biosynthesis pathway and their association with the accumulation of essential oil compounds under different environmental conditions could facilitate the production of economically valued varieties of medicinal plants through genetic engineering techniques. To our knowledge, control of phenylpropanoid biosynthesis regarding essential oil production in basil has not been investigated and published. Hence, the current study was aimed to examine the effect of drought stress on the expression levels of the genes C4H, 4CL, cinnamyl alcohol dehydrogenase (CAD), eugenol O-methyl transferase (EOMT) and CVOMT, essential oil components and their relationship in basil.
Section snippets
Effect of drought stress on essential oil compounds
GC-MS analysis was performed for identification of essential oil compounds in control and drought stressed samples. Analysis of variance was performed to assess the effect of drought stress on the content of methylchavicol, methyleugenol, eugenol, α-bergamotene, β-myrcene, and linalool. Drought stress significantly (P ≤ 0.05) affected the content of all components except for linalool (Table 1). The highest amount of methylchavicol, methyleugenol, α-bergamotene, and β-myrcene was achieved under
Discussion
Essential oil synthesis in plants is influenced by a number of factors such as light intensity, seasonal variation, climate change, plant growth regulators, and environmental stresses (Werker et al., 1993). The ecological and climate situations have a huge impact on the efficiency and variety of essential oils in plants (Zarei et al., 2015). Water stress is an important factor affecting the synthesis of natural products (Solinas et al., 1996). Changes in essential oils extracted from aromatic
Concluding remarks
In conclusion, our results showed that the expression levels of CVOMT and EOMT were increased under drought stress conditions, whereas the expression of CAD relatively remained constant. The expression levels of 4CL and C4H genes were decreased under the same conditions. Methylchavicol and methyleugenol are the important compounds of essential oil in O. basilicum that drought stress increased their contents. Our results indicated that under the application of controlled drought stress, CVOMT
Plant materials
Seeds of O. basilicum c.v. keshkeni luvelou, kindly provided by Prof. A. Hassani (Urmia University, Iran) were sown based on a completely randomized design (CRD) in plastic pots containg a mixture of soil: sand (2:1), under natural light condition in greenhouse. Then, the seedlings were thinned to eight per pot and were treated with water deficit stress under three levels of 100, 75 and 50% of FC at 6–8 leaf stage. The FC percentage of soil was determined by pressure plate extractor (Azarkhakab
Acknowledgments
The authors thank Urmia University for financial support of the work as well as Institute of Biotechnology of Urmia University for providing lab facilities.
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