Cloning, characterization and expression of a phenylalanine ammonia-lyase gene (CcPAL) from cumin (Cuminum cyminum L.)
Graphical abstract
Introduction
Cumin (Cuminum cyminum L.) is an annual, diploid cross-pollinated plant of Apiaceae family (Srinivasan, 2018). The origin of cumin is Egypt, east of the Mediterranean area and Turkistan, but today it is cultivated mostly in Iran, Turkey, Egypt, China, Central America, Spain, Mexico, Morocco, Pakistan, and India (Mehdizadeh et al., 2017). After black pepper, cumin is considered the second most popular spice in the world and well known for its aroma, therapeutic and medicinal properties (Mortazavian et al., 2018). The major production area of cumin in Iran includes arid and semi-arid regions in Central, Eastern and, South-Eastern provinces (Hashemian et al., 2013). Cumin has long been used to treat several diseases including bloating, indigestion, chronic diarrhea, and epilepsy (Ghasemi et al., 2019). These medicinal and health benefits of cumin, have been ascribed to its bioactive components like phenols, flavonoids, and terpenes (Mnif and Aifa, 2015).
Phenolics/flavonoids which have a pivotal contribution to the tolerance mechanism by inducing antioxidant responses under biotic and abiotic stresses, are the products of the phenylpropanoid pathway and PAL is a critical enzyme that controls the speed of the first step in their biosynthesis (Mishra and Sangwan, 2019). Since PALs are involved in the production of numerous bioactive compounds, recently much attention has been paid to them in medicinal herbs and has been cloned and characterized from Ephedra sinica (Okada et al., 2008), Salvia miltiorrhiza (Song and Wang, 2009), Angelica gigas (Park et al., 2010), Scutellaria baicalensis (Xu et al., 2010), Lycoris radiate (Jiang et al., 2011), Jatropha curcas (Gao et al., 2012), Rhus chinensis (Ma et al., 2013), Dracaena cambodiana (Wang et al., 2013), Solenostemon scutellarioides (Zhu et al., 2015), Ocimum basilicum (Khakdan et al., 2018). PAL proteins are encoded by a multi-gene family in higher plants. Four PAL genes have been identified in the genome of parsley, Arabidopsis, and tomato, five in Populus trichocarpa, seven in cucumber, and approximately 40–50 copies in potato (Ma et al., 2013; Yan et al., 2019). Moreover, the expression levels of various PAL isoforms present in both monocot and dicot plant species have been characterized to change substantially in response to various biotic and abiotic stresses such as UV irradiation, salinity, fungal infection, tissue wounding, low temperature, plant growth regulators, salicylic acid, jasmonic acid, and chitosan (Dixon and Paiva, 1995; Lee et al., 2003; Pawlak-Sprada et al., 2011; Xu et al., 2012; Cass et al., 2015; Nag and Kumaria, 2018; Sharma et al., 2019). To date, no information is accessible about the sequence and expression pattern of genes of the phenylpropanoid pathway including PAL in cumin. In the present study, we for the first time isolated a partial cDNA clone of the PAL gene (CcPAL) from the C. cyminum using the RT-PCR method. Bioinformatic analyses concerning the gene homology and phylogenetic relationship, amino acid sequence, structure and physiochemical properties of the deduced polypeptid were investigated. The research is also the first report about expression analysis of CcPAL in different tissues and in response to different stresses in cumin. As the phenylpropanoid pathway produces a variety of bioactive compounds, assessing the properties and expression patterns of genes participating in the production of these metabolites, such as PAL, to provide a more comprehensive understanding of the tolerance mechanisms in C. cyminum during exposure to different stresses and the biosynthesis of the compounds is beneficial. In addition, the results of this study will clarify to us how PAL affects the production and control of secondary metabolic products in cumin.
Section snippets
Plant material, growth conditions and treatments
Cumin seeds were prepared from the Pakan Bazr Company (Isfahan, Iran). They were surface-sterilized with 1% sodium hypochlorite for 15 min and washed 3–4 times with sterile distilled water. The disinfected seeds were placed in sterile petri dishes and transferred to the germinator under the optimum conditions (24 °C and 16 h of light and 8 h of darkness photoperiod). The germinated seeds were planted in 12-cm diameter pots filled with pre-washed and nutrient-free sand. The pots were kept in a
Gene cloning, sequencing and prediction of the protein domains
PAL is a kind of enzyme which found widely in plants, as well as some yeast, fungi, and bacteria and has been discovered several decades ago (MacDonald and D’Cunha, 2007). Since then, partial or full length PAL gene has been cloned and identified from several different species. However, in C. cyminum, one of the significant medicinal and aromatic plants, no previous studies have been reported describing the PAL gene so far. In this study, using highly conserved sequences of PALs, a PAL partial
Conclusions
In summary, a partial cDNA of PAL gene from cumin (designated as CcPAL), successfully was cloned, and characterized. Multiple sequence analysis and phylogenetic relationship demonstrated that CcPAL is closely related to the other Apiaceae species. Our data revealed that expression of the CcPAL gene was stimulated by different stresses, indicating that CcPAL may be a stress-responsive gene and involved in stress-related pathways. Therefore, CcPAL could be manipulated as a potential target gene
Acknowledgments
The authors are grateful to the Shahid Bahonar University of Kerman, Kerman, Iran for financially supporting this research.
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