Cloning, characterization and expression of a phenylalanine ammonia-lyase gene (CcPAL) from cumin (Cuminum cyminum L.)

https://doi.org/10.1016/j.jarmap.2020.100253Get rights and content

Highlights

  • A partial cDNA of the PAL gene from cumin (CcPAL), was cloned and characterized.

  • CcPAL sequence showed an evolutionary affinity with that of several taxa in Apiaceae.

  • CcPAL is spatially regulated at the transcription level being mostly expressed in shoots.

  • CcPAL expression was significantly induced by various biotic and abiotic elicitors.

  • The phenolic content of cumin shoots was correlated to CcPAL gene expression.

Abstract

Phenylalanine ammonia-lyase (PAL) that functions a substantial role in defense mechanisms, growth, and development of the plant, is situated at branching point from primary to secondary metabolism. In this study, using highly conserved sequences of plant PALs registered in GenBank, a partial cDNA clone of cumin (Cuminum cyminum) PAL gene (designated as CcPAL) was isolated. The relative transcript levels of CcPAL gene, its enzyme activity, and accumulation of total phenolics were also analyzed in shoots in response to different stresses. The isolated CcPAL had 414 bp in length with a maximum open reading frame (ORF) of 351 bp and encodes a peptide with 116 amino acids. The deduced peptide sequence of CcPAL has classical PAL domains and is a stable protein. Results of multiple sequence alignment indicate that CcPAL had high similarity with other plant PALs available in the GenBank. Analysis of the phylogenetic tree indicated that CcPAL categorized in the angiosperm-type PAL family and dicotyledon subfamily and clustered together with other PAL from Apiaceae species. Transcription analysis indicated that CcPAL expressed in all tested tissues, but more highly in shoots. Moreever, the expression of CcPAL was found to be induced by cadmium, wounding, salicylic acid (SA) and cold treatments. A positive correlation was found between transcript levels of CcPAL and total phenolics accumulation of cumin shoots in response to different stresses. These results suggest that the CcPAL and phenylpropanoid pathway may play a great role in the biosynthesis of the bioactive compounds and counteracting various stresses in C. cyminum.

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.

References (57)

  • S. Dehghan et al.

    Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    Bioscience Reports

    (2014)
  • R.A. Dixon et al.

    Stress-induced phenylpropanoid metabolism

    Plant Cell

    (1995)
  • M.D. Dufoo-Hurtado et al.

    Low-temperature conditioning of “Seed” cloves enhances the expression of phenolic metabolism related genes and anthocyanin content in ‘Coreano’ garlic (Allium sativum) during plant development

    Journal of Agricultural and Food Chemistry

    (2013)
  • R.S. Ejtahed et al.

    Expression analysis of phenylalanine ammonia lyase gene and rosmarinic acid production in Salvia officinalis and Salvia virgata shoots under salicylic acid elicitation

    Applied Biochemistry and Biotechnology

    (2015)
  • J. Gao et al.

    Characterization, and expression profile of a phenylalanine ammonia lyase gene from Jatropha curcas L

    Molecular Biology Reports

    (2012)
  • C. Geourjon et al.

    SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments

    Computer Applications in the Biosciences

    (1995)
  • S. Ghasemi et al.

    Changes in the expression of some genes involved in the biosynthesis of secondary metabolites in Cuminum cyminum L. under UV stress

    Protoplasma

    (2019)
  • D. Gonzalez-Mendoza et al.

    Changes of photochemical efficiency and epidermal polyphenols content of Prosopis glandulosa and Prosopis juliflora leaves exposed to cadmium and copper

    Open Life Sciences

    (2017)
  • N. Hashemian et al.

    Diversity in chemical composition and antibacterial activity of essential oils of cumin (Cuminum cyminum L.) diverse from northeast of Iran

    Australian Journal of Crop Science

    (2013)
  • M. Hashemitabar et al.

    cDNA cloning, phylogenic analysis and gene expression pattern of phenylalanine ammonia-lyase in sugarcane (Saccharum officinarum L.)

    Brazilian Archives of Biology and Technology

    (2014)
  • D.R. Hoagland et al.

    The water culture method for growing plants without soil. California Agricultural Experiment Station

    Circular

    (1950)
  • M.W. Hyun et al.

    Fungal and plant phenylalanine ammonia-lyase

    Mycobiology

    (2004)
  • M.J. Jeong et al.

    Differential expression of kenaf phenylalanine ammonia-lyase (PAL) ortholog during developmental stages and in response to abiotic stresses

    Plant Omics

    (2012)
  • Y. Jiang et al.

    Molecular cloning and characterization of a phenylalanine ammonia-lyase gene (LrPAL) from Lycoris radiata

    Molecular Biology Reports

    (2011)
  • Y. Jiang et al.

    Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata

    Molecular Biology Reports

    (2013)
  • O.I. Kosyk et al.

    Phenylalanine ammonia-lyase activity and anthocyanin content in different varieties of lettuce under the cadmium influence

    The Ukrainian Biochemical Journal

    (2017)
  • J. Kováčik et al.

    Phenylalanine ammonia-lyase and phenolic compounds in chamomile tolerance to cadmium and copper excess

    Water Air and Soil Pollution

    (2007)
  • A. Kumar et al.

    The phenylalanine ammonia-lyase gene family in raspberry. structure, expression, and evolution

    Plant Physiology

    (2001)
  • Cited by (4)

    • Insight into VvGH3 genes evolutional relationship from monocotyledons and dicotyledons reveals that VvGH3-9 negatively regulates the drought tolerance in transgenic Arabidopsis

      2022, Plant Physiology and Biochemistry
      Citation Excerpt :

      As the typical classification of plant include monocotyledons and dicotyledons, 716 Ovate Family Proteins (OFPs) from 32 species were divided into 15 subgroups, which had 5 dicotyledon and 10 monocotyledon subgroups, and gene numbers of dicotyledon were far beyond the monocotyledon (Li et al., 2019). The Phenylalanine Ammonia-lyase (PAL) genes also had similar result (Habibollahi et al., 2020). However, there were few reports on comparative analysis of GH3 genes between dicotyledon and monocotyledon.

    View full text