Document Type : Research Paper

Authors

1 M.Sc. Student of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

2 Associate Professor, Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

3 Assistant Professor, Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Abstract

Abstract
Background and Objectives
Currently, there is an immense attention towared medicinal plant and secondary motabiltes. Yarrow (Achillea millefolium L.) is a medicinal plant which consists of two important compounds; including penene and linalool. Since these two compounds have the importance values, the researchers significantly focused on them. The rate of transcript expression of penene synthesis and Linalol synthesis induced at the end of 2-c-methyl-D-erythritol-4-phosphate (MEP) pathway that caused to produce Penene and Linalol.  In order to evaluate the synthesis of Penene and Linalol, a research study was carried to exame the exogenous treatment (zero, 25 and 50 mg/l) of gibberellic acid (GA3) at different times (24, 48 and 74 hr.) on Yarrow at the greenhouse on 1395-1397. The results of R-PCR indicated that significant rate of the transcript expression was related to the Penene synthesis at 25 mg concentration of GA3 at the 48 hr after treatment. In the most samples as the rate of the Penene synthesis increased the rate of Linalol synthesis decreased. The evidences suggested that the exogenous treatment of GA3 induced a transduction signal in the plant. In which at the end, the plant molecular process respond to it and showed in overall that evaluation of GA3 implemented different encoding gene expression for theses compounds, and illustrated that when the Penene synthesis inceased in the treated plant the Linalol synthesis dcreased simontenously.
 
Materials and Methods
A greenhouse pot trial was conducted at the Experimental Research Station of College of Agriculture at Shahid Chamran University of Ahvaz. The plants used in this study were provided from the National Center for Genetic and Biological Resources of Iran, with access code P1000093. Two months after seed germination, the desired amounts of gibberellic acid were prepared with distilled water (25 mg/l and 50 mg/l), and then each potted plant was sprayed uniformly with 10cc of prepared solution. Sampling was carried out by collecting the leaves of treated plants at 24, 48 and 72 hours after the treatment. The phenol-chloroform method (Wang and Ghabrial, 2002) was conducted with some modifications to extract RNA of collected leaves at the two biological replications. Quantity and quality of extracted RNAs were measured using Nanodrop and electrophoresis of %1 agarose gel. Synthesis of cDNA was performed using the Takara cDNA synthetase kit according to its manufacturer's instructions. Partial sequences of pinene synthase and linalool synthase genes were obtained in an early experiment in our laboratory (data is not published). The coding sequence of the target genes in other plants within the same family aligned with our obtained sequences to identify conserved segments for each gene using CLUSTAL W program. The standard housekeeping actin for yarrow with accession number JX679606.1, which is available in the public GenBank database, used to normalize the expression of the genes of interest. The primers of all genes (pinene synthase, linalool synthase and actin) were designed from the conserved sequences by PrimerQuest Tool, and then verified with oligoanalyzer v.3.1. Relative expressions of target genes were determined using real-time PCR with SYBR green fluorescence detection. Relative differences in target gene expression were calculated using REST software.
 
Results
The acquired result of real-time PCR revealed that in pinene synthase gene, the highest amount of transcription occurred at 48 hours after the treatment with the concentration of 25 mg of gibberellic acid and, interestingly, the lowest expression of the transcript for linalool synthase gene observed at the same level of concentration and time. In most of the samples, with the increase in pinene synthase transcriptase, the amount of linalool synthase transcript decreased.
 
Discussion
Taking into account the fact that both pinene and linalool are synthesized from the common substrate geranyl diphosphate. Therefore, an increase in the expression one leads to reduction in the transcript expression of the other one in the same pathway. Our results also confirmed that the transcription level of pinene synthase gene has been increasingly altered in response to the applied concentrations of exogenous gibberellic acid, resulting in a reduction in the expression of linalool synthase gene. Evidence suggests that the application of gibberellic acid triggers a cascade of molecular events which ultimately constitutes the plant response to the elicitor.

Keywords

Main Subjects

References
Afkar, S. Karimzadeh, Gh. Jalali, M. Sharifi, M. and Behmanesh, M. (2013). Influence of methyljasmonate on menthol production and gene expression in peppermint (Mentha x piperita L.). Journal of Medicinal Plants and By-Products, 2(1), 75-82.
Aharoni, A., Jongsma, M. A. and Bouwmeester, H. J. (2005). Volatile science? Metabolic engineering of terpenoids in plants. Trends in Plant Science, 10(12), 594-602.
Baydar, H. and Baydar, N. G. (2005). The effects of harvest date, fermentation duration and Tween 20 treatment on essential oil content and composition of industrial oil rose (Rosa damascene Mill). Industrial Crops and Products, 21(2), 251-255.
Benedek, B. and Kopp, B. (2007). Achillea millefolium L. revisited: Recent findings confirm the traditional use. Wiener Medizinische Wochenschrift, 157(13-14), 312-314.
Bohlmann, J. and Keeling, C. I. (2008). Terpenoid biomaterials. The Plant Journal, 54(4), 656-669.
Bohlmann, J., Steele, C. L. and Croteau, R. (1997). Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization and functional expression of myrcene synthase, (−)-(4S)-limonene
synthase, and (−)-(1S,5S)-pinene synthase. The Journal of Biological Chemistry, 272(35), 21784-21792.
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology, 25(2), 169-193.
Cordoba, E., Salmi, M. and Leo, P. (2009). Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. Journal of Experimental Botany, 60(10), 2933-2943.
Deschamps, C. and Simon, J. E. (2006). Terpenoid essential oil metabolism in basil (Ocimum basilicum L.) following elicitation. Essential Oil Research, 18, 618-621.
Dixon, R. A. and Paiva, N. L. (1995). Stress-induced phenyl propanoid metabolism. Plant Cell, 7(7), 1085-1097.
Dudareva, N., Pichersky, E. and Gershenzon, J. (2004). Biochemistry of plant volatiles. Plant Physiology, 135(4), 1893-1902.
Ghani, A. and M. Azizi. (2010). The effect of different drying methods on quantity and quality characteristics of five yarrow species (achillea). Plant Productions, 32(1) 1-11. [In Farsi]
Hassani, L., Abdollahi Mandoulakani, B., Darvishzadeh, R. and Hassani, A. (2016). Increasing the expression of genes chavicol o-methyl teransferase and cinnamate 4-hydroxilase under methyl jasmonate treatment in medicinal plant basil (Ocimum basilicum L.).Scientic Journal of Agriculture, 39(3) 101-112.
Hayashi, H., Huang, P. and Inoue, K. (2003). Up-regulation of soyasponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiology, 44(4), 404-411.
Javedan Asl, M., Rajabi Memari, H., Nabati Ahmadi, D. and Rahnama, A. (2015a). Comparison the different methods of genomic RNA extraction from the medicinal plant of yarrow (Achillea millefolium). Plant Productions, 39(2), 105-114. [In Farsi]
Javedan Asl, M., Rajabi Memari, H., Nabati Ahmadi, D. and Rahnama Ghahfarokhi, A. (2015b). Isolation of linalool synthase and pinene synthase genes from yarrow (Achillea millefolium L.) medicinal plant. Plant Genetic Report, 2(1), 23-349. [In Farsi]
Kim, H. J., Chen, F., Wang, X. and Rajapakse, N. C. (2006). Effect of methyl jasmonate on secondary metabolit of Sweet Basil (Ocimum basilicum L.). Journal of Agricultural and Food Chemistry, 54(6), 2327-2332.
Liu, S., Zhou, L., Yu, S., Xie, C., Liu, F. and Song, Z. (2013). Polymerization of α-pinene using Lewis acidic ionic liquid as catalyst for production of terpene resin. Biomass and Bioenergy, 57, 238-242.
Mehdi Khanlou, K. and Van Bockstaele, E. (2012). A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.). Planta, 236(5), 1381-1393.
Nicot, N., Hausman, J. F., Hoffmann, L. and Evers, D. (2005). Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 56, 2907-2914.
Paulsen, E. (2002). Contact sensitization from Compositae-containing herbal remedies and cosmetics. Contact Dermatitis, 47(4), 189-198.
Pfaffl, M. (2001). A new mathematical for relative quantification in realtime RT-PCR. Nucleic Acids Research, 29(9), 1-6.
Pfaffl, M. W., Horgan, G. W. and Dempfle, L. (2002). Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research, 30(9), 1-10.
Pooraskari, O., Nabati Ahmadi, D., Mehdi Khanlou, K. and Nejadsadeghi, L. (2018). The comparison of
conventional phenol-chloroform method for extracting RNA with modified method in
Achllea millefolium L. 15th National Iranian Crop Science Congress, Karaj, Iran. [In Farsi]
Rahimmalek, M., Tabatabaei, B. E. S., Etemadi, N., Goli, S. A. H., Arzani, A. and Zeinali, H. (2009). Essential oil variation among and within six Achillea species transferred from different ecological regions in Iran to the field conditions. Industrial Crops and Products, 29(2-3), 348-355.
Schmiderer, C., Grausgruber-Groger, S., Grassi, P., Steinborn, R. and Novak, J. (2010). Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis). Journal of Plant Physiology, 167(10), 779-786.
Schmittgen, T. D. and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101-1108.
Soleymani, F. and Taheri, H., (2017). Relative expression of genes of menthol biosynthesis pathway in peppermint (Mentha piperita L.) after chitosan, gibberellic acid and methyl jasmonate treatments. Russian Journal of Plant Physiology, 64(1), 59-66.
Suzuki, H., Srinivasa Reddy, M. S., Naoumkina, M., Aziz, N., May, G. D., Huhman, D. V., Sumner, L. W., Blount, J. W., Mendes, P. and Dion, R. A. (2005). Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic reprogramming in cell suspension cultures of the model legume.Medicago truncatula Planta, 220(5), 696-707.
Wang, R. Y. and Ghabrial, S. A. (2002). Effect of aphid behavior on efficiency of transmission of Soybean mosaic virus by the soybean-colonizing aphid, Aphis glycines. Plant Disease, 86(11), 1260-1264.
Yu, Z. X., Li, J. X., Yang, C. Q., Hu, W. L., Wang, L. J. and Chen, X. Y. (2012). The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L. Molecular Plant, 5(2), 353-365.
 
 © 2020 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-Non Commercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/)