Enhancement of Essential Oil Production and Expression of Some Menthol Biosynthesis-Related Genes in Mentha piperita Using Cyanobacteria

Mentha piperita L. is one of the most important aromatic crops and is cultivated worldwide for essential oils (EOs).  The aim of the present study was to investigate the potential of two cyanobacteria, Anabaena vaginicola ISB42 and Nostoc spongiaeforme var. tenue ISB65, as biological-elicitors to improve the growth and essential oil production of M. piperita. In this experiment, inoculation of M. piperita with cyanobacteria was performed by adding 1% cyanobacterial suspension to the soil of treated pots on the first time of planting and every 20 days thereafter. The experiment was performed in a randomized complete block design in an experimental greenhouse condition. After 90 days planting, the vegetative growth factors, the content of photosynthetic pigments, as well as the quantity and quality of EOs of treated and control plants were evaluated. Also, quantitative changes in the expression of some menthol biosynthesisrelated genes were investigated. Cyanobacterial application led to significant increases in M. piperita growth indices including root and shoot biomass, leaf number, leaf area, node number and ramification, as well as photosynthetic pigments content. The statistical analysis showed a 41-75 % increase in some of these growth indices, especially in Nostoc-treated plants. A. vaginicola and N. spongiaeforme var. tenue inoculation led to a 13% and 25% increase in the EOs content of M. piperita, respectively. The EOs components were also affected by cyanobacterial treatments. According to the statistical analysis, Nostoc-treated plants showed the highest amount of (-)-menthone and (-)-limonene, with a 2.36 and 1.87-fold increase compared to the control. A. vaginicola and N. spongiaeforme var. tenue inoculation also led to 40% and 98% increase in transcript level of (-)-limonene synthase gene, respectively. The expression of the (-)-menthone reductase gene, was also increased by 65% and 55% in response to A. vaginicola and N. spongiaeforme var. tenue application, respectively.  Our data demonstrated that in addition to growth enhancement, these two heterocystous cyanobacteria improved the quantity and quality of EOs by up-regulating the key genes involved in the menthol biosynthetic pathway. Based on our results, these cyanobacteria can be considered valuable candidates in the formulation of low-cost and environmentally friendly biofertilizers in sustainable peppermint production