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

Document Type : Research Paper


1 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.

2 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

3 Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran


Background: Mentha piperita L. is one of the most important aromatic crops and is cultivated worldwide for essential oils (EOs). 
Objectives: 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.
Materials and Methods: 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.
Results: 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. 
Conclusions: 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


Main Subjects

1. Motesharezadeh B, Etesami H, Bagheri-Novair S, Amirmokri 
H. Fertilizer consumption trend in developing countries vs. 
developed countries. Environ Monit Assess.2017;189:103. doi: 
2. Wang Y, Zhu Y, Zhang S, Wang Y. What could promote farmers 
to replace chemical fertilizers with organic fertilizers? J Clean 
Prod. 2018;199:882-890. doi: 10.1016/j.jclepro.2018.07.222
3. Lin W, Lin M, Zhou H, Wu H, Li Z, Lin W. The effects of 
chemical and organic fertilizer usage on rhizosphere soil in tea 
orchards. PLoS One.2019;14:e0217018. doi: 10.1371/journal.
4. Ning CC, Gao PD, Wang BQ, Lin WP, Jiang NH, Cai 
KZ. Impacts of chemical fertilizer reduction and organic 
amendments supplementation on soil nutrient, enzyme activity 
and heavy metal content. J Integr Agric.2017;16(8):1819-1831. 
doi: 10.1016/S2095-3119(16)61476-4 
5. Pagnani G, Pellegrini M, Galieni A, D’Egidio S, Matteucci F, 
Ricci A, Stagnari F, Sergia M, Lo Sterzoa C, Pisantea M, Del Gallo 
M. Plant growth-promoting rhizobacteria (PGPR) in Cannabis 
sativa ‘Finola’ cultivation: an alternative fertilization strategy 
to improve plant growth and quality characteristics. Ind Crops 
Prod. 2018;123:75-83. doi: 10.1016/j.indcrop.2018.06.033
6. Tennakoon PLK, Rajapaksha RMCP, Hettiarachchi LSK. 
Tea yield maintained in PGPR inoculated field plants despite 
significant reduction in fertilizer application. Rhizosphere. 
2019;10:100146. doi: 10.1016/j.rhisph.2019.100146
7. Chittora D, Meena M, Barupal T, Swapnil P, Sharma K. Cyanobacteria as a source of biofertilizers for sustainable agriculture. Biochem Biophys. Rep.2020;22:100737. doi: 10.1016/j.
8. Singh JS, Kumar A, Rai AN, Singh DP. Cyanobacteria: 
a precious bio-resource in agriculture, ecosystem, and 
environmental sustainability. Front Microbiol.2016;7:529. doi: 
9. Shariatmadari Z, Riahi H, Seyed Hastroudi M, Ghassempour 
A, Aghashariatmadary Z. Plant growth promoting cyanobacteria and their distribution in terrestrial habitats 
of Iran. Soil Sci Plant Nutr.2013;59(4):535–547. doi: 
10. Karthikeyan N, Prasanna R, Nain L, Kaushik BD. Evaluating 
the potential of plant growth promoting cyanobacteria as 
inoculants for wheat. Eur J Soil Biol.2007;43(1):23-30. doi: 
11. Seyed Hashtroudi M, Ghassempour AR, Riahi H, Shariatmadari 
Z, Khanjir M. Endogenous auxins in plant growth promoting 
cyanobacteria – Anabaena vaginicola and Nostoc calcicola. J 
Appl Phycol.2012;25:379-386. doi: 10.1007/s10811-012-9872-7
12. Mohan A, Kumar B. Growth performance and yield potential 
of cereal crops (wheat, maize and barley) in association with 
cyanobacteria. Int J Curr Microbiol Appl Sci.2017;6(10):744–
758. doi: 10.20546/ijcmas.2017.610.091
13. Saadatnia H, Riahi H. Cyanobacteria from paddy fields in 
Iran as a biofertilizer in rice plants. Plant Soil Environ. 
2009;55(5):207-212. doi: 10.17221/384-PSE
14. Suresh A, Soundararajan S, Elavarasi S, Lewis Oscar F, 
Thajuddin N. Evaluation and characterization of the plant 
growth promoting potentials of two heterocystous cyanobacteria 
for improving food grains growth. Biocatal Agric Biotechno. 
2019;17:647-652. doi: 10.1016/j.bcab.2019.01.002
15. Song X, Zhang J, Peng C, Li D. Replacing nitrogen fertilizer 
with nitrogen-fixing cyanobacteria reduced nitrogen leaching in 
red soil paddy fields. Agric Ecosyst Environ.2021;312:107320. 
doi: 10.1016/j.agee.2021.107320
16. Santini G, Biondi N, Rodolfi L, Tredici MR. Plant biostimulants 
from cyanobacteria: an emerging strategy to improve yields and 
sustainability in agriculture. Plants (Basel).2021;10(4):643. 
doi: 10.3390/plants10040643
17. Shariatmadari Z, Riahi H, Abdi M, Seyed Hashtroudi M, 
Ghassempour AR. Impact of cyanobacterial extracts on the 
growth and oil content of the medicinal plant Mentha piperita
L. J Appl Phycol.2015;27(6):2279-2287. doi: 10.1007/s10811-
18. Cappellari LDR, Santoro MV, Schmidt A, Gershenzon J, 
Banchio E. Induction of essential oil production in Mentha 
× piperita by plant growth promoting bacteria was correlated 
with an increase in jasmonate and salicylate levels and a 
higher density of glandular trichomes. Plant Physiol Biochem. 
2019;141:142–153. doi: 10.1016/j.plaphy. 2019.05.030
19. Schwab W, Davidovich-Rikanati R, Lewinsohn E. Biosynthesis of plant-derived flavor compounds. Plant J. 2008;54:712-732. 
doi: 10.1111/j.1365-313X. 2008.03446.x
20. Guo K, Sui Y, Li Z, Huang Y, Zhang H, Wang W. Colonization 
of Trichoderma viride Tv-1511 in peppermint (Mentha
× piperita L.) roots promotes essential oil production by 
triggering ROS-mediated MAPK activation. Plant Physiol 
Biochem.2020;151:705-718. doi: 10.1016/j.plaphy.2020.03.042
21. Thakur M, Bhattacharya S, Khosla PK, Puri S. Improving 
production of plant secondary metabolites through biotic and 
abiotic elicitation. J Appl Res Med Aromat Plants.2019;12:1-12. 
doi: 10.1016/j.jarmap.2018.11.004
22. dos Santos Marques CT, Gama EVS, da Silva F, Teles S, Caiafa 
AN, Lucchese AM. Improvement of biomass and essential oil 
production of Lippia alba (Mill) N.E. Brown with green manures in succession. Ind Crops Prod.2018;112:113-118. doi:
23. Sarrou E, Ganopoulos I, Xanthopoulou A, Masuero D, 
Martens S, Madesis P, Mavromatis A, Chatzopoulou P. 
Genetic diversity and metabolic profile of Salvia officinalis
populations: implications for advanced breeding strategies. 
Planta.2017;246:201-215. doi: 10.1007/s00425-017-2666-z 
24. Shariatmadari Z, Ghorbani Nohooji M, Riahi H, Heidary F. 
Optimization of essential oils production in Mentha longifolia
L. using plant growth promoting cyanobacteria. J Med 
Plants.2022;21(83):47-59. doi:10.52547/jmp.21.83.47 
25. Araújo NAF, Brandäo RM, Barguil BM, Cardoso MdG, Pereira 
MMA, Buttrós VHT, Dória J. Plant growth-promoting bacteria 
improve growth and modify essential oil in Rose (Rosa hybrid
L.) cv. Black Prince. Front Sustain Food Syst.2020;4:606827. 
doi: 10.3389/fsufs.2020.606827
26. Bose SK, Yadav RK, Mishra S, Sangwan RS, Singh AK, 
Mishra B, Srivastava AK, Sangwan NS. Effect of gibberellic 
acid and calliterpenone on plant growth attributes, trichomes, 
essential oil biosynthesis and pathway gene expression in 
differential manner in Mentha arvensis L. Plant Physiol 
Biochem.2013;66:150-158. doi: 10.1016/j.plaphy.2013.02.011
27. Andersen RA. Algal culturing techniques. Elsevier academic 
28. Komárek J. Süßwasserflora von Mitteleuropa, Bd. 19/3: 
Cyanoprokaryota 3. Teil/3rd. Part: Heterocytous Genera.
Springer Berlin Heidelberg Dez;2013. 
29. Lichtenthaler HK, Wellburn AR. Determination of total 
carotenoids and chlorophyll a and b of leaf extract in different 
solvents. Biochem Soc Trans.1983;11(5):591-592. doi: 
30. Adams RP. Identification of essential oil components by gas 
chromatography-quadrupole mass spectroscopy. Allured 
Publishing Corporation, Carol Stream, Illinois;2001.
31. Livak KJ, Schmittgen TD. Analysis of relative gene expression 
data using real-time quantitative PCR and the 2− ΔΔCT 
method. Methods.2001;25(4):402-408. doi: 10.1006/meth. 
32. Zhang J, Cook J, Nearing JT, Zhang J, Raudonis R, Glick BR, 
Langille MGI, Cheng Z. Harnessing the plant microbiome 
to promote the growth of agricultural crops. Microbiol 
Res.2021;245:126690. doi: 10.1016/j.micres.2020.126690
33. Singh S. A review on possible elicitor molecules of cyanobacteria: their role in improving plant growth and 
providing tolerance against biotic or abiotic stress. J Appl 
Microbiol.2014;117(5):1221-1244. doi: 10.1111/jam.12612
34. Nisha R, Kaushik A, Kaushik CP. 2007. Effect of indigenous 
cyanobacterial application on structural stability and productivity 
of an organically poor semi-arid soil. Geoderma.2007;118:49-56. 
doi: 10.1016/j.geoderma.2006.10.007
35. Zarezadeh S, Riahi H, Shariatmadari Z, Sonboli A. Effects of 
cyanobacterial suspensions as bio-fertilizers on growth factors 
and the essential oil composition of chamomile, Matricaria 
chamomilla L. J Appl Phycol .2020;32:1231-1241. doi: 10. 
36. Chookalaii H, Riahi H, Shariatmadari Z, Mazarei Z, Seyed 
Hashtroudi M. Enhancement of total flavonoid and phenolic 
contents in Plantago major L. with plant growth promoting 
cyanobacteria. J Agric Sci Technol.2020;22(2):505-518.
37. Zhu XC, Song FB, Liu SQ, Liu TD, Zhou X. Arbuscular 
mycorrhiza improves photosynthesis and water status of Zea 
mays L. under drought stress. Plant Soil Environ. 2012;58:186-
191. doi: 10.17221/23/2011-PSE
38. Anbi AA, Mirshekari B, Eivazi A, Yarnia M, Behrouzyar EK. 
PGPRs affected photosynthetic capacity and nutrient uptake 
in different Salvia species. J Plant Nutr.2020;43:108-121. doi: 
39. Helaly AA, Hassan SM, Craker LE, Mady E. Effects of 
growth-promoting bacteria on growth, yield and nutritional 
value of collard plants. Ann Agric Sci.2020;65(1):77-82. doi: 
40. Cappellari LDR, Santoro MV, Reinoso H, Travaglia C, Giordano 
W, Banchio E. Anatomical, morphological, and phytochemical 
effects of inoculation with plant growth- promoting rhizobacteria 
on peppermint (Mentha piperita). J Chem Ecol. 2015;41:149-
158. doi: 10.1007/s10886-015-0549-y
41. Santoro MV, Cappellari LDR, Giordano W, Banchio E, Papen 
H. Plant growth promoting effects of native Pseudomonas
strains on Mentha piperita (peppermint): an in vitro study. 
Plant Biol.2015;17(6):1218-1226. doi: 10.1111/plb.12351
42. Mota ÍA. Variation of yield and composition of essential oils 
from Mint and Basil in response to mycorrhizae bio-elicitor 
and hydric stress. Master in Pharmacy and Chemistry of 
Natural Products, Polytechnic Institute of Bragança;2018.
43. Cappellari L, Santoro MV, Nievas F, Giordano W, Banchio 
E. Increase of secondary metabolite content in marigold by 
inoculation with plant growth-promoting rhizobacteria. Appl 
Soil Ecol.2013;70:16-22. doi:10.1016/j.apsoil.2013.04.001
44. Banchio E, Xie X, Zhang H, Paré PW. Soil bacteria elevate 
essential oil accumulation and emissions in sweet basil. J 
Agric Food Chem.2009;5:653-657. doi:10.1021/jf8020305
45. Croteau RB, Davis EM, Ringer KL, Wildung MR. (-)-Menthol 
biosynthesis and molecular genetics. Naturwissenschaften. 
2005;92(12):562-577. doi: 10.1007/s00114-005-0055-0
46. Búfalo J, Rodrigues TM, de Almeida LFR, Tozin LRDS, 
Marques MOM, Boaro CSF. PEG-induced osmotic stress 
in Mentha × piperita L.: Structural features and metabolic 
responses. Plant Physiol Biochem.2016;105:174-184. doi: 10. 
47. Soleymani F, Taheri H, Shafeinia A. Relative expression 
of genes of menthol biosynthesis pathway in peppermint 
(Mentha piperita L.) after chitosan, gibberellic acid and methyl 
jasmonate treatments. Russ J Plant Physiol. 2017;64:59-66. 
48. Davis EM, Ringer KL, McConkey ME, Croteau R. Monoterpene metabolism. Cloning, expression, and characterization of menthone reductase from peppermint. Plant 
Physiol.2005;137(3):873-881. doi: 10.1104/pp.104.053306