## 3 Discussion  
Glandular trichomes, referred to as true cell factories, are able to secrete and store large quantities of specialized metabolites. The monoterenes of mint essential oil are produced by, and stored in, the peltate glandular trichomes [13]. The number of glandular trichomes per leaf, and their production of chemical compounds, depend on environmental and developmental parameters [14]. The distribution of peppermint gland initiation reflects the basipetal pattern of leaf maturation: relatively immature regions at the leaf's base continue to produce oil glands long after gland production has stopped at the leaf's apex [13]. To accurately determine the densities of mint glandular trichomes under low light conditions, five different areas at the same stage of development, from the basal zone to the top zone of an abaxial leaf, were analyzed. Our results show remarkably reduced densities of peltate glandular trichomes in all five selected leaf zones under low light conditions, which correlates with reduced chemical compound production. The present results suggest that reduced light intensity effects not only peltate glandular trichome density, but also the production and accumulation of essential oil, in _Mentha candensis_. This is consistent with the results for _Mentra pipertia_ and _Mentha arrensis_[15; 16]. In other plant species, cultivated tomato for instance, increases in photosynthetically active radiation induce the growth of type VI leaf glandular trichomes [17]. In the case of _Varronia curassavica_ [PERSON], higher irradiance results in an increased essential oil yield and a higher frequency of glandular trichomes [18]. The conclusion is that light density regulates not only the density of mint's glandular trichomes but also the synthesis of essential oil. However, the molecular mechanisms by which light regulates monoterenes biosynthesis, as well as the reprogramming of the pathway, are still not fully understood. To study the process in more detail, the mRNA expression patterns under changing light conditions were assessed via transcriptome analysis. Transcriptome sequencing is a powerful tool for screening gene expression patterns and identifying candidate genes. In the current study, high-throughput RNA sequences were generated to analyze the transcriptomes of _M. candensis_ undergoing a 24 h darkness treatment followed by exposure to light for 24 h. Approximately 72 million high-quality clean reads were generated, and a total of 95,579 unigenes, with an N50 of 1260 bp and average length of 754 bp, were obtained. In a previous study, nine of the orthologous genes involved in the menthol synthesis pathway were identified. Darkness for 24 h down-regulated the expression levels of _McGpps-L_, _McGpps-s_, _McLs_, _McL3 OH_, _MciPD_, _MciPR_, and _McPR_ genes. The RNA-Seq study showed that the expression levels of these genes were upregulated after light exposure, and RT-PCR confirmed these results. Among the transcriptional profiles, 14 of the DEGs involved in the monoter  
Figure 10: Expression validations of eight selected genes in the control, darkness and recovery light samples using qRT-PCR. The RPKM values obtained from the RNA-Seq data are depicted at the top of each figure. The level of significance, determined via Student’s _t_-test, is depicted as follows: *\(p<0.05\); **\(p<0.01\). ns, not significant. Each value is the mean of at least three replicates. DEGs involved in the sesquiterpenoid and triterpen biosynthesis pathways showed the same transcription patterns, except for unigene0051735 and unigene001095. The significant downregulation of genes involved in the MEP pathway may explain the reduced essential oil metabolite production. Furthermore, changes in the monoterpene profiles show that low light intensity leads to an accumulation of the branchpoint intermediate pulegone and the side product piperitone. In fact, under conditions of salinity and osmotic stresses, pulegone accumulated in _Mentha_ species [19; 20; 21; 22].