## 2 Results and Discussion
### Large-Scale Profiling of Alfafla Volatile Metabolites
The quantitatively most abundant volatile metabolites in leaves were C6 metabolites (38.4%), alcohols (29.2%), and ketones (13.1%). The C6 metabolites ((Z)-3-hexenal, hexanal, 2-hexyn-1-ol, (_E_)-2-hexenal, 1-hexanol, (_E_,_E_)-2,_A_-hexadienal, and (_Z_)-3-hexenyl acetate) are the main contributors to the green sensory characteristics of many plants as green leaf volatiles (GLV), and this was the largest class of volatile metabolites in alfalfa stems and leaves [34]. GLVs including alcohols, aldehydes, and esters of C6 metabolites are synthesized from C18 unsaturated fatty acids (linoleic and linolenic acid) via the lipoxygenase activity. The aldehydes of the C6 metabolites can be converted to their isomers by alkenal isomerases, or they can be reduced to alcohols and esters by alcohol dehydrogenases and alcohol acetyltransferases, respectively [35]. (_E_)-2-Hexenal was the most abundant metabolite of the C6 family of metabolites in stems (270.85 \(\upmu\)g g\({}^{-1}\) fr. wt) and leaves (2310.32 \(\upmu\)g g\({}^{-1}\) fr. wt). The physiological roles of (_E_)-2-hexenal include high antibacterial activity against Botrytis cinerea in Arabidopsis. It also inhibits root elongation in Arabidopsis, and triggers signaling within  
Figure 3: The relative proportion of specific chemical classes of volatile metabolites emitted from glandular trichomes isolated from stems, trichome-free stems, and leaves were significantly different. Vertical bars with the same letter are not significant; however, those indicated with different letters are significant according to the [PERSON]’s multiple range test (_p_ < 0.05). and between plants to emit volatile compounds in tomato [36; 37; 38]. (_Z_)-3-Hexenyl acetate, which is released upon mechanical wounding of the leaves, was present only in leaves (23.29 \(\mathrm{\SIUnitSymbolMicro g}\)\(\mathrm{\SIUnitSymbolMicro g}\)\(\mathrm{\SIUnitSymbolMicro g}\)\(\mathrm{\SIUnitSymbolMicro f}\)\(\mathrm{\SIUnitSymbolMicro w}\)) [39]. The relative proportion of alcohols in leaves was significantly more than three times higher than in glandular trichomes or stems. The most abundant alcohol in leaves was 1-octen-3-ol, which is known as one of the major aroma-active compounds and an insect attractant. The relative proportion of aldehydes in glandular trichomes and stems was similar, and the major aldehyde metabolites were nonanal and decanal in these two samples. However, the major aldehyde in leaves was (_E_)-2-pentenal, followed by octanal, (_E_,_E_)-2,4-heptadienal, and (_E_)-2-heptenal. The relative proportion of ketones in glandular trichomes was significantly higher than in stems and leaves, 3.2 and 2.2 times higher respectively, and 2-propanone was qualitatively the primary ketone in glandular trichomes, contributing 25.1% of the total trichome ketones. Terpenoids made up 15.5% of total relative volatiles in stems, but only 5.4 and 4.7% in glandular trichomes and leaves, respectively. \(\alpha\)-Terpinol was the most abundant metabolite in glandular trichomes, followed by \(\beta\)-linalool, _cis_-linalool oxide, isoprene, _p_-cymene, D-limonene, and _p_-menth-1-en-4-ol. However, the most abundant metabolites in stems, in decreasing order of their relative concentrations, were \(\alpha\)-terpinol, \(\beta\)-linalool, _cis_-linalool oxide, ocemenol, and myrcene. The most abundant metabolites in leaves, in decreasing order of relative concentration, were \(\beta\)-linalool, \(\alpha\)-terpinol, _cis_-linalool oxide, (_E_,_E_)-2,6-dimethyl-1,3,5,7-octatetraene, _p_-menth-1-en-4-ol, and isoprene. The different metabolic profiles in glandular trichomes, stems, and leaves revealed that biosynthesis and emission of volatile metabolites varied substantially in different tissues.