An in vitro phytohormone survey reveals concerted regulation of the Cannabis glandular trichome proteome

Glandular trichomes (GTs) are major sites of cannabinoid and terpenoid metabolism in &lt;em&gt;Cannabis sativa&lt;/em&gt; L flowers. Several phytohormones, such as jasmonic acid (JAS) and salicylic acid (SAL) cause increased cannabinoid content in &lt;em&gt;Cannabis&lt;/em&gt; flowers, but how they regulate GTs to cause this change is unknown. This study aimed to understand the mechanisms of action of phytohormones on mature GTs using a novel &lt;em&gt;in vitro&lt;/em&gt; assay. Live GTs were isolated from ‘Hindu Kush’ <em>Cannabis&lt;/em&gt;, a high-tetrahydrocannabinol cultivar, and incubated on basal media plates with either kinetin (KIN), JAS, SAL, abscisic acid, ethephon, gibberellic acid, brassinolide, or sodium diethyldithiocarbamate (DIE), an inhibitor of jasmonic acid synthesis. Quantitative proteomic analysis (SWATH-MS) revealed that KIN, JAS and SAL caused the greatest number of changes in the GT proteome while the other treatments exerted much smaller changes. Surprisingly, metabolite analysis showed none of the treatments caused cannabinoids to increase in the GT, while DIE caused cannabigerolic acid content to decrease. Nor was there any concerted increased abundance of cannabinoid biosynthetic pathway proteins by any treatments. This indicates that cannabinoid increases in previous &lt;em&gt;in planta&lt;/em&gt; phytohormone studies are likely due to other processes, such as through increased GT density. As well, KIN, JAS and SAL treated GT’s had a large proportion of differentially abundant proteins in common. Several were key proteins for leucoplast differentiation and function, cuticular wax and fatty acid metabolism and primary metabolism regulation. This points towards cytokinin, jasmonic acid and salicylic acid signalling likely being important for coordinating &lt;em&gt;Cannabis&lt;/em&gt; GT differentiation and development. </em>