Fig 1.
Scanning electron microscopy and fluorescent microscopy of C. sativa trichomes.
[A] SEM micrograph of a capitate stalked glandular trichome. Glandular trichome heads are morphologically distinct from the associated multicellular stalk. Glandular trichome heads are attached to multi-cellular stalks by a narrow constriction of the stalk. [B] Fluorescent microscopy image of a glandular trichome head showing the stipe cells (red fluorescence) that subtend the discoid layer of disc cells (no red fluorescence) at 400–440 nm excitation. Remnants of the ruptured cuticular layer covering the glandular cavity are also visible. Chlorophyll autofluorescence in red. [C] SEM micrograph of capitate stalked glandular trichomes, showing apparent continuity between epidermal layer and stalk. [D] Fluorescent microscopy image of capitate stalked glandular trichome at 400–440 nm excitation with gland intact. Secondary metabolites in the glandular trichome head fluoresce blue when excited, while chlorophyll in chloroplasts in the stalk fluoresce red.
Fig 2.
Purification of glandular trichome heads and glandular trichome stalks from a heterogenous mix of C. sativa flower epidermal tissues.
[A] Heterogenous mix of epidermal tissues separated by percoll gradient centrifugation. [B] Glandular trichome heads collected at the interphase of the 0% and 30% Percoll boundary and [C] glandular trichome stalks collected at the interphase of the 30% and 45% Percoll boundary. Purity estimates for [D] glandular trichome head fraction and [E] glandular trichome stalk fraction. Boxplots for percent (%) head and stalk counts for each fraction are shown based on image analysis. N = 10.
Fig 3.
Venn diagram and cluster analysis of trichome fractions and flowers.
[A] Venn diagram comparing the proteins found within the three tissue types: glandular trichome heads, stalks, and late flowers. [B] Hierarchical clustering of stalk enriched (S1-3), glandular trichome head (H1-3), and late flower (LF1-3) total spectral counts. The horizontal dendrogram represents the relationship each sample or column has with any other sample in the data set as pertains to that samples combination of spectral signatures. The vertical dendrogram defines the relationship a given protein or row has with any other protein in the data set with respect to abundance across all samples in this dataset.
Fig 4.
Pairwise comparisons of protein abundance in trichome fractions and flowers.
Volcano plot data models (Log2 fold change plotted against–Log10 (Benjamini-Hochberg corrected p-value)) of relative protein abundance in a pairwise comparison of [A] glandular trichome heads (negative fold change) and late-stage flowers (positive fold change), [B] glandular trichome stalk (negative fold change) and late-stage flowers (positive fold change) and [C] glandular trichome heads (negative fold change) and glandular trichome stalk (positive fold change). Vertical line at x = 0 represents the 0-fold change threshold. Perforated lines intersecting x = -1 and x = 1 represent a two-fold change in abundance. Red triangles denote significant proteins found exclusively in one group but not the other. Grey triangles represent unique, yet non-significant proteins. Blue dots represent proteins found in both sample types that are significantly more abundant in one sample or the other. Grey dots represent proteins found in both sample types that are not significantly more abundant in either sample type. [D] Percent (%) representation of major metabolic categories in pairwise comparisons. Represented protein counts are indicated in brackets (N). Respective protein names and associated log-fold changes p-values for all categories across all comparisons can be found in S3.H = head, S = stalk, F = late flower.
Fig 5.
Mapman batch classification of proteins relating to metabolism in trichome heads vs late flowers.
Fold change differences in a protein’s abundance is indicated by the legend key where blue represents proteins more abundant in late- stage flowers, white indicates no change in a proteins abundance, and red indicates proteins found more abundant in glandular trichome heads.
Fig 6.
Mapman batch classification of proteins relating to metabolism in trichome stalks vs late flowers.
Fold change differences in a protein’s abundance is indicated by the legend key where blue represents proteins more abundant in glandular trichome stalks, white indicates no change in a proteins abundance, and red indicates proteins found more abundant in late-stage flowers.
Fig 7.
Mapman batch classification of proteins relating to metabolism in glandular trichome heads vs stalks.
Fold change differences in a protein’s abundance is indicated by the legend key where blue represents proteins found more abundant in glandular trichome stalks, white indicates no change in a proteins abundance, and red indicates proteins found preferentially abundant in glandular trichome heads.
Fig 8.
Carbon flow schematic of C. sativa glandular trichomes.
Mesophyll tissues of the late-stage flowers (blue) are source tissues for the photosynthetic (PS) production of sucrose (SUC). Sucrose and/or raffinose (SUC/RAF) are transported through glandular trichome stalks (yellow) to glandular trichome head sink tissue (orange) via yet unidentified mechanisms. Sugars in the glandular trichome heads are shunted into glycolysis (GLY) and the oxidative pentose phosphate pathway (OPP) to supply carbon to mitochondria and plastids. In mitochondria, catabolites enter the Krebs cycle (TCA) and respiratory chain (RES) for energy production. Resulting carbon dioxide is partially recycled through pep-carboxylase (PC) activity, and the ATP producedis used to generate proton gradients through ATPase activity and to drive primary active transport energised by ABC transporters (ABC). In plastids, catabolites can support terpenoid and cannabinoid biosynthesis (C/TBS) through the MEP pathway and, in case of cannabinoids, additionally through fatty acid metabolism (FAM). Stalks show PS and RES activity in addition to active detoxification (DETOX).