SummaryThe cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile‐like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two‐photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12–16 secretory disc cells and strongly monoterpene‐dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red‐shifted autofluorescence, eight secretory disc cells and less monoterpene‐dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co‐expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.

Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [ 3 H]phenylalanine ([ 3 H]Phe) to dissected cambium/ developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric lignin and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [ 3 H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [ 3 H]Phe. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.

There have been few studies on quantifying carotenoid accumulation in carrots, and none have taken the comparative approach. The abundance and distribution of carotenes in carrot roots of three varieties, white, orange, and high carotene mass (HCM) were compared using light and transmission electron microscopy (TEM). Light microscopy has indicated that, in all three varieties, carotenes were most abundant in the secondary phloem and this area was selected for further TEM analysis. While carotenes were extracted during the fixation process for TEM, the high-pressure freezing technique we employed preserved the spaces (CS) left behind by the extracted carotene crystals. Chromoplasts from the HCM variety contained significantly (P < 0.05) more CS than chromoplasts from the orange variety. Chromoplasts from the white variety had few or no CS. There was no significant difference between the HCM and orange varieties in the number of chromoplasts per unit area, but the white variety had significantly (P < 0.05) fewer chromoplasts than the other two varieties. A large number of starch-filled amyloplasts was observed in secondary phloem of the white variety but these were not found in the other two varieties. The results from this comparative approach clearly define the subcellular localization of carotenoids in carrot roots and suggest that while the HCM genotype was selectively bred for increased carotene content, this selection did not lead to increased numbers of carotene-containing chromoplasts but rather greater accumulation of carotene per chromoplast. Furthermore, the results confirm that roots of the white carrot variety retain residual amounts of carotene.

In winter, dormant cambial cells contain many small vacuoles interspersed throughout the cytoplasm. This differs dramatically from actively growing cambial cells whose structure is dominated by large central vacuoles. Structure reported in studies using conventional chemical fixation and transmission electron microscopy (TEM) conflicts with that described earlier for live cambial cells using light microscopy. In this study, cryofixation (high-pressure freezing/freeze substitution) was used to preserve dormant Pinus contorta fusiform cambial cells, revealing structure more consistent with that in early micrographs of live cambial cells. At the ultrastructural level, the plasmalemma was consistently smooth and tightly associated with the cell wall, contrary to the highly in-folded plasmalemma seen in chemically fixed cambial cells. In addition, both TEM and live-cell confocal microscopy demonstrated that, in some places, dormant cells were partitioned into more numerous, smaller vacuoles than were observed after chemical fixation. Populations of different vacuoles were apparent based on size, shape and membrane staining. Larger vacuoles had prominent tonoplasts and were often present as axially elongated, interconnecting networks with associated microfilament bundles. Endoplasmic reticulum fragmented during rest into numerous vesicular structures similar to small vacuoles, then with the transition to quiescence reformed into the smooth cisternal form.

Sacci of conifer pollen do not function primarily to increase the efficiency of wind pollination as is widely thought. Rather, they are bladders and cause pollen to float upwards in a liquid drop into the ovules. This observation is seemingly unsupported in the case of oriental spruce (Picea orientalis (L.) Link), which has saccate pollen. Ovulate cones are pendant at the time of pollination, which requires that pollen sink into the ovules. Pollen of oriental spruce floats at first but within 1-2 min sinks into the ovule. As sinking does not occur in saccate pollen of other Pinaceae, a variety of techniques was used to determine anatomical differences leading to this uncharacteristic tendency. Light, scanning electron, and confocal microscopy of the pollen surface yielded no significant appearing difference between pollen of oriental spruce and white spruce. However, transmission electron microscopy of freeze-fixed/freeze-substituted hydrated pollen revealed that the ektexine of oriental spruce pollen sacci is porous compared to that of white spruce. Confocal microscopy allowed examination of pollen hydration dynamics. Water enters pollen at the distal pole between sacci, and resulting rapid expansion of the tube cell forces air out of the saccate space. White spruce pollen remains buoyant because of enclosed air pockets in the saccus ektexine. Evolutionary change in pollen wall anatomy with resultant loss of saccus function is correlated with a change in ovulate strobilus orientation at pollination in oriental spruce. A suite of characters interact in the conifer pollination mechanism, and concerted change in these characters may lead to speciation.