Jocelyn C. Turner

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

QUANTITATIVE ANALYSIS OF CANNABINOIDS IN THE SECRETORY PRODUCT FROM CAPITATE-STALKED GLANDS OF CANNABIS SATIVA L. (CANNABACEAE)

Capitate-stalked glandular trichomes from pistillate bracts of Cannabis sativa L. were analyzed to determine the cannabinoid composition of secretory products within the secretory sac. Analyses were performed on cloned materials of a strain characteristically high in cannabidiol. The secretory product is accumulated in a single large secretory sac, which develops above a multicellular disk of secretory cells during gland ontogeny. The content of the secretory sac was removed with micropipets, using a micromanipulator without damaging or removing disk cells or their contents, and analyzed by gas-liquid chromatography. The cannabinoid content of the secretory sac was compared with previously quantitated cannabinoids of whole capitate-stalked glands. Results indicated that nearly all of the cannabinoid content of capitate-stalked glands was present in the secretory sac.

Effects of sample treatment on chromatographic analysis of cannabinoids in Cannabis sativa L. (Cannabaceae)

Abstract Gas—liquid chromatographic and high-performance liquid chromatographic analyses on the effects of leaf treatment as well as the conditions for cannabinoid extraction were examined in two clones of Cannabis sativa L. Cannabinoid extracts of dried leaves, when analyzed by gas—liquid chromatography, showed no significant quantitative or qualitative differences regardless of drying procedure or temperature and duration of extraction investigated. Comparable high-performance liquid chromatographic analyses, however, indicated that while extraction temperature did not influence the cannabinoid profile, drying conditions had a significant effect. High ratios of acid to neutral forms were derived only from extracts of leaves dried at 37°C as compared to 60°C. Fresh, non-dried leaf material also yielded high ratios of acid to neutral forms, but the duration of extraction was found to affect cannabinoid yield significantly. Longer extractions of fresh leaves resulted in lower amounts of cannabinoids extracted. This study determined optimal procedures for analyzing fresh plant materials.

A Temporal Study of Cannabinoid Composition in Continual Clones of Cannabis sativa L. (Cannabaceae)

Genetically and developmentally defined vegetative samples of three clones of Cannabis sativa L. were grown in a common environment and analyzed for cannabinoid production. Significant variations occurred in cannabinoid levels in each clonal population. Throughout the 2-yr study, the cannabinoid fluctuations were random rather than cyclic. Although within each clone all cannabinoids increased or decreased simultaneously, fluctuations in cannabinoid levels occurred independently from clone to clone. In addition, each clone retained its distinctive morphology and cannabinoid profile throughout the study.

Cannabinoid Occurrence in Seedlings of Cannabis sativa L.: Quantitation in Seedlings of Known Age and Primary Leaf Length

Seedlings of a Δ9-tetrahydrocannabinol (THC) dominant strain of Cannabis sativa were employed to develop a biological system to investigate cannabinoid biosynthesis. High-performance liquid chromatography was used to detect and quantify the cannabinoids. Cannabinoids were first detected in seedlings of light/dark-grown plants at 48-50 h. The first cannabinoid detected was cannabichromene (CBC) at 52-54 h. At 60-62 h both cannabigerol (CBG) and THC were detected along with CBC. A similar sequence appeared in dark-grown seedlings, but CBC became evident only at 56-58 h, and THC and CBG were first detected at 66-68 h. This pattern is significant because previously proposed pathways of cannabinoid synthesis have indicated that CBG is a precursor to CBC, yet CBG is not the first cannabinoid to be detected but occurs later with the appearance of THC. Cannabinoid concentrations were always higher in light-grown than in dark-grown plants of comparable age. Cannabinoid quantities on a dry-weight basis increased with increasing leaf length and/or increasing age.