The Chinese herb Artemisia annua possesses small 10-cell biseriate glandular trichomes on the surface of its aerial tissues. These trichomes were isolated from floral tissue using a Bead Beater based method. Expression patterns of expressed sequence tags from a trichome library whose Basic Local Alignment Search Tool (BLAST) results suggested a possible function in terpenoid metabolism were investigated by RT-PCR. Known terpenoid biosynthetic enzymes, such as amorpha-4,11-diene synthase showed a high degree of trichome-specific expression. In order to investigate cell specific gene expression within the trichome, the promoter for the gene encoding amorpha-4,11-diene synthase was isolated but the lack of an efficient transformation protocol in A. annua hindered reporter gene localization experiments. Traditional and whole-mount <i>in situ</i> hybridization techniques were used to further the study of cell specific gene expression within the glandular trichome. An RNA probe constructed from the sequence of amorpha-4,11-diene synthase localized expression to the 2nd and 3rd subapical cell pairs of the glandular trichomes. This suggests that at least part of the artemisinin biosynthetic pathway resides within the lower cell pairs. To better understand the genes involved in terpenoid biosynthesis in A. annua, the full length sequence of a short chain alcohol dehydrogenase highly represented in an expressed sequence tag library and shown to have trichome-specific expression by RT-PCR, was cloned. Heterologous expression in Escherichia coli demonstrated that the enzyme was capable of oxidizing a wide range of monoterpenols to their corresponding ketone forms. All of this data helps us to better understand the organization of expression and biochemistry of terpenoids in A. annua glandular trichomes.

The Chinese herb Artemisia annua possesses small 10-cell biseriate glandular trichomes on the surface of its aerial tissues. These trichomes were isolated from floral tissue using a Bead Beater based method. Expression patterns of expressed sequence tags from a trichome library whose Basic Local Alignment Search Tool (BLAST) results suggested a possible function in terpenoid metabolism were investigated by RT-PCR. Known terpenoid biosynthetic enzymes, such as amorpha-4,11-diene synthase showed a high degree of trichome-specific expression. In order to investigate cell specific gene expression within the trichome, the promoter for the gene encoding amorpha-4,11-diene synthase was isolated but the lack of an efficient transformation protocol in A. annua hindered reporter gene localization experiments. Traditional and whole-mount <i>in situ</i> hybridization techniques were used to further the study of cell specific gene expression within the glandular trichome. An RNA probe constructed from the sequence of amorpha-4,11-diene synthase localized expression to the 2nd and 3rd subapical cell pairs of the glandular trichomes. This suggests that at least part of the artemisinin biosynthetic pathway resides within the lower cell pairs. To better understand the genes involved in terpenoid biosynthesis in A. annua, the full length sequence of a short chain alcohol dehydrogenase highly represented in an expressed sequence tag library and shown to have trichome-specific expression by RT-PCR, was cloned. Heterologous expression in Escherichia coli demonstrated that the enzyme was capable of oxidizing a wide range of monoterpenols to their corresponding ketone forms. All of this data helps us to better understand the organization of expression and biochemistry of terpenoids in A. annua glandular trichomes.

Artemisinin is a potent antimalarial sesquiterpene lactone produced and stored in the glandular trichomes (GLTs) of Artemisia annua. Although they produce no artemisinin, nor any of the precursor compounds, A. annua roots appear to have a regulatory effect on production of the terpene in leaves. However, more information is needed to define the role of the roots in artemisinin production in the plant. Grafting among three cultivars was used to measure phenotypic responses: SAM, and #15 cultivars both have GLTs, but produce artemisinin at 1.49% and 0.57% DW, respectively; GLS cultivar produces neither GLTs nor artemisinin. Compared to ungrafted plants, all self-grafts, e.g. SAM/SAM (scion/rootstock), increased scion artemisinin probably from grafting stress. SAM/#15 grafts yielded less artemisinin than SAM/SAM, but more than either #15/#15 or ungrafted #15 and SAM suggesting rootstock inhibition of the scion. SAM/SAM also had more artemisinin than #15/SAM, which was also greater than either #15/#15 or ungrafted #15 and SAM. The #15/SAM graft also produced more artemisinin than SAM/#15, and with the other grafting results suggested that SAM roots were stimulating artemisinin production in the #15 scion. There was no appearance of either GLTs or artemisinin when GLS scions were grafted to SAM indicating that GLTs had to be present to receive putative signals from SAM rootstocks. Furthermore, artemisinic acid and arteannuin B were only present in SAM scions and not scions of #15 suggesting a block in one of the side pathways of artemisinin biosynthesis. Other artemisinic metabolites, total flavonoids, and GLTs numbers were also measured. The various phenotypes were analyzed several months after grafting indicating a persistent change and suggesting a possible epigenetic alteration of the scion. This study will provide fundamental information regarding the role that roots play in the production of artemisinin in the shoots of A. annua.

The Snedden lab has been studying a family of Ca2+-binding proteins from Arabidopsis that are related to the prototypical Ca2+ sensor calmodulin (CaM) and are termed CMLs (CaM-like proteins). Previous work on CML42 demonstrated that it displays biochemical properties typical of Ca2+ sensors and interacts in vitro with KIC (KCBP-interacting Ca2+-binding protein), a protein known to function in trichome branching. In the present study, I investigated whether CML42 is also involved in trichome branching. I examined a CML42 T-DNA insertion knockout line (cml42) and found that it exhibits a mutant trichome phenotype with increased branch numbers compared to wildtype plants. All other aspects of cml42 growth and morphology, including root hairs, appeared normal relative to wildtype plants. kic knockout plants did not show any discernible trichome phenotype when compared to wildtype plants, nor did transgenic lines overexpressing CML42. Transgenic plants lacking both CML42 and KIC expression (cml42kic) displayed a cml42 mutant phenotype. The genetic studies suggest that CML42 is a negative regulator of trichome branching. Biochemical analysis on recombinant full-length CML42, C-terminal, and N-terminal fragments, using the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), demonstrated that Ca2+-binding results in a conformational change in CML42 and the exposure of hydrophobic regions, particularly within the C-terminal lobe. Collectively, data from the Snedden Lab support the important role of Ca2+ signalling in trichome branching and morphology. / Thesis (Master, Biology) -- Queen's University, 2009-08-11 14:00:06.008

A unifying goal of plant systematics is in pursuit of a natural system that means phylpgenetic relationships. We can construct it by means of various characters. There are many arguments about generic circumscription of Pteridophyta. It¡¦s worth while to reexamine some characters, such as trichome. Trichomes have long been considered one of the most important characters by pteridologists. However, The study of trichomes in Taiwan is very rare. In the present study, trichome morphology is used to assess phylogenetic relationships among genera of Dryopteridaceae (sensu Kramer et al.) . The observation of trichomes focus on hairs on the lamina and scales on the base of stipes. Lamina surface hairs are classified into unicellular hairs, club-like unicellular glands, uniseriate hairs, spine-like hairs, appressed glandular hairs, uniseriate hairs with a glandular head and verruca. Stipe base scale margins are classified into entire, serrate, unicellular branch, uniseriate branch, multicellular branch with a glandular head, and dorsi-ventral branch. Color distribution and branch dimension of scales were also good differentiation characters. The main taxonomic conclusions are as follows: (1)Trichome characters support the distinctness of Nothoperanema, Peranema, Polystichm, Dryopsis, Ctenitis, Tectaria, Athyrium, Cystopteris, Acystopteris, and Woodsia; (2) Diplazium and Dryopteris are hererogenous and show little correaltion to exist system. The latter two genera need further research.

Specialized metabolism (secondary metabolism) in glandular trichomes of sweet basil (Ocimum basilicum L.) and accumulation of specialized metabolites (secondary metabolites) in rhizomes of turmeric (Curcuma longa L.) was investigated using proteomic and metabolomic approaches, respectively. In an effort to further clarify the regulation of metabolism in the glandular trichomes of sweet basil, we utilized a proteomics-based approach that applied MudPIT (multidimensional protein identification technology) and GeLC-MS/MS (gel enhanced LC-MS/MS) to protein samples from isolated trichomes of four different basil lines: MC, SW, SD, and EMX-1. Phosphorylation, ubiquitination and methylation of proteins in these samples were detected using X!tandem. Significant differences in distribution of the 755 non-redundant protein entries demonstrated that the proteomes of the glandular trichomes of the four basil lines were quite distinct. Correspondence between proteomic, EST, and metabolic profiling data demonstrated that both transcriptional regulation and post-transcriptional regulation contribute to the chemical diversity. One very interesting finding was that precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP (2-C-methyl-D-erythritol 4- phosphate) pathway, but not the mevolonate pathway, in basil glandular trichomes. Our results suggest that carbon flow can be readily redirected between the phenylpropanoid and terpenoid pathways in this specific cell type. To investigate the impact of genetic, developmental and environmental factors on the accumulation of phytochemicals in rhizomes of turmeric, we performed metabolomic analysis in a 2x2x4 full factorial design experiment using GC-MS, LC-MS, and LC-PDA. Our results showed that growth stage had the largest effect on levels of the three major curcuminoids. Co-regulated metabolite modules were detected, which provided valuable information for identification of phytochemicals and investigation of their biosynthesis. Based on LC-MS/MS data, 4 new diarylheptanoids were tentatively identified in turmeric rhizomes using Tandem-MSASC, a home-made software tool that automatically recognizes spectra of unknown compounds using three approaches. Based on our metabolomic results, we proposed two new strategies, “metabolomics-guided discovery” and “correlation bioassay”, to identify bioactive constituents from plant extracts based on information provided by metabolomic investigation.

Cotton, Gossypium hirsutum L., leaf grade values can significantly increase with remnants of leaf and bract materials, and can result in increased ginning costs and discounts to the producer. Cotton classed through the USDA-AMS Classing Office in Corpus Christi, Texas has reported increases in leaf grade values beginning in 2000 (USDA, 2012). The impacts of the interaction of agronomic characteristics of cotton cultivars with those of various harvest aid regimes were studied over three growing seasons, and data were used to narrow possible contributors to the observed increased leaf grade values. Multiple trials were conducted throughout the Coastal Bend and Blackland Prairie of Texas, in addition to Tifton, Georgia. Cotton was harvested, lint samples were ginned in a microgin, and lint quality was quantified with HVI. Harvest aid regimes selected provided a broad range of defoliation and desiccation, from a multiple herbicidal and hormonal modes-of-action. Defoliation levels ranged from 0 to 96% and desiccation levels ranged from 0 to 90%. Harvest aid treatments had no impact (P≤0.05) on leaf grade values for either of the years of the trials. Multiple trials were conducted in five counties in Texas, including the Lower and Upper Coastal Bend and the Blackland Prairie, and were defoliated with a uniform harvest aid treatment to identify leaf and bract morphological differences, and to determine their role in leaf grade. Multi-acre module trials were conducted with a smooth leaf cultivar and a hairy leaf cultivar to obtain leaf grade values following commercial ginning. Leaf and bract pubescence, and leaf and bract area were collected to analyze the resulting impact on cotton leaf grade values. Visual quantification of leaf and bract trichome density was quantified on 10 youngest fully-expanded leaves and 10 mid-canopy full sized bolls, respectively, when cotton was at physiological cut-out. Trichome density quantification indicated substantial variation in cultivars and discrepancies from company based rating systems. Leaf grades values generally increased with increasing trichomes densities, although not always statistically significant. In the split plot cultivar and harvest aid trial, harvest aid efficacy was similar for each of the cultivars, but cultivar trichome density was positively influence the cotton leaf grade value.

Athrixia phylicoides (bush tea) is one of many plants from the Asteraceae family used as a traditional herbal medicine. With very few cultivated plants, natural growing plants currently serve as the main resource for plant material. The plant is not yet commercialised and its medicinal value is known and used only by a few people. With the long term aim at commercial scale propagation, this study consists of three parts. Firstly we developed a protocol for in vitro propagation of A. phylicoides. Secondly, the ultrastructure and morphology of leaves were studied microscopically and thirdly, comparisons were made between in vitro and ex vitro grown plants. Nodal segments of greenhouse plants were used to establish cultures. Better growth and less wilting was recorded on explants surface sterilised with NaOCl compared to Ca(OCl)2 after establishment. The addition of growth regulators IBA (indole-3-butyric acid) and BAP (6-benzylaminopurine) to the culture medium did not seem to affect the growth response of explants during the multiplication phase. Hyperhydricity was a problem throughout our trials. The development of hyperhydricity symptoms seems to be related to seasonal changes in the stock plant material used to initiate cultures, rather than the composition of growth medium or growth room temperatures. The occurrences of hyperhydricity symptoms were inconsistent and unpredictable. A rooting medium with added BAP and decreased sucrose levels resulted in a higher rooting percentage compared to the control medium, free from BAP and with a higher sucrose concentration, which yielded no rooting. However, in another experiment, in vitro rooting occurred spontaneously after subdividing and transfer of microshoots to fresh control medium. The addition of GA3 to the establishment medium (but not to the subsequently used multiplication and rooting media) yielded a slightly higher percentage of rooting. However, cultures initially established on GA3 medium yielded fewer roots per explant and roots were shorter than those of explants established on hormone free medium. The medicinal properties of plants are often linked to the production of essential oils. We hypothesised that the medicinal value of A. phylicoides can be linked to the production of the aromatic essential oils released by leaves. A microscopic study of leaves provided some preliminary insight of the mechanisms involved in the production of medicinally active products. Electron- and light microscopic examination of leaves were used to identify and study structures that are apparently involved in the production and secretion of essential oils. Two types of trichomes were identifyed – nonglandular and glandular trichomes. These glandular trichomes are multicellular with a subcuticular storage space and are present only on the adaxial surface of leaves. In the case of medicinal plants, it is essential that the medicinal properties of the plant are not altered by the method of propagation. This was our motivation for comparing the morphology and ultrastructure of leaves of plants that were grown in their natural environment to that of plants grown in vitro. Leaf surfaces of in vitro grown plants were smaller and the number of glandular trichomes per surface area was less on in vitro grown plants. There were no noticeable changes in the morphology of glandular trichomes. / Dissertation (MSc(Agric))--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted

Orientador: Silvia Rodrigues Machado / Resumo: O gênero Jacaranda Juss. (Bignoniaceae), um representante da tribo Jacarandeae, é constituído por 47 espécies, das quais 39 são endêmicas para o Brasil. Este gênero se destaca dos demais gêneros de Bignoniaceae principalmente por apresentar estaminódio alongado glandular e forte potencial etnobotânico e fitoquímico em várias espécies ocorrentes no Brasil. Estruturas secretoras são sítios de produção de substâncias bioativas, estudos sobre distribuição, morfologia e organização anatômica e sua composição química podem contribuir para conhecimento de Jacaranda no âmbito biológico, químico e farmacológico. Neste contexto, o objetivo deste estudo foi realizar uma descrição anatômica foliar de 24 espécies de Jacaranda ressaltando características capazes de agrupar espécies e comparar com ambientes de ocorrência e evidenciar características diagnósticas para as espécies. Dentre elas, também foi objetivo desse estudo, realizar uma prospecção das estruturas secretoras foliares e florais em seis espécies associando o morfotipo das glândulas com sua localização, organização anatômica e as principais classes de compostos produzidos. Para isso, usamos técnicas rotineiras para o processamento de material herborizado e material coletado fresco para estudos anatômicos, micromorfológicos de superfície, histoquímicos e analises de agrupamento. Características morfológicas e anatômicas foliares, tais como, o tamanho, forma, tipo da margem e textura dos foliólulos, assim como a estrutura da ala... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The genus Jacaranda Juss. (Bignoniaceae), a representative of the Jacarandeae tribe, consists of 47 species, 39 of which are endemic to Brazil. This genus stands out from the other genera of Bignoniaceae mainly for presenting elongated glandular staminode and strong ethnobotanical and phytochemical potential in several species occurring in Brazil. Secretory structures are sites of production of bioactive substances, studies on distribution, morphology and anatomical organization and their chemical composition can contribute to Jacaranda knowledge in the biological, chemical and pharmacological fields. In this context, the objective of this study was to perform a leaf anatomical description of 24 species of Jacaranda highlighting characteristics capable of grouping species and comparing with occurrence environments and highlighting diagnostic characteristics for the species. Among them, it was also the aim of this study, to prospect for leaf and floral secretory structures in six species associating the gland morphotype with its location, anatomical organization and the main classes of compounds produced. For this, we use routine techniques for the processing of herborized material and fresh collected material for anatomical, surface micromorphological, histochemical and cluster analysis studies. Leaf morphological and anatomical characteristics, such as the size, shape, type of margin and texture of the leaflets, as well as the structure of the wing and edge, as well as the t... (Complete abstract click electronic access below) / Doutor

A crytological and nutritional study was conducted on Vitreoscilla stercoraria, ATCC 15218, and Vitreoscilla species, ATCC 13982. Cell and trichome morphology in broth cultures and on solid media were studied.