Glandular Trichomes on the Leaves of Nicotiana tabacum: Morphology, Developmental Ultrastructure, and Secondary Metabolites

AbstractTobacco (Nicotiana tabacum L.) plants are known to present high levels of secondary metabolites that increase with the plant age. Molecular biology techniques like restriction enzyme digestion and PCR, requires as pre-requisite the isolation of genomic DNA of suitable purity, good quality and with low levels of contaminants. Several methods to isolate pure and intact tobacco DNA for molecular research purposes have been developed. In this work, a combination between a tobacco seed germination technique using gibberellic acid and a fast and simple genomic DNA extraction method from 14-days old tobacco seedlings to reduce the secondary metabolites levels in the final samples was presented. Ten tobacco genotypes were used to evaluate this method. The DNA concentrations were in a range between 0.73 μg/μL to 1.47 μg/μL for Habana-2000 cv. and Criollo cv., respectively. The absorbance ratios values to determine DNA quality were acceptable. This method allows the obtaining of high molecular weight DNA suitable for digestion with restriction enzymes, EcoRI and BamHI. Tobacco seedlings DNA in a short period of time, in a simple way and with a low cost, was obtained with this extraction method.

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Leaf glandular trichomes in Empetrum nigrum: morphology, histochemistry, ultrastructure and secondary metabolites

Nordic Journal of Botany ◽

10.1111/j.1756-1051.2011.01322.x ◽

2012 ◽

Vol 30 (4) ◽

pp. 470-481 ◽

Cited By ~ 16

Author(s):

Lyudmila E. Muravnik ◽

Alexey L. Shavarda

Keyword(s):

Secondary Metabolites ◽

Glandular Trichomes ◽

Empetrum Nigrum

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Fertilization Following Pollination Predominantly Decreases Phytocannabinoids Accumulation and Alters the Accumulation of Terpenoids in Cannabis Inflorescences

Frontiers in Plant Science ◽

10.3389/fpls.2021.753847 ◽

2021 ◽

Vol 12 ◽

Author(s):

Carni Lipson Feder ◽

Oded Cohen ◽

Anna Shapira ◽

Itay Katzir ◽

Reut Peer ◽

...

Keyword(s):

Quantitative Analysis ◽

Secondary Metabolites ◽

Glandular Trichomes ◽

Regulation System ◽

Dioecious Plant ◽

Functional Roles ◽

Pollen Sacs ◽

Analytical Standards ◽

Main Factor ◽

Original Female

In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-Δ9-trans-tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings may point to the functional roles of secondary metabolites in Cannabis.

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Identification of two new trichome-specific promoters of Nicotiana tabacum

10.1101/620021 ◽

2019 ◽

Author(s):

Mathieu Pottier ◽

Raphaëlle Laterre ◽

Astrid Van Wessem ◽

Aldana M. Ramirez ◽

Xavier Herman ◽

...

Keyword(s):

Metabolic Engineering ◽

Nicotiana Tabacum ◽

Glandular Trichomes ◽

Regulatory Elements ◽

Promoter Regions ◽

Transgenic Lines ◽

Leaf Tissues ◽

Set Up ◽

Almost All ◽

Specific Line

AbstractMain conclusion pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were identified and their strength and specificity were compared to those of previously described promoters in this species.Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and gene expression level of NtRbcS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 is also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Almost all transgenic lines displayed GUS activity in tall glandular trichomes, indicating that the appropriate cis regulatory elements were included in the selected promoter regions. However, unlike for the other promoters, no trichome-specific line was obtained for pNtLTP1:GUS-VENUS, thus in agreement with the RT-qPCR data. These data thus provide two new transcription promoters that could be used in metabolic engineering of glandular trichomes.

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