Whole genome sequence data of the multidrug-resistant Proteus mirabilis-MN029, a model bacterial strain for the evaluation of antibacterial activity from selected native plant extracts.

Antimicrobial resistance remains a significant global and One Health threat, owing to the diminishing effectiveness of antibiotics against rapidly evolving multidrug-resistant bacteria, and the limited innovative research towards the development of new antibiotic therapeutics. In this article, we present the whole-genome sequence data of Proteus mirabilis-MN029 obtained from highly accurate long-read PacBioⓇ HiFi technology. The antibacterial activities of the selected African native plant species were also evaluated using the disk diffusion method. Acquired antibiotic resistance genes and chromosomal mutations corresponding to antibiotics of clinical importance were identified from genomic data. Using ethlyl acetate as solvent, Pterocarpus angolensis leaf extracts showed the most promising antibacterial effects against Proteus mirabilis-MN029. These datasets will be useful for future experimental research aimed at designing new antibacterial drugs from plant extracts that are effective alone or in combination with existing antibiotics to overcome multidrug-resistance mechanisms.

Amelioration of chromium toxicity in wheat plants through exogenous application of nano silicon.

Chromium (Cr) contamination in agricultural soils poses a risk to crop productivity and quality. Emerging nano-enabled strategies show great promise in remediating soils contaminated with heavy metals and enhancing crop production. The present study was aimed to investigate the efficacy of nano silicon (nSi) in promoting wheat growth and mitigating adverse effects of Cr-induced toxicity. Wheat seedlings exposed to Cr (K2Cr2O7) at a concentration of 100 mg kg-1 showed significant reductions in plant height (29.56%), fresh weight (35.60%), and dry weight (38.92%) along with enhanced Cr accumulation in roots and shoots as compared to the control plants. However, the application of nSi at a concentration of 150 mg kg-1 showcased substantial mitigation of Cr toxicity, leading to a decrease in Cr accumulation by 27.30% in roots and 35.46% in shoots of wheat seedlings. Moreover, nSi exhibited the capability to scavenge oxidative stressors, such as hydrogen peroxide (H2O2), and malondialdehyde (MDA) and electrolyte leakage, while significantly enhancing gas exchange parameters, total chlorophyll content, and antioxidant activities (enzymatic and nonenzymatic) in plants grown in Cr-contaminated soil. This study further found that the reduced Cr uptake by nSi application was due to downregulating the expression of HMs transporter genes (TaHMA2 and TaHMA3), alongwith upregulating the expression of antioxidant-responsive genes (TaSOD and TaSOD). The findings of this investigation highlight the remarkable potential of nSi in ameliorating Cr toxicity. This enhanced efficacy could be ascribed to the distinctive size and structure of nSi, which augment its ability to counteract Cr stress. Thus, the application of nSi could serve as a viable solution for production of crops in metal contaminated soils, offering an effective alternative to time-consuming and costly remediation techniques.

Nanoparticle elicitation: A promising strategy to modulate the production of bioactive compounds in hairy roots.

Hairy root culture is one of the promising biotechnological tools to obtain the stable and sustainable production of specialized metabolites from plants under controlled environment conditions. Various strategies have been adopted to enhance the accumulation of bioactive compounds in hairy roots yet their utilization at the commercial scale is restricted to only a few products. Recently, nanotechnology has been emerged as an active technique that has revolutionized the many sectors in an advantageous way. Elicitation using nanoparticles has been recognized as an effective strategy for enhancing the bioactive compounds of interest in plants. Nanoparticles elicit the activity of defense-related compounds through activation of the specific transcription factors involved in specialized metabolites production. This review discusses the recent progress in using nanoparticles to enhance specialized metabolite biosynthesis using hairy root culture system and the significant achievements in this area of research. Biotic and abiotic elicitors to improve the production of bioactive compounds in hairy roots, different types of nanoparticles as eliciting agents, their properties as dependent on shape, most widely used nanoparticles in plant hairy root systems are described in detail. Further challenges involved in application of nanoparticles, their toxicity in plant cells and risks associated to human health are also envisaged. No doubt, nanoparticle elicitation is a remarkable approach to obtain phytochemicals from hairy roots to be utilized in various sectors including food, medicines, cosmetics or agriculture but it is quite essential to understand the inter-relationships between the nanoparticles and the plant systems in terms of specifics such as type, dosage and time of exposure as well as other important parameters.

Potential of Selected African Medicinal Plants as Alternative Therapeutics against Multi-Drug-Resistant Bacteria.

Antimicrobial resistance is considered a "One-Health" problem, impacting humans, animals, and the environment. The problem of the rapid development and spread of bacteria resistant to multiple antibiotics is a rising global health threat affecting both rich and poor nations. Low- and middle-income countries are at highest risk, in part due to the lack of innovative research on the surveillance and discovery of novel therapeutic options. Fast and effective drug discovery is crucial towards combatting antimicrobial resistance and reducing the burden of infectious diseases. African medicinal plants have been used for millennia in folk medicine to cure many diseases and ailments. Over 10% of the Southern African vegetation is applied in traditional medicine, with over 15 species being partially or fully commercialized. These include the genera Euclea, Ficus, Aloe, Lippia. And Artemisia, amongst many others. Bioactive compounds from indigenous medicinal plants, alone or in combination with existing antimicrobials, offer promising solutions towards overcoming multi-drug resistance. Secondary metabolites have different mechanisms and modes of action against bacteria, such as the inhibition and disruption of cell wall synthesis; inhibition of DNA replication and ATP synthesis; inhibition of quorum sensing; inhibition of AHL or oligopeptide signal generation, broadcasting, and reception; inhibition of the formation of biofilm; disruption of pathogenicity activities; and generation of reactive oxygen species. The aim of this review is to highlight some promising traditional medicinal plants found in Africa and provide insights into their secondary metabolites as alternative options in antibiotic therapy against multi-drug-resistant bacteria. Additionally, synergism between plant secondary metabolites and antibiotics has been discussed.

Natural Plant-Derived Compounds in Food and Cosmetics: A Paradigm of Shikonin and Its Derivatives.

Shikonin and its derivatives are the natural naphthoquinone compounds produced in the roots of the Boraginaceae family. These red pigments have been used for a long time in coloring silk, as food colorants, and in the Chinese traditional system of medicines The resurgence of public interest in natural and plant-based products has led to this category of compounds being in high demand due to their wide range of biological activities including antioxidant, antitumor, antifungal, anti-inflammatory ones. Different researchers worldwide have reported various applications of shikonin derivatives in the area of pharmacology. Nevertheless, the use of these compounds in the food and cosmetics fields needs to be explored more in order to make them available for commercial utilization in various food industries as a packaging material and to enhance their shelf life without any side effects. Similarly, the antioxidant properties and skin whitening effects of these bioactive molecules may be used successfully in various cosmetic formulations. The present review delves into the updated knowledge on the various properties of shikonin derivatives in relation to food and cosmetics. The pharmacological effects of these bioactive compounds are also highlighted. Based on various studies, it can be concluded that these natural bioactive molecules have potential to be used in different sectors, including functional food, food additives, skin, health care, and to cure various diseases. Further research is required for the sustainable production of these compounds with minimum disturbances to the environment and in order to make them available in the market at an economic price. Simultaneous studies utilizing recent techniques in computational biology, bioinformatics, molecular docking, and artificial intelligence in laboratory and clinical trials would further help in making these potential candidates promising alternative natural bioactive therapeutics with multiple uses.

The use of African medicinal plants in cancer management.

Cancer is the third leading cause of premature death in sub-Saharan Africa. Cervical cancer has the highest number of incidences in sub-Saharan Africa due to high HIV prevalence (70% of global cases) in African countries which is linked to increasing the risk of developing cervical cancer, and the continuous high risk of being infected with Human papillomavirus In 2020, the risk of dying from cancer amongst women was higher in Eastern Africa (11%) than it was in Northern America (7.4%). Plants continue to provide unlimited pharmacological bioactive compounds that are used to manage various illnesses, including cancer. By reviewing the literature, we provide an inventory of African plants with reported anticancer activity and evidence supporting their use in cancer management. In this review, we report 23 plants that have been used for cancer management in Africa, where the anticancer extracts are usually prepared from barks, fruits, leaves, roots, and stems of these plants. Extensive information is reported about the bioactive compounds present in these plants as well as their potential activities against various forms of cancer. However, information on the anticancer properties of other African medicinal plants is insufficient. Therefore, there is a need to isolate and evaluate the anticancer potential of bioactive compounds from other African medicinal plants. Further studies on these plants will allow the elucidation of their anticancer mechanisms of action and allow the identification of phytochemicals that are responsible for their anticancer properties. Overall, this review provides consolidated and extensive information not only on diverse medicinal plants of Africa but on the different types of cancer that these plants are used to manage and the diverse mechanisms and pathways that are involved during cancer alleviation.

Recent Progress on Vaccines Produced in Transgenic Plants.

The development of vaccines from plants has been going on for over two decades now. Vaccine production in plants requires time and a lot of effort. Despite global efforts in plant-made vaccine development, there are still challenges that hinder the realization of the final objective of manufacturing approved and safe products. Despite delays in the commercialization of plant-made vaccines, there are some human vaccines that are in clinical trials. The novel coronavirus (SARS-CoV-2) and its resultant disease, coronavirus disease 2019 (COVID-19), have reminded the global scientific community of the importance of vaccines. Plant-made vaccines could not be more important in tackling such unexpected pandemics as COVID-19. In this review, we explore current progress in the development of vaccines manufactured in transgenic plants for different human diseases over the past 5 years. However, we first explore the different host species and plant expression systems during recombinant protein production, including their shortcomings and benefits. Lastly, we address the optimization of existing plant-dependent vaccine production protocols that are aimed at improving the recovery and purification of these recombinant proteins.

Anticancer Mechanisms of Bioactive Compounds from Solanaceae: An Update.

Plants continue to provide unlimited pharmacologically active compounds that can treat various illnesses, including cancer. The Solanaceae family, besides providing economically important food plants, such as potatoes and tomatoes, has been exploited extensively in folk medicine, as it provides an array of bioactive compounds. Many studies have demonstrated the anticancer potency of some of the compounds, but the corresponding molecular targets are not well defined. However, advances in molecular cell biology and in silico modelling have made it possible to dissect some of the underlying mechanisms. By reviewing the literature over the last five years, we provide an update on anticancer mechanisms associated with phytochemicals isolated from species in the Solanaceae plant family. These mechanisms are conveniently grouped into cell cycle arrest, transcription regulation, modulation of autophagy, inhibition of signalling pathways, suppression of metabolic enzymes, and membrane disruption. The majority of the bioactive compounds exert their antiproliferative effects by inhibiting diverse signalling pathways, as well as arresting the cell cycle. Furthermore, some of the phytochemicals are effective against more than one cancer type. Therefore, understanding these mechanisms provides paths for future formulation of novel anticancer drugs, as well as highlighting potential areas of research.

Combating Human Viral Diseases: Will Plant-Based Vaccines Be the Answer?

Molecular pharming or the technology of application of plants and plant cell culture to manufacture high-value recombinant proteins has progressed a long way over the last three decades. Whether generated in transgenic plants by stable expression or in plant virus-based transient expression systems, biopharmaceuticals have been produced to combat several human viral diseases that have impacted the world in pandemic proportions. Plants have been variously employed in expressing a host of viral antigens as well as monoclonal antibodies. Many of these biopharmaceuticals have shown great promise in animal models and several of them have performed successfully in clinical trials. The current review elaborates the strategies and successes achieved in generating plant-derived vaccines to target several virus-induced health concerns including highly communicable infectious viral diseases. Importantly, plant-made biopharmaceuticals against hepatitis B virus (HBV), hepatitis C virus (HCV), the cancer-causing virus human papillomavirus (HPV), human immunodeficiency virus (HIV), influenza virus, zika virus, and the emerging respiratory virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been discussed. The use of plant virus-derived nanoparticles (VNPs) and virus-like particles (VLPs) in generating plant-based vaccines are extensively addressed. The review closes with a critical look at the caveats of plant-based molecular pharming and future prospects towards further advancements in this technology. The use of biopharmed viral vaccines in human medicine and as part of emergency response vaccines and therapeutics in humans looks promising for the near future.

Plant-made HIV vaccines and potential candidates.

Millions of people around the world suffer from heavy social and health burdens related to HIV/AIDS and its associated opportunistic infections. To reduce these burdens, preventive and therapeutic vaccines are required. Effective HIV vaccines have been under investigation for several decades using different animal models. Potential plant-made HIV vaccine candidates have also gained attention in the past few years. In addition to this, broadly neutralizing antibodies produced in plants which can target conserved viral epitopes and neutralize mutating HIV strains have been identified. Numerous epitopes of envelope glycoproteins and capsid proteins of HIV-1 are a part of HIV therapy. Here, we discuss some recent findings aiming to produce anti-HIV-1 recombinant proteins in engineered plants for AIDS prophylactics and therapeutic treatments.

Improving zinc phytoremediation characteristics in Salix pedicellata with a new acclimation approach.

Some species of Salix sp. (willows) are a potential phytoremediator that can accumulate substantial contents of mineral elements and, therefore, to detoxify soils contaminated with pollutants and heavy metals such as the zinc (Zn). However, high concentrations of Zn inhibit plant growth and reduce biomass production in plants. In an attempt to overcome this inconvenience and to enhance plant tolerance to Zn toxicity, we tested a new tolerance induction approach by acclimation in two clones of Salix pedicellata, named SPK-12 and SP-K20. The approach comprises two successive phases. The first is a "tolerance induction phase" consisting of gradual exposure of plants to low concentrations of Zn sulfate (ZnSO4) at regular intervals until reaching DI100 (ZnSO4 inhibitory concentration). And, the second is a "tolerance maintenance phase" to uphold the acquired tolerance to Zn toxicity. The SP-K20 clone was acclimated to DI100 threshold over 33 days without noticeable symptoms of chlorosis or growth inhibition. Compared to controls, the SP-K20 clone was able to accumulate high concentrations of Zn, suggesting that phytoremediation abilities of S. pedicellata have been improved throughout the applied approach. Acclimated Salix plants might thus improve metal phytoextraction in heavily polluted soils without biomass growth inhibition.

Strigolactone biology: genes, functional genomics, epigenetics and applications.

Strigolactones (SLs) represent an important new plant hormone class marked by their multifunctional role in plant and rhizosphere interactions. These compounds stimulate hyphal branching in arbuscular mycorrhizal fungi (AMF) and seed germination of root parasitic plants. In addition, they are involved in the control of plant architecture by inhibiting bud outgrowth as well as many other morphological and developmental processes together with other plant hormones such as auxins and cytokinins. The biosynthetic pathway of SLs that are derived from carotenoids was partially decrypted based on the identification of mutants from a variety of plant species. Only a few SL biosynthetic and regulated genes and related regulatory transcription factors have been identified. However, functional genomics and epigenetic studies started to give first elements on the modality of the regulation of SLs related genes. Since they control plant architecture and plant-rhizosphere interaction, SLs start to be used for agronomical and biotechnological applications. Furthermore, the genes involved in the SL biosynthetic pathway and genes regulated by SL constitute interesting targets for plant breeding. Therefore, it is necessary to decipher and better understand the genetic determinants of their regulation at different levels.

Recent Patents in Agricultural Biotechnology; Focus on Health.

BACKGROUND: Agricultural biotechnology, including the generation of genetically modified food crops, has been the subject of much controversy over the last few years. Initially serving the basic needs of farmers, Ag Biotech has more recently gained much appeal for its opportunities with respect to both the nutritional and pharmaceutical sciences. METHODS: The following review describes a number of recently approved patents that could have direct implications for the field of medicine. Topics range from the development of pharmaceuticals in plants using hairy roots or virus expression vectors, to the role of epigenetics for improving the nutritional value of food crops. RESULTS: Many of these patents were developed by smaller companies or publically funded research institutes, disproving the perception that intellectual property in Ag Biotech is restricted to only large multinational corporations. CONCLUSION: The review concludes with a discussion of the future of these technologies in the face of the current negative political climate.

Fatty acid ω-hydroxylases from Solanum tuberosum.

KEY MESSAGE: Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1. Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L(-1) 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L(-1)). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L(-1) of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days.

Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots.

Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli.

Molecular farming on rescue of pharma industry for next generations.

Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.

A simplified Protocol to Induce Callogenesis in Protoplasts of Date Palm (Phoenix dactylifera L.) Cultivars.

BACKGROUND: In Algeria, date palm is currently confronted to the Bayoud disease. Biotechnological tools such as protoplastsfusion can appear as an alternative to ensure rapid multiplication and improvement of this species. OBJECTIVES: Callogenesis induction in protoplasts isolated from embryogenic callus of three date palm cultivars. MATERIALS AND METHODS: Some factors influencing the isolation and culture of protoplasts segregated from the calli of three date palm (Phoenix dactylifera L.) cultivars (Deglet Nour, Akerbouch and Degla Beida) were studied. Protoplasts of each cultivar were cultured on a semi-solid medium supplemented with various hormonal balances. RESULTS: Maceration with an enzymatic solution containing 1.5% cellulase and 1% macerozyme R10 in the presence of 0.5 M mannitol for more than 16 h with gentle agitation allows isolation of a great number of viable protoplasts. In addition, purification of protoplasts on a cushion of 21 or 25% sucrose was effective in cell debris removal and maximum recovery. The culture of isolated protoplasts on a semi-solidified Murashige and Skoog medium, with 0.3% agarose, 2 mg. L-1 2,4-D and 0.5 mg.L-1 BAP allowed good viable protoplast maintenance as well as cell wall regeneration. After more than two months of culture, cell divisions were still occurring and microcalli became visible to the naked eye, containing a large number of cells. CONCLUSIONS: The developed protocol can be useful for application of somatic hybridization to improve date palm cultivars.

Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

Functional analysis of the DAT gene promoter using transient Catharanthus roseus and stable Nicotiana tabacum transformation systems.

The Catharanthus roseus DAT gene encodes the enzyme acetyl-CoA:deacetylvindoline-4-O-acetyltransferase involved in the last step of the indole alkaloid pathway leading to vindoline. This gene is characterized by specific cell type expression in idioblasts and laticifers. To understand the specific transcriptional regulation mechanism(s) of DAT, several DAT promoter GUS constructs were cloned into pCAMBIA1305.1. Agroinfiltration of different explant types of C. roseus resulted in organ-specific accumulation of GUS, albeit at various levels. Heterologous accumulation of GUS in transgenic tobacco revealed both general and non-specific expression with the exception of a stomata-specific expression when 2.3 kb of the DAT promoter was coupled with a portion of the DAT ORF. These results suggest that in addition to the 2.3 kb upstream of the DAT transcriptional start site, additional cis-acting elements may be responsible for the specific spatial expression of DAT in vivo. Furthermore, hairy roots transformed with DAT promoter GUS constructs demonstrated GUS expression in root tissues (visualized through GUS enzyme activity), even though DAT is repressed in non-transformed roots.