R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.)

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA)

Genetic deficiency of acid α-glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe disease (PD) encompassing at least four clinical subtypes of varying severity (infantile; childhood, juvenile and late onset). Our objective is to develop an innovative and affordable approach for enzyme replacement therapy (ERT) via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life for people living with Pompe disease. A consensus at a 2019 US Acid Maltase Deficiency (AMDA) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of Pompe disease. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. Previously, we have shown that a lysate from transgenic tobacco seeds expressing human GAA (tobrhGAA) was enzymatically active and can correct enzyme deficiency in cultured PD cells and in adult lymphocytes of Pompe patients and in vivo in disease-relevant tissues in GAA knockout (KO) mice when administered IP. We have extended these pre-clinical studies in PD knockout (KO) mice with ground tobrhGAA seeds that supports proof-of-concept for Oral-ERT for future clinical trials. Briefly in GAA KO mice, Oral-ERT with ground tobrhGAA seeds showed significant reversal of fore-limb and hind-limb muscle weakness, increased motor coordination/balance/strength and mobility, improved spontaneous learning, increased GAA baseline activity in tissues, reduced glycogen in tissues and negible serum titers to GAA. Pharmacokinetics showed maximum serum GAA concentration (Cs) at 8-10 hr and peak urine excretion at 10-12 hr. The tobrhGAA was taken up in PD fibroblast, lymphoid and myoblast cells. Enzyme kinetics compared favorably or superior to placental hGAA, plus alglucosidase alfa or other rhGAAs for Km, Vmax, pH optima, thermal heat stability and IC50 for inhibitors. The tobrhGAA in seeds was extremely stable stored for 15 years at room temperature. NGS-genome sequencing of the tobrhGAA and wild-type plants and RNA expression profiles was performed and will be posted on our website. Thus, Oral-ERT with ground tobrhGAA seeds is an innovative approach that overcomes some of the challenges of alglucosidase alfa-ERT and provides a more effective, safe and significantly less expensive treatment.

Influence of the methanol extract and fractions of Smilax brasiliensis Sprengel on development in vitro of Nicotiana tabacum and Alllium cepa

Smilax brasiliensis (Smilacaceae) is a native Brazilian plant found in the Cerrado biome and commonly used in folk medicine. The aim of this study was to evaluate the influence of the methanol extract and fractions from S. brasiliensis leaves on development in vitro of Nicotiana tabacum (tobacco) and Allium cepa (onion) seeds. In vitro germination protocol of onion seeds was established. Tobacco and onion seeds were placed to germinate on basal medium added of extract or fractions of S. brasiliensis leaves and dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and gibberellic acid (GA) at different concentrations in the presence of light. The germination ranged from 40 to 100% for tobacco and from 60 to 100% for onion seeds. The results indicated that the extract and fractions promoted higher growth than 2,4-D and GA when analyzed number of nodes, leaves, root and root size for germinated tobacco seeds, and higher growth when analyzed number roots and stem size for onion seeds. Regarding BAP, the extract and fractions inhibited leaf growth, root and root size of tobacco seeds. Further studies are needed to evaluate the possible use of the methanol extract and fractions from S. brasiliensis leaves as natural sources of hormones and/or bioherbicides.

Development of a mathematical model of vibratory non-lift movement of light seeds taking into account the aerodynamic forces and moments

The modern practice of using vibratory machines when working with fine-size light-weight seeds is faced with such an undesirable phenomenon as the impact of aerodynamic forces and moments on the kinematics of vibrational movement of particles of the seed mixture fractions. According to the results of scientific studies devoted to the solution of this problem, only mathematical models of vibrational movement are used, where the aerodynamic factor is taken into account as taking the seeds by airflow. This is typical only for cleaning modes with the rebound of seeds from the vibrating surface. Aerodynamic forces and moments are present in them only as a force of aerodynamic resistance. The action of lateral aerodynamic forces and their moments are not taken into account. Their consideration allows to extend the range of action of the aerodynamic factor on the modes of vibration cleaning (vibroseparation) without rebound (but with sliding and rolling) which are of greater interest in terms of improving the efficiency of processing fine-size seeds. A mathematical model of seed vibration movement taking into account the action of a complete set of aerodynamic forces (dynamic resistance forces and lateral aerodynamic forces) and moments was proposed. This makes it possible to simulate non-lifting modes of vibrational movement of seeds. A system of algebraic equations that are linear with respect to the kinematic parameters of seed movement which was obtained by translating differential equations of movement into a finite-difference form was presented. The possibility of numerical solution of equations of movement by the Euler method was shown. The results of the evaluation of the model adequacy for the processes of vibration separation of tobacco seeds and false flax were presented. As shown by the results of calculations and experiments, the developed model provides an increase in the adequacy of the simulation results by 30 % in comparison with the model where the aerodynamic factor is not taken into account

THE EFFECT OF BIOREGULATOR REGOPLANT ON TOBACCO PRODUCTIVITY DURING THE SEEDING AND FIELD PERIODS

We studied the effect of the growth regulator Regoplant on the mass of seedlings, the quality of seedlings and the productivity of tobacco. Soaking tobacco seeds before sowing in a 0.0001 % solution of the preparation at an exposure of 6 hours, and then spraying the plants at the stage of “auricles” and “seedling ready for planting” with a working solution of an agrochemical with a 0.00001 % concentration significantly improved the quality of seedlings. This technique increases the length of tobacco seedlings to the apical point by 66 %, to the end of elongated leaves by 29 %, the mass of stems by 53 %, the mass of roots by 32 %, the diameter of the stem by 38 %. After planting the plants in the field, developed under the influence of the Regoplant stimulator, we noted an increase in productivity by 16 % and an improvement in the quality of raw materials due to an increase in carbohydrates and a decrease in proteins. The economic effect against the background of the application of Regoplant preparation amounted to 106 thousand rubles.

Analyses and evaluation of the main chemical components in different tobacco (Nicotiana tabacum L.) genotypes

The nicotine, reducing sugar and ion contents from the threshing of tobacco can re-used from the industry. The crude oil and fatty oil compositions of tobacco seeds can be considered as an alternative source of raw material for biodiesel. In this study, the nicotine, reducing sugar content, crude oil, fatty acid composition and ion content were determined in 29 genotypes and 1 cultivar of tobacco. The genetic diversity was determined among the tobacco cultivar and genotypes base on examined properties. The nicotine content varied between 0.10-0.87%, reducing sugar ranged from 9.70-21.30%, crude oil varied between 24.33-47.00% and fatty acid compositions was found in the range of 77.94-100%. Linoleic (13.92-75.04%) and butyric (0.33-64.98%) acids were the major components. Overall, the BSR-5 (52.56 mg/g) and ESR-5 (44.58 mg/g) genotypes exhibited the highest potassium contents and ESR-7 (6.54 mg/g) and ESR-8 (1.28 mg/g) genotypes had the lowest chlorine contents. As a result of this study, the highest nicotine content, reducing sugar and crude oil of tobacco were found in ESR-4, ESR-11 and BSR-5 genotypes, respectively. The dendrogram analysis divided the tobacco into two main groups and most of the same origin genotypes fell into the same group. The results indicated that the different tobacco leaves and seeds can be evaluated as an alternative source in the industry as cigarettes, biodiesel and different industrial applications such as cosmetic, oil paints and varnishes based on their chemical properties.

Modelling of Nicotiana Tabacum L. Oil Biodiesel Production: Comparison of ANN and ANFIS

Among the modern computational techniques, Artificial Neural Network (ANN) and Adaptive Neuro-Fuzzy Inference System (ANFIS) are preferred because of their ability to deal with non-linear modelling and complex stochastic dataset. Nondeterministic models involve some computational complexities while solving real-life problems but would always produce better outcomes. For the first time, this study utilized the ANN and ANFIS models for modelling tobacco seed oil methyl ester (TSOME) production from underutilized tobacco seeds in the tropics. The dataset for the models was obtained from an earlier study which focused on design of the experiment on TSOME production. This study is an an exposition of the influence of transesterification parameters such as reaction duration (T), methanol/oil molar ratio (M:O), and catalyst dosage on the TSOME/biodiesel yield. A multi-layer ANN model with ten hidden layers was trained to simulate the methanolysis process. The ANFIS approach was further implemented to model TSOME production. A comparison of the formulated models was completed by statistical criteria such as coefficient of determination (R2), mean average error (MAE), and average absolute deviation (AAD). The R2 of 0.8979, MAE of 4.34468, and AAD of 6.0529 for the ANN model compared to those of the R2 of 0.9786, MAE of 1.5311, and AAD of 1.9124 for the ANFIS model. The ANFIS model appears to be more reliable than the ANN model in predicting TSOME production in the tropics.