Rapid enhancement of α-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes

Abstract

Agrobacterium-mediated transient gene expression have become a method of choice over stable plant genetic transformation. Tocopherols are a family of vitamin E compounds, which are categorized along with tocotrienols occurring naturally in vegetable oils, nuts and leafy green vegetables. This is the first report involving AtTC and AtHPT transient expression in Nicotiana benthamiana and this system can be used efficiently for large scale production of vitamin E. Agroinfiltration studies were carried out in N.benthamiana for the expression of Arabidopsis thaliana (At) genes encoding homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) individually and in combination (HPT + TC). The transgene presence was analyzed by reverse transcription PCR, which showed the presence of both the vitamin E biosynthetic pathway genes. The gene expression analysis was carried out by (reverse transcription quantitative real-time polymerase chain reaction) RT-qPCR and α-tocopherol content was quantified using high performance liquid chromatography (HPLC). The relative gene expression analysis by RT-qPCR confirmed an increased expression pattern where TC + HPT combination recorded the highest of 231 fold, followed by TC gene with 186 fold, whereas the HPT gene recorded 178 fold. The α-tocopherol content in leaves expressing HPT, TC, and HPT + TC was increased by 4.2, 5.9 and 11.3 fold, respectively, as compared to the control. These results indicate that the transient expression of HPT and TC genes has enhanced the vitamin E levels and stable expression of both A. thaliana genes could be an efficient strategy to enhance vitamin E biosynthesis in agricultural crop breeding.

Metabolic effects of agro-infiltration on N. benthamiana accessions

“…The precursors for this metabolites could be increased by the chlorophyll degradation observed in the present study. Another high value product naturally produced during this degradation process was α-tocopherol (Sathish et al 2018 ). Further potential candidate molecules could be lipids and hydrocarbons for biofuels and industrial use.…”

“…This Nicotiana species is amenable to agroinfiltration and high level transient gene expression. In addition to pharmaceutical proteins, N. benthamiana has also been exploited as a cell factory for high value or speciality chemicals (Sathish et al 2018 ). Fundamentally, it is also an invaluable resource for determining subcellular localisation and protein interactions.…”

Enhanced vitamin E content in an Indica rice cultivar harbouring two transgenes from Arabidopsis thaliana involved in tocopherol biosynthesis pathway

“…Conferring the genetic trait to improve vital nutrient accumulation in the edible parts of staple food crops, such as rice, through metabolic engineering is considered a fast, sustainable and cost‐effective alternative to conventional breeding (Maestre et al, 2017). In earlier reports from our laboratory, enhanced α‐tocopherol levels in the stable transformants of Nicotiana tabacum (Harish et al, 2013a, 2013b) and in Nicotiana benthamiana adopting a transient expression system using A. thaliana tocopherol cyclase (TC) and homogentisate phytyl transferase (HPT) (Sathish et al, 2018) were shown. In the present study, Agrobacterium ‐mediated transformation of Indica rice ASD16 with two genes involved in tocopherol biosynthesis, viz ., TC and HPT, was carried out and the transgenic plants were analysed for the vitamin E (α‐tocopherol) content.…”

Constitutive Overexpressing At.TC Improves Drought-mediated Oxidative Tolerance in Transgenic Brassica Napus L.

“…In the past few years, attempts to genetically overexpressing the tocopherol biosynthetic genes in model plants in order to increase the accumulation of tocopherol and their diverse function have been reported [11,12]. Genetic manipulation of genes encoding individual enzymes in this pathway such as γ-tocopherol methyltransferase (γ-TMT) [13,14], TC [15,16], and Homogentisate phytyl transferase (HPT) [17,18,15]. Kanwischer and co-workers demonstrated that the overexpression of TC/VTE1 enhance the tocopherols level by 7-folds [19].…”