Abstract
Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 μg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.
Highlights
Some genera of the family Solanaceae, including Hyoscyamus, Duboisia, Atropa, and Scopolia, produce biologically active tropane alkaloids (TAs)[1]
Our results demonstrate that targeted Vitreoscilla soluble hemoglobin (VHb) expression significantly increased the Tropane alkaloids (TAs) content in the H. niger hairy roots, and resulted in the up-regulation of key genes involved in the TA biosynthetic pathway
H. niger, a 20-amino acid (AA) N-terminal mitochondrial localization signal from subunit IV of yeast CoxIV, an N-terminal extension with features characteristic of mitochondrial and chloroplast transit peptides from Arabidopsis (AtHRS1)[27], and an A. thaliana gene that encodes a Mg2+- and ATP-dependent RecA homologue targeted to plastids[28], were selected, respectively
Summary
Some genera of the family Solanaceae, including Hyoscyamus, Duboisia, Atropa, and Scopolia, produce biologically active tropane alkaloids (TAs)[1]. Similar results were reported for hairy roots of Atropa belladonna overexpressing the same genes, wherein the best transgenic line showed 7.3-fold higher content of scopolamine than that in the control line[7]. The constitutive expression of VHb improved the germination rate of transgenic seeds and affected the levels of some endogenous genes of Arabidopsis that are involved in oxygen metabolism and biosynthesis of antioxidants. The VHb transgenic plants were more tolerant to photo-oxidative damage because of the increased production of antioxidants, such as ascorbate[16] These results suggested that the VHb gene might potentially be used in molecular breeding for improving the production of TAs in H. niger hairy roots
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