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Heterologous expression of cyanobacterial PCS confers augmented arsenic and cadmium stress tolerance and higher artemisinin in Artemisia annua hairy roots.

ABSTRACT: The present study provides the first report of heterologous expression of phytochelatin synthase from Anabaena PCC 7120 (anaPCS) into the hairy roots of Artemisia annua. Transformed hairy roots of A. annua expressing anaPCS gene showed better tolerance to heavy metals, viz., arsenic (As) and cadmium (Cd) owing to 143 and 191% more As- and Cd-accumulation, respectively, as compared to normal roots with a bioconcentration factor (BCF) of 9.7 and 21.1 for As and Cd, respectively. Under As and Cd stresses, transformed hairy roots possessed significantly higher amounts of phytochelatins and thiols probably due to the presence of both AaPCS (Artemisia annua PCS) and anaPCS. In addition, artemisinin synthesis was also induced in transformed hairy roots under heavy metals stresses. In-silico analysis revealed the presence of conserved motifs in both AaPCS and anaPCS sequences as well as structural modelling of PCS functional domain was conducted. Interaction of AaPCS and anaPCS proteins with CdCl₂ and sodium arsenate gene ontology analysis gave insights to anaPCS functioning in transformed hairy roots of A. annua. The study provides transformed hairy roots of A. annua as an efficient tool for effective phytoremediation with added advantages of artemisinin extraction from hairy roots used for phytoremediation.

Supplementary information

The online version contains supplementary material available at 10.1007/s11816-021-00682-5.

SUBMITTER: Pandey N

PROVIDER: S-EPMC8180384 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

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Heterologous expression of cyanobacterial PCS confers augmented arsenic and cadmium stress tolerance and higher artemisinin in Artemisia annua hairy roots.

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