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.

Keywords: Anabaena PCC 7120; Arsenic; Artemisia annua; Cadmium; Hairy root; Phytochelatin synthase.

Fig. 1

a Normalized gene expression analysis. b RT-PCR for expression analysis of rolB gene in hairy root, normal root and transformed hairy roots expressing anaPCS gene

Fig. 2

Heavy metal accumulation in normal roots, hairy roots and transformed hairy roots (expressing anaPCS gene). a Arsenic content and b cadmium content. Vertical bars denote mean ± SD and different alphabets show statistical significance among treatments (control/As-treatment/Cd-treatment) at p =  < 0.05

Fig. 3

Bioconcentration factor (BCF) of arsenic and cadmium-stressed normal roots, hairy roots and transformed hairy roots (expressing anaPCS gene). Vertical bars denote mean ± SD and different alphabets show statistical significance among treatments (control/As-treatment/Cd-treatment) at p =  < 0.05

Fig. 4

Phytochelatin content and phytochelatin synthase expression in normal roots (NR), hairy roots (HR) and transformed hairy roots (THR) (expressing anaPCS gene) under arsenic and cadmium stress. a Phytochelatin content; b RT-PCR for expression analysis of Anabaena PCC 7120 PCS gene (anaPCS); c RT-PCR for expression analysis of Artemisia annua PCS (AaPCS)

Fig. 5

a Thiol content and b GSH content in normal roots, hairy roots and transformed hairy roots (expressing anaPCS gene) under arsenic and cadmium stress. Vertical bars denote mean ± SD and different alphabets show statistical significance among treatments (control/As-treatment/Cd-treatment) at p =  < 0.05

Fig. 6

Antioxidative enzyme activities of a Catalase, b ascorbate peroxidase, c glutathione reductase, and d peroxidases, in normal roots, hairy roots and transformed hairy roots (expressing anaPCS gene) under arsenic and cadmium stress. Vertical bars denote mean ± SD and different alphabets show statistical significance among treatments (control/As-treatment/Cd-treatment) at p =  < 0.05

Fig. 7

a Artemisinin content and b expression analysis of artemisinin biosynthetic enzymes (ADS, CYP71AV1, DBR2 and ALDH1) through RT-PCR, in normal roots, hairy roots and transformed hairy roots (expressing anaPCS gene) under arsenic and cadmium stress. In a vertical bars denote mean ± SD and different alphabets show statistical significance among treatments (control/As-treatment/Cd-treatment) at p =  < 0.05

Fig. 8

Phylogenetic tree: a UPGMA method and b maximum parsimony method showing origin, evolutionary relationship and functional homology of phytochelatin synthase protein in A. annua, S. tuberosum, A. thaliana, B. juncea, O. sativa and Anabaena PCC 7120. The trees were generated using 1000 boot replication values based on maximum-likelihood methods

Fig. 9

Comparative analysis of similarities and differences in phytochelatin synthase (PCS) protein among A. annua, S. tuberosum, A. thaliana, B. juncea, O. sativa and Anabaena PCC 7120. The Circos software was used to generate circular map based on similarity percentage matrices using Clustal W algorithm

Fig. 10

The motif scan analysis showing distribution and presence/absence of common and uncommon motifs found in A. annua, S. tuberosum, A. thaliana, B. juncea, O. sativa and Anabaena PCC 7120 discovered through MEME and MAST analysis. a The block diagram showing the sequence of discovered motifs for AaPCS. The red arrows indicate the presence of uncommon motif in O. sativa and black arrows indicate the presence of uncommon motifs in Anabaena PCC 7120 which are absent in other members. b The sequential logo of the motif 1 showing consensus sequences present in all the representatives’ members

Fig. 11

Predicted structures of functional domain of a A. annua PCS (AaPCS) and b Anabaena PCC 7120 PCS (anaPCS) generated and visualized through MODELLER module of Discovery Studio 3.0

Fig. 12

a Comparative analysis of docked complex with experimentally resolved X-ray diffraction structures of functional domain of AaPCS and anaPCS with cadmium chloride (CdCl₂) and sodium arsenate. b, c Structure of docked complexes in A. annua as visualized in Discovery Studio 3.0. d, e Structure of docked complex in Anabaena PCC 7120 as visualized in Discovery Studio 3.0

Fig. 13

Functional interactive network of a AaPCS and b anaPCS with other protein family members as found on STRING server where the colored nodes describe query proteins from first shell interactors and white nodes form second shell interactors. The large node size represents characterized proteins and smaller nodes for uncharacterized proteins