Cloning of Full-length AaMYC2-LIKE and AaMYC2 bHLH TFs from A. annua
The full-length CDS of both bHLH transcription factors were cloned in the pTZ57R/T vector by performing a PCR reaction using Leaf cDNA as a template. The sequence of the AaMYC2-LIKE as characterised by (23) was 1272bp and the sequence of AaMYC2 as characterised by (25) was 1878bp. The CDS of AaMYC2-LIKE codes for 424 amino acids with a molecular weight of 46.6 KD while as the CDS of AaMYC2 codes for 626 amino acids with a molecular weight of 68.49 KD. The 424 and 626 amino acid sequences were analysed by SWISS PROSITE software and the structure was depicted as shown in Fig. 1.
AaMYC2-LIKE and AaMYC2 synergistically enhance the transactivation activity within yeast cells.
As AaMYC2-LIKE and AaMYC2 are transcriptionally active proteins, we were curious to know their combined effect on the transactivation activity. For this we analysed co-transformed transactivation potential of AaMYC2 and AaMYC2-LIKE together as well as individual constructs in yeast cells harbouring respective transformants. AaMYC2-LIKE and AaMYC2 were cloned in a pGBKT (BD) vector separately having a GAL4 DNA binding domain and transformed into yeast strain Y187 as per manual’s instructions (Clontech). After confirming the yeast transformations, the constructs were streaked on SD/-Trp plates containing D-raffinose, D-galactose and X-gal. As depicted in (Fig. 2A), only the cells transformed with individual BD-AaMYC2 and BD-AaMYC2-LIKE, positive control (Y187 cells co-transformed with pGBKT7-p53 and pGADT7-T plasmids) and co-transformed BD-AaMYC2/BD-AaMYC2-LIKE could develop the blue colour which is ascribed to activation of β- galactosidase reporter gene in host yeast strain. The Y187 cells transformed with only the pGBKT7 vector, treated as a negative control, did not show any blue colouration. Further, in quantitative assay the measurement of β-galactosidase activity using ONPG as substrate revealed the presence of significantly higher β-galactosidase units in Y187 cells co-transformed with BD-AaMYC2 and BD-AaMYC2-LIKE constructs in comparison to individual transformation of BD-AaMYC2 and BD-AaMYC2-LIKE and negative control yeast cells. The positive control yeast cells exhibited higher β-galactosidase activity. As depicted in Fig. 2B all the constructs developed yellow colour except for the negative control and the intensity of the colour being slightly higher in the co-transformed BD-AaMYC2 and BD-AaMYC2-LIKE yeast cells. As shown in Fig. 2C, the β-galactosidase units are significantly higher in the co-transformed yeast cells as compared to individual transformation. Taken together, our findings suggest that both BD-AaMYC2 and BD-AaMYC2-LIKE proteins being transcriptionally active act synergistically and further enhance the transactivation.
Aamyc2-like Physically Interacts With Aamyc2
The bHLH transcription factors have the potential to form homo or hetero dimers by virtue of their typical helix-loop-helix domain. So we performed in-vivo protein-protein interaction between AaMYC2 and AaMYC2-LIKE by a yeast two-hybrid assay within yeast cells and a Bimolecular Fluorescent complementation assay in onion epidermal cells. For the yeast-two hybrid assay, AaMYC2-LIKE was cloned in the yeast bait vector pGBKT7 fused with Gal4 DNA-binding domain (DNA-BD) while as AaMYC2 was cloned in the prey vector pGADT7 fused with Gal4 activation domain (AD). Both the transcription factors cloned in yeast vectors along with kit positive and negative control were transformed into Y2HGold Yeast Strain and allowed to grow on a double auxotrophic dropout medium as per manual’s instructions (Clontech). To confirm the protein-protein interactions between AaMYC2 and AaMYC2-LIKE, the transformed colonies were re-streaked on Quadruple Dropout (QDO) media lacking amino acids (Ade -His -Leu –Trp). As shown in Fig. 3A the growth of transformed colonies in the QDO media confirmed the protein-protein interaction between the above-mentioned bHLH transcription factors. To further confirm the interaction of AaMYC2 and AaMYC2-LIKE within plant cells, in-vivo Bimolecular Fluorescent complementation assay was performed. For this assay, AaMYC2-LIKE was cloned in the c-terminal of Yellow fluorescent protein (c-YFP) and AaMYC2 was cloned in the N-terminal of YFP (n-YFP). Both the constructs were co-transformed in Onion epidermal cells by agrobacterium-mediated transformation and after proper incubation at 28°C, the transformed onion epidermal cells were visualised under a fluorescent microscope as described by (26). The fluorescence (Fig. 3B) was seen in the nucleus of the co-transformed onion epidermal cells which confirmed that AaMYC2 interacts with AaMYC2-LIKE within the nucleus which was counter stained with DAPI confirming the nuclear localised protein-protein interaction. The empty transformed c-YFP and n-YFP taken as control did not show any fluorescence. Altogether these results confirm that AaMYC2 physically interacts with AaMYC2-LIKE within the yeast cells and nucleus of onion epidermal cells.
Aamyc2 Activates The Transcript Levels Of Aamyc2-like Within Planta
To further confirm the collective role of AaMYC2 and AaMYC2-LIKE within plants, transgenic lines in A. annua were generated under the 35S promoter of pBI-121. The transcript level of AaMYC2 and AaMYC2-LIKE were analysed in AaMYC2, AaMYC2-LIKE, AaMYC2-RNAi/AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 transiently transgenic overexpression lines. Our results from Fig. 4A depict the transcript level of AaMYC2 is downregulated in AaMYC2-RNAi/ AaMYC2-LIKE line while being elevated 3 times in the AaMYC2 overexpression line, 2.5 times in the AaMYC2-LIKE overexpression line and 6 times in co-transformed AaMYC2/AaMYC2-LIKE overexpression line as compared to the wildtype. It can be seen from Fig. 4B the transcript level of AaMYC2-LIKE was 0.5 times elevated in the AaMYC2-RNAi/AaMYC2-LIKE line as compared to the wildtype revealing the negative impact of downregulating AaMYC2 on AaMYC2-LIKE. While the transcript level of AaMYC2-LIKE was elevated 2.5 in the AaMYC2 overexpression line, 4.5 in the AaMYC2-LIKE overexpression line, and 6 times in the co-transformed AaMYC2-LIKE/AaMYC2 lines as compared to the wildtype. The expression levels of both AaMYC2 and AaMYC2-LIKE are significantly higher in the co-transformed overexpression lines suggesting these transcription factors positively regulate their transcript levels within plants.
AaMYC2 acts synergistically with AaMYC2-LIKE and elevates transcript levels of both Artemisinin biosynthetic and trichome development genes
The overexpression of AaMYC2 and AaMYC2-LIKE under 35S promoter in pBI-121 in A. annua transgenic plants harbouring overexpression of AaMYC2 and AaMYC2-LIKE constructs were confirmed by the RT-PCR in which the transcript levels of key Artemisinin Biosynthetic genes like cytochrome P-450 dependent hydroxylase (CYP71AV1), double bond reductase (DBR2) and aldehyde dehydrogenase (ALDH1) were analysed. As seen in Fig. 5A the transcript level of CYP was elevated 2,5, and 5.5 times in AaMYC2, AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines respectively while the transcript level of DBR2 was elevated 2.7, 4.7, and 5.2 times in AaMYC2, AaMYC2-LIKE and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines as compared to wildtype. The transcript level of ALDH1 was elevated 6.9, 11, and 12.6 times in AaMYC2, AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines as compared to wildtype. These results suggest that AaMYC2 and AaMYC2-LIKE act synergistically in regulation of transcript levels of artemisinin biosynthetic genes. As trichomes are main sites for storage and secretion of artemisinin, we proceeded to analyse the transcript levels of key Trichome development genes in transgenic overexpression lines of A. annua. Previous studies in A. annua show the main genes involved in trichomes as, HOMEODOMAIN PROTEIN 1 (AaHD1) which positively controls both glandular and non-glandular trichome initiations (27), Homeodomain-leucine zipper (HD-ZIP) IV (AaHD8) which directly promotes the expression of AaHD1 for glandular trichome initiation (28), AaMIXTA1 which regulates cuticle biosynthesis and prompted GST initiation (29) and Trichome-specific fatty acyl-CoA reductase 1 (TAFR1) which is involved in both trichome development and sesquiterpenoid biosynthesis (30). As depicted in Fig. 5B, The transcript level of HDA1 was elevated 4.1, 3.9, and 6.7 times in AaMYC2, AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines respectively as compared to wildtype while as the transcript level of HDA8 was elevated 3, 2.5, and 5.5 times in AaMYC2, AaMYC2-LIKE and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines as compared to wildtype. The transcript level of MIXTA was elevated 4.7, 3.7, and 5.5 times in AaMYC2, AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines while as the transcript level of TFAR1 was elevated 2.5, 2.4, and 4.6 times in AaMYC2, AaMYC2-LIKE and co-transformed AaMYC2-LIKE/AaMYC2 overexpression lines as compared to wildtype. Our results suggest that co-transformed overexpression lines of AaMYC2 and AaMYC2-LIKE acting synergistically have enhanced transcripts levels of significant Trichome development genes as well.
Synergistic interaction of AaMYC2-LIKE-AaMYC2 elevates artemisinin content within the transgenic lines of A. annua
In order to determine the synergistic effect of AaMYC2 and AaMYC2-LIKE on the final accumulation of artemisinin content within planta, transgenic overexpression lines of A. annua were subjected to HPLC analysis. The quantification of artemisinin content in the transgenic overexpression lines of AaMYC2, AaMYC2-LIKE, AaMYC2-RNAi/ AaMYC2-LIKE, and co-transformed AaMYC2-LIKE/AaMYC2 was performed by the HPLC analysis using artemisinin from Sigma as standard. As expected (Fig. 6) the artemisinin content was significantly higher in the co-transformed overexpression line of AaMYC2 and AaMYC2-LIKE than in individual overexpression lines of AaMYC2 and AaMYC2-LIKE as compared to wildtype while as the artemisinin content was reduced in the AaMYC2-RNAi/AaMYC2-LIKE expression line than in the individual overexpression line of AaMYC2. The artemisinin content was improved to 8.82 mg/G DW in AaMYC2, 12mg/G DW in AaMYC2-LIKE, and 15mg/G DW in co-transformed AaMYC2/AaMYC2-LIKE as compared to 4.8 mg/G DW in Vector control and 3.4 mg/G DW in wildtype. Artemisinin content was reduced to 5.1mg/G DW in AaMYC2-RNAi/AaMYC2-LIKE overexpression plants. Our study suggests the synergistic interaction of AaMYC2 and AaMYC2-LIKE in co-transformed transgenic lines has a constructive impact within planta as the accumulation of artemisinin content was enhanced.