Identification and the potential involvement of miRNAs in the regulation of artemisinin biosynthesis in A. annua

Abstract

Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by Artemisia annua (A. annua). The lower in planta content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular. Abbreviations ART Artemisinin A. annua Artemisia annua miRNA MicroRNA TF Transcription factor Artemisia annua (A. annua), an important medicinal herb is the only natural source of artemisinin (ART). ART, a sesquiterpene lactone is the most reliable and widely used drug to quell Malaria, which is one of the most devastating diseases and a leading cause of deaths worldwide. According to WHO, there were a staggering 228 million cases and 405,000 deaths reported in 2018 alone, despite the disease being preventable as well as curable 1. WHO has recommended the use of ART only in combination of other drugs, to slow down the development of resistance in Plasmodium sps. against it and to maintain its efficacy. The combination treatment is widely known as artemisinin-based combination therapies (ACTs). In wild type A. annua, ART is present in the aerial parts, in extremely low quantities (0.02-1.07%) 2. Numerous efforts were made to generate ART synthetically or semi-synthetically, but does not meet industrial practices due to high production cost. The insufficient supply of ART produced by plants or synthetic methods, resulted in the lack of ACTs available to the patients and causes approximately half a million mortalities every year 3. Despite various efforts to produce semi-synthetic ART at low cost, the natural source of ART remains the main provider. Thus, researchers are investigating effective strategies to enhance in planta content of ART. The Ian Grahm lab and Bill and Malinda gates foundation invested huge fund in production of high ART yielding hybrids an...

Aging affects artemisinin synthesis in Artemisia annua

“…1 B). Luckily miR156 and several AaSPL s have been reported by other groups 18 , 19 . The SPL genes could be targeted or non-targeted by miR156, the reported AaSPL s and those from the our RNAseq data were aligned with SPL s from Arabidopsis ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ), AaSPL7 , AaSPL9 , AaSPL12 , AaSPL13 , SPL4 and SPL5 showing more similarities to miR156-target-SPLs in Arabidopsis were further confirmed (Fig.…”

“…( B – D ) qRT-PCR analysis of miRNAs and genes in in leaves of A. annua in different age . miR156 , AaSPL7 , AaSPL9 , AaSPL12 and AaSPL13 were reported 18 , 19 , miR845b , miR1134 , SPL4 and SPL5 were found from the sequencing data. …”

“…2 ). The level of miR156 18 and two novel miRNAs, miR845b and miR1134 declined with age, and the picked SPLs 19 enhanced to varying degrees (Fig. 1 B–D).…”

“…Indicating miR156 and targeted SPLs works as age cue in A. annua too. Because of the low abundance of miR156 in A. annua 18 , there may be other miRNAs to complement its role.

Figure 2 Phylogenetic tree of the SBP domain of SPLs from A. annua .and Arabidopsis .

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Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis

“…Deep sequencing technology has led to the identification of numerous miRNAs in plant genomes from species such as Italian sweet pepper [20], tea, mulberry, Chinese cabbage, turnip, longan (Dimocarpus longan Lour. ), bread wheat, Artemisia annua, Populus euphratica, Moringa oleifera, and Solanum lycopersicum [21][22][23][24][25][26][27][28][29][30]. The following are some examples of these miRNAs, many of which are involved in specific signaling pathways.…”