Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production.

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Summary Six transcription factors of APETALA2/ethylene‐response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real‐time quantitative polymerase chain reaction (RT‐Q‐PCR) showed that AaORA exhibited similar expression patterns to those of amorpha‐4,11‐diene synthase gene (ADS), cytochrome P450‐dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. AaORA is a trichome‐specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T‐shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up‐regulated or down‐regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN‐LIKE PROTEIN (HEL) and BASIC CHITINASE (B‐CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

Lignocellulosic biomass was used for thousands of years as animal feed and is now considered a great sugar source for biofuels production. It is composed mostly of secondary cell walls built with polysaccharide polymers that are embedded in lignin to reinforce the cell wall structure and maintain its integrity. Lignin is the primary material responsible for biomass recalcitrance to enzymatic hydrolysis. During plant development, deep reductions of lignin cause growth defects and often correlate with the loss of vessel integrity that adversely affects water and nutrient transport in plants. The work presented here describes a new approach to decrease lignin content while preventing vessel collapse and introduces a new strategy to boost transcription factor expression in native tissues. We used synthetic biology tools in Arabidopsis to rewire the secondary cell network by changing promoter-coding sequence associations. The result was a reduction in lignin and an increase in polysaccharide depositions in fibre cells. The promoter of a key lignin gene, C4H, was replaced by the vessel-specific promoter of transcription factor VND6. This rewired lignin biosynthesis specifically for vessel formation while disconnecting C4H expression from the fibre regulatory network. Secondly, the promoter of the IRX8 gene, secondary cell wall glycosyltransferase, was used to express a new copy of the fibre transcription factor NST1, and as the IRX8 promoter is induced by NST1, this also created an artificial positive feedback loop (APFL). The combination of strategies—lignin rewiring with APFL insertion—enhances polysaccharide deposition in stems without over-lignifying them, resulting in higher sugar yields after enzymatic hydrolysis.