Promoter variations in DBR2-like affect artemisinin production in different chemotypes of Artemisia annua

Artemisia annua is the only known plant source of the potent antimalarial artemisinin,which occurs as the low-and high-artemisinin producing(LAP and HAP)chemotypes.Nevertheless,the different mechanisms of artemisinin producing between these two chemotypes were still not fully understood.Here,we performed a comprehensive analysis of genome resequencing,metabolome,and transcriptome data to systematically compare the difference in the LAP chemotype JL and HAP chemotype HAN.Metabolites analysis revealed that 72.18%of sesquiterpenes was highly accumulated in HAN compared to JL.Integrated omics analysis found a DBR2-Like(DBR2L)gene may be involved in artemisinin biosynthesis.DBR2L was highly homologous with DBR2,belonged to ORR3 family,and had the DBR2 activity of catalyzing artemisinic aldehyde to dihydroartemisinic aldehyde.Genome resequencing and promoter cloning revealed that complicated variations existed in DBR2L promoters among different varieties of A.annua and were clustered into three variation types.The promoter activity of diverse variant types showed obvious differences.Furthermore,the core region(-625 to 0)of the DBR2L promoter was identified and candidate transcription factors involved in DBR2L regulation were screened.Thus,the result indicates that DBR2L is another key enzyme involved in artemisinin biosynthesis.The promoter variation in DBR2L affects its expression level,and thereby may result in the different yield of artemisinin in varieties of A.annua.It provides a novel insight into the mechanism of artemisinin-producing difference in LAP and HAP chemotypes of A.annua,and will assist in a high yield of artemisinin in A.annua.

N-glucosyltransferase GbNGT1 from ginkgo complements the auxin metabolic pathway

As auxins are among the most important phytohormones,the regulation of auxin homeostasis is complex.Generally,auxin conjugates,especially IAA glucosides,are predominant at high auxin levels.Previous research on terminal glucosylation focused mainly on the O-position,while IAA-N-glucoside and IAA-Asp-N-glucoside have been neglected since their discovery in 2001.In our study,IAA-Asp-N-glucoside was found to be specifically abundant(as high as 4.13 mg/g)in the seeds of 58 ginkgo cultivars.Furthermore,a novel N-glucosyltransferase,termed GbNGT1,was identified via differential transcriptome analysis and in vitro enzymatic testing.It was found that GbNGT1 could catalyze IAA-Asp and IAA to form their corresponding N-glucosides.The enzyme was demonstrated to possess a specific catalytic capacity toward the N-position of the IAA-amino acid or IAA from 52 substrates.Docking and site-directed mutagenesis of this enzyme confirmed that the E15G mutant could almost completely abolish its N-glucosylation ability toward IAA-Asp and IAA in vitro and in vivo.The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana.The effect of GbNGT1 on IAA distribution promotes root growth in Arabidopsis thaliana.

The APETALA2–MYBL2 module represses proanthocyanidin biosynthesis by affecting formation of the MBW complex in seeds of Arabidopsis thaliana

Proanthocyanidins(PAs)are the second most abundant plant phenolic natural products.PA biosynthesis is regulated by the well-documented MYB/bHLH/WD40(MBW)complex,but how this complex itself is regu-lated remains ill dened.Here,in situ hybridization and b-glucuronidase staining show that APETALA2(AP2),a well-dened regulator offlower and seed development,is strongly expressed in the seed coat endothelium,where PAs accumulate.AP2 negatively regulates PA content and expression levels of key PA pathway genes.AP2 activates MYBL2 transcription and interacts with MYBL2,a key suppressor of the PA pathway.AP2 exerts its function by directly binding to the AT-rich motifs near the promoter region of MYBL2.Molecular and biochemical analyses revealed that AP2 forms AP2–MYBL2–TT8/EGL3 com-plexes,disrupting the MBW complex and thereby repressing expression of ANR,TT12,TT19,and AHA10.Genetic analyses revealed that AP2 functions upstream of MYBL2,TT2,and TT8 in PA regulation.Our work reveals a new role of AP2 as a key regulator of PA biosynthesis in Arabidopsis.Overall,this study sheds new light on the comprehensive regulation network of PA biosynthesis as well as the dual regulatory roles of AP2 in seed development and accumulation of major secondary metabolites in Arabidopsis.