Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata

Camptotheca acuminata produces camptothecin(CPT),a monoterpene indole alkaloid(MIA)that is widely used in the treatment of lung,colorectal,cervical,and ovarian cancers.Its biosynthesis pathway has attracted significant attention,but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor(AP2/ERF)transcription factors(TFs)remains unclear.In this study,a systematic analysis of the AP2/ERF TFs family in C.acuminata was performed,including phylogeny,gene structure,conserved motifs,and gene expression profiles in different tissues and organs(immature bark,cotyledons,young flower,immature fruit,mature fruit,mature leaf,roots,upper stem,and lower stem)of C.acuminata.A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies,including AP2(26 genes),DREB(61 genes),ERF(92 genes),RAV(18 genes),and Soloist(one gene).The combination of gene expression patterns in different C.acuminata tissues and organs,the phylogenetic tree,the co-expression analysis with biosynthetic genes,and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C.acuminata might be involved in CPT synthesis regulation,which exhibit relatively high expression levels in the upper stem or immature bark.Among these,four genes(Cac AP2/ERF123,Cac AP2/ERF125,Cac AP2/ERF126,and Cac AP2/ERF127)belong to the ERF–B2 subgroup;two genes(Cac AP2/ERF149 and Cac AP2/ERF152)belong to the ERF–B3 subgroup;and two more genes(Cac AP2/ERF095 and Cac AP2/ERF096)belong to the DREB–A6 subgroup.These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C.acuminata.

The Jasmonate-Responsive AP2/ERF Transcription Factors AaERF1 and AaERF2 Positively Regulate Artemisinin Biosynthesis in Artemisia annua L.

Plants of Artemisia annua produce artemisinin, a sesquiterpene lactone widely used in malaria treatment. Amorpha-4,11-diene synthase （ADS）, a sesquiterpene synthase, and CYP71AV1, a P450 monooxygenase, are two key enzymes of the artemisinin biosynthesis pathway. Accumulation of artemisinin can be induced by the phytohormone jasmonate （JA）. Here, we report the characterization of two JA-responsive AP2 family transcription factors-AaERF1 and AaERF2-from A. annua L. Both genes were highly expressed in inflorescences and strongly induced by JA. Yeast one- hybrid and electrophoretic mobility shift assay （EMSA） showed that they were able to bind to the CRTDREHVCBF2 （CBF2） and RAVlAAT （RAA） motifs present in both ADS and CYP71AV1 promoters. Transient expression of either AaERF1 or AaERF2 in tobacco induced the promoter activities of ADS or CYP71AV1, and the transgenic A. annua plants overexpressing either transcription factor showed elevated transcript levels of both ADS and CYP71AV1, resulting in increased accumulation of artemisinin and artemisinic acid. By contrast, the contents of these two metabolites were reduced in the RNAi transgenic lines in which expression of AaERF1 or AaERF2 was suppressed. These results demonstrate that AaERF1 and AaERF2 are two positive regulators of artemisinin biosynthesis and are of great value in genetic engineering of arte- misinin production.

Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice

Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in plants. However, the genetic and molecular mechanisms underlying trichome formation （i.e., initiation and elongation） in rice remain largely unclear. Here, we report an AP2/ERF transcription factor, Hairy Leaf 6 （HL6）, which controls trichome formation in rice. Functional analyses revealed that HL6 transcriptionally regulates trichome elongation in rice, which is dependent on functional OsWOX3B, a homeodomain-containing protein that acts as a key regulator in trichome initiation. Biochemical and molecular genetic analyses demonstrated that HL6 physically interacts with OsWOX3B, and both of them regulate the expression of some auxin-related genes during trichome formation, in which OsWOX3B likely enhances the binding ability of HL6 with one of its direct target gene, OsYUCCA5. Popu- lation genetic analysis indicated that HL6 was under negative selection during rice domestication. Taken together, our findings provide new insights into the molecular regulatory network of trichome formation in rice.

ERF5 enhances protocorm-like body regeneration via enhancement of STM expression in Dendrobium orchid

Orchid plants develop protocorms upon germination and produce protocorm-like structures called protocorm-like bodies(PLBs)from protocorms and somatic cells via tissue culture.Protocorm-like bodies have broad technical application potential in the orchid industry and their regeneration is a distinct developmental process in the plant kingdom.However,little is known about this unparalleled developmental program.In this study,we identified a PLB-abundant gene,ethylene response factor(ERF),and a transcription factor named Do ERF5,and determined its important role in PLB regeneration in Dendrobium orchid.Overexpression of Do ERF5 in Dendrobium greatly enhanced the PLB regeneration from PLB and stem explants,and upregulated the expression of WOUND-INDUCED DEDIFFERENTIATION(Do WIND)homologs and SHOOT MERISTEMLESS(Do STM),as well as the genes involved in cytokinin biosynthesis(Do IPT)and the cytokinin response factors(Do ARRs).However,silencing Do ERF5 reduced the regeneration rate of PLBs,and downregulated the expression of Do WIND homologs,Do STM and Do ARRs.We demonstrated that Do ERF5 is directly bound to the Do STM promoter and regulates its expression.In addition,overexpression of Do STM in Dendrobium orchid resulted in favorable regeneration of PLBs.Our results clarify that Do ERF5 regulates the regeneration of PLB by enhancing Do STM expression.Our findings provide new insights into how Do ERF5 mediates PLB regeneration and offers technical potential in improving clonal propagation,preservation,and the bioengineering of orchids.