A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua

A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua Yinkai Yang, Yongpeng Li, Pengyang Li, Qin Zhou, Miaomiao Sheng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3978505/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Aug, 2024 Read the published version in BMC Genomics → Version 1 posted 3 You are reading this latest preprint version Abstract Background Transcription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In Artemisia annua , various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in A. annua (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin. Results We found five AaSHI genes ( AaSHI1 to AaSHI5 ) in the A. annua genome. The expression levels of AaSHI1 , AaSHI2 , AaSHI3 , and AaSHI4 genes were higher in trichomes and young leaves, and decreased when the plants were subjected to dark treatment. The expression pattern of these four AaSHI genes was consistent with the expression pattern of four artemisinin biosynthetic genes and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in A. annua provided the evidence that AaSHI1 could directly bind to the promoters of artemisinin biosynthetic genes AaADS and AaCYP71AV1 , and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in A. annua . Conclusions Overall, a genome-wide analysis of the AaSHI TF family of A. annua was conducted. Five AaSHIs were identified in A. annua genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of artemisinin biosynthetic genes including AaADS and AaCYP71AV1 . These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new A. annua germplasms. Artemisia annua artemisinin biosynthesis transcriptional regulation SHI family transcription factor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Malaria is a parasitic disease caused by Plasmodium infection and results in over 200 million cases globally every year [ 1 ]. Artemisinin, a sesquiterpene lactone that contains a peroxy bridge, has demonstrated remarkable effectiveness in the treatment of malignant malaria. Chinese scientist Youyou Tu was awarded the Nobel Prize in Physiology or Medicine in 2015 for her discovery of the anti-malarial property of artemisinin. While artemisinic acid, the precursor of artemisinin, can be synthesized in yeast by synthetic biology technology, its production cost remains high and is insufficient meet the substantial market demand [ 2 ]. Currently, the primary source of the artemisinin is still the Compositae family plant Artemisia annua . Artemisinin is mainly synthesized and stored in glandular secretory trichomes (GSTs), which are specialized ten-cell epidermal structure found in young leaves and flower buds of A. annua . As a typical sesquiterpene compound, a series of metabolic and regulatory genes in artemisinin biosynthetic pathway have been thoroughly characterized [ 3 , 4 ]. Within GSTs, farnesyl pyrophosphate (FPP), a common precursor for sesquiterpene synthesis, is converted to dihydroartemisinic acid (DHAA) through the catalytic action of four enzymes that are specifically localized in GSTs (AaADS, AaCYP71AV1, AaDBR2, and AaALDH1) [ 5 ] (Fig. 1 ). In the sub-epidermal space of GSTs, DHAA can be turned into artemisinin [ 6 ] (Fig. 1 ). It is considered as an effective metabolic engineering approach to enhance the production of specific high-value metabolites in plants through manipulation of transcription factors (TFs). It has been reported that various TFs from different families, including WRKY, AP2/ERF, TCP, bHLH, bZIP, and MYB, are involved in light- and phytohormone-mediated regulation of artemisinin biosynthesis [ 7 – 13 ]. Two TFs of different families, WRKY-family AaGSW1 and AP2/ERF-family AaORA, are specifically expressed in the GSTs, and promote artemisinin biosynthesis [ 7 , 10 ]. SHI family TFs are unique to plants and exhibit functional diversity, encompassing the regulation of growth, development, metabolism, and response to stresses [ 14 ]. SHI family proteins are characterized by the presence of two functional domains: a circular zinc finger domain at the N-terminal and an IGGH domain at the C-terminal [ 15 ]. The circular zinc finger domain consists of two fingers, which adapt a transverse palm arrangement, similar to the DNA-binding domain. The IGGH domain contains certain acidic residues that play a role in mediating both homologous and heterogeneous dimerization among the SHI proteins [ 15 ]. There are ten SHI family proteins in Arabidopsis thaliana , of which AaSRS8 is reported to be a pseudogene [ 16 ]. STYLISH1, which is related to the development of shoot apical meristems, can directly bind to the YUCCA4 promoter to regulate auxin synthesis [ 15 ]. SRS5 negatively regulates lateral root formation through inhibiting the expression of LBD16 and LBD29 genes [ 17 ]. In addition, light-induced SRS5 can also be ubiquitinated and degraded by COP1 protein and positively modulate the photomorphogenesis in seedlings by directly activating target genes expression [ 18 ]. Moreover, the maize LRP1 is auxin-responsive and associated with the initiation of lateral and seminal roots in maize [ 19 ]. In rice, OsSHI1 regulates tillering and panicle branching depending on the physical interaction with IPA1 [ 20 ]. There have been no reports on the AaSHI TFs in A. annua , and the regulatory function of AaSHIs in relation to artemisinin biosynthesis in A. annua remains largely unexplored. In this study, we identified five AaSHI genes in the A. annua genome. Expression analysis conducted in various tissues and under different light conditions revealed a positive correlation among AaSHI1 , AaSHI2 and AaSHI4 , the four artemisinin biosynthetic genes, and two GST-specific TF genes, AaGSW1 and AaORA . Yeast one-hybrid assays and dual-luciferase (Dual-LUC) reporter assays showed that AaSHI1 could directly activate the expression of AaADS and AaCYP71AV1 . Transient transformation in A. annua leaves confirmed that AaSHI1 positively regulated artemisinin biosynthesis. The data present in this study demonstrated that AaSHI1 were positive regulators of artemisinin biosynthetic pathway. Taken together, this study provided a theoretical basis for informing the function of AaSHI family members and cultivating higher artemisinin content A. annua through molecular biotechnology in the future. Results Identification of AaSHI members in the A. annua genome To obtain sequence information of AaSHI, nine AtSHI family proteins of A. thaliana were used to blast with four sets of haplotype chromosome genomes of A. annua (Supplementary Table 1). Multiple sequence alignments and protein integrity alignment analysis found that unctg_3838g01590241 (LQ-9_phase0) was half the length of the unctg_3207g01555681 and lacked the zinc finger domains, while chr5g00243221 (LQ-9_phase1) also lacked the zinc finger domains (Supplementary Figue 1). As a result, five AaSHI genes in the HAN1_phase0 genome were selected for subsequent analyses, named AaSHI1 ( chr1g04025621 ), AaSHI2 ( chr3g03131551 ), AaSHI3 ( chr5g03332101 ), AaSHI4 ( chr6g00096641 ), AaSHI5 ( chr6g00640341 ), respectively. Then we analyzed the physical and chemical properties of these identified genes. These genes encoded 276–390 amino acids with the molecular weight (MW) ranged from 68.19 kDa to 95.67 kDa, and their isoelectric points ranged from 5.05 to 5.13. Table 1 Detailed information for five AaSHI members in the A. annua genome. NAME Gene ID ORF length (bp) Protein length (aa) Mw (kDa) pI AaSHI1 chr1g04025621 1,005 334 83.54 5.08 AaSHI2 chr3g03131551 1,041 346 85.71 5.07 AaSHI3 chr5g03332101 972 323 79.64 5.11 AaSHI4 chr6g00096641 1,173 390 95.67 5.05 AaSHI5 chr6g00640341 831 276 68.19 5.13 Phylogenetic tree construction, multiple sequence alignment and gene structure analysis To further investigate the evolutionary relationships and their evolutionary conservation among individual members of the AaSHI family, a phylogenetic tree of 42 SHI members in A. annua and 5 other species was constructed using MEGAX software (Fig. 2 A). Phylogenetic tree showed that these amino acid sequences formed three branches, and the members of AaSHI were distributed in two branches, and were most closely related to the dicotyledonous plant A. thaliana , followed by V. vinifera , while they were further related to Z. mays , O. sativa . Amino acid sequence alignment and conserved domain analysis indicated that all five AaSHI proteins contained the circular zinc finger domains and IGGH domains (Fig. 2 B). Meanwhile, MEME online software was used to predict conservative structural domains (Fig. 2 C, Supplementary Table 3), and 5 motifs were identified. Among them, motif4 and motif5 were distributed in five AaSHI proteins, which just corresponded to the circular zinc finger domain and IGGH domain. Gene structure analysis shows that they all have two exons and one intron, proving that the five AaSHI sequences share the same structure. Chromosome localization and synteny analysis Based on the genome information of A. annua , the chromosome location analysis of AaSHI genes showed that AaSHI1 , AaSHI2 and AaSHI3 were distributed on chr1, chr3 and chr5 respectively, while AaSHI4 and AaSHI5 were distributed on chromosome 6, and there were no tandem duplication events among members of A. annua SHI gene family during the evolution (Fig. 3 A). Synteny analysis detected that two AaSHI genes, AaSHI3 and AaSHI4 , participated in a segmental duplication event. Large-scale comparative synteny maps of AaSHI and AtSHI , SlSHI , VvSHI genes showed that A. annua and V. vinifera had the highest synteny with 8 pairs of genes, but only 6 pairs of synteny genes with S. lycopersicum (Fig. 3 B, Supplementary Table 4). Analysis of cis -acting elements of AaSHI genes promoter To analyze the potential biological functions of the AaSHI gene, cis -acting element analysis of the promoter region located 3.0 kb upstream of the start codon of the AaSHIs was performed via the PlantCARE online website (Supplementary Table 5). As shown in Fig. 4 A, the promoter regions of AaSHIs mainly contained four major types of cis -acting elements: Plant growth and development, phytohormone responsive, light responsive, abiotic and biotic stress. MYB binding sites were the most common in the AaSHI genes promoter, followed by MYC binding elements were also more distributed except for AaSHI5 , which indicated that AaSHI may interact with MYB and MYC TFs to regulate the growth of A. annua . Interestingly, the ABRE element and W-box on the AaSHI5 promoter were significantly more than the other four AaSHI genes, suggesting that its function may differ from the others. In addition, some jasmonic acid response elements could also be found in the all AaSHI genes promoter. To intuitively illustrate the distribution of cis -acting elements, we counted the number of elements of each type and presented them in a bar graph (Fig. 4 B). These data indicated that the AaSHI genes potentially have diverse roles in the phytohormone and environmental response of A. annua. The expression profile of AaSHI gene family by RNA-seq database Using different tissues, light and dark treatment and phyllotaxy RNA-seq database to analyze the gene expression profile [ 21 ]. Evaluate the expression level of AaSHI gene family with artemisinin biosynthetic genes and GST-specific TFs by TPM, visual mapping was performed after normalization using the TBtools software. The results showed that AaSHI1 , AaSHI2 , AaSHI3 and AaSHI4 were all specifically expressed in the trichome, which was consistent with the expression pattern of artemisinin, while AaSHI5 was specifically expressed in the stem (Fig. 5 A). The results of gene expression in distinct leaf positions indicated that except for AaCYP71AV1 , other genes showed significant leaf order, that is, they were highly expressed in tender leaves and showed a decreasing trend with the order of leaf arrangement (Fig. 5 B). Meanwhile, AaSHI genes with artemisinin biosynthetic genes and GST-specific TFs were all induced by light, AaSHI5 was not detected due to its low transcript level (Fig. 5 C). The expression pattern of AaSHIs The expressions of AaSHI genes were evaluated by qRT-PCR. As shown in Fig. 6 A- 6 D, AaSHI1-4 were highly expressed in the young leaves but weakly expressed in root, which is in line with the artemisinin biosynthetic genes. AaSHI5 showed the highest expression in the stem (Fig. 6 E). In addition, the expression levels of artemisinin biosynthetic pathway genes are highest in the tender leaves and gradually decreases as leaf age. Similar expression pattern was observed in AaSHI1 , AaSHI2 , AaSHI4 , and AaSHI5 (Fig. 6 F- 6 J). Considering that AaSHI5 was preferentially expressed in stem rather than young leaves, we speculated that the AaSHI1, AaSHI2, and AaSHI4 are related to the biosynthesis of artemisinin. Correlation analysis with artemisinin biosynthetic genes Next, we conducted co-expression analysis of AaSHIs , artemisinin biosynthetic genes, and two key regulators of artemisinin biosynthetic pathway AaGSW1 and AaORA , based on their tissue/organ and light-treated transcriptome data. The Pearson coefficient was used to calculate the correlation between the AaSHI TFs and artemisinin biosynthetic genes. Pairs of genes that meet the criteria will be screened and then visualized using Cytoscape software. TPM of AaSHIs in GST was indicated by a color gradient. Correlation analysis showed that three genes ( AaSHI1 , AaSHI2 and AaSHI4 ) had a positive correlation of all four artemisinin biosynthetic genes as well as GST-specific TFs. However, AaSHI3 and AaSHI5 were not correlated with these key genes (Fig. 7 , Supplementary Table 6). Therefore, we chose AaSHI1, AaSHI2 and AaSHI4 for further functional study. Subcellular localization of the AaSHI proteins The subcellular localization prediction was conducted on the Plant mPLoc website, and the all three TFs were predicted to be localized to the nucleus. Subsequently, the C-ternimal of AaSHIs were fused with YFP and expressed in N. benthamiana leaves to experimentally verify the subcellular localization. As shown in Fig. 8 , pHB-AaSHI1/2/4-YFP were detected in the nucleus exclusively, while the control YFP (Yellow Fluorescent Protein) displayed in both nucleus and cytoplasm. This is consistent with website predictions and their potential functional localization as a TF. AaSHIs transactivated the expression of artemisinin biosynthetic genes To verify the activation effect of AaSHIs on four artemisinin biosynthetic genes, we used tobacco leaves for transient transformation. The reporter vector was obtained by inserting the successfully cloned promoter into the pGreenⅡ 0800-LUC vector with homologous recombination method and transferred into A. tumefaciens GV3101 (pSoup) for dual-LUC assay. Meanwhile, AaSHIs were inserted in pHB vector driven by the 35S promoter (Fig. 9 A). The results indicated that AaSHI1 has significant activation effect on the promoters of AaADS and AaCYP71AV1 , while AaSHI2 could solely activate the expression of AaADS gene (Fig. 9 B- 9 E). In contrast, AaSHI4 had no activation effect on these artemisinin pathway genes. In order to explore the binding sites of AaSHI members on four specific artemisinin biosynthetic genes, we analyzed the SHI-binding sites in the 3,000 bp upstream promoter regions of these four genes. It was reported that the binding sites for SHI proteins were ACTCTAC, ACTCCAT, ACTCAAC and ACTCTAA [ 15 , 17 , 18 , 20 ], so we concluded that the possible binding site for SHI TFs was ACTCnAn. The analysis results showed that there were six potential binding sites on the promoters of AaADS and three on AaCYP71AV1 (Supplementary Fig. 2). Yeast one-hybrid assays were performed to further examine the binding ability. The ORF of AaSHI1 and AaSHI2 were inserted into the pB42AD effector vector. Each predicted binding site motif along with the four nucleotide sequences on both sides was artificially synthesized into three repeat fragment and inserted into the pLacZ reporter vector. As shown in Fig. 9 F and 9 G, AaSHI1 could directly bind to AaADS and AaCYP71AV1 promotors while AaSHI2 could only bind to AaADS promotor. These data suggested AaSHI1 and AaSHI2 served as direct positive regulators of artemisinin biosynthetic genes AaSHI1 promote the expression of artemisinin biosynthetic genes Considering that AaSHI1 had better transcriptional activation effects on artemisinin biosynthetic genes as compared with AaSHI2, transient transformation was conducted to verify the function of AaSHI1 in regulating artemisinin biosynthesis in vivo . pHB-AaSHI1-YFP constructs in Agrobacterium strain cell GV3101 was injected into the back of the first pair of true leaves of A. annua (Fig. 10 A). After 48 hours of cultivation, samples were collected and qRT-PCR was used to detect the expression level of artemisinin biosynthetic genes. The results showed that AaSHI1 significantly promoted AaADS expression in A. annua , about 8.6-fold of that in the empty control (Fig. 10 B). Meanwhile, AaSHI1 also significantly activated the expression of AaCYP71AV1 , with about 2.5-fold that of the control. These observations were consistent with previous dual-LUC assays. In general, these results indicated that AaSHI1 had positive roles in regulating artemisinin biosynthesis. Materials and methods Identification of SHI-family TFs of A. annua Four sets of haplotype chromosome genomes of A. annua were downloaded [ 22 ]. Nine sequences of SHI gene family in A. thaliana were used as baits to align four sets of haplotype chromosomal genomes of A. annua using the blastp program of TBtools software [ 23 ], and four sets of AaSHI gene family sequences of A. annua were obtained. Five AaSHI gene sequences in the HAN 1_phase0 genome were finally selected for subsequent analyses after performing the protein alignment in the NCBI database. The physiochemical properties of the AaSHI proteins were analyzed using the online tool ExPASy program [ 24 ]. Phylogenetic tree construction, protein alignment and gene structure analysis A phylogenetic tree of the SHI-family proteins from A. annua , A. thaliana , Oryza sativa , Solanum lycopersicum , Vitis vinifera and Zea mays , was constructed using the Neighbor-Joining method. The Bootstrap value was set to 1,000, and the other parameters were maintained to their default values. The sequences used in the phylogenetic tree were downloaded from Phytozome13. The alignment of AtSHIs with AaSHIs was performed and visualized by ClustalW and Genedoc software, respectively. The MEME online tool was employed to predict the conserved motifs of AaSHIs with the number of the motifs set to 5. The conserved motifs and structure of the genes were visualized using TBtools. Chromosomal localization, intraspecies and interspecies synteny analysis Chromosomal locations and replication events of AaSHI genes as well as self-alignment of the whole genome sequence were visualized by TBtools. The whole genome sequence file and gene structure annotation file were obtained from EnsemblPlants database. A synonymous relationship analysis was conducted between AaSHIs and SHIs from other three species ( A. thaliana , V. vinifera , S. lycopersicum ). Analysis of cis -regulatory elements The cis -regulatory elements were predicted in promoter sequences (3,000 bp upstream of first ATG) of AaSHI genes and four artemisinin biosynthetic genes using the PlantCARE online website [ 25 ]. Subsequently, the obtained cis -acting elements on the promoter of AaSHIs were classified and sorted. Due to the limited coverage of SHI TFs at present, online prediction cannot obtain SHI binding sites (SBS) on the promoter four artemisinin biosynthetic genes. Therefore, we manually searched based on existing research. Correlation analysis of AaSHI family genes with artemisinin biosynthetic and regulatory genes We retrieved the transcriptome data of five AaSHI genes, four artemisinin biosynthetic genes and two GST-specific TF genes. The correlation between five AaSHI family genes with four artemisinin biosynthetic genes and two GST-specific artemisinin regulatory genes was calculated by Pearson coefficient. The correlation coefficients R and p -value would be obtained. The positive and negative correlation coefficients R represent the promoting and inhibitory effects of AaSHI TFs on artemisinin biosynthetic and regulatory genes, respectively. The screening threshold was set as follows: the absolute value of correlation coefficient R > 0.8 and the p -value < 0.05 [ 26 , 27 ]. Finally, the network diagram of the co-expression of five AaSHI family genes, four artemisinin biosynthetic genes and two GST-specific artemisinin regulatory genes was visualized by Cytoscape_v3.7.2 software. Different presentation effects can be achieved by modifying parameters in the software. qRT-PCR analysis Two-month-old plants of A. annua variety Huhao 1 [ 3 ] were grown at 25°C. Total RNAs of A. annua were isolated from various tissues of the plants using the RNApure Plant Kit (Tiangen, China). cDNA synthesis was carried out using the HiScript III 1st Strand cDNA Synthesis Kit with gDNA Wiper (Vazyme, China). qPCR amplification was performed as previously reported [ 13 ]. Actin was used as an internal control. Each sample has three biological replicates. Subcellular localization analysis The Plant-mPLoc website was used to predict subcellular localization of AaSHI proteins. To further analyze the subcellular localization, high-Fidelity DNA polymerase KOD-Plus was used to clone AaSHI genes (Toyobo, Osaka, Japan). And then the full-length coding sequences of candidate AaSHIs ( AaSHI1 , AaSHI2 and AaSHI4 ) were inserted into the plant expression pHB-YFP vector. The constructed plasmids pHB-AaSHIs-YFP and pHB-YFP (empty vector) were transformed into the Agrobacterium tumefaciens strain GV3101 to transient infect the 5-week-old Nicotiana benthamiana leaves. The DAPI signal and the YFP signal were observed using confocal microscopy after culturing N. benthamiana plants in dark for 24 h and then in light condition for 24 h at a constant temperature of 25°C [ 28 ]. Dual-LUC assay To generate reporter constructs used in the Dual-LUC assays, the promoters of fourartemisinin biosynthetic genes were cloned and constructed into pGreenII0800 plasmid. The pHB-AaSHIs-YFP constructs were considered as effectors and pHB-YFP construct was considered as control. Effectors and reporters were transformed into GV3101 respectively. Reporter strains and effector strains were mixed with ratio of 1:1 and transiently transformed the N. benthamiana leaves. The culture condition of tobacco used for dual-LUC assays was the same as those for subcellular localization analysis [ 29 ]. After two days, the samples were harvested to analyze the LUC/REN ratio (Promega, Madison, WI, USA) [ 30 ]. Three biological replicates were performed for dual-LUC assays. Yeast one-hybrid assay The ORF of AaSHI1 and AaSHI2 were inserted into the pB42AD effector vector. The sequences containing predicted binding site motif along with the four nucleotide sequences on both sides were inserted into the pLacZ reporter vector. Effector vector and reporter vector were cotransferred into the yeast EGY48 strain by LiAc mediated method [ 28 ]. The positively transformed clones were grown on SD/-Ura-Trp medium with X-gal at 30°C. The discoloration of yeast plaque was observed after 24h. Transient expression in the leaves of A. annua The leaves of 2-week-old A. annua seedlings mentioned earlier were used for transient transformation [ 31 ]. Agrobacterium strain cells GV3101 containing pHB-AaSHI1-YFP constructs were injected into the back of the first pair of true leaves. The injected leaves were dried with absorbent paper and covered with a transparent plastic lid to maintain humidity. The seedings were cultivated in the dark for 24 hours and then transfer to light conditions for another 24 hours. The samples were collected for qRT-PCR. Conclusions Malaria remains a great threat to global security and caused about 247 million infections and 619,000 deaths worldwide in 2021 (World Malaria Report 2022). Artemisinin and its derivatives which show potent anti-malarial activity have been widely used for the treatment of malaria and have significantly reduced its fatality. Artemisinin is originally isolated and purified from A. annua , a Chinese medicinal plant. Currently, because of the low artemisinin production by using heterologous systems and such as tobacco and Physcomitrella patens , the main source of artemisinin was still the cultivated A. annua [ 32 , 33 ]. It is essential to increase the artemisinin production in A. annua , thereby meeting the large-scale global demand. Increasing the GST density and enhancing the expression levels of artemisinin biosynthetic genes are the most effective strategies to improve the artemisinin content in A. annua . Recently, many TFs have been confirmed to have important roles in regulating GST formation and artemisinin biosynthesis. For example, MYB TF family members such as AaMIXTA1, AaMYB5, AaMYB16, AaMYB17, AaTLR1 and AaTLR2 are related to GST initiation, while AaMYB15 and AaMYB108 are involved in the regulation of artemisinin biosynthesis [ 34 – 39 ]. Similarly, several members from WRKY TF family have proved to be involved in GST formation or artemisinin biosynthesis. AaGSW1, AaWRKY9, AaWRKY14 and AaWRKY17 were found to modulate artemisinin biosynthesis while AaGSW2 acts as a key regulator of GST initiation [ 12 , 40 – 43 ]. In addition, two MADS-box members AaSEP1 and AaSEP4 were reported to regulate GST formation and artemisinin biosynthesis, respectively [ 44 , 45 ]. These findings demonstrated there were functional differences among the members of one TF family. Given the advantages of A. annua genome, WRKY, bHLH and B-box TF family members were genome-wide characterized [ 46 – 48 ]. However, the regulatory roles of SHI TF family in artemisinin production in A. annua remain largely unknown. The SHI TF family is an ancient plant gene family, in which AtLRP1 is the first cloned SHI/STY gene, named LRP (Lateral Root Primordia) due to its activation and expression during the formation of lateral root primordia (Smith and Fedoroff 1995). It serves as a molecular marker gene for studying the early stages of lateral root primordia formation and development. At present, there have been some studies on the function of the SHI TF family in A. thaliana , and in other species such as Z. mays , O. sativa , Hordeum vulgare , and Glycine max [ 19 , 20 , 49 , 50 ]. It is proved that SHI TF family members can participate in the formation of A. thaliana roots and the development of organs such as leaves and flowers as well as regulate hormone biosynthesis and signal transduction [ 51 , 52 ], while the regulation of secondary metabolism by the SHI family has not been reported. To investigate whether AaSHI members could regulate the synthesis of artemisinin, a genome-wide analysis of AaSHI in A. annua was conducted. After a series of sequence analysis, five TFs of the AaSHI family were ultimately screened and named AaSHI1-5 according to their sequence numbers (Table 1 ). The number of SHI family TFs varies from 0 to 28 in different species [ 14 , 52 ], indicating that AaSHI was a small family within this range. The phylogenetic tree analysis divided AaSHIs into two groups and was recently related to AtSHIs and VvSHIs, while the remaining group was the SHI proteins of monocot Z. mays , O. sativa . The circular zinc finger domain and IGGH domain are typical conserved domains in the SHI family [ 53 , 54 ], both of which are present in AaSHIs, demonstrating the conservation of the AaSHI gene in A. annua (Fig. 2 ). Chromosome mapping and intra-species synteny analysis showed that five SRS genes were distributed on four chromosomes, and no tandem repeats were found, but there was a fragment replication event (Fig. 3 ). Intraspecies synteny analysis found that there was one fragment replication event in the AaSHI family. Unlike some AtSRSs and OsSHI1 , which are mainly expressed in roots and flowers and almost not expressed in leaves [ 16 , 55 ], AaSHIs expressed in leaves, and the expression level of AaSHI2 in leaves was higher than that in flowers and roots (Fig. 5 B). Moreover, the expression of AaSHIs in leaves also exhibits leaf order, with the highest expression level in youngest leaves and decreasing with leaf order (Fig. 5 B). We speculated that AaSHI may have functional differences with A. thaliana , or may have other potential regulatory functions. Analysis of cis -acting elements on the AaSHIs promoter revealed its possible involvement in plant growth and development as well as multiple signaling pathways (Fig. 4 ). The co-expression network revealed the correlation between AaSHIs with four artemisinin biosynthetic genes and GST-specific TFs, which indicated that AaSHI1, AaSHI2, AaSHI4 were positively correlated with these genes and could be used as candidate genes for further functional studies (Fig. 7 ). Subcellular localization experiments revealed that all these three candidate genes localize in the nucleus, consistent with their function as TFs (Fig. 8 ). Dual-LUC assays (Fig. 9 B- 9 E) and yeast one-hybrid assays (Fig. 9 F, 9 G) indicated AaSHI1 and AaSHI2 had direct transcriptional activation effects on artemisinin biosynthetic gene AaADS . In addition, AaSHI1 could directly activate the expression of AaCYP71AV1 . Accordingly, transient expression assays in A. annua further demonstrated that AaSHI1 could significantly upregulate the expression of AaADS and AaCYP71AV1 in vivo (Fig. 10 B). Taken together, we concluded that AaSHI1 was positive regulator of artemisinin biosynthesis by activating artemisinin biosynthetic genes AaADS and AaCYP71AV1 . Numerous studies have demonstrated that TFs increase the artemisinin yield via activating the expression level of four artemisinin biosynthetic genes. For example, AaWRKY1 could activate AaADS and AaCYP71AV1 expression and enhance the artemisinin production [ 56 ]. AaMYC2 has the ability to bind to the G-box motifs on the promoters of AaCYP71AV1 and AaDBR2 , and overexpression of AaMYC2 leads to an increase in artemisinin production [ 9 ]. AaTCP15, as a TF capable of responding to both JA and ABA signals, can directly bind and activate the promoter of AaDBR2 and AaALDH1 . Meanwhile, AaORA, a positive regulatory factor, can interact with and activate the transcriptional activity of AaTCP15 by forming an AaORA-AaTCP15 module to synergistically activate AaDBR2 [ 57 ]. In this study, we demonstrated the strong activation effects of AaSHI1 on AaADS and AaCYP71AV1 . Declarations Acknowledgements We appreciate the experimental support from the Public Platform of Pharmaceutical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University. Authors ’ contributions Xiaolong Hao and Guoyin Kai conceived and designed the project. Yinkai Yang, Pengyang Li performed the experiments. Yinkai Yang, Yongpeng Li, Qin Zhou Miaomiao Sheng and Xiaojing Ma analyzed the data. Yinkai Yang, Yongpeng Li and Xiaolong Hao wrote the manuscript. Tsubasa Shoji, Xiaolong Hao and Guoyin Kai revised the manuscript. All authors read and approved the final manuscript. Funding This work was supported by National Key Research and Development Program of China (2023YFC3503900), National Natural Science Foundation of China (82003889, 82073963, 82304651), Zhejiang Provincial Natural Science Foundation of China (LQ21H280004), China-Japan Youth Exchange Program in Science, Technology and Humanities Seminar on “Twinning Short-Term Exchange Project”, Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), National “Ten-thousand Talents Program” for Leading Talents of Science and Technology Innovation in China, National Young Qihuang Scholars Training Program, The Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province (2021C02074), Research Project of Zhejiang Chinese Medical University (2021JKZDZC06, 2022RCZXZK23, 2023JKZKTS08) and China Postdoctoral Science Foundation (2022M722851). Availability of data and materials The datasets generated and analyzed during the current study are included in this article. The sequencing data that support the findings of this study are openly available inthe global pharmacopoeia genome database (http://www.gpgenome.com/species/92.).Raw reads for RNA-Seq were downloaded from the NCBI database with accessionnumber SRP129502 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP129502) and SRP092562 (https://www.ncbi.nlm.nih.gov/sra/SRP092562). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References White NJ. Qinghaosu (Artemisinin). The price of success. Science. 2008;320(5874):330–4. Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013;496(7446):528. 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Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 09 Aug, 2024 Read the published version in BMC Genomics → Version 1 posted Editor assigned by journal 28 Mar, 2024 Submission checks completed at journal 21 Mar, 2024 First submitted to journal 22 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:05:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3978505/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3978505/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-024-10683-7","type":"published","date":"2024-08-09T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53427381,"identity":"f8f4c84a-d652-4e2b-a660-3d794ea35809","added_by":"auto","created_at":"2024-03-25 21:10:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":338323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe artemisinin biosynthetic pathway and its regulatory network in light conditions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/986cf98320812dfeafb479a2.jpg"},{"id":53426884,"identity":"80d7eb13-977b-46a6-8f66-728ed161d3e2","added_by":"auto","created_at":"2024-03-25 20:54:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":635691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis and Amino acid sequence alignment of AaSHI members.\u003c/strong\u003e (A) Using six species (\u003cem\u003eA. annua, A. thaliana, Z. mays, V. vinifera, S. lycopersicum, O. sativa\u003c/em\u003e) for phylogenetic tree analysis, different species were labeled with different symbols. (B) Protein sequence alignment of AaSHIs and AtSHI/STY family members. The range of nuclear localization signal (NLS) and IGGH domain were marked with horizontal lines and RING zinc finger domain was marked with asterisks. (C) The distribution of conserved motifs and gene structure of the \u003cem\u003eAaSHI\u003c/em\u003e gene. Five different motifs are marked with different colors. In the gene structure map, the green part was the UTR region, the yellow part was the CDS region, and the intron was the vacant part.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/76eb8c5c21b2999fed0ce13b.jpg"},{"id":53427114,"identity":"d067db91-ee7e-47fa-8c48-e7812280b633","added_by":"auto","created_at":"2024-03-25 21:02:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":329849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe chromosomal location and synteny analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAaSHI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e (A) Circos diagram illustrated the chromosomal locations of \u003cem\u003eAaSHI\u003c/em\u003e genes and their synteny. The blue line indicated the presence of replication events between two genes. (B) Synteny analysis of \u003cem\u003eAaSHI\u003c/em\u003e genes between \u003cem\u003eA. annua\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e,\u003cem\u003eS. lycopersicum \u003c/em\u003eand \u003cem\u003eV. vinifera\u003c/em\u003e,\u003cem\u003e \u003c/em\u003erespectively. The red line represented the synteny of \u003cem\u003eSHI\u003c/em\u003e genes between two species.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/788f33adc2dc3f4b6914bb25.jpg"},{"id":53426890,"identity":"7124db12-509e-411b-b66a-50bef764b41a","added_by":"auto","created_at":"2024-03-25 20:54:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":213858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromoter \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-regulatory elements analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAaSHI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e (A) Classified and statistically analyzed the data exported from plantcare, and visualize it in the form of heat maps. The color depth was consistent with the number of cis acting elements. (B) The sum of \u003cem\u003ecis\u003c/em\u003e-acting elements of each major type. The color of the column corresponds to the chart A.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/d9b6c277adc6613724626af4.jpg"},{"id":53426894,"identity":"ae970529-7c61-4953-acca-60413813cdd4","added_by":"auto","created_at":"2024-03-25 20:54:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAaSHI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes, artemisinin biosynthetic genes and GST-specific TFs by RNA-seq database. \u003c/strong\u003eThe transcriptional expression levels of eleven\u003cem\u003e \u003c/em\u003egenes in seven tissues including root, stem, old leaf, young leaf, bud, flower and trichome (A), leaf 0, leaf 1, leaf 3, leaf 4, leaf 5 and leaf 7 in \u003cem\u003eA. annua\u003c/em\u003e plants from top to bottom (B), and treated with darkness and light (C).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/45b2b6efb5ad6519d8f08c10.jpg"},{"id":53426892,"identity":"782bd8c0-58f7-4533-8231-3ca2565f935e","added_by":"auto","created_at":"2024-03-25 20:54:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":321183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eqRT-PCR validation of five \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAaSHI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes. \u003c/strong\u003e(A-E) The relative expression levels of AaSHI genes in young leaf, old leaf, stem and root were detected by qRT-PCR. The relative expression in young leaf of each \u003cem\u003eAaSHI\u003c/em\u003egene was set to 1. (F-J) The relative expression levels of AaSHI genes of leaf 0, leaf 1, leaf 3, leaf 5 and leaf 7 in \u003cem\u003eA. annua \u003c/em\u003eplants from top to bottom were detected by qRT-PCR. The relative expression of each\u003cem\u003e AaSHI\u003c/em\u003e genein leaf 0 was set to 1.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/f7f78d3780a48f24b22a5bc1.jpg"},{"id":53426888,"identity":"5a8fb53d-f73c-45a3-97c8-4826ef41c723","added_by":"auto","created_at":"2024-03-25 20:54:14","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":211058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAaSHI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene family with artemisinin biosynthetic genes and GST-specific TFs. \u003c/strong\u003eFive \u003cem\u003eAaSHIs\u003c/em\u003e were labeled with ellipses, six genes that have been reported to regulate artemisinin biosynthesis were labeled with rectangles. Different color gradients were assigned to \u003cem\u003eAaSHIs\u003c/em\u003e based on their expression levels in GST. Regulatory relationships were all represented by solid line due to their positive regulation.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/b337689ffbd2fd1c58e0a468.jpg"},{"id":53426885,"identity":"4fab72bc-5815-4337-86ed-c9c27d3c122f","added_by":"auto","created_at":"2024-03-25 20:54:14","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":221003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe subcellular localization of AaSHI1, AaSHI2, AaSHI4 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e leaves. \u003c/strong\u003eYFP was observed at 488 nm. Determined the nucleus by DAPI staining and observed at 405 nm. YFP is used as a negative control. Scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/85a1141a4d945aef23d79afa.jpg"},{"id":53427125,"identity":"66c3c0a4-68be-4085-8089-5ebf01e8db98","added_by":"auto","created_at":"2024-03-25 21:02:14","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":550105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual-LUC assays verified the activation effect of AaSHI proteins on artemisinin biosynthesis.\u003c/strong\u003e (A) Schematic diagram of vetor construction of and pHB-AaSHI1/2/4, pGREEN0800-promoter-LUC. (B-E) The results of dual-LUC assay in tobacco leaf cells. Error bars indicate the mean ± standard deviation (SD). Student’s t-test was used to evaluate the significant difference of AaSHIs activation on artemisinin biosynthetic genes. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (F) Schematic diagram of the binding sites on the promoter and the nucleotide sequence of the binding sites. (G) Yeast one-hybrid assay for interaction between AaSHI1/2 protein with binding site motifs, the triple fragments used were presented in F.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/567fe043a447bcf6b62c28f1.jpg"},{"id":53426891,"identity":"99d0c891-8b1c-4e88-9996-19c84b02a4d0","added_by":"auto","created_at":"2024-03-25 20:54:15","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":111221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient expression assays indicated that AaSHI1 promoted the expression level of artemisinin biosynthetic genes. \u003c/strong\u003e(A) Schematic diagram for the first pair of true leaves of \u003cem\u003eA. annua \u003c/em\u003einjected with \u003cem\u003eAgrobacterium\u003c/em\u003e strain.\u003cem\u003e \u003c/em\u003e(B) The relative expression of four artemisinin biosynthetic genes with transient expression assays in \u003cem\u003eA. annua\u003c/em\u003e leaf cells. Error bars indicate the mean ± SD. Student’s t-test was used to evaluate the significant difference of AaSHI1 activation on artemisinin biosynthetic genes. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/d497319d1ecd957ba1811980.jpg"},{"id":62298337,"identity":"76b16574-13e5-48f6-afd4-8f92d739eee4","added_by":"auto","created_at":"2024-08-12 16:12:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4225774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/e0fa6fc1-6db4-4266-b441-d1e7ac1d1f56.pdf"},{"id":53426895,"identity":"2c84c6ad-7c69-453f-a9c5-d0b700050c19","added_by":"auto","created_at":"2024-03-25 20:54:15","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":656926,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3978505/v1/ae2741f51070548471be1c4e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria is a parasitic disease caused by \u003cem\u003ePlasmodium\u003c/em\u003e infection and results in over 200\u0026nbsp;million cases globally every year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Artemisinin, a sesquiterpene lactone that contains a peroxy bridge, has demonstrated remarkable effectiveness in the treatment of malignant malaria. Chinese scientist Youyou Tu was awarded the Nobel Prize in Physiology or Medicine in 2015 for her discovery of the anti-malarial property of artemisinin. While artemisinic acid, the precursor of artemisinin, can be synthesized in yeast by synthetic biology technology, its production cost remains high and is insufficient meet the substantial market demand [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, the primary source of the artemisinin is still the Compositae family plant \u003cem\u003eArtemisia annua\u003c/em\u003e. Artemisinin is mainly synthesized and stored in glandular secretory trichomes (GSTs), which are specialized ten-cell epidermal structure found in young leaves and flower buds of \u003cem\u003eA. annua\u003c/em\u003e. As a typical sesquiterpene compound, a series of metabolic and regulatory genes in artemisinin biosynthetic pathway have been thoroughly characterized [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Within GSTs, farnesyl pyrophosphate (FPP), a common precursor for sesquiterpene synthesis, is converted to dihydroartemisinic acid (DHAA) through the catalytic action of four enzymes that are specifically localized in GSTs (AaADS, AaCYP71AV1, AaDBR2, and AaALDH1) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the sub-epidermal space of GSTs, DHAA can be turned into artemisinin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is considered as an effective metabolic engineering approach to enhance the production of specific high-value metabolites in plants through manipulation of transcription factors (TFs). It has been reported that various TFs from different families, including WRKY, AP2/ERF, TCP, bHLH, bZIP, and MYB, are involved in light- and phytohormone-mediated regulation of artemisinin biosynthesis [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Two TFs of different families, WRKY-family AaGSW1 and AP2/ERF-family AaORA, are specifically expressed in the GSTs, and promote artemisinin biosynthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSHI family TFs are unique to plants and exhibit functional diversity, encompassing the regulation of growth, development, metabolism, and response to stresses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. SHI family proteins are characterized by the presence of two functional domains: a circular zinc finger domain at the N-terminal and an IGGH domain at the C-terminal [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The circular zinc finger domain consists of two fingers, which adapt a transverse palm arrangement, similar to the DNA-binding domain. The IGGH domain contains certain acidic residues that play a role in mediating both homologous and heterogeneous dimerization among the SHI proteins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There are ten SHI family proteins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, of which AaSRS8 is reported to be a pseudogene [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. STYLISH1, which is related to the development of shoot apical meristems, can directly bind to the YUCCA4 promoter to regulate auxin synthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. SRS5 negatively regulates lateral root formation through inhibiting the expression of \u003cem\u003eLBD16\u003c/em\u003e and \u003cem\u003eLBD29\u003c/em\u003e genes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, light-induced SRS5 can also be ubiquitinated and degraded by COP1 protein and positively modulate the photomorphogenesis in seedlings by directly activating target genes expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, the maize LRP1 is auxin-responsive and associated with the initiation of lateral and seminal roots in maize [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In rice, OsSHI1 regulates tillering and panicle branching depending on the physical interaction with IPA1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. There have been no reports on the AaSHI TFs in \u003cem\u003eA. annua\u003c/em\u003e, and the regulatory function of AaSHIs in relation to artemisinin biosynthesis in \u003cem\u003eA. annua\u003c/em\u003e remains largely unexplored.\u003c/p\u003e \u003cp\u003eIn this study, we identified five \u003cem\u003eAaSHI\u003c/em\u003e genes in the \u003cem\u003eA. annua\u003c/em\u003e genome. Expression analysis conducted in various tissues and under different light conditions revealed a positive correlation among \u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e and \u003cem\u003eAaSHI4\u003c/em\u003e, the four artemisinin biosynthetic genes, and two GST-specific TF genes, \u003cem\u003eAaGSW1\u003c/em\u003e and \u003cem\u003eAaORA\u003c/em\u003e. Yeast one-hybrid assays and dual-luciferase (Dual-LUC) reporter assays showed that AaSHI1 could directly activate the expression of \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e. Transient transformation in \u003cem\u003eA. annua\u003c/em\u003e leaves confirmed that AaSHI1 positively regulated artemisinin biosynthesis. The data present in this study demonstrated that AaSHI1 were positive regulators of artemisinin biosynthetic pathway. Taken together, this study provided a theoretical basis for informing the function of AaSHI family members and cultivating higher artemisinin content \u003cem\u003eA. annua\u003c/em\u003e through molecular biotechnology in the future.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of AaSHI members in the\u003c/b\u003e \u003cb\u003eA. annua\u003c/b\u003e \u003cb\u003egenome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo obtain sequence information of AaSHI, nine AtSHI family proteins of \u003cem\u003eA. thaliana\u003c/em\u003e were used to blast with four sets of haplotype chromosome genomes of \u003cem\u003eA. annua\u003c/em\u003e (Supplementary Table\u0026nbsp;1). Multiple sequence alignments and protein integrity alignment analysis found that unctg_3838g01590241 (LQ-9_phase0) was half the length of the unctg_3207g01555681 and lacked the zinc finger domains, while chr5g00243221 (LQ-9_phase1) also lacked the zinc finger domains (Supplementary Figue 1). As a result, five \u003cem\u003eAaSHI\u003c/em\u003e genes in the HAN1_phase0 genome were selected for subsequent analyses, named \u003cem\u003eAaSHI1\u003c/em\u003e (\u003cem\u003echr1g04025621\u003c/em\u003e), \u003cem\u003eAaSHI2\u003c/em\u003e (\u003cem\u003echr3g03131551\u003c/em\u003e), \u003cem\u003eAaSHI3\u003c/em\u003e (\u003cem\u003echr5g03332101\u003c/em\u003e), \u003cem\u003eAaSHI4\u003c/em\u003e (\u003cem\u003echr6g00096641\u003c/em\u003e), \u003cem\u003eAaSHI5\u003c/em\u003e (\u003cem\u003echr6g00640341\u003c/em\u003e), respectively. Then we analyzed the physical and chemical properties of these identified genes. These genes encoded 276\u0026ndash;390 amino acids with the molecular weight (MW) ranged from 68.19 kDa to 95.67 kDa, and their isoelectric points ranged from 5.05 to 5.13.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetailed information for five AaSHI members in the \u003cem\u003eA. annua\u003c/em\u003e genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAME\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eORF length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein length (aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMw (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAaSHI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003echr1g04025621\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAaSHI2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003echr3g03131551\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAaSHI3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003echr5g03332101\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAaSHI4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003echr6g00096641\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAaSHI5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003echr6g00640341\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction, multiple sequence alignment and gene structure analysis\u003c/h2\u003e \u003cp\u003eTo further investigate the evolutionary relationships and their evolutionary conservation among individual members of the AaSHI family, a phylogenetic tree of 42 SHI members in \u003cem\u003eA. annua\u003c/em\u003e and 5 other species was constructed using MEGAX software (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Phylogenetic tree showed that these amino acid sequences formed three branches, and the members of AaSHI were distributed in two branches, and were most closely related to the dicotyledonous plant \u003cem\u003eA. thaliana\u003c/em\u003e, followed by \u003cem\u003eV. vinifera\u003c/em\u003e, while they were further related to \u003cem\u003eZ. mays\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e. Amino acid sequence alignment and conserved domain analysis indicated that all five AaSHI proteins contained the circular zinc finger domains and IGGH domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Meanwhile, MEME online software was used to predict conservative structural domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Supplementary Table\u0026nbsp;3), and 5 motifs were identified. Among them, motif4 and motif5 were distributed in five AaSHI proteins, which just corresponded to the circular zinc finger domain and IGGH domain. Gene structure analysis shows that they all have two exons and one intron, proving that the five \u003cem\u003eAaSHI\u003c/em\u003e sequences share the same structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChromosome localization and synteny analysis\u003c/h2\u003e \u003cp\u003eBased on the genome information of \u003cem\u003eA. annua\u003c/em\u003e, the chromosome location analysis of \u003cem\u003eAaSHI\u003c/em\u003e genes showed that \u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e and \u003cem\u003eAaSHI3\u003c/em\u003e were distributed on chr1, chr3 and chr5 respectively, while \u003cem\u003eAaSHI4\u003c/em\u003e and \u003cem\u003eAaSHI5\u003c/em\u003e were distributed on chromosome 6, and there were no tandem duplication events among members of \u003cem\u003eA. annua SHI\u003c/em\u003e gene family during the evolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Synteny analysis detected that two \u003cem\u003eAaSHI\u003c/em\u003e genes, \u003cem\u003eAaSHI3\u003c/em\u003e and \u003cem\u003eAaSHI4\u003c/em\u003e, participated in a segmental duplication event. Large-scale comparative synteny maps of \u003cem\u003eAaSHI\u003c/em\u003e and \u003cem\u003eAtSHI\u003c/em\u003e, \u003cem\u003eSlSHI\u003c/em\u003e, \u003cem\u003eVvSHI\u003c/em\u003e genes showed that \u003cem\u003eA. annua\u003c/em\u003e and \u003cem\u003eV. vinifera\u003c/em\u003e had the highest synteny with 8 pairs of genes, but only 6 pairs of synteny genes with \u003cem\u003eS. lycopersicum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-acting elements of\u003c/b\u003e \u003cb\u003eAaSHI\u003c/b\u003e \u003cb\u003egenes promoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo analyze the potential biological functions of the \u003cem\u003eAaSHI\u003c/em\u003e gene, \u003cem\u003ecis\u003c/em\u003e-acting element analysis of the promoter region located 3.0 kb upstream of the start codon of the \u003cem\u003eAaSHIs\u003c/em\u003e was performed \u003cem\u003evia\u003c/em\u003e the PlantCARE online website (Supplementary Table\u0026nbsp;5). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the promoter regions of \u003cem\u003eAaSHIs\u003c/em\u003e mainly contained four major types of \u003cem\u003ecis\u003c/em\u003e-acting elements: Plant growth and development, phytohormone responsive, light responsive, abiotic and biotic stress. MYB binding sites were the most common in the \u003cem\u003eAaSHI\u003c/em\u003e genes promoter, followed by MYC binding elements were also more distributed except for \u003cem\u003eAaSHI5\u003c/em\u003e, which indicated that AaSHI may interact with MYB and MYC TFs to regulate the growth of \u003cem\u003eA. annua\u003c/em\u003e. Interestingly, the ABRE element and W-box on the \u003cem\u003eAaSHI5\u003c/em\u003e promoter were significantly more than the other four \u003cem\u003eAaSHI\u003c/em\u003e genes, suggesting that its function may differ from the others. In addition, some jasmonic acid response elements could also be found in the all \u003cem\u003eAaSHI\u003c/em\u003e genes promoter. To intuitively illustrate the distribution of \u003cem\u003ecis\u003c/em\u003e-acting elements, we counted the number of elements of each type and presented them in a bar graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These data indicated that the \u003cem\u003eAaSHI\u003c/em\u003e genes potentially have diverse roles in the phytohormone and environmental response of \u003cem\u003eA. annua.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression profile of\u003c/b\u003e \u003cb\u003eAaSHI\u003c/b\u003e \u003cb\u003egene family by RNA-seq database\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing different tissues, light and dark treatment and phyllotaxy RNA-seq database to analyze the gene expression profile [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Evaluate the expression level of AaSHI gene family with artemisinin biosynthetic genes and GST-specific TFs by TPM, visual mapping was performed after normalization using the TBtools software. The results showed that \u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e, \u003cem\u003eAaSHI3\u003c/em\u003e and \u003cem\u003eAaSHI4\u003c/em\u003e were all specifically expressed in the trichome, which was consistent with the expression pattern of artemisinin, while \u003cem\u003eAaSHI5\u003c/em\u003e was specifically expressed in the stem (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results of gene expression in distinct leaf positions indicated that except for \u003cem\u003eAaCYP71AV1\u003c/em\u003e, other genes showed significant leaf order, that is, they were highly expressed in tender leaves and showed a decreasing trend with the order of leaf arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Meanwhile, \u003cem\u003eAaSHI\u003c/em\u003e genes with artemisinin biosynthetic genes and GST-specific TFs were all induced by light, \u003cem\u003eAaSHI5\u003c/em\u003e was not detected due to its low transcript level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe expression pattern of AaSHIs\u003c/h2\u003e \u003cp\u003eThe expressions of \u003cem\u003eAaSHI\u003c/em\u003e genes were evaluated by qRT-PCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cem\u003eAaSHI1-4\u003c/em\u003e were highly expressed in the young leaves but weakly expressed in root, which is in line with the artemisinin biosynthetic genes. \u003cem\u003eAaSHI5\u003c/em\u003e showed the highest expression in the stem (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In addition, the expression levels of artemisinin biosynthetic pathway genes are highest in the tender leaves and gradually decreases as leaf age. Similar expression pattern was observed in \u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e, \u003cem\u003eAaSHI4\u003c/em\u003e, and \u003cem\u003eAaSHI5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Considering that \u003cem\u003eAaSHI5\u003c/em\u003e was preferentially expressed in stem rather than young leaves, we speculated that the AaSHI1, AaSHI2, and AaSHI4 are related to the biosynthesis of artemisinin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation analysis with artemisinin biosynthetic genes\u003c/h2\u003e \u003cp\u003eNext, we conducted co-expression analysis of \u003cem\u003eAaSHIs\u003c/em\u003e, artemisinin biosynthetic genes, and two key regulators of artemisinin biosynthetic pathway \u003cem\u003eAaGSW1\u003c/em\u003e and \u003cem\u003eAaORA\u003c/em\u003e, based on their tissue/organ and light-treated transcriptome data. The Pearson coefficient was used to calculate the correlation between the AaSHI TFs and artemisinin biosynthetic genes. Pairs of genes that meet the criteria will be screened and then visualized using Cytoscape software. TPM of \u003cem\u003eAaSHIs\u003c/em\u003e in GST was indicated by a color gradient. Correlation analysis showed that three genes (\u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e and \u003cem\u003eAaSHI4\u003c/em\u003e) had a positive correlation of all four artemisinin biosynthetic genes as well as GST-specific TFs. However, \u003cem\u003eAaSHI3\u003c/em\u003e and \u003cem\u003eAaSHI5\u003c/em\u003e were not correlated with these key genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplementary Table\u0026nbsp;6). Therefore, we chose AaSHI1, AaSHI2 and AaSHI4 for further functional study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization of the AaSHI proteins\u003c/h2\u003e \u003cp\u003eThe subcellular localization prediction was conducted on the Plant mPLoc website, and the all three TFs were predicted to be localized to the nucleus. Subsequently, the C-ternimal of \u003cem\u003eAaSHIs\u003c/em\u003e were fused with YFP and expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves to experimentally verify the subcellular localization. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, pHB-AaSHI1/2/4-YFP were detected in the nucleus exclusively, while the control YFP (Yellow Fluorescent Protein) displayed in both nucleus and cytoplasm. This is consistent with website predictions and their potential functional localization as a TF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAaSHIs transactivated the expression of artemisinin biosynthetic genes\u003c/h2\u003e \u003cp\u003eTo verify the activation effect of AaSHIs on four artemisinin biosynthetic genes, we used tobacco leaves for transient transformation. The reporter vector was obtained by inserting the successfully cloned promoter into the pGreenⅡ 0800-LUC vector with homologous recombination method and transferred into \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 (pSoup) for dual-LUC assay. Meanwhile, \u003cem\u003eAaSHIs\u003c/em\u003e were inserted in \u003cem\u003epHB\u003c/em\u003e vector driven by the \u003cem\u003e35S\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The results indicated that AaSHI1 has significant activation effect on the promoters of \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e, while AaSHI2 could solely activate the expression of \u003cem\u003eAaADS\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). In contrast, AaSHI4 had no activation effect on these artemisinin pathway genes. In order to explore the binding sites of AaSHI members on four specific artemisinin biosynthetic genes, we analyzed the SHI-binding sites in the 3,000 bp upstream promoter regions of these four genes. It was reported that the binding sites for SHI proteins were ACTCTAC, ACTCCAT, ACTCAAC and ACTCTAA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], so we concluded that the possible binding site for SHI TFs was ACTCnAn. The analysis results showed that there were six potential binding sites on the promoters of \u003cem\u003eAaADS\u003c/em\u003e and three on \u003cem\u003eAaCYP71AV1\u003c/em\u003e (Supplementary Fig.\u0026nbsp;2). Yeast one-hybrid assays were performed to further examine the binding ability. The ORF of \u003cem\u003eAaSHI1\u003c/em\u003e and \u003cem\u003eAaSHI2\u003c/em\u003e were inserted into the \u003cem\u003epB42AD\u003c/em\u003e effector vector. Each predicted binding site motif along with the four nucleotide sequences on both sides was artificially synthesized into three repeat fragment and inserted into the \u003cem\u003epLacZ\u003c/em\u003e reporter vector. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, AaSHI1 could directly bind to \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e promotors while AaSHI2 could only bind to \u003cem\u003eAaADS\u003c/em\u003e promotor. These data suggested AaSHI1 and AaSHI2 served as direct positive regulators of artemisinin biosynthetic genes\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAaSHI1 promote the expression of artemisinin biosynthetic genes\u003c/h2\u003e \u003cp\u003eConsidering that AaSHI1 had better transcriptional activation effects on artemisinin biosynthetic genes as compared with AaSHI2, transient transformation was conducted to verify the function of AaSHI1 in regulating artemisinin biosynthesis \u003cem\u003ein vivo\u003c/em\u003e. \u003cem\u003epHB-AaSHI1-YFP\u003c/em\u003e constructs in \u003cem\u003eAgrobacterium\u003c/em\u003e strain cell GV3101 was injected into the back of the first pair of true leaves of \u003cem\u003eA. annua\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). After 48 hours of cultivation, samples were collected and qRT-PCR was used to detect the expression level of artemisinin biosynthetic genes. The results showed that AaSHI1 significantly promoted \u003cem\u003eAaADS\u003c/em\u003e expression in \u003cem\u003eA. annua\u003c/em\u003e, about 8.6-fold of that in the empty control (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Meanwhile, AaSHI1 also significantly activated the expression of \u003cem\u003eAaCYP71AV1\u003c/em\u003e, with about 2.5-fold that of the control. These observations were consistent with previous dual-LUC assays. In general, these results indicated that AaSHI1 had positive roles in regulating artemisinin biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eIdentification of SHI-family TFs of\u003c/b\u003e \u003cb\u003eA. annua\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFour sets of haplotype chromosome genomes of \u003cem\u003eA. annua\u003c/em\u003e were downloaded [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nine sequences of SHI gene family in \u003cem\u003eA. thaliana\u003c/em\u003e were used as baits to align four sets of haplotype chromosomal genomes of \u003cem\u003eA. annua\u003c/em\u003e using the blastp program of TBtools software [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and four sets of AaSHI gene family sequences of \u003cem\u003eA. annua\u003c/em\u003e were obtained. Five \u003cem\u003eAaSHI\u003c/em\u003e gene sequences in the HAN 1_phase0 genome were finally selected for subsequent analyses after performing the protein alignment in the NCBI database. The physiochemical properties of the AaSHI proteins were analyzed using the online tool ExPASy program [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction, protein alignment and gene structure analysis\u003c/h2\u003e \u003cp\u003eA phylogenetic tree of the SHI-family proteins from \u003cem\u003eA. annua\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eVitis vinifera\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e, was constructed using the Neighbor-Joining method. The Bootstrap value was set to 1,000, and the other parameters were maintained to their default values. The sequences used in the phylogenetic tree were downloaded from Phytozome13. The alignment of AtSHIs with AaSHIs was performed and visualized by ClustalW and Genedoc software, respectively. The MEME online tool was employed to predict the conserved motifs of AaSHIs with the number of the motifs set to 5. The conserved motifs and structure of the genes were visualized using TBtools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChromosomal localization, intraspecies and interspecies synteny analysis\u003c/h2\u003e \u003cp\u003eChromosomal locations and replication events of \u003cem\u003eAaSHI\u003c/em\u003e genes as well as self-alignment of the whole genome sequence were visualized by TBtools. The whole genome sequence file and gene structure annotation file were obtained from EnsemblPlants database. A synonymous relationship analysis was conducted between \u003cem\u003eAaSHIs\u003c/em\u003e and \u003cem\u003eSHIs\u003c/em\u003e from other three species (\u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eV. vinifera\u003c/em\u003e, \u003cem\u003eS. lycopersicum\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory elements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ecis\u003c/em\u003e-regulatory elements were predicted in promoter sequences (3,000 bp upstream of first ATG) of \u003cem\u003eAaSHI\u003c/em\u003e genes and four artemisinin biosynthetic genes using the PlantCARE online website [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Subsequently, the obtained \u003cem\u003ecis\u003c/em\u003e-acting elements on the promoter of \u003cem\u003eAaSHIs\u003c/em\u003e were classified and sorted. Due to the limited coverage of SHI TFs at present, online prediction cannot obtain SHI binding sites (SBS) on the promoter four artemisinin biosynthetic genes. Therefore, we manually searched based on existing research.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation analysis of\u003c/b\u003e \u003cb\u003eAaSHI\u003c/b\u003e \u003cb\u003efamily genes with artemisinin biosynthetic and regulatory genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe retrieved the transcriptome data of five \u003cem\u003eAaSHI\u003c/em\u003e genes, four artemisinin biosynthetic genes and two GST-specific TF genes. The correlation between five \u003cem\u003eAaSHI\u003c/em\u003e family genes with four artemisinin biosynthetic genes and two GST-specific artemisinin regulatory genes was calculated by Pearson coefficient. The correlation coefficients R and \u003cem\u003ep\u003c/em\u003e-value would be obtained. The positive and negative correlation coefficients R represent the promoting and inhibitory effects of AaSHI TFs on artemisinin biosynthetic and regulatory genes, respectively. The screening threshold was set as follows: the absolute value of correlation coefficient R\u0026thinsp;\u0026gt;\u0026thinsp;0.8 and the \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Finally, the network diagram of the co-expression of five \u003cem\u003eAaSHI\u003c/em\u003e family genes, four artemisinin biosynthetic genes and two GST-specific artemisinin regulatory genes was visualized by Cytoscape_v3.7.2 software. Different presentation effects can be achieved by modifying parameters in the software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR analysis\u003c/h2\u003e \u003cp\u003eTwo-month-old plants of \u003cem\u003eA. annua\u003c/em\u003e variety Huhao 1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] were grown at 25\u0026deg;C. Total RNAs of \u003cem\u003eA. annua\u003c/em\u003e were isolated from various tissues of the plants using the RNApure Plant Kit (Tiangen, China). cDNA synthesis was carried out using the HiScript III 1st Strand cDNA Synthesis Kit with gDNA Wiper (Vazyme, China). qPCR amplification was performed as previously reported [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eActin\u003c/em\u003e was used as an internal control. Each sample has three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization analysis\u003c/h2\u003e \u003cp\u003eThe Plant-mPLoc website was used to predict subcellular localization of AaSHI proteins. To further analyze the subcellular localization, high-Fidelity DNA polymerase KOD-Plus was used to clone \u003cem\u003eAaSHI\u003c/em\u003e genes (Toyobo, Osaka, Japan). And then the full-length coding sequences of candidate \u003cem\u003eAaSHIs\u003c/em\u003e (\u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e and \u003cem\u003eAaSHI4\u003c/em\u003e) were inserted into the plant expression \u003cem\u003epHB-YFP\u003c/em\u003e vector. The constructed plasmids \u003cem\u003epHB-AaSHIs-YFP\u003c/em\u003e and \u003cem\u003epHB-YFP\u003c/em\u003e (empty vector) were transformed into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 to transient infect the 5-week-old \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. The DAPI signal and the YFP signal were observed using confocal microscopy after culturing \u003cem\u003eN. benthamiana\u003c/em\u003e plants in dark for 24 h and then in light condition for 24 h at a constant temperature of 25\u0026deg;C [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDual-LUC assay\u003c/h2\u003e \u003cp\u003eTo generate reporter constructs used in the Dual-LUC assays, the promoters of fourartemisinin biosynthetic genes were cloned and constructed into pGreenII0800 plasmid. The \u003cem\u003epHB-AaSHIs-YFP\u003c/em\u003e constructs were considered as effectors and \u003cem\u003epHB-YFP\u003c/em\u003e construct was considered as control. Effectors and reporters were transformed into GV3101 respectively. Reporter strains and effector strains were mixed with ratio of 1:1 and transiently transformed the \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The culture condition of tobacco used for dual-LUC assays was the same as those for subcellular localization analysis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After two days, the samples were harvested to analyze the LUC/REN ratio (Promega, Madison, WI, USA) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Three biological replicates were performed for dual-LUC assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eYeast one-hybrid assay\u003c/h2\u003e \u003cp\u003eThe ORF of \u003cem\u003eAaSHI1\u003c/em\u003e and \u003cem\u003eAaSHI2\u003c/em\u003e were inserted into the \u003cem\u003epB42AD\u003c/em\u003e effector vector. The sequences containing predicted binding site motif along with the four nucleotide sequences on both sides were inserted into the \u003cem\u003epLacZ\u003c/em\u003e reporter vector. Effector vector and reporter vector were cotransferred into the yeast EGY48 strain by LiAc mediated method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The positively transformed clones were grown on SD/-Ura-Trp medium with X-gal at 30\u0026deg;C. The discoloration of yeast plaque was observed after 24h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient expression in the leaves of\u003c/b\u003e \u003cb\u003eA. annua\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe leaves of 2-week-old \u003cem\u003eA. annua\u003c/em\u003e seedlings mentioned earlier were used for transient transformation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eAgrobacterium\u003c/em\u003e strain cells GV3101 containing \u003cem\u003epHB-AaSHI1-YFP\u003c/em\u003e constructs were injected into the back of the first pair of true leaves. The injected leaves were dried with absorbent paper and covered with a transparent plastic lid to maintain humidity. The seedings were cultivated in the dark for 24 hours and then transfer to light conditions for another 24 hours. The samples were collected for qRT-PCR.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eMalaria remains a great threat to global security and caused about 247\u0026nbsp;million infections and 619,000 deaths worldwide in 2021 (World Malaria Report 2022). Artemisinin and its derivatives which show potent anti-malarial activity have been widely used for the treatment of malaria and have significantly reduced its fatality. Artemisinin is originally isolated and purified from \u003cem\u003eA. annua\u003c/em\u003e, a Chinese medicinal plant. Currently, because of the low artemisinin production by using heterologous systems and such as tobacco and \u003cem\u003ePhyscomitrella patens\u003c/em\u003e, the main source of artemisinin was still the cultivated \u003cem\u003eA. annua\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It is essential to increase the artemisinin production in \u003cem\u003eA. annua\u003c/em\u003e, thereby meeting the large-scale global demand. Increasing the GST density and enhancing the expression levels of artemisinin biosynthetic genes are the most effective strategies to improve the artemisinin content in \u003cem\u003eA. annua\u003c/em\u003e. Recently, many TFs have been confirmed to have important roles in regulating GST formation and artemisinin biosynthesis. For example, MYB TF family members such as AaMIXTA1, AaMYB5, AaMYB16, AaMYB17, AaTLR1 and AaTLR2 are related to GST initiation, while AaMYB15 and AaMYB108 are involved in the regulation of artemisinin biosynthesis [\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similarly, several members from WRKY TF family have proved to be involved in GST formation or artemisinin biosynthesis. AaGSW1, AaWRKY9, AaWRKY14 and AaWRKY17 were found to modulate artemisinin biosynthesis while AaGSW2 acts as a key regulator of GST initiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In addition, two MADS-box members AaSEP1 and AaSEP4 were reported to regulate GST formation and artemisinin biosynthesis, respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These findings demonstrated there were functional differences among the members of one TF family. Given the advantages of \u003cem\u003eA. annua\u003c/em\u003e genome, WRKY, bHLH and B-box TF family members were genome-wide characterized [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, the regulatory roles of SHI TF family in artemisinin production in \u003cem\u003eA. annua\u003c/em\u003e remain largely unknown.\u003c/p\u003e \u003cp\u003eThe SHI TF family is an ancient plant gene family, in which AtLRP1 is the first cloned SHI/STY gene, named LRP (Lateral Root Primordia) due to its activation and expression during the formation of lateral root primordia (Smith and Fedoroff 1995). It serves as a molecular marker gene for studying the early stages of lateral root primordia formation and development. At present, there have been some studies on the function of the SHI TF family in \u003cem\u003eA. thaliana\u003c/em\u003e, and in other species such as \u003cem\u003eZ. mays\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, \u003cem\u003eHordeum vulgare\u003c/em\u003e, and \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. It is proved that SHI TF family members can participate in the formation of \u003cem\u003eA. thaliana\u003c/em\u003e roots and the development of organs such as leaves and flowers as well as regulate hormone biosynthesis and signal transduction [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], while the regulation of secondary metabolism by the SHI family has not been reported. To investigate whether AaSHI members could regulate the synthesis of artemisinin, a genome-wide analysis of AaSHI in \u003cem\u003eA. annua\u003c/em\u003e was conducted. After a series of sequence analysis, five TFs of the AaSHI family were ultimately screened and named AaSHI1-5 according to their sequence numbers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of SHI family TFs varies from 0 to 28 in different species [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], indicating that AaSHI was a small family within this range. The phylogenetic tree analysis divided AaSHIs into two groups and was recently related to AtSHIs and VvSHIs, while the remaining group was the SHI proteins of monocot \u003cem\u003eZ. mays\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e. The circular zinc finger domain and IGGH domain are typical conserved domains in the SHI family [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], both of which are present in AaSHIs, demonstrating the conservation of the \u003cem\u003eAaSHI\u003c/em\u003e gene in \u003cem\u003eA. annua\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Chromosome mapping and intra-species synteny analysis showed that five SRS genes were distributed on four chromosomes, and no tandem repeats were found, but there was a fragment replication event (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Intraspecies synteny analysis found that there was one fragment replication event in the AaSHI family. Unlike some \u003cem\u003eAtSRSs\u003c/em\u003e and \u003cem\u003eOsSHI1\u003c/em\u003e, which are mainly expressed in roots and flowers and almost not expressed in leaves [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], \u003cem\u003eAaSHIs\u003c/em\u003e expressed in leaves, and the expression level of \u003cem\u003eAaSHI2\u003c/em\u003e in leaves was higher than that in flowers and roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, the expression of \u003cem\u003eAaSHIs\u003c/em\u003e in leaves also exhibits leaf order, with the highest expression level in youngest leaves and decreasing with leaf order (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We speculated that AaSHI may have functional differences with \u003cem\u003eA. thaliana\u003c/em\u003e, or may have other potential regulatory functions. Analysis of \u003cem\u003ecis\u003c/em\u003e-acting elements on the \u003cem\u003eAaSHIs\u003c/em\u003e promoter revealed its possible involvement in plant growth and development as well as multiple signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The co-expression network revealed the correlation between \u003cem\u003eAaSHIs\u003c/em\u003e with four artemisinin biosynthetic genes and GST-specific TFs, which indicated that AaSHI1, AaSHI2, AaSHI4 were positively correlated with these genes and could be used as candidate genes for further functional studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Subcellular localization experiments revealed that all these three candidate genes localize in the nucleus, consistent with their function as TFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Dual-LUC assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE) and yeast one-hybrid assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG) indicated AaSHI1 and AaSHI2 had direct transcriptional activation effects on artemisinin biosynthetic gene \u003cem\u003eAaADS\u003c/em\u003e. In addition, AaSHI1 could directly activate the expression of \u003cem\u003eAaCYP71AV1\u003c/em\u003e. Accordingly, transient expression assays in \u003cem\u003eA. annua\u003c/em\u003e further demonstrated that AaSHI1 could significantly upregulate the expression of \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1 in vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Taken together, we concluded that AaSHI1 was positive regulator of artemisinin biosynthesis by activating artemisinin biosynthetic genes \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that TFs increase the artemisinin yield \u003cem\u003evia\u003c/em\u003e activating the expression level of four artemisinin biosynthetic genes. For example, AaWRKY1 could activate \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e expression and enhance the artemisinin production [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. AaMYC2 has the ability to bind to the G-box motifs on the promoters of \u003cem\u003eAaCYP71AV1\u003c/em\u003e and \u003cem\u003eAaDBR2\u003c/em\u003e, and overexpression of AaMYC2 leads to an increase in artemisinin production [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AaTCP15, as a TF capable of responding to both JA and ABA signals, can directly bind and activate the promoter of \u003cem\u003eAaDBR2\u003c/em\u003e and \u003cem\u003eAaALDH1\u003c/em\u003e. Meanwhile, AaORA, a positive regulatory factor, can interact with and activate the transcriptional activity of AaTCP15 by forming an AaORA-AaTCP15 module to synergistically activate \u003cem\u003eAaDBR2\u003c/em\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this study, we demonstrated the strong activation effects of AaSHI1 on \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the experimental support from the Public Platform of Pharmaceutical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaolong Hao and Guoyin Kai conceived and designed the project. Yinkai Yang, Pengyang Li performed the experiments. Yinkai Yang, Yongpeng Li, Qin Zhou Miaomiao Sheng and Xiaojing Ma analyzed the data. Yinkai Yang, Yongpeng Li and Xiaolong Hao wrote the manuscript. Tsubasa Shoji, Xiaolong Hao and Guoyin Kai revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;National Key Research and Development Program of China (2023YFC3503900),\u0026nbsp;National Natural Science Foundation of China (82003889, 82073963, 82304651), Zhejiang Provincial Natural Science Foundation of China (LQ21H280004),\u0026nbsp;China-Japan Youth Exchange Program in Science, Technology and Humanities Seminar on \u0026ldquo;Twinning Short-Term Exchange Project\u0026rdquo;,\u0026nbsp;Key project at central government\u0026nbsp;level: The ability establishment of sustainable use for valuable\u0026nbsp;Chinese medicine resources (2060302),\u0026nbsp;National \u0026ldquo;Ten-thousand Talents Program\u0026rdquo; for Leading Talents of Science and Technology Innovation in China, National Young Qihuang Scholars Training Program,\u0026nbsp;The Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province (2021C02074), Research Project of Zhejiang Chinese Medical University (2021JKZDZC06, 2022RCZXZK23,\u0026nbsp;2023JKZKTS08)\u0026nbsp;and\u0026nbsp;China Postdoctoral Science Foundation (2022M722851).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are included in this article. The sequencing data that support the findings of this study are openly available inthe global pharmacopoeia genome database (http://www.gpgenome.com/species/92.).Raw reads for RNA-Seq were downloaded from the NCBI database with accessionnumber SRP129502 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP129502) and SRP092562 (https://www.ncbi.nlm.nih.gov/sra/SRP092562).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWhite NJ. Qinghaosu (Artemisinin). The price of success. 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Front Plant Sci. 2022; 13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFridborg I, Kuusk S, Moritz T, Sundberg E. The Arabidopsis dwarf mutant shi exhibits reduced gibberellin responses conferred by overexpression of a new putative zinc finger protein. Plant Cell. 1999;11(6):1019\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFridborg I, Kuusk S, Robertson M, Sundberg E. The Arabidopsis protein SHI represses gibberellin responses in Arabidopsis and barley. Plant physiol. 2001;127(3):937\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SG, Lee S, Kim YS, Yun DJ, Woo JC, Park CM. Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development. Plant Mol Biol. 2010;74(4\u0026ndash;5):337\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang W, Fu X, Pan Q, Tang Y, Shen Q, Lv Z, Yan T, Shi P, Li L, Zhang L et al. Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua. Biomed Res Int. 2016; 2016:7314971.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa YN, Xu DB, Yan X, Wu ZK, Kayani SI, Shen Q, Fu XQ, Xie LH, Hao XL, Hassani D, et al. Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua. Plant Biotechnol J. 2021;19(7):1412\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Artemisia annua, artemisinin biosynthesis, transcriptional regulation, SHI family, transcription factor","lastPublishedDoi":"10.21203/rs.3.rs-3978505/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3978505/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTranscription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In \u003cem\u003eArtemisia annua\u003c/em\u003e, various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in \u003cem\u003eA. annua\u003c/em\u003e (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found five \u003cem\u003eAaSHI\u003c/em\u003e genes (\u003cem\u003eAaSHI1\u003c/em\u003e to \u003cem\u003eAaSHI5\u003c/em\u003e) in the \u003cem\u003eA. annua\u003c/em\u003e genome. The expression levels of \u003cem\u003eAaSHI1\u003c/em\u003e, \u003cem\u003eAaSHI2\u003c/em\u003e, \u003cem\u003eAaSHI3\u003c/em\u003e, and \u003cem\u003eAaSHI4\u003c/em\u003e genes were higher in trichomes and young leaves, and decreased when the plants were subjected to dark treatment. The expression pattern of these four \u003cem\u003eAaSHI\u003c/em\u003e genes was consistent with the expression pattern of four artemisinin biosynthetic genes and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in \u003cem\u003eA. annua\u003c/em\u003e provided the evidence that AaSHI1 could directly bind to the promoters of artemisinin biosynthetic genes \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e, and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in \u003cem\u003eA. annua\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, a genome-wide analysis of the AaSHI TF family of \u003cem\u003eA. annua\u003c/em\u003e was conducted. Five \u003cem\u003eAaSHIs\u003c/em\u003e were identified in \u003cem\u003eA. annua\u003c/em\u003e genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of artemisinin biosynthetic genes including \u003cem\u003eAaADS\u003c/em\u003e and \u003cem\u003eAaCYP71AV1\u003c/em\u003e. These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new \u003cem\u003eA. annua\u003c/em\u003e germplasms.\u003c/p\u003e","manuscriptTitle":"A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 20:54:09","doi":"10.21203/rs.3.rs-3978505/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-03-28T08:33:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-21T20:03:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-02-22T11:58:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a1bb09e-ca3d-41e9-9a59-213953e8708e","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:02:23+00:00","versionOfRecord":{"articleIdentity":"rs-3978505","link":"https://doi.org/10.1186/s12864-024-10683-7","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2024-08-09 15:57:32","publishedOnDateReadable":"August 9th, 2024"},"versionCreatedAt":"2024-03-25 20:54:09","video":"","vorDoi":"10.1186/s12864-024-10683-7","vorDoiUrl":"https://doi.org/10.1186/s12864-024-10683-7","workflowStages":[]},"version":"v1","identity":"rs-3978505","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3978505","identity":"rs-3978505","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}