The protein sequences of 22 Arabidopsis ARF were downloaded from the Arabidopsis Information Resource (TAIR) (http://www.aabidopsis.org/). The cannabis genome, protein, and genome annotation files (GCA_900626175.2) were obtained from the NCBI for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/) (Laverty et al., 2019). To obtain the ARF family candidate genes in the cannabis genome, 22 Arabidopsis ARF proteins were used as a query for comparison with the cannabis protein database by BLAST sequence alignment (E-value <1E^-5) performed using the TBtools software (Chen et al., 2020). Next, the protein sequences encoded by the ARF family candidate genes were submitted to the NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/cdd/) to examine whether they contained the conserved ARF (AUX_RESP) and B3 DNA-binding domains. Various physicochemical parameters of the ARF genes were analyzed using ProtParam (http://web.expasy.org/protparam/), and their subcellular locations were predicated by Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).

MEGA7.0 was used to construct a phylogenetic tree using the neighbor-joining method, which was further edited using FigTree software. MEME software (http://meme-suite.org) was used to analyze the conserved motifs in the CsARF genes. The coding sequence and untranslated region (UTR) of CsARFs were extracted from the cannabis genome annotation file using TBtools software, which was also used to combine the gene conserved motif, coding sequence and UTR.

TBtools software was used to extract the 2000 bp sequence upstream of the upstream transcription start site of the 22 CsARF genes (Chen et al., 2020), and then these sequence were applied to analyzed cis-regulatory elements using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Information on the chromosomal location of the CsARF genes was extracted from the cannabis genome file and gene annotation file using TBTools. Next, the physical location of the CsARF genes on the chromosomes was determined using TBtools. TBtools, MCscanX and Circos were used to calculate and draw collinear genes in the cannabis genome and among the different species.

The coding sequence (CDS) of CsARF2 and AtARF6 was amplified using the ‘H8’ leaf and the ‘Col’ leaf cDNA as templates, respectively. After PCR amplification and detection by gel electrophoresis, the target sequence was recovered and ligated to the linearized pBI121-GFP vector. The recombinant plasmid was then transferred in T1 cell, and the positive clones were further confirmed by PCR.

The recombinant vector was transformed into GV3101 cell, and the positive clones confirmed by PCR amplification were incubated in LB medium and shaken until the OD value of the bacterial solution is 0.6, and it was further collected by centrifugation at 5000 r/min for 10 min. The bacteria solution was then suspended twice into the MS liquid medium containing 10 mM MES, 150 μM acetosytingone and 10 mM MgCl_2. After standing for 3 h at room temperature, it was injected into tobacco leaves with a 1 mL needle and observed in laser scanning confocalmicroscopy (LSM880, Zesiss) after 48 h.

Seeds of the ‘H8’ variety were sown in a greenhouse at the Institute of Bast Fiber Crops at the Chinese Academy of Agricultural Sciences. Greenhouse growth conditions have been previously reported (Jiang et al., 2022). Auxin (indole-3-acetic acid, IAA) (coolaber, Beijing, China) was dissolved in ethanol and diluted to the required concentrations (20 µM). After spaying with IAA, the leaves of ‘H8’ were sampled at 0, 1, 6 and 12 h for RNA extraction. Once the plant entered the reproductive stage, tissues including male flowers at different stages, roots, stems and leaves collected from male plants of the ‘H8’ variety were used for RNA extraction.

The two varieties, ‘H8’ and ‘Y7’, were grown in the greenhouse, and the growth conditions have been previously reported (Jiang et al., 2022). When the plants were at the flowering stage in the greenhouse, the CBD content of male and female flowers was measured as previously reported (Mandrioli et al., 2019).

Total RNA was extracted using EASYspin Plus Plant RNA Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to previously published protocols (Zhu et al., 2020). After assessing RNA quality on agarosegels, reverse transcription were performed according to the instructions of the Evo M-MLV RT Premix for qPCR Kit (Accurate, Changsha, China), and the SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate, Changsha, China) was used for real-time quantitative reverse transcription PCR (qRT-PCR) according to published protocols (Zhou et al., 2022). The primers used for qRT-PCR analysis are listed in Supplementary Table S1 . Data analysis was performed using the 2^−ΔΔCt method.

Statistical analysis was performed using SPSS 18.0 software. Values are the mean and SD of three independent experiments with three replicates each. For statistical analysis, data were analyzed by one-way analysis of variance (ANOVA), and different letters indicate significant difference at P < 0.05. The correlation coefficient between the expression levels of genes and CBD content was analyzed via Pearson’s correlation coefficient and Student’s t-test.

We used 22 Arabidopsis ARF proteins as a query for BLAST with the cannabis protein database and identified 46 ARF candidate genes. After examining the conserved domains using the NCBI CDD databases, 22 cannabis ARF proteins were obtained. Their physicochemical properties were analyzed using ProtParam (http://web.expasy.org/protparam/). As shown in Table 1 , the lengths of the CsARF proteins ranged from 353 (CsARF22) to 1147 (CsARF9) aa, molecular weights ranged from 39.62 to 128.29 kDa, pI varied from 5.25 to 9.46, the grand average of hydropathicity ranged from -0.738 to -0.408, and the aliphatic index varied from 65.25 to 81.14 ( Table 1 ).

To investigate the structural diversity of CsARF genes, we analyzed the gene structure of CsARF using TBtool and MEME software. As shown in Figure 1 , all CsARF genes except for CsARF1, CsARF2 and CsARF3 contained 3′ and 5′ UTR regions, and their proteins contained 1~15 introns and 2~16 exons. Motif analysis revealed that the number of motifs ranged from 2 to 10 ( Figure 2 ). To analyze the evolutionary relationships between the CsARF genes of different species, a phylogenetic tree was constructed using ARFs from Arabidopsis and rice. A total of 69 ARF proteins were observed, including 44 genes from dicotyledonous plants (e.g., Arabidopsis and cannabis) and 25 from monocotyledonous plants (e.g., rice). As shown in Figure 3 , the ARF proteins were grouped into four major subgroups: subgroups I, II, III, and IV. Subgroup I was the largest subfamily, containing 34 members, whereas subgroup IV contained only two members ( Figure 3 ).

Cis-regulatory elements located the promoter of a gene and acted as regulators of functional genes by binding to transcription factors. Therefore, the distribution of Cis-regulatory elements was further studied. As shown in Figure 4 , nine different types of Cis-regulatory element were identified, among which 5 Cis-regulatory element related to hormones including auxin (IAA), Methyl Jasmonate (MeJA), gibberellin (GA), abscisic acid (ABA) and salicylic acid (SA), and light responsiveness was the main type of Cis-regulatory element in the promoter of CsARF gene. In addition, among the CsARF genes, CsARF3 contains the most types of response elements (7), while CsARF5 contains the least types (2) ( Figure 4 ).

Chromosomal distribution analysis revealed that 22 CsARF genes were unevenly distributed across the nine chromosomes of the cannabis genome, with the exception of chromosome 9 ( Figure 5 ). Among these nine chromosomes, chromosome 1 contained the highest number of CsARF genes, while chromosomes 4, 5, and X contained only one gene ( Figure 5 ). Three pairs of tandem duplication genes were identified on chromosomes 1 (CsARF10/CsARF11), 2 (CsARF19/CsARF21), and 6 (CsARF1/CsARF2/CsARF3), indicating that the expansion of the ARF family in cannabis was associated with tandem duplication events. Duplication events of ARF genes were also investigated in the cannabis genome. Only one pair of duplicated genes (CsARF10/CsARF11) was identified in the cannabis genome ( Figure 6 ). To further understand the evolutionary mechanism of the ARF family in cannabis, collinearity diagrams of the ARF family from two dicotyledonous plants (soybeans and cannabis) and one monocotyledonous plant (rice) were constructed. As a result, 4 pairs of orthologous genes were identified between cannabis and rice ( Figure 7 ), fewer than those identified in soybean and rice (29 pairs). Further, among these genes, the orthologs of two CsARF genes, CsARF4 and CsARF19, were identified in both rice and soybean. CsARF18 was only identified in rice, whereas 11 CsARF genes (CsARF5, CsARF6, CsARF8, CsARF9, CsARF10, CsARF11, CsARF14, CsARF15, CsARF16, CsARF17 and CsARF20) were only present in soybean. The remaining genes were not identified in the duplicated blocks ( Figure 7 ).

Subcellular localization predication of the CsARFs was performed, and the results showed that most of the CsARF proteins were localized in the nucleus, except for the single protein CsARF22, which localized in the cytoplasm ( Table 1 ). To verify the results of subcellular localization predication, CsARF2 was then randomly selected for subcellular localization assay in tobacco leaves. As shown in Figure 8 , CsARF2 was localized in the nucleus of tobacco leaf cells compared with the control (the empty vector pBI121-GFP) and the positive control (AtARF6 in Arabidopsis). These results indicate that CsARF2 functions as a transcriptional regulator in the nucleus, and may involve in the regulation of growth and development in cannabis.

To explore the potential role of CsARF genes in cannabis development, the expression profile of CsARF genes was analyzed in five tissues obtained from the transcriptome data of ‘USO14’ monoecious cultivars in our laboratory (unpublished). As a result, 22 CsARF genes exhibited differential expression patterns in different tissues. Six CsARF genes, including CsARF1, CsARF2, CsARF3, CsARF13, CsARF19 and CsARF21, were preferentially expressed in male flowers; CsARF5 and CsARF16 were highly expressed in female flowers; five CsARF genes (CsARF6, CsARF7, CsARF9, CsARF11 and CsARF15) showed high expression levels in the stem; and the transcript levels of CsARF4 and CsARF8 were higher in the roots than those in the other four tissues ( Figure 9 ).

To gain insight into the role of CsARF genes in male development, we investigated the expression patterns of the six genes with the highest transcript levels at different stages of male flower development ( Figure 10A ). As shown in Figure 10B , three CsARF genes including CsARF3, CsARF13 and CsARF21, were found to be highly expressed at all female flower stages compared to the other four tissues, suggesting that these genes may be involved in the female flower development.

To investigate the possible role of CsARF genes in the regulation of CBD biosynthesis, the expression levels of all CsARF genes in female and male cannabis varieties with high CBD content ‘H8’ and low CBD content ‘Y7’ were detected by qRT-PCR ( Figure 11A ). The CBD content of female and male tissues of ‘H8’ was higher than that of ‘Y7’ ( Figure 11A ). Eleven genes were more highly expressed in ‘H8’ female flowers (H8-F) than in Y7 female flowers (Y7-F), and 15 genes showed higher transcript levels in ‘H8’ male flowers (H8-MF) than in Y7 male flowers (Y7-MF) ( Figure 11B ). Furthermore, correlation analysis between the expression level of CsARF genes and CBD content in male and female flower tissues of two cultivars ‘H8’ and ‘Y7’ showed that the expression level of CsARF13 was negatively correlated with CBD content, while the expression levels of six genes (CsARF5, CsARF10, CsARF11, CsARF14, CsARF21 and CsARF22) were positively correlated with CBD content ( Figure 11C ).

ARFs are important regulators of auxin signaling as they are sensitive to auxin. To investigate whether ARFs respond to auxin, cannabis ‘H8’ leaves were treated with IAA and used for gene expression analysis at 0, 1, 6 and 12 h. Half of all CsARF genes were randomly selected for qRT-PCR analysis. As a result, nine genes were induced by IAA treatment except for CsARF11 and CsARF12 ( Figure 12 ). However, their expression patterns were significantly different. CsARF6 and CsARF9 were strongly induced at 1 h after IAA treatment, and the expression of CsARF1, CsARF2, CsARF3 and CsARF22 increased at 1 h and peaked at 6 h after IAA treatment, whereas the transcript levels of the remaining IAA-induced genes peaked at 12 h after IAA treatment ( Figure 12 ).