miR156, one of the most conserved miRNA families, plays very important roles in the process of plant growth and development by direct cleavage of SPL transcripts [42]. In the model plant Arabidopsis, 10 of 16 AtSPLs are direct targets of AtmiR156 [42]. In rice, 11 of 19 OsSPL genes are targeted by OsmiR156 [9]. We predicted CpSPLs targeted by AtmiR156 using the on line plant target prediction tool. The prediction result indicated that a total of 10 CpSPLs were the targets of AtmiR156, eight of which (including CpSPL3, CpSPL6, CpSPL9-13, and CpSPL15) were targeted in the coding regions, while two (CpSPL4 and CpSPL5) in 3′ UTR regions (Fig. 1).
We constructed a phylogenetic tree of 15 CpSPLs and 16 AtSPLs using MEGA X with ML method. As shown in Fig. 2, 31 SPLs from two species were classfied into seven groups, named from G1 to G7, and each group consisted of at least one SPL from C. pilosula and one from A. thaliana. There are only two members in G3 (AtSPL8 and CpSPL8) and G7 (AtSPL7 and CpSPL7). G5 is the largest group with three AtSPLs (AtSPL6/9/15) and four CpSPLs (CpSPL3/6/9/15). It was interesting that all the members in G1, G3, and G7 belong to non-miR156-targeted SPLs, including six AtSPLs (AtSPL1/7/8/12/14/16) and four CpSPLs (CpSPL1/7/8/14). Except for CpSPL2, All the members in G2, G4, G5, and G6 were miR156-targeted SPLs. In Arabidopsis, AtSPL2/10/11, three members closely related, regulate root regeneration by inhibiting auxin biosynthesis [43]. Phylogenetic tree clustered CpSPL2, CpSPL10, AtSPL2, AtSPL10, and AtSPL11 in G4, indicating CpSPL2 and CpSPL10 are probably involved in root growth.
Gene structure and conserved motif analysis
To clarify the structural diversities of 15 CpSPLs, we performed gene exon/intron structure analysis. The result displayed that the number of introns had a high variation and ranged from one to ten (Fig. 3). Interestingly, we found that most CpSPLs in the same group share similar structure. For instance, CpSPL1 and CpSPL14, belonging to G1, have ten introns, respectively. CpSPL4 and CpSPL5, members in G6, have only one intron, respectively (Fig. 3).
To explore the conserved motifs, 15 CpSPLs were subjected to analysis with MEME program. Among the 12 conserved motifs identified (Fig. 4, Table S3), motif 1, motif 2, and motif 3 existed in all the 15 CpSPLs and formed the conserved SBP domain. Similar motif composition existed in the same group. For example, CpSPL2 and CpSPL10 in G4 all consisted of five conserved motifs (motif 1/2/3/10/11). The motif composition in CpSPL9 was completely consistent with that in CpSPL15, suggesting that CpSPL9 and CpSPL15 probably have similar and redundant functions in plant development.
Cis-acting elements analysis of CpSPLs promoter regions
We analyzed the cis-acting elements of 15 CpSPLs promoter regions and light responsive elements (including G-box, GATA-motif, GTGGC-motif, AE-box, TCT-motif, and chs-CMA2a), hormone responsive elements [such as gibberellin (GARE-motif), MeJA (CGTCA- and TGACG-motif), and abscisic acid (ABA) (ABRE)], stress responsive elements [such as drought (MBS), low-temperature (LTR), and anaerobic induction (ARE)], and CAT box related to meristem expression were found in their promoter regions (Table S4). Among these cis-elements, MeJA-responsive elements existed in the promoter regions of almost all the CpSPLs except for CpSPL9 and CpSPL13, and ABA-responsive element (ABRE) existed in the promoter regions of 10 CpSPLs (including CpSPL1, CpSPL6-10, CpSPL12, and CpSPL14-15).
Spatiotemporal expression analysis of CpSPL genes
We investigated the expression patters of 15 CpSPLs in the leaves, stems, and roots from one-, two-, and three-month-old seedlings, and the flower and calyx from the plants at the flowering stage by qRT-PCR assay. The results showed that most CpSPLs expressed in almost all the tissues (Fig. 5). Compared with other genes, the expression level of CpSPL7 was more constant in all the tissues tested. CpSPL8 showed highest level in calyx. CpSPL5 was expressed at relatively higher levels in leaf and calyx. The expression levels of CpSPL3, CpSPL8, CpSPL10, CpSPL12, and CpSPL13 in the stems gradually decreased with the maturation of the seedlings. CpSPL1 and CpSPL14, two members in G1, showed similar expression patterns and their expression levels in the root increased gradually with the maturation of the seedlings. In addition, the expression patterns of CpSPL9 and CpSPL15 were highly similar, with higher levels in flowers and 3-month-old roots. In summary, spatiotemporal expression analysis results indicated that CpSPL genes exhibited various expression patterns, which provide preliminary information for understanding their potential functions in the development of C. pilosula.
Expression profiles of CpSPLs under various conditions
To assess the expression profiles of 15 CpSPL genes under various treatments (NaCl, MeJA, and ABA), a histogram was generated using the relative expression level (Fig. 6). When treated with NaCl, the transcript levels of eight CpSPLs (CpSPL1, CpSPL2, CpSPL4, CpSPL6, CpSPL7, CpSPL11, CpSPL14 and CpSPL15) and four CpSPLs (CpSPL5, CpSPL8, CpSPL10, and CpSPL13) were significantly up-regulated and down-regulated, respectively. Among those, CpSPL2, CpSPL6, and CpSPL11 increased to 6.02, 5.66, and 7.94 times than the control, respectively, while CpSPL5 and CpSPL8 decreased to 20.00 and 12.50 times than the control, respectively (Fig. 6A). For MeJA treatment, the transcript levels of CpSPL4, CpSPL6, CpSPL14 and CpSPL15 significantly increased, with the highest change folds in CpSPL15 (3.03 folds). The transcript levels of five CpSPL genes (CpSPL3, CpSPL5, CpSPL8, CpSPL10, and CpSPL11) significantly decreased, with 14.28, and 10.00 change folds in CpSPL5 and CpSPL8, respectively (Fig. 6B). Under ABA treatment, eight CpSPLs (CpSPL1, CpSPL4, CpSPL6, CpSPL7, CpSPL9, CpSPL12, CpSPL14 and CpSPL15) responded positively to the treatment, while three genes (CpSPL3, CpSPL5 and CpSPL8) responded negatively to ABA treatment. Among those genes, CpSPL15 and CpSPL8 exhibited highest upregulation and downregulation, respectively (Fig. 6C).
Overexpression of CpSPL2 or CpSPL10 promotes the growth of C. pilosula hairy root
To investigate the function of CpSPL2 and CpSPL10 in root development, we generated CpSPL2-overexpressing or CpSPL10-overexpressing transgenic hairy roots. The expression level of CpSPL2 or CpSPL10 in the transgenics was examined by qRT-PCR (Fig. 7A, B). Two independent CpSPL2-overexpressing lines (CpSPL2-OE3 and CpSPL2-OE5) and CpSPL10-overexpressing lines (CpSPL10-OE2 and CpSPL2-OE3) with dramatically elevated CpSPL2 or CpSPL10 expression were selected for further analysis. In comparison to the control, the hairy roots overexpressing CpSPL2 or CpSPL10 grew faster (Fig. 7C, D, and E). When the transgenic hairy roots with the length about 1.0 cm were cultured for one month, the biomass of CpSPL2-OE3, CpSPL2-OE5, CpSPL10-OE2, and CpSPL10- OE3 was 2.19, 1.98, 3.15, and 2.83 times that of the control (EV2), respectively (Fig. 7F). Our results indicated that both CpSPL2 and CpSPL10 promote the growth of hairy roots.
Overexpression of CpSPL2 or CpSPL10 promotes accumulation of lobetyolin and total saponins in C. pilosula hairy root
To evaluate the impact of CpSPL2 or CpSPL10 on active ingredients, HPLC and UV spectrophotometer were used to determine the concentrations of lobetyolin and total saponins in those transgenic lines, respectively. It was surprising that the production of both lobetyolin and total saponins were greatly increased in CpSPL2-OE or CpSPL10-OE lines. The concentration of lobetyolin in CpSPL2-OE3, CpSPL2-OE5, CpSPL10-OE2, and CpSPL10-OE3 was 6.43, 6.25, 6.29, and 7.03 times that of the control (EV2), respectively (Fig. 8A). The concentration of total saponins in CpSPL2-OE3, CpSPL2-OE5, CpSPL10-OE2, and CpSPL10-OE3 was 3.18, 2.72, 1.81, and 1.94 times that of the control (EV2), respectively (Fig. 8B). In summary, CpSPL2 and CpSPL10 promote not only the growth of hairy roots but also accumulations of lobetyolin and total saponins.