M. xiaojinensis (Mx) was used in these experiments because of its high apomictic rate to ensure stable and robust juvenile materials (Li et al., 2004). The apomictic origin of donor trees used for leafy cutting collection has been confirmed by simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers (Wang et al., 2012). Cuttings were excised from basal suckers (juvenile phase, Mx-J), shoots from the canopy of reproductively mature trees (adult phase, Mx-A), and rejuvenated plants via in vitro apical meristem culture (rejuvenated or juvenile like, Mx-R; Xiao et al., 2014).

Semi-lignified leafy cuttings (8–10 cm in length) were excised from actively growing shoots of donor plants, dipped 1 cm in depth into a solution containing 3000 mg/L indole butyric acid (IBA) (V900325, Sigma-Aldrich, St. Louis, MO, USA) for 1 min, and then the cuttings were plugged into 50 cell trays containing fine sand as a rooting medium. The cuttings were incubated in a solar greenhouse under 90–95% relative humidity (Xiao et al., 2014). Cuttings treated with an IBA-free medium solution were used as controls. The experimental errors were managed by using a complete randomized design for three biological replicates, each including at least 50 leafy cuttings. Rooting parameters were scored at 35 d after application (Xiao et al., 2014).

Nine miR156 precursor genes were previously identified in apple genome (Ma et al., 2014). MdMIR156a6 (Genome location: MDC018927.245), which relative transcript level was higher than the other members, was chosen for overexpression study (Supplementary Figure 1). The apple MdMIR156a6 DNA fragment was amplified from Malus domestica genomic DNA. Artificial target mimics were generated by modifying the sequence of the AtIPS1 gene to knock-down miR156 expression (Franco-Zorrilla et al., 2007). The PCR products were sequenced by BGI (Shenzhen, China). All constructs were cloned behind the constitutive CaMV 35S promoter in the pBI121 vector, and then introduced into Nicotiana benthamiana by Agrobacterium tumefaciens-mediated transformation (Horsch et al., 1985).

MxSPLs coding sequences were amplified from M. xiaojinensis cDNA. The miR156-resistant SPLs (rSPLs) were made by two rounds of mutagenic PCR using KOD-Plus Nero DNA polymerase (KOD-401, TOYOBO LIFE SCIENCE, Japan), and sequencing confirmed the mutations by BGI (Shenzhen, China). The rSPLs variants were driven by the CaMV 35S promoter in the pBI121 vector. These constructs were introduced into 35S:MdMIR156a6 transgenic tobacco leaves by A. tumefaciens-mediated transformation. The infected leaves were selected on MS medium supplemented with 100 mg/L kanamycin and 300 mg/L cefotaxime sodium to generate rSPL and miR156 co-overexpressing transgenic lines. All primers are listed in Supplementary Table 1. Transgenic tobacco plants were proliferated by subculture on hormone-free MS medium. Rooting rate and the number of adventitious roots were recorded at 5, 7, 9, 11, and 13 d after subculture. Plants were incubated at 25°C in long-day light conditions.

Mx-J, Mx-A, and Mx-R cutting samples were collected at 0, 14, and 28 d after IBA treatment; cuttings from 35S:MdMIR156a6 and 35S:MIM156 transgenic tobacco lines were excised at 0, 3, and 6 d after subculture on hormone-free MS medium. All samples were collected from three biological replicates and n = 10 in each replicate. A one-centimeter section from the bottom of each cutting was excised and fixed in a 5:50:5 (v/v/v) formaldehyde/ethanol/acetic acid (FAA) solution overnight at room temperature. Cuttings were dehydrated in an ethanol series (70, 85, 95, and 100%), infiltrated with xylene, and embedded in paraffin. Then, 15 μm-thick transverse sections were cut with a rotatory microtome (KD-2258, KEDEE, China) and stained with toluidine blue (Rigal et al., 2012).

For relative gene expression assays with M. xiaojinensis, stem bark from 0.5 to 1 cm basal sections of 20 cuttings were frozen in liquid nitrogen at 0, 6, 12, 24, 72, 120, and 168 h after IBA treatment. For gene expression profile analysis in tobacco, stems from 0.5 to 1 cm basal sections of 20 cuttings were sampled at 0, 3, 6, 12, 24, 48, and 72 h after subculture on hormone-free MS medium. Total RNA was isolated from ~500 mg of frozen tissue using a modified cetyltrimethylammonium bromide (CTAB) method (Gasic et al., 2004). The RNA was digested by DNaseI (2313A, Takara, Dalian, China) and reverse-transcribed using oligo-dT18 primers and reverse transcriptase according to the manufacturer's instructions (2641A, Takara, Dalian, China). Semi-quantitative RT-PCR results were quantified by using ImageJ 1.47v (Wayne Rasband, National Institutes of Health, USA) according to the commands in “gels submenu” to analyze one-dimensional electrophoretic gels (https://imagej.nih.gov/ij/docs/menus/analyze.html#gels). Quantitative RT-PCR were performed using SYBR green reagents (RR820A, Takara, Dalian, China) in an Applied Biosystems 7500 real-time PCR system. M. xiaojinensis EF1α or N. benthamiana EF1α was used as the expression control for these experiments. The relative expression level was calculated according to the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Three independent biological replicates and technical replicates were performed.

MicroRNA was extracted by using the RNAiso for Small RNA kit (9753Q, Takara, Dalian, China) according to the manufacturer's instruction. MiR156 expression level was analyzed by qRT-PCR as described previously (Xiao et al., 2014). The apple PIN and ARF family genes were previously identified (Devoghalaere et al., 2012; Zhang H. et al., 2015). The apple and N. benthamiana RTCS-like gene were selected by using a BLASTP search of known Maize RTCS (GenBank accession number EF051732) gene against the Apple Genome Database (https://www.rosaceae.org/) and Tobacco Genome Database (https://www.solgenomics.net/), respectively. N. benthamiana PIN and ARF genes were selected by using a BLASTP search of known Arabidopsis auxin-related genes against the Tobacco Genome Database (https://www.solgenomics.net/). All primers used for qRT-PCR are listed in Supplementary Tables 2,3.

Statistical analysis was performed using the Statistical Product and Service Solutions (SPSS) software (IBM Co., Armonk, USA). All experimental data were tested by Student's t-test or Duncan's multiple-range test.

Juvenile cuttings (Mx-J) exhibited a highly adventitious rooting ability in M. xiaojinensis (Figure 1A). Leafy Mx-J cuttings exhibited a 78.63% rooting percentage at day 35 after IBA treatment (Figure 1B). In contrast, the rooting percentage of adult cuttings (Mx-A) was significantly lower (4.38%) than that of Mx-J. The adventitious root number per cutting was also lower in Mx-A cuttings than in Mx-J cuttings (Figure 1C). Similarly, the adventitious root length in Mx-J was significantly longer than that in Mx-A cuttings. Mx-R cuttings exhibited significantly enhanced adventitious rooting percentage and adventitious root number than Mx-A or Mx-J cuttings (Figure 1).

To determine whether adult leafy cuttings have defects in the initiation of adventitious root formation primordia, cuttings were processed for histological analysis after IBA treatment. There were no obvious differences in anatomical structure between juvenile and adult softwood cuttings before IBA treatment (Supplementary Figure 2). At 14 d after IBA treatment, root primordia appeared in Mx-J and Mx-R but not Mx-A cuttings. At 28 d, root primordia penetrated the stem cortex and epidermis, and projected well beyond the stem surface in Mx-J and Mx-R (Figure 1E). In contrast, although meristematic cambium cells were observed between the phloem and xylem layers, except for callus, there was almost no differentiated root primordia detected in Mx-A cuttings even after 28 d of auxin treatment (Figure 1E).

Consistent with the rooting ability, the expression level of miR156 in Mx-J and Mx-R cuttings were significantly higher than that in Mx-A cuttings (Figure 2). To validate if miR156 regulates adventitious root formation, we generated transgenic tobacco plants expressing 35S:MdMIR156a6 or 35S:MIM156 to enhance or inhibit miR156 activity, respectively (Figure 3A). Mature miR156 expression levels were significantly up-regulated in 35S:MdMIR156a6 plants and significantly reduced in 35S:MIM156 plants (Figure 3B). Similarly, the expression levels of some miR156 target genes, including NbSPL2a, NbSPL2b, NbSPL5a, NbSPL5b, NbSPL15a, and NbSPL15b were down-regulated at least 2-fold in 35S:MdMIR156a6 stems, but at least 5-fold up-regulated in 35S:MIM156 stems (Figure 3C). 35S:MdMIR156a6 plants developed almost 20 adventitious roots per cutting, which was 4-fold more than the wild type after 13 days of culture on MS medium. Almost no adventitious roots were observed in 35S:MIM156 cuttings (Figure 3D). In addition to the difference in adventitious root number, the rate of adventitious root development in 35S:MdMIR156a6 plants was significantly faster than that measured in the wild-type plants (Figure 3E).

To check whether miR156 affects the initiation of adventitious root primordia, serial cross sections of the stems of wild-type, 35S:MdMIR156a6 expressing transgenic lines, and 35S:MIM156 expressing transgenic tobacco plants were stained with toluidine blue. When compared with the wild-type, the initiation of adventitious root primordia was accelerated and well-developed in 35S:MdMIR156a6 stem cuttings only 3 days after subculture on MS (Figure 4). By contrast, no adventitious root primordia were observed in 35S:MIM156 stem cuttings throughout the experiment (Figure 4).

To determine whether miR156 regulates adventitious root formation through modulating endogenous auxin levels or auxin polar transport, we examined adventitious rooting capacity in wild-type, 35S:MdMIR156a6, and 35S:MIM156 stem cuttings grown on MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or 1-N-naphthylphthalamic acid (NPA). The percent rooting was significantly increased in wild-type and 35S:MdMIR156a6 stem cuttings when cultured in the presence of 0.1 or 1 μM IAA (Figures 5A,B). The 10 μM IAA treatment delayed adventitious root formation in both wild-type and 35S:MdMIR156a6 plants; however, 35S:MdMIR156a6 plants exhibited a significantly higher adventitious rooting percent and adventitious root number than wild-type. In addition, with IAA application, the adventitious root number was clearly increased in both wild-type and 35S:MdMIR156a6 plants (Figures 5A,C) but IAA treatment did not rescue the adventitious rooting capacity defect in 35S:MIM156 plants (Figure 5).

Treatments with 20 μM NPA significantly inhibited the adventitious root formation in wild-type (Supplementary Figure 3); adventitious rooting was obviously delayed and adventitious root number was reduced in both wild-type and 35S:MdMIR156a6 plants treated with 20 μM NPA (Figure 6A). Indeed, rooting percent and root number in 35S:MdMIR156a6 plants were still consistently higher than that in wild-type (Figures 6B,C).

The expression levels of MxPIN3, MxPIN4, MxPIN6, MxPIN8, MxPIN9, MxPIN12, and MxPIN13 did not show obvious differences between Mx-A and Mx-R leafy cuttings during the adventitious root-induction process (Figure 7A and Supplementary Figure 4). The expression of MxPIN2, MxPIN5, MxPIN7, and MxPIN11 was not detected in any of the RNA samples tested. As shown in Supplementary Figure 4, the expression of MxPIN1 and MxPIN10 was higher in Mx-R than in Mx-A with or without IBA treatment. Both MxPIN1 and MxPIN10 expression was induced more than 2-fold after 6 h in the presence of IBA treatment in Mx-R, but not in Mx-A (Figure 7B). The relatively high expression level of MxPIN1 and MxPIN10 may contribute to adventitious rooting competitive in Mx-R cuttings. However, the relationship between miR156 and PIN gene expression was not robust in transgenic tobacco; no distinct changes were detected in the expression of any NbPIN members between wild-type, 35S:MdMIR156a6, and 35S:MIM156 tobacco plants (Figure 7C).

The MxARF4, MxARF7, MxARF14, MxARF15, MxARF16, MxARF17, MxARF19, MxARF20, and MxARF28 genes showed higher expression in Mx-A than in Mx-R cuttings with or without IBA treatment during the adventitious root formation process (Supplementary Figure 6). In contrast, expression of MxARF3 and MxARF8 was higher in Mx-R than in Mx-A cuttings (Supplementary Figure 6). MxARF21, MxARF23 and MxARF25 were down regulated in plants treated with IBA in Mx-R but not Mx-A cuttings (Figure 8A). However, no distinct changes were detected in 16 putative NbARF gene family members between wild-type, 35S:MdMIR156a6, and 35S:MIM156 tobacco plants during the adventitious rooting process (Figure 8B). This indicates that miR156 may not be associated with the expression of ARF transcription factors during the regulation of adventitious root formation.

In M. xiaojinensis, the MxRTCS-like gene was up-regulated 6–24 h after IBA treatment in both Mx-A and Mx-R cuttings, but the maximum induction of MxRTCS was observed in Mx-R cuttings at 120–168 h, which was 4-fold higher than that measured in Mx-A cuttings (Figure 9). Similarly, the expression of NbRTCS was also significantly induced 24–72 h after subculture in hormone-free medium in 35S:MdMIR156a6 plants, but was delayed 72 h subculture in hormone-free medium in wild-type. However, the expression of NbRTCS did not change throughout the experiment in 35S:MIM156 plants (Figure 9).

Of the 13 putative miR156-regulated SPL gene family members in apple genome, nine were actively expressed in both Mx-J and Mx-A plant cuttings, but did not include MxSPL3, MxSPL10, MxSPL11, and MxSPL12. To investigate which M. xiaojinensis SPL gene family member was involved in adventitious root formation, mutants of SPL genes, indicated here as resistant SPLs (rSPLs), were designed to no longer be targeted by miR156 (Supplementary Figure 8; Schwab et al., 2005). MxrSPL4a&4b, MxrSPL18, MxrSPL19, MxrSPL20, MxrSPL21&22, MxrSPL24, and MxrSPL26, driven by CaMV 35S promoter, were transformed into 35S:MdMIR156a6 transgenic tobacco plants (Supplementary Figure 9). In independent bivalent transgenic lines, 35S:rSPL4a&4b/35S:MdMIR156a6, 35S:rSPL18/35S:MdMIR156a6, 35S:rSPL19/35S:MdMIR156a6, and 35S:rSPL24/35S:MdMIR156a6 the adventitious rooting rate and adventitious root number did not significantly differ from that of plants expressing 35S:MdMIR156a6 (Supplementary Figure 10), indicating that MxSPL4a&4b, MxSPL18, MxSPL19, and MxSPL24 are not involved in adventitious rooting. In contrast, the bivalent transformants 35S:rSPL20/35S:MdMIR156a6, 35S:rSPL21&22/35S:MdMIR156a6, and 35S:rSPL26/35S:MdMIR156a6 exhibited reduced adventitious rooting ability (Figure 10A). 35S:rSPL20/35S:MdMIR156a6 and 35S:rSPL21&22/35S:MdMIR156a6 produced significantly fewer adventitious roots than 35S:MdMIR156a6 plants, but still more than wild-type plants (Figure 10B). The adventitious root number in 35S:rSPL26/35S:MdMIR156a6 plants was significantly fewer than that in 35S:MdMIR156a6 plants (Figure 10B). In addition, the adventitious root development rate in 35S:rSPL20/35S:MdMIR156a6, 35S:rSPL21&22/35S:MdMIR156a6, and 35S:rSPL26/35S:MdMIR156a6 plants were similar as that measured in wild-type, but slower than that observed in 35S:MdMIR156a6 plants 7 d after culture on MS medium (Figure 10C). These results indicate that MxSPL20, MxSPL21&22, and MxSPL26 not only affected adventitious root number but also delayed rooting rate.

The MxSPL20, MxSPL21&22, and MxSPL26 expression profile was detected at 0, 6, 12, 24, 72, 120, and 168 h after IBA treatment (Figure 11). There were no obviously differences in the expression of MxSPL20 and MxSPL21&22 between Mx-A and Mx-R cuttings; however, the expression of MxSPL26 was significantly higher in Mx-A than in Mx-R cuttings, indicating MxSPL26 could be a key SPL family member involved in adventitious rooting of leafy cuttings.

For some rooting recalcitrant woody plants, juvenility is necessary for efficient adventitious rooting. In general, cuttings from juvenile nodes root more readily than cuttings from adult nodes in woody plants (Greenwood, 1995). Indeed, miR156 expression levels were significantly higher in Mx-J and Mx-R cuttings than Mx-A cuttings (Figure 2), and the rooting ability of Mx-R and Mx-J cuttings were consistently higher than that of Mx-A cuttings (Figure 1). A significant decrease in miR156 expression was observed in shoots taken from 1.4 m trunk above ground compared to shoots taken from below 1.4 m in M. xiaojinesis seedlings (Ji et al., 2016). In our previous data, the micro-shoots from adult phase M. xiaojinnesis explants were rejuvenated successfully after 15 passages of in vitro subculture, marked by the elevated expression of miR156 expression in leaves of the micro-shoots, and coupled with recovered adventitious rooting ability and leaf lobes (Xiao et al., 2014).

When miR156 level was manipulated via transformation with a 35S:MIR156 construct in tomato, tobacco, or Arabidopsis, the adventitious rooting increased (Zhang et al., 2011; Feng et al., 2016; Massoumi et al., 2017). In the present study, the miR156 expression level was manipulated in transgenic tobacco to analyze how miR156/SPL modules are involved in adventitious rooting. Consistent with the previous reports, the adventitious rooting capacity also varied with 35S:MIM156 and 35S:MdMIR156a6 overexpressing transgenic tobacco plants (Figure 3). However, the uncoupling of miR156 expression and adventitious root formation was reported in E. grandis (Levy et al., 2014); therefore, miR156 may be necessary but not sufficient for adventitious rooting in woody plants. Except for high expression of miR156, auxin was also integrant for adventitious root formation. In absence of IBA treatment, even Mx-J and Mx-R leafy cuttings exhibit adventitious root defects (Figure 1). In agreement to this result, the adventitious rooting ability was reduced in all tobacco lines upon treatment with the polar auxin transport inhibitor NPA (Figure 6).

Auxin and miR156 are both involved in adventitious root development (Zhang et al., 2011; Feng et al., 2016; Steffens and Rasmussen, 2016; Massoumi et al., 2017). However, the interaction of miR156 and auxin signaling pathway is unexplored during adventitious root development. In comparison to the wild-type samples, NbPINs and NbARFs expressions was not substantially modulated in 35S:MdMIR156a6 and 35S:MIM156 plants (Figures 7, 8), indicating that miR156 promoted adventitious root formation independently from changing PIN and ARF genes expression. These results are supported by the transcriptome data, which show that PIN and ARF genes in Medicago sativa are not significantly differentially expressed between miR156 overexpression and wild-type plants (Gao et al., 2016). Similarly, the auxin response was not changed in either Pro35S:MIR156 or Pro35S:MIM156 Arabidopsis, indicating that miR156 does not modulate the auxin response during regulating shoot regeneration (Zhang T. Q. et al., 2015). Collectively, these findings suggest that miR156 does not modulate the early auxin response.

Conversely, auxin can induce the expressions of two MIR156 genes and two SPL genes during lateral root development in transgenic Arabidopsis (Yu et al., 2015). GUS staining revealed that MIR156B was specifically expressed in primary and lateral root primordia, MIR156D was especially active in primary and lateral root tip, and two SPL genes were also highly or specifically expressed in root (Yu et al., 2015). Hence, auxin inducing MIR156 and SPL expression may be tissue/organ specific during lateral root development. Our results revealed that the expression level of MxSPL26 or MxSPL20 or MxSPL21&22 was not obviously affected under IBA treatment (Figure 11). Similarly, miR156 expression was not affected by IBA application during the induction of adventitious root development in both juvenile and adult E. grandis stem cuttings (Levy et al., 2014). Thus, miR156/SPL modules were not downstream targets of auxin during adventitious root formation.

Rtcs and Rtcl in maize are orthologs of CRL1 in rice (O. sativa) and of AtLBD29 in A. thaliana, and all of these genes are involved in the initiation of adventitious root formation and are targets of the ARF gene family (Inukai et al., 2005; Taramino et al., 2007; Liu J. C. et al., 2014). Although MxRTCS-like gene expression was induced in both Mx-A and Mx-R cuttings after IBA treatment, the distinct fold-change in expression occurred only in Mx-R cuttings (Figure 9). In agreement with these finding, the NbRTCS expression was significantly induced in 35S:MdMIR156a6 transgenic tobacco plants, but no substantial changes were detected in 35S:MIM156 transformants (Figure 9). These data suggest that high expression level of miR156 is required for auxin inducing expression of RTCS-like during adventitious root formation in M. xiaojinesis and transgenic tobacco. Therefore, we speculate that decline of miR156 expression in adult phase leafy cuttings may inhibit transcript abundance of RTCS-like gene induced by auxin, thereby reducing the adventitious rooting capacity (Figure 12). The histological features showed that Mx-A leafy cuttings and 35S:MIM156 transgenic tobacco plants exhibited defect in adventitious root primordia initiation (Figures 1E,4), which provided an evidence for above mentioned speculation. In agreement with our observations, maize mutant rtcs and rice mutant rtcl both failed in adventitious root primordia initiation (Inukai et al., 2005; Muthreich et al., 2013). However, the molecular mechanism underlying miR156 modulate auxin induced RTCS-like gene expression remains unknown.

Although, the expressions of PIN and ARF genes were independently from the miR156 expression level, the response of MxPIN1 and MxPIN10 gene expression to auxin treatment differed in Mx-R and Mx-A samples. The different responses of PIN family members to auxin treatment between Mx-R and Mx-A cuttings may be caused by other developmental signals, such as glutathione content. In apple seedlings, the glutathione content and glutathione/glutathione disulfide ratio were much higher in the juvenile phase than in the adult phase, and modulating glutathione content caused concomitant changes in miR156 expression levels (Du et al., 2015). It has been proved that the reduction of glutathione availability by L-buthionine-(S,R)-sulfoximine (BSO) treatment reduced the expression of PIN1, PIN2, and PIN3 in Arabidopsis (Bashandy et al., 2010).

In addition, MxARF4, MxARF7, MxARF14, MxARF15, MxARF16, MxARF17, MxARF19, MxARF20, and MxARF28 showed relative higher expression levels in Mx-A than in Mx-R cuttings during adventitious root formation (Supplementary Figure 6). Interestingly, the peptide sequences of these ARFs, except for ARF20, are enriched in serine, proline, and leucine in their middle regions (Supplementary Figure 11). Arabidopsis ARFs with this middle region are usually transcriptional repressors (Guilfoyle and Hagen, 2007). The differential expression of MxPIN and MxARF might partly cause Mx-A recalcitrant to root formation.

MiR156 and its targeted SPL genes were demonstrated to control plant phase transition and related traits such as cell size and number, trichome development, anthocyanin synthesis, leafy morphology, flowering time, and lateral root development (Wang et al., 2008; Shikata et al., 2009; Yu et al., 2010, 2015; Gou et al., 2011). Yet, the function of SPL genes involving in adventitious rooting are largely unexplored. It was reported that hypocotyls of spl2/9/11/13/15 mutants, as well as plants transformed with 35S:MIR156A, produced the same number of adventitious roots as wild-type plants (Xu et al., 2016). However, it is not clear which SPL gene involved in regulating adventitious rooting in Arabidopsis, because the expression level of miR156 targeted SPLs had no significantly difference among spl2/9/11/13/15 mutants, 35S:MIR156A plants and wild-type young seedlings (Xu et al., 2016). Here, MxSPL26 was experimentally confirmed to be involved in inhibiting adventitious root formation most possibly in function redundancy with SPL20 and SPL21&22, but SPL26 seemed to play a major role. The expression of MxSPL26 was constantly higher than that of MxSPL20 and MxSPL21&22 in M. xiaojinensis cuttings. MxSPL26 relative expression exhibited a 5- to 10-fold increase in stem bark from Mx-A cuttings than in that from Mx-R after 72~120 h from plugging into the media, but no obvious differences in the expression of MxSPL20 and MxSPL21&22 genes were found between Mx-A and Mx-R (Figure 11). The number of roots per plant was significantly reduced in all the 35S:rMxSPLs/35S:MdMIR156a6 lines but MxSPL26 resistant form transgenic tobacco lines produced the least number of adventitious roots compared with rMxSPL20, rMxSPL21&22 and 35S:MdMIR156a6 transgenic lines (Figure 10 and Supplementary Figure 10). According to the phylogenetic tree, MxSPL20 and MxSPL21&22 exhibited close evolutionary, but quite far phylogenetic relationship from MxSPL26 (Supplementary Figure 12). Notably, although SPL3, SPL9, and SPL10 were all involved in repressing Arabidopsis lateral root development, SPL10 seemed to play a major role (Yu et al., 2015). Similarly, AtSPL3 and AtSPL9 exhibited close evolutionary, but quite far phylogenetic relationship from AtSPL10 (Supplementary Figure 12).

In conclusion, in semi-lignified leafy cuttings of M. xiaojinensis, a relatively higher expression of the miR156 is necessary for adventitious root formation. MiR156 functions via its target gene MxSPL26 and rooting-related genes such as MxRTCS-like; but acts independently of MxPIN or MxARF family members in response to auxin. These results provided a potential strategy for the improvement of the adventitious rooting ability of perennial woody plants via manipulating miR156 and SPL gene expression.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.