Abstract Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L^−1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1–1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants. Keywords: tetraploid Robinia pseudoacacia L., dark pretreatment, adventitious rooting, miRNA-seq, RNA-seq 1. Introduction Tetraploid Robinia pseudoacacia L. (R. pseudoacacia L.) is a leguminous, deciduous, ornamental tree species that is artificially produced by doubling the chromosomes, with the application of colchicine, in diploid cells of the congeneric species, known as black locust (2n = 92). R. pseudoacacia L. is native to south Korea, and was introduced into China in 1997 [[42]1,[43]2]. Economically, this species has a significant role in supplying wood production, honey production, and feed for animals; it also favors the rapid fixation of elite genotypes [[44]3,[45]4]. Furthermore, tetraploid R. pseudoacacia L. is highly adaptable to harsh environments, including cold, drought, salt, pest infestation, and nutrient deficiencies [[46]5], which increases its economic value for further research studies [[47]6]. The propagation of these species is difficult due to its long generation periods, and thus life cycle, which is a limiting factor. Currently, clonal propagation through stem cuttings is the only way to deploy genetically improved tetraploid R. pseudoacacia L. varieties [[48]7]. Several reports have also shown that in vitro propagation of tetraploid R. pseudoacacia L. is an effective method to produce large numbers of clonal plants [[49]8], because woody species are usually more difficult to root than herbaceous plants [[50]9]. There are different types of roots, such as the primary root, secondary root, crown root, and adventitious roots (AR). Roots that are formed spontaneously, or from non-rooted tissues, are known as AR. AR formation is an inheritable quantitative trait in plants controlled by multiple endogenous and environmental factors, such as the genetic background of the maternal plant; the application of exogenous hormones; and environmental conditions, such as light and etiolation [[51]10,[52]11]. The partial or complete absence of light for a specific time period is termed etiolation [[53]12]. Etiolation is known to significantly induce AR formation in different species, causing partial rejuvenation [[54]11,[55]13,[56]14,[57]15]. Etiolation changes the physiology, anatomy, and molecular mechanisms of different tree species [[58]16]. Moreover, etiolation regulates essential hormone-related genes and hormone levels [[59]17]. For example, increased Indole-3-acetic acid (IAA) levels and expression of indole-3-pyruvate monooxygenase YUCCA (YUC) and auxin efflux carrier (PIN) genes were observed in Arabidopsis seedlings [[60]18]. CYP90 levels increased under weak light throughout brassinosteroid (BR) biosynthesis during petiole elongation in Arabidopsis [[61]19]. Hormonal signaling induces many metabolic processes that depend on light signals during plant growth and development [[62]20]. During the light signal transduction pathway, hormone action occurs in a downstream manner [[63]21]. Additionally, plant hormones are also the most important modulators of AR formation [[64]22]. Auxins play an essential role during AR development in many plants. Among auxins, indole-3-butyric acid (IBA) is the most widely employed exogenous auxin used for the stimulation of AR formation. However, the molecular mechanisms by which auxin regulates the process of AR formation are poorly understood. In cuttings of many plants, auxins (IBA) play a crucial role in inducing AR [[65]23]; it is experimentally proven in Arabidopsis that IBA acts mainly via conversion to the biologically active IAA in the cuttings [[66]24]. However, in some plant species, such as petunia [[67]25], adventitious roots are produced without chemical stimulation, and are instead controlled by polar auxin transport and local accumulation of the endogenous auxin (IAA) in the rooting zones. The treatment of cuttings with the IBA has been reported to significantly improve the rooting rate in tetraploid Robinia pseudoacacia L. [[68]12], Juglans regia L. [[69]26], Pinus contorta [[70]27,[71]28], Malus pumila [[72]29], and Pinus radiata [[73]30]. Regarding in vitro induction of AR formation, IBA is considered to be more stable and effective than IAA, and is widely used in vegetative propagation [[74]22]. Moreover, additional phytohormones, including ethylene, cytokinins, jasmonate, abscisic acid, and gibberellin, act in concert with auxin in a complex regulatory network of AR formation [[75]31]. Overall, these observations have revealed the essential role of auxin in the complex process of AR formation. Deep sequencing [[76]32,[77]33] has improved our understanding of these complex biological processes of AR formation at a different levels, especially at the genetic level. However, information at the level of miRNA and their targets is still limited; analyzing gene expression is an effective way to improve understanding of the development of AR at the molecular level. MicroRNAs are universal, highly conserved, 20–24 nucleotides (nt) long non-coding RNAs, first revealed in eukaryotic organisms. Transcriptomic and transgenic methods have been employed to discover the functions of these microRNAs, as negative regulators of their target genes, during AR development in different plant species [[78]34,[79]35]. Individual miRNAs, however, cannot regulate AR formation; they must be associated with their target genes. For example, some auxin responsive factors, such as ARF17, ARF6, and ARF8, are regulated by miR160 and miR167 during AR development in Arabidopsis [[80]36]; miR390 and ARFs form an auxin-responsive regulatory network that regulates lateral root initiation and growth from the main root [[81]37]. In addition, a novel regulatory pathway, involving bidirectional cell signaling mediated by miR165 and miR166 and the transcription factors SHR (SHORT ROOT) and SCR (SCARECROW), has been recognized as establishing root cellular functions [[82]38]. Hou et al. (2019) report that the overexpression of miR171 and miR390 in tomato plants can increase the lateral root number compared to wild-type plants [[83]39]. Therefore, microRNAs are believed to be important regulators of plant growth and development. Elevated levels of miR156 promote AR development in tomato, tobacco, and maize [[84]40]. Comprehensively, the formation of the root is a dynamic process that involves the integration of plant hormones, transcriptional regulators, and microRNAs to produce the correct AR formation [[85]41]. Currently, clonal propagation through stem cuttings is the only way to deploy genetically improved tetraploid R. pseudoacacia L. varieties [[86]7]. However, the AR mechanism, and key molecular factors that control AR formation under in vitro conditions, have not been fully explored. This study was intended to investigate the in vitro adventitious root formation of tetraploid R. pseudoacacia L. using dark-pretreated micro-shoot cuttings as plant materials. The main objectives of this study were to analyse (1) the effects of auxin on AR formation in dark-pretreated micro-shoot cuttings, (2) which genes are playing a role during IBA-dependent AR formation, while setting a particular focus on miRNAs and their interaction with putative target genes during the whole process of AR formation. To date, there is no investigation that describes how miRNAs and their targets modulate AR formation in tetraploid R. pseudoacacia L. Sequenced transcriptome and miRNA data were used to study the molecular mechanisms during etiolation-induced AR formation in tetraploid R. pseudoacacia L. It was anticipated that a better understanding of the underlying mechanisms in dark-pretreated micro-shoot cuttings will accelerate the genetic improvement of tetraploid R. pseudoacacia L. and provide the basis for further study on other tree species. 2. Materials and Methods 2.1. Plant Material, Growth Conditions, and Sample Collection In the present study, tissue culture-grown micro-shoots (donor plants) of “tetraploid R. pseudoacacia L. clone-38” (recalcitrant clones) were used to study AR formation. Donor plants were propagated for four months in tissue culture medium in Murashige and Skoog (MS) [[87]42], supplemented with 0.2 mg L^−1 IBA, 30 g L^−1 sugar, and 6 g L^−1 agar at pH 5.8 in the laboratory at Beijing Forestry University, Beijing, China. Temperatures were maintained at 24 ± 1 °C with a 16/8-h photoperiod (photosynthetic photon flux density (PPFD) = 40–50 μmol m^−2 s^−1). These donor plants were used for the further experiment, in which donor plants underwent complete dark pretreatment for five days. Dark-pretreated donor plants were further divided into two categories, control (non-IBA) and treated (IBA), in which treated plants were supplemented with auxin IBA 0.2 mg L^−1. Furthermore, lower portions, approximately 1–1.5 cm, of control and treated plants were evaluated to identify specific timing of AR formation after completion of five days of dark treatment. Treatment method was conducted according to Munir et al. [[88]43], with slight modifications. On the basis of the specific timing of AR formation, samples for RNA-seq and miRNA-seq were collected from both non-IBA and IBA-treated micro-shoot cuttings and immediately stored in liquid nitrogen at −80 °C. 2.2. Paraffin Section Preparation and Microscopic Examination At least 20 basal stem tissues (0.5 cm) were collected, with three replicates, for each time point from both control (non-IBA) and treated (IBA) plants to examine histological changes during AR formation at specific time points, i.e., 0, 12, 36, 48, and 72 h after excision (HAE). The collected tissues were fixed in formaldehyde acetic acid (FAA) solution (50% ethanol, 38% formaldehyde, 5% acetic acid) and treated as previously described [[89]44]. Thin sections of 8 μm thickness were cut with a rotary microtome (LEICA RM2235) for histological observation. The cross-sections were placed on slides, stained with safranin (1%) and fast-green (0.5%) solution, and examined under a light microscope to observe the AR histology. All sections were photographed by LEICA DMI40008 microscope at the different time points. 2.3. RNA and Small RNA Library Construction and Sequencing The same methodology was applied to collect samples for miRNA-seq and RNA-seq as that conducted for the collection of histological samples from the basal portion (0.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. First, total RNA extraction (15 samples) from the cuttings at the time of excision (0 HAE) and of the IBA treatment of “tetraploid R. pseudoacacia L. clone-38” micro-shoot cutting bases collected at 12, 36, 48, and 72 HAE, was carried out according to the protocol described by Munir (2021) [[90]43], in which RNA purity (OD260/280) was ensured using ultraviolet spectrophotometer Nanodrop. Total miRNA was also extracted from the basal portion of “tetraploid R. pseudoacacia L. clone 38” micro-shoot cuttings (0.5 cm) at the above-mentioned time points. After that, seven different comparison libraries (0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72 HAE) were constructed for RNA and miRNA sequencing. The final quality of the cDNA library was ensured using an Agilent2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA was fractionated on a 15% denaturing polyacrylamide gel. The miRNA regions corresponding to 18–30 nt were excised and recovered. These sRNAs were then 5’ and 3’ RNA adapter-ligated using T4 RNA ligase (Takara, Dalian, China). Ligated products were purified using an Oligotex mRNA mini kit (Qiagen, Hilden, Germany) and subsequently transcribed into cDNAs via a SuperScript Ⅱ RT (Invitrogen, Carlsbad, CA, USA). PCR amplifications were performed with primers annealed to the ends of the adapters. 2.4. Analysis of Differentially Expressed miRNAs and Their Target Genes To further reveal the possible functions of differentially expressed (DE) miRNAs and their associated target genes, all target genes were investigated against Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases ([91]http://www.genome.jp/kegg/, accessed on 15 August 2021). FDR ≤ 0.05 was the threshold for significant enrichment in GO and KEGG enrichment analyses. The expression profiles of the differentially expressed miRNAs and their target genes were visualized with TBtools software, Guangdong, China [[92]45]. 2.5. miRNA Identification during AR Formation Tag sequences derived from deep sequencing were treated via Phred and Cross-match ([93]http://www.phrap.org/phredphrapconsed.html, last accessed on 30 November 2020) in which clean reads were obtained by the removal of adapter sequences; alternatively, reads in which the presence of unknown base N was greater than 5%, and low-quality sequences (in which the percentage of low-quality bases with quality value ≤ 10 was greater than 20%) were filtered, and low-quality tags and contamination were removed from adaptor sequences not ligated to any other sequences. The high-quality miRNA reads were then trimmed from their adapter sequences. scRNAs, snRNAs, rRNAs, tRNAs, and snoRNAs were detached from the miRNA sequences by BLASTn search using the NCBI Genbank database ([94]http://www.ncbi.nlm.nih.gov/blast/Blast.cgi/ accessed on 30 November 2020) and Rfam (11.0) database ([95]http://www.sanger.ac.uk/resources/databases/rfam.html accessed on 30 November 2020). Using tag2 annotation, all annotations were summarized by BGI software using the following series of preferences: