MicroRNA319-TCP19-IAA3.2 Module Mediates Lateral Root Growth in Populus tomentosa

Abstract

MicroRNA319 (miR319) and its targets TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are well-characterized regulators of leaf and flower development, yet their role in root development remains elusive. Here, we demonstrated that overexpression of miR319a led to a decrease in the number and density of lateral roots in poplar, while repressing miR319a by short tandem target mimics (STTM) promoted lateral root (LR) development. The auxin signaling repressors IAA3.1 and IAA3.2 were upregulated in miR319a-OE plants but downregulated in miR319a-STTM plants. After exogenous applications of naphthaleneacetic acid (NAA), which exhibited the characteristics and physiological functions of the endogenous auxin indole-3-acetic acid, the number and density of LR in WT increased by 30% and 44%, respectively. In miR319a-OE plants, the LR number increased by 23% and 48%, and the LR density increased by 10% and 26%. NAA treatment can partially compensate for the phenotype of inhibited LR development caused by the overexpression of miR319a. After N-1-naphthylphthalamic acid (NPA) treatment, which is a key inhibitor of the directional (polar) transport of the auxin hormone in plants, the LR number in WT decreased by 70%. In the overexpression plants, the number of lateral roots decreased by 85–87%, and in the STTM plants, the number of lateral roots decreased by about 83%. It was proved that NPA treatment could reverse the phenotype of increased LR number in miR319a-STTM plants. Expression analysis revealed that miR319a significantly inhibited the expression of the key auxin-regulated genes IAA3.1 and IAA3.2, suggesting that auxin signaling might mediate its effects on lateral root formation. Additionally, we compared the fluorescence signal in the reporter line with GFP expression driven by the auxin-responsive DR5 promoter within the genetic backgrounds of WT, miR319a-OE, and miR319a-STTM plants, which revealed that auxin signaling was stronger in the epidermal cells and elongation zone cells in the LR of miR319a-OE plants, whereas in LR of WT and miR319a-STTM plants, auxin signaling was more pronounced in the root tip meristematic cells. Furthermore, transactivation assays and expression analysis indicated that IAA3.2 was a downstream target of TCP19. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) confirmed that TCP19 directly bound to the promoter region of IAA3.2. These findings establish that miR319a targeted and cleaved TCP19, and TCP19 further directly and negatively regulates the expression of IAA3.2, thereby controlling LR development in Populus tomentosa (P. tomentosa). The formation of LR can expand the plant root system, which is of great significance for the vegetative propagation of plants and the in-vitro regeneration of explants. Moreover, the formation of LR is an important strategy for plants to cope with environmental stresses. This study provides a theoretical basis for breeding poplars more suitable for vegetative propagation.

1. Introduction

Lateral root morphogenesis constitutes a pivotal process in tree growth and development, with particular significance in vegetative propagation, including cutting propagation [1,2]. Furthermore, lateral roots play an essential role in drought stress adaptation by enhancing hydraulic conductivity and nutrient acquisition efficiency [3]. In poplars, the development of lateral roots has a very important effect on their response to environmental stress. Studies demonstrated that poplars enhance their tolerance to stress such as drought, salt, and low-phosphorus conditions by promoting lateral root formation [4,5,6,7].

Despite its fundamental importance, the molecular regulatory networks and phytohormonal signaling pathways governing lateral root formation in plants remain inadequately characterized. While substantial mechanistic insights have been gained from model herbaceous species, notably Arabidopsis [8], the genetic and physiological mechanisms controlling lateral root initiation and elongation in economically important woody species, particularly Populus, remain largely unexplored.

Auxin signaling has been unequivocally established as a core regulatory module governing lateral root morphogenesis across diverse plant taxa [8,9]. However, the intricate crosstalk between auxin signaling cascades and other regulatory networks, particularly microRNA-mediated post-transcriptional regulation, remains poorly elucidated. Considering the multifaceted nature of lateral root organogenesis and its critical implications for tree propagation efficiency and stress resilience, comprehensive investigations into the genetic determinants and molecular mechanisms underlying this developmental process are imperative.

MicroRNAs (miRNAs), a class of small non-coding RNAs, emerged as crucial regulators of plant growth, development, and stress responses to both biotic and abiotic stimulus [10]. Among the numerous miRNAs computationally predicted and experimentally validated across plant species [11], the miR319 family represents one of the most evolutionarily ancient and phylogenetically conserved miRNA families [12,13]. MiR319 exerts its regulatory function primarily through post-transcriptional repression of TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are integral to multiple plant developmental pathways [14]. While miR319 has been extensively characterized in leaf morphogenesis and growth regulation [15,16], its role in root development remains underexplored. Previous studies have shown that miR319 participated in the development of reproductive structures, including petals and stamens [17], and contributes to secondary cell wall biosynthesis in stems [18]. In Populus, miR319a has been shown to regulate salt tolerance through regulation of xylem vessel morphology, including the vessel number and lumen area, as well as ion transport dynamics [19]. Despite these organ-specific functional characterizations, the precise role of miR319 in root development, particularly in lateral root formation and root system architecture establishment, remains to be elucidated.

Recent studies revealed that miR319 participates in complex cross-talk with multiple phytohormonal signaling pathways to orchestrate plant growth and development. Notably, TCP19, a direct target of miR319a, has been demonstrated to physically interact with RGA, a key component of the gibberellin (GA) signaling pathway, thereby regulating trichome initiation [20]. Furthermore, miR319-mediated regulation of TaPCF8 in wheat has been shown to integrate auxin signaling and biosynthesis pathways, ultimately controlling plant height determination [21]. TCP transcription factors have been increasingly recognized as crucial modulators of auxin signaling dynamics during cellular differentiation and organ maturation processes [22]. Specifically, TCPs influence auxin biosynthesis and response by regulating genes such as YUC5, IAA3, and PIN1 [16,23]. Given the central role of auxin signaling in root development [8,24], it remains unclear whether miR319-TCP regulatory networks influence auxin metabolism and signaling to influence lateral root development.

In poplars, current research on the regulation of lateral root development by small RNA-mediated hormone signaling is still limited. In this study, we identified that miR319a/TCP19/IAA3.2 module functioned as a key regulator for LR development in P. tomentosa. Genetic modification of miR319a/TCP19 expression and detection of auxin signaling showed the antagonism between the miR319a and auxin pathway in modulating LR growth in P. tomentosa. Molecular and biochemical evidence indicate that TCP19, as a downstream target of miR319a, directly bound to the promoter of IAA3.2 and negatively regulated its expression level, thereby altering auxin signaling. These findings establish a novel link between miR319a and lateral root formation, providing new insights into the genetic and molecular regulation of tree growth and propagation.

2. Results

2.1. MiR319a Modulates Lateral Root Development in P. tomentosa

The miR319 family in Populus trichocarpa, consisting of nine distinct members, was distinguished by a highly conserved 20-nucleotide mature sequence [25]. Previous studies implicated miR319a in diverse biological processes in woody plants [20]. To investigate its expression pattern in P. tomentosa, we quantified miR319a transcript levels in WT plants across various tissues using stem-loop RT-qPCR. MiR319a was ubiquitously expressed, with the highest levels detected in the stem (Figure 1a).

Histochemical staining of miR319a_pro:GUS transgenic plants corroborated the RT-qPCR results, revealing weak GUS activity in the root, lateral root (LR), and root base (Figure 1b,c,e), but stronger signals in the stem and leaf (Figure 1d,f). These findings suggest that miR319a regulated LR development through modulation of auxin source and canalization pathways. The results of tissue expression implicate the possible role of miR319a in LR regulation.

We subsequently performed tissue propagation of the miR319a-OE and miR319a-STTM transgenic P. tomentosa plants and analyzed the relative expression level of miR319a in root (Figure S1a). Shoots from transgenic plants and WT plants were rooted on woody plant medium (WPM), and root traits were assessed after 14 days (Figure 2a). The number of adventitious roots increased significantly in the miR319a-STTM plants, but the difference was not obvious in the overexpression lines (Figure 2b). There were also no significant differences in lateral root length among the WT, miR319a-OE, and miR319a-STTM transgenic poplars (Figure 2c). However, compared with WT, the number of lateral roots in the miR319a-OE L1 and L2 lines decreased by 40% and 69%, respectively (Figure 2d), and the LR density decreased by 57% and 67%, respectively (Figure 2e). In contrast, in miR319a-STTM L1 and L2 lines, both the LR number and LR density approximately doubled. Additionally, 10-week-old miR319a-OE plants cultivated in soil exhibited higher fresh (Figure S1b,c) and dry root weights (Figure S1b,d) compared to WT plants. These data collectively demonstrate that miR319a acted as a negative regulator of LR development in P. tomentosa.

2.2. MiR319a Regulates Lateral Root Formation by Modulating Auxin Signaling

Auxin has been widely proven to be involved in regulating the development of lateral roots [8,9]. To explore the functional connection between miR319a and auxin signaling during LR development, we analyzed the expression of genes involved in auxin biosynthesis, signaling, and transport in miR319a-OE and miR319a-STTM plants (Figure 3a–c). For auxin biosynthesis, YUC2 and TAA1a expression levels were reduced in one of the miR319a-OE lines and enhanced in one of the miR319a-STTM lines (Figure 3a). Further, the auxin signaling repressors IAA3.1 and IAA3.2 were upregulated in miR319a-OE plants but downregulated in miR319a-STTM plants (Figure 3b). The relative expression level of auxin transporters PIN1, PIN2, PIN5a, and PIN5b did not show an obvious expression trend in WT and miR319a transgenic plants (Figure 3c). To investigate whether miR319a affects the lateral root development by regulating the auxin synthesis or signaling in poplar roots, the auxin content (IBA, MeIAA, and IAA) was detected of WT, miR319a-OE, and miR319a-STTM roots using liquid chromatography-coupled mass spectrometry (LC-MS) (Figure S2). The results show that compared to WT, in the root of miR319a-OE lines, the IBA content was decreased but the IAA content was significantly increased, while both IBA and IAA contents were increased in miR319a-STTM plants. Previous studies found that IBA promotes the production of lateral roots in rice more effectively than IAA [26]. In addition, exogenous application of IBA promotes the rooting of poplar tissue-cultured plants [27], while there was no significant difference in the MeIAA content among the WT, miR319a-OE, and miR319a-STTM plants. Since multiple factors, such as auxin synthesis, transport, and translocation, could affect the changes in auxin content, we speculated that miR319a might influence lateral root formation by modulating auxin levels in the roots.

To further validate the role of miR319a in auxin signaling, we therefore compromised auxin homeostasis by treating explants with 0.1 µM NAA, a plant growth regulator with auxin-like activity and 1 µM NPA, an inhibitor of polar auxin transport (Figure 4a). After exogenous applications of NAA, the LR number and LR density in WT increased by 30% and 44%, respectively. In the miR319a-OE L1 and L2 plants, the LR number increased by 23% and 48%, and the LR density increased by 10% and 26%, respectively. In the miR319a-STTM L1 and L2 plants, after NAA treatment, the number of LR increased by 9% and 14%, and the LR density increased by 34% and 42%, respectively. These results indicate that NAA treatment could partially rescue the phenotypes of reduced LR number and density in miR319a-OE plants. Conversely, NPA treatment inhibited LR formation across all genotypes, though the inhibitory effect was less pronounced in miR319a-STTM plants compared to WT and miR319a-OE lines (Figure 4a–c). After NPA treatment, the LR number in WT decreased by 70%. In the miR319a-OE plants, the LR number decreased by 85–87%, and in the miR319a-STTM plants, the LR number decreased by approximately 83%. This suggested that NPA treatment could reverse the phenotype of increased lateral root number in miR319a-STTM plants. Together, these findings demonstrate that miR319a regulated LR formation in P. tomentosa by modulating auxin signaling, with TCP19 likely playing a pivotal role in this process.

IAA3 has been shown to suppress the auxin response [16]. The IAA3.1 and IAA3.2 exhibited opposite expression patterns in the miR319a-OE lines and miR319a-STTM lines, which was consistent with the reduced auxin signaling in the miR319a-OE plants (Figure 3b). Furthermore, we transformed the nuclear-localized DR5rev::GFP reporter vector, a biosensor of in vivo auxin signaling, into the genetic backgrounds of WT, miR319a-OE, and miR319a-STTM, respectively, to investigate the role of miR319a in regulating the auxin pathway during LR formation (Figure 5). The results reveal that auxin signaling was stronger in the epidermal cells and elongation zone cells in the LR of miR319a-OE plants, whereas in LR of WT and miR319a-STTM plants, auxin signaling was more pronounced in the root tip meristematic cells. This suggested that miR319a might influence lateral root formation by modulating auxin signaling in the root tip meristematic region.

2.3. TCP19 Acts as a Target of miR319a

TCP transcription factors were well-established primary targets of miR319, including TCP2/3/4/10/24 in Arabidopsis [15]. In P. tomentosa, six TCP genes (TCP4, TCP9, TCP19, TCP20, TCP24, and TCP34) were identified based on sequence homology, all of which were confirmed as direct targets of miR319a [20]. TCP transcription factors have also been increasingly implicated in auxin-related pathways in regulating diverse developmental processes [28,29,30]. To explore the functional connection between miR319a-TCPs and auxin signaling during LR development, we firstly analyzed the expression changes of these TCP genes in the lateral roots of WT and miR319a transgenic plants. RT-qPCR analysis demonstrated that the expression of all six TCP genes was significantly reduced in the roots of miR319a-OE plants, whereas only TCP19 was markedly upregulated in the roots of miR319a-STTM plants. This finding highlights that TCP19 acted as a key target of miR319a in roots (Figure 6a).

To further explore the role of TCP19 in LR development, we conducted transcriptional activation assays using the GAL4/VP16 system in yeast cells. Yeast cells expressing either GAL4BD-TCP19 or GAL4BD-VP16-TCP19 recombinant proteins did not grow on selective SD–Trp–His–Ade medium (Figure 6b). In contrast, the GAL4BD-VP16-positive control grew normally and turned blue upon overlay with X-α- gal (Figure 6b), indicating that TCP19 might function as a transcriptional repressor. We also investigated the subcellular localization of TCP19 by fusing its full-length CDS to GFP under the control of the CaMV35S promoter. The 35Spro:GFP and 35Spro:TCP19-GFP constructs were transiently expressed in Nicotiana benthamiana. Confocal microscopy showed that TCP19-GFP fluorescence was exclusively localized in the nucleus, as indicated by co-localization with nucleus-specific DAPI staining. In contrast, GFP alone exhibited a ubiquitous distribution throughout the cell (Figure 6c). We then performed analysis of histological GUS staining on GUS reporter lines driven by the TCP19 promoter. The results show that it was expressed at relatively high levels in both leaves and lateral roots (Figure S3). These findings preliminarily verify that TCP19 was a nuclear-localized transcriptional repressor, which acted as a target gene of miR319a to regulate LR development.

To assess the functional role of TCP19 in LR formation, we overexpressed a mutated version of TCP19 (TCP19m) in P. tomentosa under the control of the CaMV 35S promoter. Two independent transgenic lines with high expression levels of TCP19m (L7 and L9) were selected for further analysis (Figure 7b). Compared to WT plants, TCP19m-overexpressing lines displayed a significantly higher number of LR (Figure 7a,c) and increased LR density (Figure 7a,d), confirming that TCP19 promoted LR growth in poplar.

2.4. TCP19 Directly Binds to the Promoter of IAA3.2

Given that TCP19 acts downstream of miR319a in poplar and its overexpression promoted LR development, we hypothesized that TCP19 might regulate auxin-related genes to influence LR development. Previous studies have shown that TCP transcription factors regulate genes involved in auxin biosynthesis, transport, and signaling [16,23,31], and it has been demonstrated that class-I and class-II TCPs preferentially bind to the DNA motifs GGNCCCAC and GTGGNCCC, respectively [32]. Promoter analysis revealed TCP binding elements in the regulatory regions of IAA3.1 and IAA3.2, suggesting potential direct regulation by TCP19 (Figure S4a). Expression pattern analysis further demonstrated that IAA3.1 is predominantly expressed in young stems (Figure S4b), while IAA3.2 is mainly expressed in roots (Figure S4c).

To establish whether TCP19 directly regulates IAA3.2, we analyzed the expression of IAA3.1 and IAA3.2 in the root of TCP19m-overexpressing (TCP19m-OE) lines. Only IAA3.2 expression was significantly reduced in these plants (Figure 8a), indicating a TCP19-specific regulation of IAA3.2. We further confirmed this interaction using transient expression assays in transiently transformed tobacco leaves. Reporter constructs containing a 2.5-kb promoter fragment of IAA3.2 driving a GUS reporter gene were co-transfected with TCP19m effector constructs under the control of the CaMV 35S promoter. The result reveals that TCP19 significantly suppressed the promoter activity of IAA3.2. As the result shows, in the presence of TCP19, the activity of β-glucuronidase (GUS) decreased by 77%, providing functional evidence of TCP19-dependent repression of IAA3.2 (Figure 8b).

To confirm the direct binding of TCP19 to the IAA3.2 promoter, we conducted chromatin immunoprecipitation (ChIP) followed by RT-qPCR using TCP19m-OE plants. The ChIP enrichment analysis revealed significant binding of TCP19 to the IAA3.2 promoter regions containing the GGNCCC motif (P1 and P2), in contrast to negative controls (Figure 8c). These results validate that TCP19 directly interacted with the IAA3.2 promoter, leading to the repression of its expression.

Collectively, these findings establish a regulatory module in which the miR319a-TCP19 pathway suppressed IAA3.2 expression to regulate auxin-dependent LR development in P. tomentosa. This study provided mechanistic insights into the role of miR319a in modulating root architecture through the modulation of auxin signaling in poplar.

3. Discussion

MiR319 and its target transcription factors, TCPs, are well-documented regulators of plant development, primarily in aerial tissues. The miR319/TCP module has been implicated in leaf morphogenesis, senescence, and cellular differentiation [33]. In floral development, miR319 mutants exhibit defects in petal and stamen morphology, including impaired anther formation and altered petal size [17]. While significant advances have been made in understanding the role of miR319/TCPs in above-ground organs, their involvement in the regulation of underground tissues, particularly lateral root (LR) development, remains underexplored. Though several studies suggested that miR319 was involved in root responses to various abiotic stresses [34,35], the molecular mechanisms underlying the role of miR319 in LR development have not been fully elucidated. As a herbaceous plant, Arabidopsis mainly has fibrous roots. The formation of lateral roots is weakly associated with secondary growth, and auxin relies on short-distance transport in the root tip [8]. In contrast, P. tomentosa is a woody plant with a complex root system composed of a taproot and lateral roots. The development of lateral roots in P. tomentosa requires coordinated secondary growth. Auxin primarily depends on long-distance transport in vascular tissues, and there are species-specific signal responses [36].

Previous studies on the regulation of lateral roots by miR319/TCP were mostly based on Arabidopsis, which made it difficult to reflect the specific mechanisms of woody plants. In this study, we revealed a molecular pathway in which miR319a regulated LR development by targeting TCP19 to inhibit the auxin signaling repressor IAA3.2 in P. tomentosa. These findings fill the gap in the species differences of root regulation mechanisms between herbaceous and woody plants.

3.1. Spatial and Temporal Regulation of LR Development by miR319a

MicroRNAs are recognized as key regulators of lateral root development, influencing processes such as initiation, patterning, and emergence. In Arabidopsis, miR156 was found to positively regulate lateral root development [37,38]. In poplars, miR390 was demonstrated to mediate salt stress-induced LR formation, enhancing their salt tolerance by promoting root elongation and density. Furthermore, microRNA sequencing in Citrus revealed that miR319, in combination with miR171, contributes to root development under long-term boron toxicity [39]. Our analysis of transgenic plants expressing miR319apro:GUS confirmed that miR319a was expressed at low levels in LR (Figure 1). This temporal expression pattern suggested that miR319a might play a regulatory role in LR growth.

3.2. MiR319a Module Modulates LR Development via Auxin Signaling

Previous studies demonstrated that miR319 modulated various hormone signaling pathways, including gibberellin (GA) [20], jasmonic acid (JA) [40], and ethylene (ETH) [35]. Additionally, miR319 has been implicated in regulating auxin signaling in the context of wheat growth [21]. Auxin plays a central role in all stages of LR development, including initiation, patterning, and emergence [41,42,43]. In our study, we observed that overexpression of miR319a resulted in a reduction in LR number and density, whereas knockdown of miR319a promoted LR formation (Figure 2 and Figure S2). Instead, it might focus more on influencing genes related to auxin synthesis and signal transduction. Notably, among the genes related to auxin transport, the relative expression levels of members of the PIN protein family, such as PIN1, PIN2, PIN5a, and PIN5b, did not show obvious regular changes between WT and miR319a transgenic plants (Figure 3c). This implied that the regulation of lateral root development by the miR319a/TCP19 module might not be mainly mediated through the regulation of auxin transport by these PIN proteins. Moreover, we found that auxin signaling was enhanced in the roots of miR319a-STTM (Figure 3b and Figure 4), suggesting that miR319a negatively regulated LR development by modulating auxin signaling.

3.3. Regulation of LR Development via TCP19 and IAA3.2

Although the targets of miR319a include six TCP genes, only TCP19 was found by us to be significantly up-regulated in miR319a-STTM plants, while the expression of other TCP genes showed no obvious changes (Figure 6a). Overexpression of TCP19 could increase the number and density of LR (Figure 7). These characteristics distinguished it from other TCP genes and made it the core target for miR319a to regulate lateral root development.

TCPs are known to regulate plant development by influencing phytohormone biosynthesis and signaling, particularly auxin [16,35]. In Arabidopsis, mutation in TCP15 results in upregulation of YUCCA (YUC) genes, which modulates auxin biosynthesis and affects various developmental processes, including gynoecium and silique development [31]. TCP proteins are involved in regulating auxin homeostasis by directly binding to the promoters of auxin-related genes, such as IAA3/SHY2, and modulating their expression [44]. Similarly, TCPs modulated auxin homeostasis and respond to affect LR patterning in Arabidopsis [45]. In this study, we observed that both TCP19 and IAA3.2 were highly expressed in lateral roots, suggesting that they might regulate the LR development in poplars (Figures S3 and S4). Notably, overexpression of TCP19 increased LR number and density, which correlated with the down regulation of IAA3.2 (Figure 8a). Motif analysis, transcription activation, and ChIP-qPCR analysis further confirmed that TCP19 directly bound to the promoter of IAA3.2, modulating its expression and contributing to LR development (Figure S4a and Figure 8a–c). Interestingly, our work also aligned with Kong et al. [46], who found that TCPs regulate GH3.3 expression to maintain auxin homeostasis in roots. Additionally, TCPs were demonstrated to coordinate root development in response to environmental cues, such as nutrient availability [31]. Our results extend these findings by providing direct evidence that TCP19 regulates IAA3.2 expression in poplar, influencing lateral root formation through auxin-mediated pathways.

In summary, we demonstrated that miR319a negatively regulated lateral root development by targeting TCP19 at the post-transcriptional level. Among the six TCP genes identified, TCP19 appeared to be the primary mediator of miR319a’s effect on lateral root growth. The miR319a/TCP19 module regulated auxin signaling by directly binding to the promoter of IAA3.2, influencing lateral root formation in Populus (Figure 8d). This work established a novel link between miR319a/TCPs and auxin signaling in lateral root development, providing new insights into the molecular mechanisms controlling root architecture. Future studies should explore the role of other TCP transcription factors and IAA proteins in this regulatory pathway.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions

All P. tomentosa (P. tomentosa) cutting-propagated plants used in this study were cultivated on woody plant medium (WPM; Coolaber, Beijing, China) in a controlled tissue culture room (Yanghui Instrument, Ningbo, China) under conditions of 16 h of light and 8 h of dark, with 5000 lux supplemental light, 25 °C, and 60% relative humidity.

4.2. Poplar Transformation

Agrobacterium-mediated transformation was performed using leaf disks as described by Jia et al. [47]. Hygromycin (HYG)-resistant plants were selected using specific HYG primers (Table S1) to identify positive transgenic lines. Ten positive mTCP19-OE transgenic plants were screened out via PCR with the gene-specific primers of hygromycin-resistant genes. One-month-old rooted plantlets were transferred to a glasshouse and grown under the same long-day conditions. After one month of cultivation in the greenhouse, the roots of WT and mTCP19-OE transgenic plants were used to detect the expression level of TCP19.

4.3. Gene Cloning and Vector Construction

The miR319a target sites are located within the coding sequence (CDS) of TCP19. The TCP19 gene was amplified from P. tomentosa cDNA using gene-specific primers (Table S1) and cloned into the pCXDG vector [48]. Full-length TCP19 was amplified using specific primer pairs (BD/BD-VP16) and cloned into the BD vector. A 2.5-kb promoter region of Pro-IAA3.2 was amplified from genomic DNA of P. tomentosa and inserted into the pCXGUS-P vector for GUS reporter gene analysis [48], using a TA cloning kit for sequencing. Previous studies generated TCP19pro:GUS, miR319a pro:GUS, miR319a-OE, and miR319a-STTM-positive transgenic plants, which were selected for this study [20].

4.4. Histochemical Analysis

GUS staining was performed as described by Jefferson [49]. Tissues from 15-day-old plants were fixed in 90% acetone for 30 min at 4 °C, then washed three times with double-distilled water (ddH2O). Fixed tissues were incubated in GUS staining solution (0.5 M Tris, pH 7.0, 10% Triton X-100, and 1 mM X-Gluc) for 30 min at 37 °C in the dark. After staining, roots were cleared in 75% ethanol and observed using an Olympus 566 SZX16 microscope (Tokyo, Japan). The GUS staining assays were performed in three biological replicates.

4.5. RNA Extraction and Quantitative RT-qPCR

Total RNA was extracted from tissues of root, young stem, mature stem, young leaf, shoot, xylem, and bark in poplars using the Plant RNeasy Mini Kit (Qiagen, Beijing, China). Furthermore, electrophoresis was used to confirm whether the RNA degraded, and the NanoDrop 2000 (Thermo Fisher Scientific, Shanghai, China) was employed to measure the RNA concentration. The DNA in the RNA samples was removed using the DNA eraser in the reverse transcription kit and then reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer’s instructions. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Premix ExTaq™ (Takara) in a qTOWER3G IVD Real-time PCR machine (Analytik Jena AG, Germany). The reaction mixture consisted of 5 μL SYBR Premix ExTaq™, 1 μL cDNA (10 ng), and 0.2 μM of each primer. The UBQ gene from P. tomentosa was used as a reference gene. Specific primers for qRT-PCR were designed according to Shi et al. [50] (Table S1). The RT-qPCR assays were performed in three biological replicates.

4.6. Yeast Transactivation Assay

The full-length open reading frame (ORF) of TCP19 was amplified using gene-specific primers (Table S1) and cloned into the pGBKT7(BD) and BD-VP16 vector. The pGBKT7 vector and BD-VP16 vector were also transformed into yeast cells, respectively, serving as negative and positive controls. The recombinant plasmids were introduced into the Saccharomyces cerevisiae strain Gold2 following the method described by Xu et al. [51]. Yeast transformants were initially grown on SD medium (Coolaber, Beijing, China) lacking tryptophan (Trp) and then further selected on SD medium lacking Trp, histidine (His), and adenine (Ade) for transactivation assays. Transactivation activity was assessed by adding X-α-gal.

4.7. Subcellular Localization Analysis

The full-length TCP19 gene was cloned into the pCXDG vector and fused with GFP to create a TCP19-GFP fusion protein. Then, the recombinant plasmid 35Spro:TCP19-GFP was transferred into tobacco leaves using the agroinfiltration method under the condition of 35Spro:GFP used as a control. The Nicotiana benthamiana leaves were stained with 40, 6-diamidino-2-phenylindole (DAPI). Fluorescent signals of GFP and DAPI were documented using a confocal laser microscope (Leica TCS SP8 X; Leica, Wetzlar, Germany).

4.8. Morphological Characterization of Roots

For root analysis, 15-day-old WT plants grown on woody plant medium (WPM) were transferred for observation. The number of the adventitious roots and lateral roots of the plants was determined by counting. The poplar plants were photographed, and using the scale used during photography as a parameter, the length of adventitious roots and lateral roots was calculated with the ImageJ software version 1.46r (https://imagej.net/ij, accessed on 1 May 2023) (Bethesda, MD, USA), and the lateral root density of the plant using the known number and length of lateral roots was calculated. Five biological replicates were used for morphological characterization of roots.

4.9. Transient Expression and GUS Activity Assay

The full-length TCP19 fragment was cloned under the control of the 35S CaMV promoter to drive expression as an effector, and the 35Spro:GFP vector was used as a control. The GUS reporter gene was driven by the IAA3.2 promoter to serve as the reporter. GUS activity was quantitatively measured by monitoring the cleavage of the β-glucuronidase substrate 4-methylumbelliferyl β-D-glucuronide (MUG), which produces the fluorescent product 4-methylumbelliferone (4MU) upon hydrolysis [49]. The transient expression and GUS activity assays were performed in three biological replicates.

4.10. Chromatin Immunoprecipitation Quantitative PCR (ChIP-qPCR)

Two-month-old TCP19m transgenic plants containing a GFP epitope were used for ChIP-qPCR assays. Immunoprecipitation was carried out using GFP antibody and normal mouse IgG for negative control. ChIP analysis was performed as described by Yang et al. [52]. The primers used for ChIP-qPCR are listed in Table S1. The ChIP-qPCR assays were performed in three biological replicates.

4.11. Determination of Auxin Content by Liquid Chromatography–Mass Spectrometry (LC–MS)

To extract and analyze IAA, IBA, and MeIAA contents in WT and miR319a transgenic plants, the roots were flash-frozen with liquid nitrogen in a mortar right after they were detached from the plants to avoid wounding-induced hormone changes. Then, the frozen tissues were ground into powder. Each sample was accurately weighed at 1.5 g and transferred to 15 mL screw-cap tubes while being kept in liquid nitrogen. In total, 8 μL of 1 μg/mL internal standard working solution was added to each tube. Next, 10 mL of extraction solvent (2-propanol/H₂O/concentrated HCl at 2:1:0.002, vol/vol/vol) was added, and the volume was adjusted if over 1 g of fresh tissue was used. The tubes were shaken at 100 rpm. for 30 min at 4 °C because plant hormones were unstable. After that, 5 mL dichloromethane was added, and the mixture was shaken in a 4 °C cold room for 30 min. The samples were centrifuged at 4 °C and 13,000× g for 5 min. The lower-phase solvent was transferred to a screw-cap vial, concentrated using a nitrogen evaporator, and redissolved in 0.4 mL methanol. The samples were stored at −20 °C until analysis and kept at 8 °C in an autosampler tray during analysis. In total, 2 µL of the sample was injected into a reverse-phase C18 Gemini HPLC column for HPLC–ESI–MS/MS analysis. Finally, quantitative analysis was performed by calculating the ‘correction factor’ of each plant hormone relative to its internal standard. The hormone amounts were normalized to the mass of fresh tissue, and the Analyst 1.5 software was used if an Applied Biosystems triple quadrupole MS was applied.

4.12. Statistical Analyses

Quantitative data for expression level analyses, GUS activity measurements, fluorescence intensity measurements, and phenotype data measurements were determined for statistical significance using Student’s t-test performed to distinguish significant differences between pairwise samples (*, p < 0.05; **, p < 0.01; and ***, and p < 0.001).

4.13. Accession Numbers

The sequences used in this study are available in Phytozome (version 11.0, https://phytozome-next.jgi.doe.gov/blast-search, accessed on 1 May 2023) under the following accession numbers: TCP19 (Potri.011G083100.4), TCP9 (Potri.004G065800.5), TCP4 (Potri.001G375800.1), TCP20 (Potri.011G096600.2), TCP24 (Potri.013G119400.1), TCP34 (Potri.019G091300.1), miR319a (PRJNA611665), IAA3.1 (Potri.005G053800.1), IAA3.2 (Potri.013G041300.1), GH3.2 (Potri.001G298300.1), GH3.5 (Potri.001G410400.1), PIN1a (Potri.012G047200.1), PIN2 (Potri.018G139400.1), PIN5a (Potri.004G124200.1), PIN5b (Potri.017G078300.1), YUC1 (Potri.006G248200), and TAA1a (Potri.012G083300).

5. Conclusions

In this study, we identified that the miR319a/TCP19/IAA3.2 module regulated lateral root development in poplar by altering auxin signaling. The detection of the expression levels of auxin-related genes and the observation of DR5rev::GFP fluorescent reporter lines demonstrated that miR319a inhibited auxin signaling in lateral roots, thereby suppressing LR development. Further investigation revealed that TCP19, a transcription repressor localized in the nucleus, was the target gene through which miR319a regulates lateral root development. Overexpression of TCP19 could promote lateral root development in poplar. In addition, TCP19 suppressed the expression of the auxin repressor IAA3.2 by directly binding to its promoter, altering auxin signaling and consequently regulating lateral root development in poplar.