The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla

The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla Huang Peng, Zhang Hongrui, An Jiaqian, Yuan Zhongjia, Duan Huilei, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9102551/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Drought is one of the major abiotic stress factors affecting plant growth and productivity. The miR156/SPL module plays a crucial role in plant growth, development, and responses to abiotic stress; however, its regulatory mechanism in mediating drought adaptation in woody plants such as birch remains incompletely understood. In this study, we used transgenic plants overexpressing bp-miR156c and BpSPL2 as experimental materials and employed GUS staining, RNA-seq, yeast one-hybrid assay, ChIP-PCR and dual-luciferase reporter assays to investigate the mechanism by which the miR156/SPL module regulates drought tolerance in birch. GUS staining results indicated that BpSPL2 is a target gene of bp-miR156c and subject to its cleavage. Compared with wild-type plants, bp-miR156c -overexpressing transgenic plants exhibited reduced drought tolerance under drought stress, whereas plants overexpressing its target gene BpSPL2 showed enhanced drought resistance. Specifically, BpSPL2-OE lines under drought stress displayed alleviated photodamage in both PSII and PSI, along with reduced oxidative damage. Moreover, overexpression of BpSPL2 significantly promoted root system development, particularly lateral root growth, in birch. RNA-seq analysis revealed that, compared with the wild type, differentially expressed genes (DEGs) in BpSPL2-OE plants under drought stress were significantly enriched not only in photosynthesis-related pathways but also in tryptophan metabolism, redox processes, and glutathione metabolism. We speculate that the alleviation of photosynthetic inhibition and oxidative damage in birch leaves under drought stress by BpSPL2 may be related to its regulation of ROS metabolism, while the promotion of lateral root development may be associated with activation of the tryptophan metabolic pathway and subsequent accumulation of IAA. Further studies demonstrated that the BpSPL2 transcription factor recognizes the GTAC motif and binds to the promoters of glutathione-S-transferase BpGSTF3 and the key rate-limiting enzyme in tryptophan synthesis gene BpASA1 , thereby enhancing their transcription. On one hand, this upregulates GST and antioxidant enzyme activities, mitigating drought-induced photodamage and oxidative injury; on the other hand, it promotes IAA accumulation, stimulating lateral root formation and ultimately improving drought tolerance in birch. In summary, our findings demonstrate that the miR156/SPL module enhances drought tolerance in birch by modulating ROS homeostasis and lateral root development, providing novel molecular insights and a theoretical foundation for drought resistance research in birch trees. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Drought is one of the major abiotic stress factors that limit plant growth, development, yield and quality(Jaleel et al. 2009 ; Gupta, Rico-Medina, and Caño-Delgado 2020 ). Currently, approximately 45% or more of global agricultural land is affected by persistent or frequent drought as a result of climate change(Liang et al. 2024 ). Drought stress affects all aspects of plant growth, development, and reproduction, encompassing morphological, physiological, and biochemical changes as well as the regulation of specific genes(Hussain et al. 2018 ; Ji et al. 2025 ). Plants primarily employ two strategies to cope with drought stress: drought escape and drought tolerance. Drought tolerance, in particular, is enhanced through multiple mechanisms. These include morphological adjustments, especially increased biomass allocation to the shoot; active accumulation of osmolytes to promote water uptake; reduction of stomatal conductance to improve water use efficiency; enhanced antioxidant enzyme activity to mitigate reactive oxygen species (ROS) damage; activation of hormone signaling pathways; and various transcriptional regulations and post-translational protein modifications(Sun et al. 2025 ; Chen et al. 2025 ). In the aforementioned biological processes of drought response, the regulation by specific miRNAs and transcription factors plays a pivotal role. During evolution, plants have developed multiple regulatory networks to cope with adverse environmental conditions, with the miRNA/SPL regulatory network being one of them(Shriram et al. 2016 ), it can adapt to environmental stresses by regulating SPL transcription factor genes via miR156 under various stress conditions(Wang and Wang 2015 ). SPLs are key inducers of plant stress resistance, mediating the plant's ability to withstand various stresses, such as drought and low temperature(Ma et al. 2021 ; Qi et al. 2025 ). However, the regulatory mechanism of the miR156/SPL module in the drought tolerance process of birch remains poorly understood. MicroRNAs are a class of small non-coding RNAs, typically 20–24 nucleotides in length, that play important roles in processes such as leaf development, flower development and resistance to both abiotic and biotic stresses(Chen 2004 ; Mallory et al. 2004 ; Sunkar and Zhu 2004 ; Khraiwesh, Zhu, and Zhu 2012 ; Sunkar, Li, and Jagadeeswaran 2012 ). Recently, an increasing number of studies have revealed that miRNAs are involved in plant tolerance to a wide range of abiotic and biotic stresses, including pathogen infection(Jones-Rhoades and Bartel 2004 ), salinity, drought, cold, oxidative stress(Li et al. 2017 ; Covarrubias and Reyes 2010 ; Yin et al. 2015 ; Koroban et al. 2016 ), UV radiation(Zhou, Wang, and Zhang 2007 ), nutrient deficiency(Panda and Sunkar 2015 ), and heavy metals(Huang et al. 2009 ), among others. In plants, miRNAs play a crucial regulatory role in response to drought stress, particularly the well-conserved miR156/SPL module. This module contributes to drought tolerance by participating in phytohormone signaling pathways, synthesis of osmoprotectant metabolites, enhancement of antioxidant enzyme activities and maintenance of ROS homeostasis. For instance, in Cassava ( Manihot esculenta Crantz ), MeSPL9 reduces drought resistance by regulating JA signaling, whereas the miR156-targeted resistant line rMeSPL9-SRDX confers drought tolerance without compromising overall plant growth(Li et al. 2022 ). In tomato ( Solanum lycopersicum L.), under drought conditions, the overexpression of miR156 and strigolactone treatment lead to enhanced drought tolerance, which is associated with ABA accumulation and reduced stomatal conductance(Visentin et al. 2020 ). MicroRNA156ab regulates plant growth and drought resistance by targeting the transcription factor MsSPL13 ; heterologous expression of MsSPL13 reduces auxin content and inhibits its growth in Arabidopsis under both normal and stress conditions(Feng et al. 2023 ). Osmoprotectant metabolites serve as key osmoregulators: in cassava, the rMeSPL9-SRDX line confers drought tolerance not only through JA signaling but also by promoting the synthesis of osmoprotectant metabolites such as proline and anthocyanins(Li et al. 2022 ). Csn-miR156f-2-5p and CsSPL14 are involved in the drought response in tea ( Camellia sinensis ) plants. Knockdown of csn-miR156f-2-5p significantly reduces both the maximum photochemical efficiency of PSII ( Fv/Fm ) and chlorophyll content in tea leaves, while lead to a substantial accumulation of ROS(Wen et al. 2024 ). Photosynthesis is one of the processes most sensitive to drought in plants. Drought stress leads to chlorophyll degradation and reduced photosynthetic capacity(Bhusal, Han, and Yoon 2019 ; Ji et al. 2025 ), causing damage to the reaction centers of both Photosystem II (PSII) and Photosystem I (PSI) in most plants and inhibiting electron transfer along the photosynthetic electron transport chain(Yao et al. 2023 ; Ohashi et al. 2006 ). When photosynthesis is suppressed under stress, the excess energy and electrons in chloroplasts are a major cause of reactive oxygen species (ROS) burst. ROS are by-products formed during normal oxygen metabolism and play crucial roles in cellular signaling and homeostasis maintenance; however, their excessive accumulation can cause damage to plant cells, with severe cases potentially leading to cell death. The homeostasis of ROS levels in plants is maintained by antioxidant enzymes that scavenge the excess ROS produced under stress conditions. The plant ROS scavenging system primarily consists of an enzymatic system, including superoxide dismutase (SOD), catalase (CAT) and peroxidases (POD), as well as enzymes in the ascorbate-glutathione (AsA-GSH) cycle such as ascorbate peroxidase (APX) and glutathione peroxidases (GPX), and peroxiredoxins (Prxs) in the thioredoxin-thioredoxin peroxidase (Trx-Prx) pathway(Wang et al. 2023 ; Zhang et al. 2020 ). In the non-enzymatic antioxidant system, small molecules such as GSH, AsA, phenolic compounds, flavonoids and carotenoids play important roles in protecting plant cells from oxidative damage(Pan et al. 2020 ). Studies have shown that the miR156/SPL module can participate in plant resistance to abiotic stress by regulating photosynthesis or ROS balance. In alfalfa ( Medicago sativa L.) SPL13RNAi plants, the maintained or slightly increased transcripts related to photorespiration may act as an energy sink to prevent over-reduction of NADPH and photoinhibition(Feyissa et al. 2020 ). Transgenic Arabidopsis thaliana overexpressing apple MsSPL13 exhibited inhibited antioxidant enzyme activities, resulting in reduced drought tolerance(Feng et al. 2023 ). Overexpression of TaSPL6 -A increased sensitivity to drought stress in wheat ( Triticum aestivum L.); after drought treatment, transgenic plants showed significantly higher leaf water loss, malondialdehyde, and ROS content, along with markedly lower antioxidant enzyme activities compared to wild-type plants. Conversely, silencing of the TaSPL6 gene enhanced drought tolerance in wheat, reflected in better growth performance(Zhao et al. 2024 ). Variation in OsSPL10 confers drought tolerance in rice ( Oryza sativa L.) by directly regulating the expression of OsNAC2 and ROS production(Li et al. 2023 ). Glutathione-S-transferases (GSTs; EC 2.5.1.18) are a class of important multifunctional enzymes involved in detoxification, defense against biotic and abiotic stresses, and transport of secondary metabolites(Hu et al. 2024 ). They detoxify both endogenous and exogenous compounds by conjugating glutathione (GSH) to hydrophobic substrates(Öztetik 2008 ), a process that involves three phases: transformation, conjugation, and compartmentalization(Light et al. 2005 ). GSTs influence plant growth and development by participating in physiological metabolism, stress resistance, and cellular signaling(Song et al. 2021 ). Functionally similar to glutathione peroxidases, GSTs can reduce peroxide production during oxidative processes, thereby enhancing plant adaptability to environmental stresses. A series of studies have shown that the expression of GST genes is induced by drought stress in various plant species, including Hordeum vulgare L. (Rezaei et al. 2013 ), Solanum lycopersicum L.(Xu et al. 2015 ), Triticum aestivum L.(Wang et al. 2019 ), Solanum tuberosum L.(Islam et al. 2018 ), Capsicum annuum L.(Islam et al. 2019 ) and Oryza sativa L.(Jain, Ghanashyam, and Bhattacharjee 2010 ). Overexpression of GST in tobacco ( Nicotiana tabacum L.) increased GST enzyme activity and GSH content under low temperature, high temperature, and salt stress, alleviating oxidative damage caused by abiotic stresses(P et al. 2000). In wheat, TaERF3 activated TaGST6 expression is enhanced by ABA in response to salt and drought stress(Rong et al. 2014 ). Heterologous overexpression of a pear GST gene in tobacco improved the scavenging capacity for O 2 − under drought conditions, thereby enhancing drought tolerance(Liu et al. 2013 ). Overexpression of the Tamarix chinensis L. GST gene ( ThGSTZ1 ) improved drought resistance by enhancing ROS scavenging ability(Yang et al. 2014 ). Transgenic Arabidopsis plants overexpressing tomato LeGSTU2 exhibited increased resistance to drought stress through elevated antioxidant enzyme activities that scavenge excess ROS(Xu et al. 2015 ). In tea plants, CsWRKY48 was found to activate the expression of CsGSTU8 by directly binding to the W-box region in its promoter; overexpression of CsGSTU8 in Arabidopsis improved ROS scavenging capacity and enhanced drought tolerance(Zhang et al. 2021 ). OsGSTU4 is induced by ABA and participates in ABA-dependent processes that confer stress resistance in transgenic plants(Sharma et al. 2014 ). To cope with water deficit induced by drought, plants have evolved a series of adaptive strategies. Among these, the plasticity of root system architecture (RSA), particularly the proliferation and development of lateral roots (LRs), is crucial for enhancing water and nutrient uptake(Ranjan et al. 2022 ; Comas et al. 2013 ; Koevoets et al. 2016 ; Karlova et al. 2021 ). Lateral root formation not only expands the root surface area(Du, Spalding, and Gray 2020 ), but is also precisely regulated by endogenous hormone networks, especially the synthesis, transport, and distribution of auxin(Teale, Paponov, and Palme 2006 ; Lavenus et al. 2013 ). Notably, auxin in roots is not only derived from polar transport from the shoots(Grieneisen et al. 2007 ; Zhao 2018 ), but is also significantly produced through local biosynthesis in root tissues(Ljung et al. 2005 ; Chen et al. 2014 ; Zhao 2018 ). Anthranilate synthase (AS) is a key rate-limiting enzyme in the tryptophan (Trp) biosynthesis pathway, catalyzing the conversion of chorismate to anthranilate (ANT)(Zhao and Last 1996 ; Niyogi and Fink 1992 ), where Trp serves as the major precursor for auxin biosynthesis in plants(Cohen, Slovin, and Hendrickson 2003 ; Zhao 2010 ; Tu et al. 2021 ). Research indicates that local auxin synthesis mediated by the AS α-subunit 1 (ASA1) plays a central role in root stress responses. Under arsenate stress, the expression of ASA1 in root tips is activated, promoting auxin synthesis and cooperating with transporters such as AUX1 / PIN2 to regulate auxin transport from the root tip to the elongation zone, thereby influencing root growth and development(Tu et al. 2021 ). In Arabidopsis leaf explants, jasmonic acid (JA) biosynthesis occurs within two hours after wounding, and the transcriptionally activated ERF109 protein upregulates ASA1 expression, contributing to auxin biosynthesis and promoting de novo root organogenesis(Zhang et al. 2019 ). Under drought stress, the Populus euphratica transcription factor PeFUS3 positively regulates lateral root growth and enhances drought resistance by activating the expression of auxin transport genes PIN2 , PIN6a , and AUX1 (Liu et al. 2025 ). Additionally, drought signals regulate lateral root formation through crosstalk between ABA and auxin pathways; for instance, ABA receptors PYL8 and PYL9(Zhao et al. 2014 ; Xing et al. 2016 ), as well as transcription factors such as MYB96 and WRKY46 (Seo et al. 2009 ; Ding et al. 2015 ), have been shown to integrate ABA and auxin signaling to control LR development. These findings suggest that ASA1 may act as a molecular hub linking drought stress signaling to root morphological remodeling, mediating lateral root initiation by modulating local auxin synthesis and thereby improving plant drought tolerance. Previous studies have reported that the miR156/SPL module participates in lateral root formation. Overexpression of apple MdmiR156n significantly increased primary root length and lateral root number in transgenic Arabidopsis plants under drought stress(Chen et al. 2023 ), although its molecular mechanism remained unclear. Here, our findings demonstrate that in birch, the miR156/SPL module promotes lateral root development by activating the expression of the ASA1 gene, thereby influencing drought tolerance. While the mechanisms by which the miR156/SPL module influences abiotic stress tolerance have been reported in species such as alfalfa(Arshad, Feyissa, et al. 2017 ; Arshad, Gruber, et al. 2017 ), rice(Cui et al. 2014 ), and wheat(Zhao et al. 2024 ), its regulatory role in the molecular basis of drought tolerance in woody plants, particularly birch, remains unclear. In this study, we employed genetic engineering techniques to obtain transgenic birch plants overexpressing bp-miR156c and BpSPL2 . By analyzing the physiological and biochemical changes in these transgenic plants under drought stress and conducting RNA-seq to investigate the molecular regulatory mechanisms mediated by BpSPL2 , we found that the miR156/SPL module enhances drought tolerance in birch by activating the expression of the BpGSTF3 and BpASA1 genes. This activation increases antioxidant enzyme activity, alleviates photoinhibition under drought stress, and promotes lateral root formation. Our study reveals the potential value of the bp-miR156c / BpSPL2 module for the genetic improvement of drought resistance in woody plants and provides a theoretical foundation for further refining the drought stress response network. Materials and methods Plant materials and treatment Transgenic birch plants overexpressing bp-miR156c and BpSPL2 , preserved from previous laboratory work, were subjected to subculture. The growth conditions were as follows: temperature at 24 ± 2°C, relative humidity at 65–75%, with light intensity and photoperiod set at 46 µmol m⁻² s⁻¹ and 16 h light/8 h dark, respectively. The culture medium consisted of: 2.14 g/L WPM powder, 20 g/L sucrose, 0.56 g/L calcium salt, 0.4 mg/L IBA, 8 g/L plant agar, adjusted to 1 L with distilled water, and pH adjusted to 5.8 using 5 M NaOH. After 40 days of growth, tissue-cultured seedlings of each transgenic line were transplanted into a soil matrix with a vermiculite:flower soil:peat soil ratio of 1:1:1 (Note: the culture medium on the roots must be thoroughly washed off during transplantation). The plants were then acclimatized and grown indoors until stable growth was achieved (i.e., surviving and growing normally after transfer from the medium to the soil matrix). After three months, both transgenic and wild-type (WT) plants were subjected to natural drought treatment. Prior to treatment, all plant materials were thoroughly watered to achieve saturated soil moisture content. The drought treatment group underwent natural drought stress for 7 days, while the control group was watered every 3 days to maintain normal soil moisture levels. After 7 days of drought treatment, growth phenotypes were observed and photographed, followed by physiological index measurements. Each treatment was performed with 15 biological replicates. Measurement of chlorophyll fluorescence parameters in BpSPL2 transgenic plants. The fully expanded third leaf from the apex of both control and treated plants from the wild-type (WT) and BpSPL2 transgenic lines was selected and dark-adapted for 30 minutes. Subsequently, following the method described by Wang et al.(Wang et al. 2025 ), the OJIP transients and 820 nm reflection ( MR820 ) kinetics of birch leaves under different treatments were measured using a Multifunctional Plant Efficiency Analyzer (M-PEA, Hansatech, UK). The curves were induced by a saturating red light pulse (1000 µmol m⁻² s⁻¹). Fluorescence signals were recorded starting at 0.01 ms and ending at 2 s. On the OJIP curve, the O, J, I and P steps correspond to time points of 0.01, 2, 30, and 1000 ms, respectively, with fluorescence intensities denoted as F 0 , F J , F I and F m . The following parameters were derived: the maximum quantum yield of PSII photochemistry, Fv / Fm = 1-( F 0 /F m ), and Δ I/I₀ , reflecting PSI activity. Here, I₀ represents the maximum value of the MR820 curve, and ΔI is the difference between the maximum and minimum values of the MR820 curve. To analyze the changes in relative variable fluorescence at specific characteristic points, the segments of the OJIP transients between O-P, O-J, and O-K were normalized as follows. The normalized curves from the drought-treated plants of the three genotypes were then subtracted from those of their respective controls. The calculation methods are detailed below: O-P segment normalization: V O−P =( F t - F 0 )/( F m - F 0 ), Δ V O−J = Δ[( F t - F 0 )/( F J - F 0 )], The relative variable fluorescence at the J step (2 ms) is obtained as: V J =( F J - F 0 )/( F m - F 0 ). O-J segment normalization: V O−J =( F t - F 0 )/( F J - F 0 ), Δ V O−J = Δ[( F t - F 0 )/( F J - F 0 )], The relative variable fluorescence at the K step (0.3 ms) is obtained as: V K =( F K - F 0 )/( F J - F 0 ). O-K segment normalization: V O−k =( F t - F 0 )/( F k - F 0 ), Δ V O−k = Δ[( F t - F 0 )/( F k - F 0 )], The relative variable fluorescence at the L step (0.15 ms) is obtained as: V L =( F L - F 0 )/( F k - F 0 ). Determination of ROS content and physiological parameter quantification Leaves from the same position of each plant were selected for physiological measurements. First, the fresh weight (M1) of the leaves was recorded. The leaves were then immersed in distilled water for 4–6 hours until fully saturated, blotted dry on the surface, and weighed to obtain the turgid weight (M2). Subsequently, the leaves were placed in an oven and dried at 65°C to a constant weight to obtain the dry weight (M3). The leaf relative water content (RWC) was calculated using the formula: RWC (%) = [(M1 – M3) / (M2 – M3)] × 100%. Other physiological and biochemical indices were measured using commercial assay kits (Griess Biotechnology Co, Ltd, Nanjing, China) according to the manufacturer's instructions. Specifically, the activities of peroxidase (POD), superoxide dismutase (SOD), and glutathione S-transferase (GST), as well as the contents of superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA), were determined using the corresponding kits. All measurements were performed with three independent biological replicates. bp-miR156c targets and cleaves BpSPL2 To validate the targeting and cleavage of BpSPL2 by bp-miR156c in birch, we utilized the online prediction tool psRNATarget ( http://plantgrn.noble.org/psRNATarget/?function=3 ) to predict BpSPL genes targeted by miR156 (see Supplementary Material). Constructs 35S::BpSPL2-GUS, 35S::bp-miR156c-GUS and 35S::bp-miR156c were generated (primer sequences and schematic diagrams of the constructs are provided in the Supplementary Material). Transient transformation in 4–6 weeks old tobacco leaves was performed following the protocol described by Lu et al. ( 2017 )(Lu et al. 2017 ). Agrobacterium tumefaciens strain GV3101 harboring the respective constructs was cultured in 20 ml of fresh LB liquid medium supplemented with 100 µg/mL kanamycin and 50 µg/mL rifampicin at 28°C with shaking at 200 rpm until the OD600 reached 0.6–0.8. For co-infiltration, Agrobacterium cultures containing GV3101-pBI121-bp-miR156c and GV3101-pBI121-BpSPL2-GUS were mixed in equal volumes and injected into tobacco leaves. The infiltrated plants were kept in the dark at room temperature for 48 hours, followed by GUS staining. Transcriptome analysis of BpSPL2 For each treatment, leaves from the same position of three biological replicate birch seedlings were collected, flash-frozen in liquid nitrogen, and stored at -80°C. RNA sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. on the Illumina NovaSeq 6000 platform. After quality control of the raw data, clean data were obtained for subsequent analysis. Based on the alignment results from HISAT2, transcripts were reconstructed using StringTie, and gene expression levels for each sample were calculated using RSEM. Genes satisfying the criteria of |log₂FC| ≥ 1 and adjusted P -value < 0.05 were identified as significantly differentially expressed genes. Functional annotation and enrichment analysis of the differentially expressed genes were then conducted using GO and KEGG databases. Yeast one-hybrid assay Promoter sequences containing the predicted GTAC-box motif (approximately 200 bp) from the target genes were cloned into the pAbAi vector to generate bait reporter constructs (pAbAi-proBpGSTF3, pAbAi-proBpASA1). The coding sequence of BpSPL2 (without the stop codon) was cloned into pGADT7-Rec to produce the effector plasmid pGADT7 -BpSPL2 . Subsequently, the minimum inhibitory concentration of Aureobasidin A (AbA) for each bait vector was determined. Yeast one-hybrid assays were performed following the manufacturer's instructions (BD Matchmaker™ Library Construction and Screening Kit User Manual). Yeast cells were plated onto selective dropout medium (SD/-Ura/-Leu) containing the corresponding concentration of AbA to screen for positive clones. Primers used are listed in the Supplementary Materials. Dual-LUC transient expression assays To analyze the regulatory role of BpSPL2 on its target genes, we constructed reporters by cloning the promoters of the target genes to drive the Luc gene in the pGreenII-0800-LUC vector (pGreenII-proBpGSTF3-LUC and pGreenII-proBpASA1-LUC), and used pGreen-62-SK to drive BpSPL2 as the effector (pGreen-BpSPL2-SK). The effector and reporter plasmids were individually transformed into Agrobacterium tumefaciens strain GV3101 harboring the pSoup helper plasmid. Following the method described by Zang et al.(Zang et al. 2017 ), bacterial cultures carrying the effector and reporter vectors were mixed in the required ratio to prepare the infiltration solution. Healthy, pre-flowering soil-grown tobacco plants at a vigorous growth stage were selected. The prepared bacterial suspension was infiltrated into tobacco leaves using a 1 mL needleless syringe. Each experimental group included at least three biological replicates. After infiltration, plants were kept in the dark for 12 hours and then transferred to normal growth conditions for 48 hours prior to fluorescence observation. Subsequently, the activities of REN and LUC were measured using a Dual-Luciferase Reporter Assay Kit (Beyotime, Shanghai, China). ChIP-PCR assay Chromatin immunoprecipitation (ChIP) was performed following the method described by Zhao et al.(Zhao et al. 2020 )Proteins and DNA were cross-linked using 3% (w/v) formaldehyde. After nuclei isolation and purification, chromatin was sheared by sonication into fragments of 200–500 bp. Following sonication, chromatin was immunoprecipitated using an anti-GFP antibody (ChIP+). Sonicated chromatin immunoprecipitated with a nonspecific antibody (IgG) served as the negative control (ChIP–). Cross-linking was reversed by digestion with proteinase K, and the ChIP products were purified using the TIANgel Maxi Purification Kit (Qiagen, Hilden, Germany). The ChIP-PCR program was as follows: 94°C for 3 min; 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 20 s; followed by a final extension at 72°C for 5 min and holding at 4°C. All primers used for ChIP-PCR are listed in the Supplementary Materials. Statistical analysis Significant differences were analyzed using Student's t-test, one-way ANOVA combined with Tukey’s multiple comparison test in GraphPad Prism 9.0. An asterisk “ * ” indicates a significant difference at p < 0.05, and identical letters denote no significant difference. Data are presented as mean ± standard deviation. Results bp-miR156c negatively regulates plant drought tolerance Under normal conditions, the bp-miR156c -OE lines exhibited superior growth compared to the WT, the plant height significantly greater than that of the wild-type (by approximately 20%, Fig. S1 A). The leaf size (measured as the area of the third mature leaf) was also significantly larger than in wild-type plants (by about 52.3%, Fig. S1 B). However, there were no significant differences in physiological indicators such as leaf water content, MDA, H₂O₂ and O₂⁻ levels, or in the activities of SOD and POD. After 7 days of drought stress, the leaves of the bp-miR156c -OE lines showed obvious wilting and water loss (Fig. 1 A). The leaf water content of WT plants was 35.1% higher than that of the bp-miR156c -OE lines ( P < 0.001), and the plants remained upright (Fig. 1 A, B). Measurement of oxidative stress-related parameters under drought conditions revealed that the bp-miR156c -OE lines had significantly higher levels of MDA, H₂O₂ and O₂⁻ (increased by approximately 29.4%, 70.1% and 39.3%, respectively) compared to the WT lines (Fig. 1 C, D, E). Furthermore, the activities of the antioxidant enzymes POD and SOD were significantly lower in the transgenic birch plants after drought stress compared to the WT plants (Fig. 1 F, G). These results indicate that overexpression of bp-miR156c in birch increases sensitivity to drought and reduces drought tolerance. BpSPL2 is a direct target of bp - miR156c While the canonical mechanism of miR156-targeted cleavage of SPL transcripts has been widely validated, the efficiency of cleavage by different miR156 family members on specific SPL genes varies considerably across species. In this study, wild-type (WT) tissue-cultured plantlets were subjected to drought stress simulated by 20% PEG treatment. The results showed that with prolonged stress duration, the expression of bp-miR156c was significantly down-regulated, whereas the expression pattern of BpSPL2 exhibited an opposite trend (Fig. 2 A). Furthermore, we predicted the target cleavage site (Fig. 2 B) and experimentally validated the bp-miR156c mediated cleavage of BpSPL2 using GUS staining assays. The constructs 35S::bp-miR156c-GUS, 35S::BpSPL2-GUS and 35S::bp-miR156c (without GUS tag) were generated. Following agrobacterium mediated transient expression in tobacco leaves and GUS staining, positive controls (35S::bp-miR156c-GUS and 35S::BpSPL2-GUS fused with the GUS tag) showed blue coloration, while the negative control (35S::bp-miR156c without GUS tag) remained colorless. In the experimental group, due to the cleavage of BpSPL2 by bp-miR156c , GUS activity was suppressed, resulting in significantly lighter or nearly undetectable staining compared to the positive controls (Fig. 2 C). These results demonstrate that bp-miR156c can specifically target and cleave BpSPL2 transcripts. BpSPL2 positively regulates drought tolerance in birch To investigate the drought tolerance phenotype conferred by BpSPL2 , we compared the physiological performance of two independent 35S::BpSPL2-OE lines (OE3 and OE5) with that of WT plants under both normal and drought conditions. Under normal growth conditions, all lines exhibited similar growth phenotypes with no significant difference. However, under drought stress, leaves of WT plants lost water more severely and showed wilting earlier than those of the overexpressing lines (Fig. 3 A). Measurement of leaf relative water content revealed that, after drought stress, both OE lines maintained significantly higher water content (approximately 2-fold) compared to the WT plants (Fig. 3 B). Subsequently, we observed and statistically analyzed the belowground root phenotypes of these three lines before and after drought treatment. The results showed that under both normal and drought conditions, the root length and fresh weight of the two OE lines were significantly greater than WT line. Specifically under drought stress, the root lengths of BpSPL2-OE 3 and OE5 lines were 45.5% and 49.5% longer than that of the WT, respectively, while their root fresh weights were 30.5% and 54.6% higher than the WT, respectively (Fig. 3 C, D). As lateral roots are a major component of the root system, we examined the root architecture of tissue-cultured seedlings (where the root structure is simpler and easier to observe) of BpSPL2-OE 3, OE5 and WT lines. The length and density of lateral roots were significantly higher in both OE lines compared to the WT (Fig. 3 E, F, G). This explains why the root systems of the two BpSPL2-OE lines were more developed than that of the WT during soil-based drought treatment. Collectively, these results indicate that BpSPL2 positively regulates drought tolerance in birch by promoting lateral root growth. BpSPL2 regulates glutathione metabolism in response to drought stress RNA-seq analysis of leaves from BpSPL2 transgenic birch plants before and after drought treatment yielded approximately 70 Gb of clean data. Each sample produced over 5.81 Gb clean data, with Q30 base percentages exceeding 95.86%. Comparative analysis between the drought stressed BpSPL2-OE and WT lines identified 5597 differentially expressed genes (DEGs), comprising 2286 upregulated and 2771 downregulated genes (Fig. S2). When comparing the same genotype before and after stress, the WT line exhibited 2946 upregulated and 3324 downregulated DEGs, whereas the BpSPL2-OE line showed only 1086 upregulated and 1148 downregulated DEGs (Fig. 4 A, B). The substantially less number of DEGs in the OE line indicates it was less perturbed by drought, consistent with its enhanced tolerance. GO and KEGG enrichment analyses were performed on the DEGs identified between the OE and WT lines under drought conditions. GO enrichment revealed that these DEGs were primarily associated with processes such as photosynthesis (GO:0009765), light harvesting (GO:0009765), and oxidoreductase activity (GO:0016491) (Fig. 4 C). KEGG pathway analysis showed significant enrichment for pathways including MAPK signaling (map04016), photosynthesis - antenna proteins (map00196), photosynthesis (map00195), tryptophan metabolism (map00380), glutathione metabolism (map00480), and biosynthesis of secondary metabolites (map00999) (Fig. 4 D). These results suggest that BpSPL2 modulates drought resistance in birch mainly by influencing photosynthesis, hormone signal transduction, and cellular redox regulation. Therefore, we subsequently focused our analysis on key processes: photosynthesis (including light harvesting and electron transport), ROS scavenging related to glutathione metabolism, and the tryptophan metabolism pathway along with its downstream IAA signaling. Overexpression of BpSPL2 stabilizes photosynthetic electron transport and the expression of related genes under drought stress Drought stress leads to water loss and wilting in birch leaves, which significantly impacts the plant's photosystems. We investigated the effects of drought stress on PSII and PSI activity in birch leaves using parameters derived from prompt chlorophyll fluorescence transients (OJIP) and 820-nm reflection ( MR820 ). Under normal conditions, the overall shapes of the OJIP fluorescence induction curves and MR820 kinetics curves showed no significant differences among the WT, BpSPL2-OE 3 and BpSPL2-OE 5 lines. However, drought stress altered the curve morphology, primarily manifested as an increase in the relative fluorescence intensity at the O step, a decrease at the P step of the OJIP curve, and a reduction in the amplitude of the MR820 curve (Fig. 5 A, C). Analysis of the chlorophyll fluorescence kinetics (OJIP) curve via the JIP-test revealed that the maximum quantum yield of PSII photochemistry ( Fv/Fm ) under drought stress was significantly higher in BpSPL2 transgenic plants compared to WT plants (Fig. 5 B). The MR820 curve reflects PSI activity. Under drought stress, the curve for WT plants was overall significantly lower than that for BpSPL2-OE plants. Quantitative data for Δ I/I₀ showed a significant decrease under drought, but overexpression of BpSPL2 partially alleviated this suppression (Fig. 5 C, D). Under drought conditions, the OJIP curves of birch leaves exhibited significant distortion, mainly characterized by a marked decrease in fluorescence intensity at the P phase. Compared to WT plants, BpSPL2-OE transgenic plants partially restored the OJIP curve morphology under drought stress, particularly improving performance at the P phase. Based on the normalized curves ( V O−P , V O−J , V O−K ) and double-normalized plots derived from the OJIP transients, BpSPL2 transgenic plants displayed distinct negative peaks, especially at the J (2 ms), K (0.3 ms) and L (0.15 ms) steps. The relative variable fluorescence values V J , V K , V L were also significantly lower in BpSPL2 transgenic plants than in WT plants (Fig. 5 E-J). These results indicate that overexpression of the BpSPL2 gene can mitigate the drought-induced inhibition of both PSII and PSI, particularly by facilitating photosynthetic electron transport in PSII. Based on the analysis of the drought transcriptome, we identified significant changes in a total of 18 genes encoding photosynthesis‑antenna proteins, including 6 Lhca and 12 Lhcb genes. Under drought stress, all these genes were down‑regulated; however, in the BpSPL2 ‑OE5 line, the extent of down‑regulation for most antenna‑protein genes was less pronounced compared to the WT plants (Fig. 5 K). Moreover, three Lhca‑encoding genes (BpChr06G30386, BpChr06G30442, BpChr08G02997) showed significantly lower expression in WT than in OE‑5 under drought conditions, while Lhcb‑encoding genes overall exhibited the highest expression in OE‑5. Additionally, 25 differentially expressed genes related to the photosynthetic photosystem pathway (map00195) were identified in both OE‑5 and WT birch after drought treatment. Under non‑stress conditions, the expression levels of all PSII and PSI reaction‑center genes showed no significant differences. After stress, these genes were expressed significantly higher in OE‑5 plants than in WT, although compared to the pre‑drought levels, their expression was down‑regulated in both lines—yet the down‑regulation was less severe in OE‑5 plants (Fig. 5 K). These results indicate that drought stress suppresses the expression of genes associated with PSI and PSII proteins. The transcriptome data are consistent with the corresponding phenotypic observations, demonstrating that overexpression of BpSPL2 reduces drought‑induced damage to photosynthetic antenna proteins and thereby alleviates photosynthetic inhibition. The BpSPL2 gene enhances ROS scavenging under drought stress and up-regulates the expression of genes involved in the glutathione metabolism pathway. Drought stress severely impacts the plant's photosynthetic system, disrupting electron transport balance and leading to excessive accumulation of ROS. In situ staining of O₂⁻ and H₂O₂ with NBT and DAB, respectively, in birch leaves before and after drought stress showed no difference in staining intensity between BpSPL2-OE and WT lines under normal conditions. After stress, however, the blue and brown staining was significantly deeper in WT leaves compared to BpSPL2-OE leaves, indicating that overexpression of BpSPL2 reduced the accumulation of O₂⁻ and H₂O₂ under drought stress (Fig. 6 A). Physiological measurements aligned with the staining results: under drought stress, the contents of O₂⁻ and H₂O₂ were significantly lower in both BpSPL2-OE lines than in the WT (Fig. 6 B, C), resulting in MDA content being 45.07% and 45.58% lower in the BpSPL2-OE 3 and BpSPL2-OE 5 lines, respectively, compared to the WT (Fig. 6 D). As indicated by the earlier RNA‑seq results, BpSPL2 likely regulates ROS metabolism in birch leaves under drought stress mainly by affecting cellular redox processes, with the glutathione metabolism pathway being significantly enriched in the KEGG analysis. We performed a heatmap analysis of the transcriptome expression for genes significantly enriched in the glutathione metabolism process. The results revealed that among the 28 differentially expressed genes (DEGs), 24 were GST genes, while the remaining included 1 GPX , 2 DHARs , and 1 GR . In the BpSPL2-OE 5 transgenic plants, 10 GST members were significantly up‑regulated compared to the WT before and after drought stress, with three genes—BpChr14G12602, BpChr05G19178, and BpChr05G19184—showing particularly prominent induction (Fig. 6 E). After screening, we selected BpChr05G19178 ( BpGSTF3 ), whose expression was markedly induced by BpSPL2 and whose promoter region contains multiple GTAC binding motifs for SPL transcription factors, for further investigation. BpSPL2 positively activates BpGSTF3 and BpASA1 , enhancing GST enzyme activity and promoting IAA accumulation. Earlier, transcriptome data analysis predicted potential downstream regulatory pathways of the BpSPL2 gene under drought stress. We performed expression pattern analysis on key genes within these pathways, including BpGSTF3 , BpChr14G12602 and BpChr05G19184 from the GST family, as well as BpASA1 , a key rate-limiting enzyme gene in the tryptophan synthesis pathway. RT-qPCR results showed tissue-specific expression: BpGSTF3 and BpChr05G19184 were significantly induced by BpSPL2 mainly in leaves, while BpChr14G12602 expression was significantly higher in roots than in leaves (Fig. 7 A, Fig S3. A, B). In contrast, BpASA1 was primarily and significantly induced in roots (Fig. 7 B). Promoter element analysis revealed that their promoters all contain the GTAC-box, a binding motif for SPL transcription factors. To determine whether BpSPL2 can bind these DNA motifs to regulate gene expression, we performed ChIP-PCR assays on fragments of proBpGSTF3 and proBpASA1 containing the GTAC-box. The results showed that BpSPL2 binds to the P4 site of proBpASA1 and the P3 site of proBpGSTF3, confirming its ability to regulate gene expression by binding these DNA motifs (Fig. 7 C, D). Subsequently, the promoter binding site fragments (200–300 bp) identified by ChIP-PCR were cloned into the pAbAi vector for yeast one-hybrid (Y1H) validation. The results indicated that sequences containing the GTAC-box can be bound by BpSPL2, enabling growth on selective minimal medium containing Aureobasidin A (AbA) (Fig. 7 E). Furthermore, dual-luciferase (LUC) reporter assays confirmed that BpSPL2 binds to the proBpGSTF3 and proBpASA1 promoters and activates their expression (Fig. 7 F, G, H). Drought stress leads to excessive ROS production in plants, triggering severe oxidative stress. Previous experiments verified that birch BpSPL2 can target and regulate BpGSTF3 , activating its expression and increasing the activity of the antioxidant enzyme GST under drought stress (by approximately 36.7% and 29.6%, Fig. 7 J). This enhances ROS scavenging capacity, reduces oxidative damage, and thereby improves drought tolerance in birch. We also measured the activities of other antioxidant enzymes involved in ROS scavenging (POD and SOD). The results showed no significant differences and generally low activity among the lines under normal conditions. However, after drought stress treatment, SOD (by 55%) and POD (by approximately 80%) activities were significantly higher in BpSPL2-OE plants compared to WT plants (Fig. 7 K, L). These results indicate that BpSPL2 can increase the antioxidant capacity in birch, reducing the degree of oxidative damage under drought stress, thereby enhancing drought tolerance. On the other hand, we also confirmed that BpSPL2 regulates BpASA1 , activating its expression (Fig. 7 G, H) and inducing IAA accumulation (Fig. 7 I), which in turn promotes lateral root development in birch and enhances its drought tolerance. Discussion Drought stress severely restricts plant growth and development, leading to premature leaf drop, delayed growth, and impaired nutrient accumulation, thereby causing significant economic losses(Luo 2010 ). Plants have evolved complex transcriptional regulatory networks to respond to drought stress, such as the miR156/SPL transcriptional regulatory module, which can be significantly induced under stress conditions to regulate a series of downstream stress responses. This study is the first to demonstrate that in birch, the miR156c/SPL2 module enhances drought tolerance by activating the key rate-limiting enzyme for auxin biosynthesis, BpASA1 and glutathione-S-transferase, BpGSTF3 , thereby promoting lateral root development and mediating the scavenging of ROS. While studies in herbaceous plants have largely shown the miR156/SPL module to act as a negative regulator of stress resistance, our findings reveal that BpSPL2 , targeted by bp-miR156c , functions as a transcriptional activator playing a crucial role in the drought stress response of birch. This novel discovery provides a fresh perspective on the drought resistance mechanisms in birch and potentially other perennial woody plants. The miR156/SPL module is a key regulator that imparts plasticity to plant growth in response to light and other environmental factors(Sang et al. 2023 ). For instance, in tomato and Arabidopsis seedlings, overexpression of miR156 and suppression of SPL lead to elongated hypocotyls seeking light and activate light-responsive mechanisms(Sang et al. 2023 ). In response to drought and other environmental stresses, the miR156/SPL module can regulate plant stress tolerance by modulating anthocyanin production(Cui et al. 2014 ), auxin content(Feng et al. 2023 ), antioxidant enzyme activity(Zhao et al. 2024 ), Fv/Fm values, chlorophyll content(Wen et al. 2024 ), ABA accumulation, and stomatal conductance(Visentin et al. 2020 ). Drought stress affects both the morphology and physiology of birch. In this study, under PEG-simulated drought stress, the BpSPL2 gene was significantly induced, while bp-miR156c was suppressed (Fig. 2 A). This aligns with findings on the miR156/SPL module in apple under stress, where adversity leads to downregulation of miR156, releasing its inhibition of the downstream gene MdSPL13 , ultimately enhancing the expression of the stress-responsive gene MdWRKY100 and improving stress resistance in apple(Ma et al. 2021 ). Interestingly, however, bp-miR156c-OE plants exhibited reduced drought tolerance (Fig. 1 ), whereas BpSPL2-OE plants showed enhanced drought tolerance (Fig. 3 ). This phenotypic observation contrasts with conclusions in herbaceous plants (such as alfalfa and rice), where miR156 often enhances stress resistance by silencing SPLs . For example, heterologous expression of OsamiR156b/c simultaneously increases both biomass and stress tolerance in alfalfa(Arshad, Feyissa, et al. 2017 ; Wang et al. 2021 ). In cassava, a perennial woody starch crop often grown as an annual, MeSPL9 also negatively regulates drought tolerance(Li et al. 2022 ). However, in the study by Ning (Ning et al. 2017 ), birch BpSPL9 was described to improve ROS scavenging ability under salt and drought stress, although the molecular mechanisms were not investigated in depth. We speculate that this may reflect functional diversity of SPL family transcription factors across different species. To further explore the molecular regulatory mechanisms underlying these morphological and physiological changes, we analyzed the transcriptomes before and after drought stress. A total of 5597 differentially expressed genes (DEGs) were identified between the BpSPL2-OE and WT lines after stress. GO and KEGG enrichment analyses of these DEGs revealed that they are primarily involved in pathways such as photosynthesis, oxidoreductase activity, MAPK signaling, tryptophan metabolism, glutathione metabolism, and biosynthesis of secondary metabolites (Fig. 4 ). Drought stress led to dry and wilted birch leaves, significantly reduced water content (Fig. 3 A, B), and inhibited photosynthesis (Fig. 5 ). When photosynthesis is suppressed, excess electrons in the photosynthetic electron transport chain result in overproduction of ROS in chloroplasts, which in turn exacerbates photosynthetic inhibition, forming a vicious cycle. Moreover, excessive ROS induces lipid peroxidation and damages membranes, proteins, chlorophyll, nucleic acids, ultimately leading to cell death(Scandalios 2005 ; Smirnoff 1993 ). By examining the effects of drought on the photosystems and several ROS-related indicators in BpSPL2-OE and WT lines, we found that the BpSPL2-OE lines better maintained ROS homeostasis (Fig. 6 ), thereby protecting birch plants and reducing oxidative damage caused by drought stress. Combining phenotypic and transcriptomic analyses, we identified glutathione metabolism as a key pathway through which BpSPL2 regulates ROS balance in birch. Expression analysis of related genes in this pathway showed that most DEGs belonged to the BpGSTs family, with BpGSTF3 being particularly prominent. GSTs detoxify ROS via glutathione conjugation, and their induced expression under drought stress can reduce peroxide production during oxidation(Hu et al. 2024 ; Edwards and Dixon 2005 ), thereby alleviating photoinhibition. Previous studies have reported that SPL genes regulate downstream gene expression by recognizing and binding to GTAC motifs(Ma et al. 2021 ). In this study, Y1H, ChIP and LUC assays confirmed that BpSPL2 directly binds to the GTAC motif in the BpGSTF3 promoter, activating its expression and enhancing GST enzyme activity (Fig. 7 ). In BpSPL2-OE plants, the activities of GST, SOD, and POD were significantly increased (Fig. 7 ), effectively reducing H₂O₂ and O₂⁻ levels (Fig. 5 B, C) and thereby mitigating membrane lipid peroxidation (reduced MDA content, Fig. 5 D). This aligns with reports that overexpression of pear GST or tamarisk ThGSTZ1 enhances ROS scavenging capacity(Liu et al. 2013 ; Yang et al. 2014 ). BpSPL2-OE plants maintained higher PSII efficiency ( Fv/Fm ) and expression of light-harvesting protein genes under drought (Fig. 5 ), indicating that GST-mediated ROS scavenging alleviated drought-induced photoinhibition, consistent with the protective role of the chloroplastic ROS scavenging system on photosynthetic machinery(Sun et al. 2013 ). Additionally, studies have shown that SPL genes can regulate the accumulation of osmoregulatory substances in plants, suggesting that BpSPL2 may also enhance drought tolerance in birch through osmotic adjustment. miR156-targeted SPLs regulate anthocyanin synthesis(Gou et al. 2011 ), which acts as both an osmoregulant and a ROS scavenger(Landi, Tattini, and Gould 2015 ). In cassava, SPL9 regulates proline and anthocyanin synthesis via JA signaling(Li et al. 2022 ), implying that BpSPL2 might participate in osmoprotection through similar pathways, although this requires further experimental validation. Furthermore, under drought stress, plants finely modulate their root system architecture to enhance water uptake capacity, among which the formation and development of lateral roots (LRs) is a key adaptive response. LR formation is a major determinant of root system architecture, significantly influencing water absorption efficiency, nutrient acquisition, and plant anchorage(Péret et al. 2009 ). The initiation and development of LRs are inextricably linked to hormonal regulation, with auxin (IAA) being one of the primary regulatory hormones. Anthranilate synthase α-subunit 1 ( ASA1 ), a key rate-limiting enzyme in the tryptophan-dependent IAA biosynthetic pathway, plays a central role in IAA biosynthesis(Niyogi and Fink 1992 ; Zhao and Last 1996 ). ASA1 catalyzes the conversion of chorismate to anthranilate, a precursor for tryptophan synthesis, which in turn leads to auxin production via the tryptophan-dependent IPA pathway. This pathway is widespread in plants and plays a crucial role in local auxin accumulation in root tips(Miles 2001 ; Zhao 2010 ; Teale, Paponov, and Palme 2006 ). Under drought stress, auxin synthesis and distribution in roots undergo significant alterations. The upregulation of ASA1 expression may promote auxin synthesis, thereby stimulating the initiation and development of lateral root primordia, increasing lateral root density and root surface area, and consequently enhancing water absorption capacity. Additionally, auxin synthesized locally in roots also plays a vital role in responding to environmental stress, coordinating plastic root development in concert with shoot-derived auxin(Chen 2004 ; Zhao 2018 ). The expression of ASA1 is regulated by upstream transcription factors. For instance, in leaf explants, jasmonic acid (JA) biosynthesis occurs within two hours after wounding and transcriptionally activates ERF109 , which encodes a protein that upregulates ASA1 expression to promote auxin biosynthesis and facilitate de novo root organogenesis(Lee et al. 2024 ). The SPL transcription factor family, as target genes of miR156, is widely involved in regulating lateral root development. Niu et al(Yu et al. 2015 ), found that miR156 is differentially expressed in specific cells and tissues of lateral roots in Arabidopsis and regulates SPL3 , SPL9 , and SPL10 genes in response to hormone signaling pathways to suppress lateral root growth. In transgenic potatoes overexpressing Stu-miR156 , the expression levels of the target gene StSPL9 were downregulated. Compared to controls, transgenic plants showed significantly reduced plant height and root length but a significant increase in lateral root number(Luo et al. 2024 ). Apple MdSPL23 binds to the promoter of MdNLP7 to inhibit its expression, thereby negatively regulating nitrogen uptake and suppressing lateral root development in apple(Xu et al. 2025 ). Rice OsSPL14 activates the expression of OsSHR1 ; the expressed OsSHR1 further promotes the transcription of OsSWEET16 , reducing the accumulation of glucose and fructose in tiller buds and decreasing fructose and sucrose in roots, ultimately leading to fewer tillers and crown roots but increased root length(Feng et al. 2025 ). In reported results, SPLs often function as negative regulators of lateral root development. However, we observed that birch BpSPL2 positively regulates lateral root development (Fig. 3 ), acting as a positive regulator to activate auxin synthesis and promote lateral root formation. Subsequent results also confirmed that in birch, BpSPL2 can positively activate the expression of the BpASA1 gene. The direct activation of BpASA1 by BpSPL2 significantly promoted auxin biosynthesis (Fig. 7 G), thereby stimulating lateral root formation and enhancing birch's adaptive capacity under drought conditions. This finding not only provides new evidence for the functional diversity of the SPL protein family but also reveals the critical role of the SPL2-ASA1-auxin signaling pathway in birch lateral root development under drought stress. Furthermore, extensive crosstalk exists among multiple hormone signaling pathways in drought stress responses. ABA, a key hormone in drought response, interacts with the auxin signaling pathway through its receptors PYL8 and PYL9 to co-regulate lateral root growth and recovery(Zhao et al. 2014 ; Xing et al. 2016 ). Transcription factors such as MYB96 and WRKY46 also integrate ABA and auxin signals to regulate lateral root initiation under drought conditions(Seo et al. 2009 ; Ding et al. 2015 ). The miR156-SPL2-ASA1 regulatory module identified in this study may also interact with ABA signaling to coordinate root system plasticity in response to drought, but this requires further investigation. In summary, our study further reveals that in birch, SPL2 can directly activate ASA1 expression to promote auxin synthesis and lateral root development, thereby enhancing drought tolerance. This finding expands the understanding of SPL family functions and provides a novel perspective on their role in drought resistance mechanisms in tree species. Conclusion This study systematically elucidates the molecular mechanism by which the bp-miR156c / BpSPL2 module enhances drought tolerance in birch by promoting lateral root development and activating the antioxidant pathway. Drought stress significantly suppresses the expression of bp-miR156c , thereby releasing its inhibition on the target gene BpSPL2 . Overexpression of bp-miR156c reduces drought tolerance in birch, while its target gene BpSPL2 directly binds to the GTAC motifs in the promoters of the target genes BpASA1 and BpGSTF3 , activating their transcription. On the one hand, it induces IAA accumulation to promote lateral root development, thereby enhancing drought resistance. On the other hand, it increases GST enzyme activity to reduce ROS-induced oxidative damage and alleviate photosynthetic inhibition. Declarations Acknowledgements This work was supported by the National Key Research and Development Program of China (No2021YFD2200304); Doctoral Student Independent Innovation Fund (2572025AW49); the Fundamental Research Funds for the Central Universities (No2572022DQ08); the National Natural Science Foundation of China (No32171738). Author Contributions: H.P. performed most of the experiments, analysed the data and drafted the manuscript. 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Zhang. 2007. 'UV-B responsive microRNA genes in', Molecular Systems Biology, 3. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 25 Apr, 2026 Reviews received at journal 21 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviews received at journal 29 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 14 Mar, 2026 Submission checks completed at journal 14 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9102551","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608385695,"identity":"8a7cbd07-e2a2-43da-93b5-86feb1f63408","order_by":0,"name":"Huang Peng","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Huang","middleName":"","lastName":"Peng","suffix":""},{"id":608385698,"identity":"5fd711cd-1a7c-47ae-b9a5-1e12e1979f1e","order_by":1,"name":"Zhang Hongrui","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Hongrui","suffix":""},{"id":608385699,"identity":"f83573b8-f735-4f0e-8196-eb7f60c1e110","order_by":2,"name":"An Jiaqian","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"An","middleName":"","lastName":"Jiaqian","suffix":""},{"id":608385700,"identity":"3531d7fe-918b-4932-bca3-e31c3ba6cc2b","order_by":3,"name":"Yuan Zhongjia","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhongjia","suffix":""},{"id":608385701,"identity":"9f35e733-aa81-4dae-8b84-581119bb0de8","order_by":4,"name":"Duan Huilei","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Duan","middleName":"","lastName":"Huilei","suffix":""},{"id":608385702,"identity":"8b7af166-8b07-40aa-8190-5cfc7ebc7c4d","order_by":5,"name":"Zhang Huihui","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Huihui","suffix":""},{"id":608385703,"identity":"1c43ad64-5f61-470f-9c60-b67027083d3c","order_by":6,"name":"Liu Xuemei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIie3QsQrCMBCA4ZNCdLiaTSJi+wqRLg6Cr9IsTgo+giJ00l3Rh9DNMeVAl7oLXRRnoWMdRMVRhMbNIf98H9wdgM32h1UdKOnSqIPcJ62z3IAwB+BFel59zlS8mJoQeBMK5BEDqjATUkapb1tHjdJpRoDg85ouWgxlPEuYGq8Oaxq2obVYhsVEuxGqCQzWNEcIZWpA4nskVAT9EyEzJORGMkDRB1PChtRMQk/gTr6eLIpv4Zw25+v2gd395JJlecfnjQLymfht3Gaz2WzfewLJp0XRYyyQMwAAAABJRU5ErkJggg==","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Liu","middleName":"","lastName":"Xuemei","suffix":""}],"badges":[],"createdAt":"2026-03-12 08:56:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9102551/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9102551/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105035065,"identity":"0f7acfed-e7c0-436d-b9b4-b01502a1e8eb","added_by":"auto","created_at":"2026-03-20 07:25:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1778810,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003ebp-miR156c\u003c/em\u003e impairs drought tolerance in birch. (A) Phenotypes of wild-type (WT) and \u003cem\u003ebp-miR156c\u003c/em\u003e-OE birch plants under normal growth conditions and after 7 days of drought stress. (B) Leaf relative water content (RWC). (C) MDA content. (D) O₂⁻ content. (E) H₂O₂content. (F) SOD activity. (G) POD activity. Note: In panel A, the \u003cem\u003ebp-miR156c \u003c/em\u003eoverexpressing plants shown belong to the same transgenic line. Scale bar ≈ 10 cm. Error bars represent the mean ± SD from three independent biological replicates (each replicate consisted of 15 plants). Asterisks indicate significant differences between the WT and transgenic plants as determined by one-way ANOVA with Tukey's multiple comparisons test. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/24ea9cfd8a791d69e729f5b0.png"},{"id":104984029,"identity":"8a0f86b0-3ede-4f7e-b37d-ba799df96e59","added_by":"auto","created_at":"2026-03-19 14:01:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1132174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ebp-miR156c\u003c/em\u003e targets and cleaves \u003cem\u003eBpSPL2\u003c/em\u003e transcripts. (A) Expression patterns of \u003cem\u003ebp-miR156c\u003c/em\u003eand \u003cem\u003eBpSPL2 \u003c/em\u003efollowing PEG-simulated drought treatment. (B) Prediction of the miR156/SPL2 cleavage site. (C) GUS staining assay validating the \u003cem\u003ebp-miR156c\u003c/em\u003e-mediated cleavage of \u003cem\u003eBpSPL2\u003c/em\u003e. Error bars represent mean ± SD from three independent biological replicates. Asterisks indicate significant differences as determined by one-way ANOVA with Tukey's multiple comparisons test. (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/b24e242db7f5e201dfd96e4d.png"},{"id":104984048,"identity":"92e1dced-7296-442b-b8ab-6746c00a737e","added_by":"auto","created_at":"2026-03-19 14:01:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3425906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBpSPL2\u003c/em\u003e enhances drought tolerance in birch. (A) Phenotypes of WT and\u003cem\u003e BpSPL2-OE \u003c/em\u003etransgenic birch plants after 7 days of drought stress. (B) Leaf relative water content. (C) Root length. (D) Root fresh weight. (E) Lateral root phenotypes of tissue-cultured seedlings of WT and \u003cem\u003eBpSPL2-OE\u003c/em\u003e lines. (F) Lateral root density. (G) Lateral root length. Error bars represent the mean ± SD from three independent biological replicates. Asterisks indicate significant differences between the WT and transgenic plants as determined by one-way ANOVA with Tukey's multiple comparisons test.(*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/0168b65a88f081f673092d4c.png"},{"id":104984047,"identity":"48b9392b-10fa-4776-9b3e-1f5b6bafc980","added_by":"auto","created_at":"2026-03-19 14:01:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1145839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBpSPL2 \u003c/em\u003edrought stress transcriptome analysis\u003cstrong\u003e \u003c/strong\u003e(A, B) MA plots of differentially expressed genes (DEGs) between WT and \u003cem\u003eBpSPL2‑OE5\u003c/em\u003e lines before and after drought treatment. (C) Gene Ontology (GO) enrichment analysis of the DEGs. (D) KEGG pathway enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/3447e986b36c5fce5e632c4e.png"},{"id":104984030,"identity":"d69ccf04-97d4-4f60-a692-d3e7790a93ce","added_by":"auto","created_at":"2026-03-19 14:01:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1886232,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll fluorescence parameters and expression of photosynthetic electron transport-related genes in BpSPL2 transgenic and WT birch under drought stress. (A) OJIP transients of \u003cem\u003eBpSPL2-OE \u003c/em\u003eand WT lines before and after drought stress. (B) Maximum photochemical efficiency of PSII (\u003cem\u003eFv/Fm\u003c/em\u003e). (C) \u003cem\u003eMR820\u003c/em\u003e kinetics of \u003cem\u003eBpSPL2-OE\u003c/em\u003e and WT lines before and after drought stress. (D) Δ\u003cem\u003eI/I₀\u003c/em\u003e values. (E) Normalized O-P curves (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO-P\u003c/em\u003e\u003c/sub\u003e). (F) \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eJ\u0026nbsp; \u003c/em\u003e\u003c/sub\u003evalues before and after drought stress. (G) Normalized O-J curves (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO-J\u003c/em\u003e\u003c/sub\u003e). (H) \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eK\u003c/em\u003e\u003c/sub\u003e values before and after drought stress. (I) Normalized O-K curves (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO-K\u003c/em\u003e\u003c/sub\u003e). (J) \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eL \u0026nbsp;\u003c/em\u003e\u003c/sub\u003evalues before and after drought stress. (K) Effects of drought stress on the expression levels of genes encoding light-harvesting antenna proteins and proteins involved in photosynthetic electron transport in leaves of different birch lines.Error bars represent the mean ± SD from three independent biological replicates. Asterisks indicate significant differences between the WT and transgenic plants as determined by one-way ANOVA with Tukey's multiple comparisons test. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/f0a40e6531cb26db963ee536.png"},{"id":104984038,"identity":"e75abcfb-9346-4f79-b6a2-b41e6bbf4e4a","added_by":"auto","created_at":"2026-03-19 14:01:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":623172,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of drought stress on ROS levels in WT and \u003cem\u003eBpSPL2-OE\u003c/em\u003e birch. (A) NBT and DAB staining for visualizing O₂⁻ and H₂O₂ accumulation, respectively. (B) H₂O₂ content. (C) O₂⁻ content. (D) Malondialdehyde (MDA) content. (E) Heatmap of differentially expressed genes in the glutathione metabolism-related gene families. Scale bar ≈5 cm.Error bars represent mean ± SD from three independent biological replicates. Asterisks indicate signific -ant differences between the WT and transgenic plants as determined by one-way ANOVA with Tukey's multiple comparisons test. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/6089c9f7610530b000e6660c.png"},{"id":104984035,"identity":"d64382f2-7053-4a6d-94c0-5abd3db0ede1","added_by":"auto","created_at":"2026-03-19 14:01:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2467572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBpSPL2\u003c/em\u003e targets and transcriptionally activates \u003cem\u003eBpGSTF3\u003c/em\u003e and \u003cem\u003eBpASA1\u003c/em\u003e. (A, B) \u003cem\u003eBpGSTF3\u003c/em\u003eand \u003cem\u003eBpASA1\u003c/em\u003e are significantly induced in \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants. (C, D) ChIP-PCR assays. (E) Yeast one-hybrid (Y1H) assay. (F) , (G), (H) Dual-luciferase reporter assay. (I) IAA content. (J) GST enzyme activity. (K) SOD enzyme activity. (L) POD enzyme activity. Error bars represent mean ± SD from three independent biological replicates. Asterisks indicate significant differences between the WT and transgenic plants. Data in panel (H) were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). All other data were analyzed by one-way ANOVA with Tukey's multiple comparisons test. \u0026nbsp;(*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/e5048a2a3caf6b2fedf16797.png"},{"id":104984032,"identity":"c1d174c9-5eef-4679-9e35-9afb9b6da0f1","added_by":"auto","created_at":"2026-03-19 14:01:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":638789,"visible":true,"origin":"","legend":"\u003cp\u003eWorking model of the \u003cem\u003ebp-miR156c/BpSPL2 \u003c/em\u003emodule under drought conditions.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/b275283069c37fff0c466f65.png"},{"id":105037570,"identity":"b40352f9-6be7-4a44-9f6f-f23b71c218f9","added_by":"auto","created_at":"2026-03-20 07:39:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13366903,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/123a008d-aea1-4a29-ac12-a20ebd5dda27.pdf"},{"id":104984031,"identity":"47038a86-2517-4976-a834-a02e9b81f300","added_by":"auto","created_at":"2026-03-19 14:01:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":659560,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102551/v1/a53d50e4274a2d23a73fcfd7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrought is one of the major abiotic stress factors that limit plant growth, development, yield and quality(Jaleel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gupta, Rico-Medina, and Ca\u0026ntilde;o-Delgado \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, approximately 45% or more of global agricultural land is affected by persistent or frequent drought as a result of climate change(Liang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Drought stress affects all aspects of plant growth, development, and reproduction, encompassing morphological, physiological, and biochemical changes as well as the regulation of specific genes(Hussain et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Plants primarily employ two strategies to cope with drought stress: drought escape and drought tolerance. Drought tolerance, in particular, is enhanced through multiple mechanisms. These include morphological adjustments, especially increased biomass allocation to the shoot; active accumulation of osmolytes to promote water uptake; reduction of stomatal conductance to improve water use efficiency; enhanced antioxidant enzyme activity to mitigate reactive oxygen species (ROS) damage; activation of hormone signaling pathways; and various transcriptional regulations and post-translational protein modifications(Sun et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the aforementioned biological processes of drought response, the regulation by specific miRNAs and transcription factors plays a pivotal role. During evolution, plants have developed multiple regulatory networks to cope with adverse environmental conditions, with the miRNA/SPL regulatory network being one of them(Shriram et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), it can adapt to environmental stresses by regulating SPL transcription factor genes via miR156 under various stress conditions(Wang and Wang \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eSPLs\u003c/em\u003e are key inducers of plant stress resistance, mediating the plant's ability to withstand various stresses, such as drought and low temperature(Ma et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qi et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, the regulatory mechanism of the miR156/SPL module in the drought tolerance process of birch remains poorly understood.\u003c/p\u003e \u003cp\u003eMicroRNAs are a class of small non-coding RNAs, typically 20\u0026ndash;24 nucleotides in length, that play important roles in processes such as leaf development, flower development and resistance to both abiotic and biotic stresses(Chen \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mallory et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sunkar and Zhu \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Khraiwesh, Zhu, and Zhu \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sunkar, Li, and Jagadeeswaran \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Recently, an increasing number of studies have revealed that miRNAs are involved in plant tolerance to a wide range of abiotic and biotic stresses, including pathogen infection(Jones-Rhoades and Bartel \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), salinity, drought, cold, oxidative stress(Li et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Covarrubias and Reyes \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yin et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Koroban et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), UV radiation(Zhou, Wang, and Zhang \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), nutrient deficiency(Panda and Sunkar \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and heavy metals(Huang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), among others. In plants, miRNAs play a crucial regulatory role in response to drought stress, particularly the well-conserved miR156/SPL module. This module contributes to drought tolerance by participating in phytohormone signaling pathways, synthesis of osmoprotectant metabolites, enhancement of antioxidant enzyme activities and maintenance of ROS homeostasis. For instance, in Cassava (\u003cem\u003eManihot esculenta Crantz\u003c/em\u003e ), \u003cem\u003eMeSPL9\u003c/em\u003e reduces drought resistance by regulating JA signaling, whereas the miR156-targeted resistant line \u003cem\u003erMeSPL9-SRDX\u003c/em\u003e confers drought tolerance without compromising overall plant growth(Li et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.), under drought conditions, the overexpression of \u003cem\u003emiR156\u003c/em\u003e and strigolactone treatment lead to enhanced drought tolerance, which is associated with ABA accumulation and reduced stomatal conductance(Visentin et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eMicroRNA156ab\u003c/em\u003e regulates plant growth and drought resistance by targeting the transcription factor \u003cem\u003eMsSPL13\u003c/em\u003e; heterologous expression of \u003cem\u003eMsSPL13\u003c/em\u003e reduces auxin content and inhibits its growth in Arabidopsis under both normal and stress conditions(Feng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Osmoprotectant metabolites serve as key osmoregulators: in cassava, the \u003cem\u003erMeSPL9-SRDX\u003c/em\u003e line confers drought tolerance not only through JA signaling but also by promoting the synthesis of osmoprotectant metabolites such as proline and anthocyanins(Li et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eCsn-miR156f-2-5p\u003c/em\u003e and \u003cem\u003eCsSPL14\u003c/em\u003e are involved in the drought response in tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e) plants. Knockdown of \u003cem\u003ecsn-miR156f-2-5p\u003c/em\u003e significantly reduces both the maximum photochemical efficiency of PSII (\u003cem\u003eFv/Fm\u003c/em\u003e) and chlorophyll content in tea leaves, while lead to a substantial accumulation of ROS(Wen et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhotosynthesis is one of the processes most sensitive to drought in plants. Drought stress leads to chlorophyll degradation and reduced photosynthetic capacity(Bhusal, Han, and Yoon \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), causing damage to the reaction centers of both Photosystem II (PSII) and Photosystem I (PSI) in most plants and inhibiting electron transfer along the photosynthetic electron transport chain(Yao et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ohashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). When photosynthesis is suppressed under stress, the excess energy and electrons in chloroplasts are a major cause of reactive oxygen species (ROS) burst. ROS are by-products formed during normal oxygen metabolism and play crucial roles in cellular signaling and homeostasis maintenance; however, their excessive accumulation can cause damage to plant cells, with severe cases potentially leading to cell death. The homeostasis of ROS levels in plants is maintained by antioxidant enzymes that scavenge the excess ROS produced under stress conditions. The plant ROS scavenging system primarily consists of an enzymatic system, including superoxide dismutase (SOD), catalase (CAT) and peroxidases (POD), as well as enzymes in the ascorbate-glutathione (AsA-GSH) cycle such as ascorbate peroxidase (APX) and glutathione peroxidases (GPX), and peroxiredoxins (Prxs) in the thioredoxin-thioredoxin peroxidase (Trx-Prx) pathway(Wang et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the non-enzymatic antioxidant system, small molecules such as GSH, AsA, phenolic compounds, flavonoids and carotenoids play important roles in protecting plant cells from oxidative damage(Pan et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies have shown that the miR156/SPL module can participate in plant resistance to abiotic stress by regulating photosynthesis or ROS balance. In alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) \u003cem\u003eSPL13RNAi\u003c/em\u003e plants, the maintained or slightly increased transcripts related to photorespiration may act as an energy sink to prevent over-reduction of NADPH and photoinhibition(Feyissa et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e overexpressing apple \u003cem\u003eMsSPL13\u003c/em\u003e exhibited inhibited antioxidant enzyme activities, resulting in reduced drought tolerance(Feng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overexpression of \u003cem\u003eTaSPL6\u003c/em\u003e-A increased sensitivity to drought stress in wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.); after drought treatment, transgenic plants showed significantly higher leaf water loss, malondialdehyde, and ROS content, along with markedly lower antioxidant enzyme activities compared to wild-type plants. Conversely, silencing of the \u003cem\u003eTaSPL6\u003c/em\u003e gene enhanced drought tolerance in wheat, reflected in better growth performance(Zhao et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Variation in \u003cem\u003eOsSPL10\u003c/em\u003e confers drought tolerance in rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) by directly regulating the expression of \u003cem\u003eOsNAC2\u003c/em\u003e and ROS production(Li et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlutathione-S-transferases (GSTs; EC 2.5.1.18) are a class of important multifunctional enzymes involved in detoxification, defense against biotic and abiotic stresses, and transport of secondary metabolites(Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They detoxify both endogenous and exogenous compounds by conjugating glutathione (GSH) to hydrophobic substrates(\u0026Ouml;ztetik \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), a process that involves three phases: transformation, conjugation, and compartmentalization(Light et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eGSTs\u003c/em\u003e influence plant growth and development by participating in physiological metabolism, stress resistance, and cellular signaling(Song et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Functionally similar to glutathione peroxidases, \u003cem\u003eGSTs\u003c/em\u003e can reduce peroxide production during oxidative processes, thereby enhancing plant adaptability to environmental stresses. A series of studies have shown that the expression of \u003cem\u003eGST\u003c/em\u003e genes is induced by drought stress in various plant species, including \u003cem\u003eHordeum vulgare\u003c/em\u003e L. (Rezaei et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.(Xu et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eTriticum aestivum\u003c/em\u003e L.(Wang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eSolanum tuberosum\u003c/em\u003e L.(Islam et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003eCapsicum annuum\u003c/em\u003e L.(Islam et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eOryza sativa\u003c/em\u003e L.(Jain, Ghanashyam, and Bhattacharjee \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Overexpression of \u003cem\u003eGST\u003c/em\u003e in tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e L.) increased GST enzyme activity and GSH content under low temperature, high temperature, and salt stress, alleviating oxidative damage caused by abiotic stresses(P et al. 2000). In wheat, \u003cem\u003eTaERF3\u003c/em\u003e activated \u003cem\u003eTaGST6\u003c/em\u003e expression is enhanced by ABA in response to salt and drought stress(Rong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Heterologous overexpression of a pear \u003cem\u003eGST\u003c/em\u003e gene in tobacco improved the scavenging capacity for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e under drought conditions, thereby enhancing drought tolerance(Liu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Overexpression of the \u003cem\u003eTamarix chinensis\u003c/em\u003e L. \u003cem\u003eGST\u003c/em\u003e gene (\u003cem\u003eThGSTZ1\u003c/em\u003e) improved drought resistance by enhancing ROS scavenging ability(Yang et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Transgenic Arabidopsis plants overexpressing tomato \u003cem\u003eLeGSTU2\u003c/em\u003e exhibited increased resistance to drought stress through elevated antioxidant enzyme activities that scavenge excess ROS(Xu et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In tea plants, \u003cem\u003eCsWRKY48\u003c/em\u003e was found to activate the expression of \u003cem\u003eCsGSTU8\u003c/em\u003e by directly binding to the W-box region in its promoter; overexpression of \u003cem\u003eCsGSTU8\u003c/em\u003e in Arabidopsis improved ROS scavenging capacity and enhanced drought tolerance(Zhang et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eOsGSTU4\u003c/em\u003e is induced by ABA and participates in ABA-dependent processes that confer stress resistance in transgenic plants(Sharma et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo cope with water deficit induced by drought, plants have evolved a series of adaptive strategies. Among these, the plasticity of root system architecture (RSA), particularly the proliferation and development of lateral roots (LRs), is crucial for enhancing water and nutrient uptake(Ranjan et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Comas et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Koevoets et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Karlova et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lateral root formation not only expands the root surface area(Du, Spalding, and Gray \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but is also precisely regulated by endogenous hormone networks, especially the synthesis, transport, and distribution of auxin(Teale, Paponov, and Palme \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lavenus et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Notably, auxin in roots is not only derived from polar transport from the shoots(Grieneisen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhao \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), but is also significantly produced through local biosynthesis in root tissues(Ljung et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhao \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Anthranilate synthase (AS) is a key rate-limiting enzyme in the tryptophan (Trp) biosynthesis pathway, catalyzing the conversion of chorismate to anthranilate (ANT)(Zhao and Last \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Niyogi and Fink \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), where Trp serves as the major precursor for auxin biosynthesis in plants(Cohen, Slovin, and Hendrickson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhao \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tu et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Research indicates that local auxin synthesis mediated by the AS α-subunit 1 (ASA1) plays a central role in root stress responses. Under arsenate stress, the expression of \u003cem\u003eASA1\u003c/em\u003e in root tips is activated, promoting auxin synthesis and cooperating with transporters such as \u003cem\u003eAUX1\u003c/em\u003e/\u003cem\u003ePIN2\u003c/em\u003e to regulate auxin transport from the root tip to the elongation zone, thereby influencing root growth and development(Tu et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In Arabidopsis leaf explants, jasmonic acid (JA) biosynthesis occurs within two hours after wounding, and the transcriptionally activated ERF109 protein upregulates \u003cem\u003eASA1\u003c/em\u003e expression, contributing to auxin biosynthesis and promoting de novo root organogenesis(Zhang et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Under drought stress, the \u003cem\u003ePopulus euphratica\u003c/em\u003e transcription factor \u003cem\u003ePeFUS3\u003c/em\u003e positively regulates lateral root growth and enhances drought resistance by activating the expression of auxin transport genes \u003cem\u003ePIN2\u003c/em\u003e, \u003cem\u003ePIN6a\u003c/em\u003e, and \u003cem\u003eAUX1\u003c/em\u003e(Liu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, drought signals regulate lateral root formation through crosstalk between ABA and auxin pathways; for instance, ABA receptors PYL8 and PYL9(Zhao et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xing et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), as well as transcription factors such as \u003cem\u003eMYB96\u003c/em\u003e and \u003cem\u003eWRKY46\u003c/em\u003e(Seo et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), have been shown to integrate ABA and auxin signaling to control LR development. These findings suggest that \u003cem\u003eASA1\u003c/em\u003e may act as a molecular hub linking drought stress signaling to root morphological remodeling, mediating lateral root initiation by modulating local auxin synthesis and thereby improving plant drought tolerance. Previous studies have reported that the miR156/SPL module participates in lateral root formation. Overexpression of apple \u003cem\u003eMdmiR156n\u003c/em\u003e significantly increased primary root length and lateral root number in transgenic Arabidopsis plants under drought stress(Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), although its molecular mechanism remained unclear. Here, our findings demonstrate that in birch, the miR156/SPL module promotes lateral root development by activating the expression of the \u003cem\u003eASA1\u003c/em\u003e gene, thereby influencing drought tolerance.\u003c/p\u003e \u003cp\u003eWhile the mechanisms by which the miR156/SPL module influences abiotic stress tolerance have been reported in species such as alfalfa(Arshad, Feyissa, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Arshad, Gruber, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), rice(Cui et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and wheat(Zhao et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), its regulatory role in the molecular basis of drought tolerance in woody plants, particularly birch, remains unclear. In this study, we employed genetic engineering techniques to obtain transgenic birch plants overexpressing \u003cem\u003ebp-miR156c\u003c/em\u003e and \u003cem\u003eBpSPL2\u003c/em\u003e. By analyzing the physiological and biochemical changes in these transgenic plants under drought stress and conducting RNA-seq to investigate the molecular regulatory mechanisms mediated by \u003cem\u003eBpSPL2\u003c/em\u003e, we found that the miR156/SPL module enhances drought tolerance in birch by activating the expression of the \u003cem\u003eBpGSTF3\u003c/em\u003e and \u003cem\u003eBpASA1\u003c/em\u003e genes. This activation increases antioxidant enzyme activity, alleviates photoinhibition under drought stress, and promotes lateral root formation. Our study reveals the potential value of the \u003cem\u003ebp-miR156c\u003c/em\u003e/\u003cem\u003eBpSPL2\u003c/em\u003e module for the genetic improvement of drought resistance in woody plants and provides a theoretical foundation for further refining the drought stress response network.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and treatment\u003c/h2\u003e \u003cp\u003eTransgenic birch plants overexpressing \u003cem\u003ebp-miR156c\u003c/em\u003e and \u003cem\u003eBpSPL2\u003c/em\u003e, preserved from previous laboratory work, were subjected to subculture. The growth conditions were as follows: temperature at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity at 65\u0026ndash;75%, with light intensity and photoperiod set at 46 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; and 16 h light/8 h dark, respectively. The culture medium consisted of: 2.14 g/L WPM powder, 20 g/L sucrose, 0.56 g/L calcium salt, 0.4 mg/L IBA, 8 g/L plant agar, adjusted to 1 L with distilled water, and pH adjusted to 5.8 using 5 M NaOH. After 40 days of growth, tissue-cultured seedlings of each transgenic line were transplanted into a soil matrix with a vermiculite:flower soil:peat soil ratio of 1:1:1 (Note: the culture medium on the roots must be thoroughly washed off during transplantation). The plants were then acclimatized and grown indoors until stable growth was achieved (i.e., surviving and growing normally after transfer from the medium to the soil matrix). After three months, both transgenic and wild-type (WT) plants were subjected to natural drought treatment. Prior to treatment, all plant materials were thoroughly watered to achieve saturated soil moisture content. The drought treatment group underwent natural drought stress for 7 days, while the control group was watered every 3 days to maintain normal soil moisture levels. After 7 days of drought treatment, growth phenotypes were observed and photographed, followed by physiological index measurements. Each treatment was performed with 15 biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of chlorophyll fluorescence parameters in\u003c/b\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003etransgenic plants.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fully expanded third leaf from the apex of both control and treated plants from the wild-type (WT) and \u003cem\u003eBpSPL2\u003c/em\u003e transgenic lines was selected and dark-adapted for 30 minutes. Subsequently, following the method described by Wang et al.(Wang et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the OJIP transients and 820 nm reflection (\u003cem\u003eMR820\u003c/em\u003e) kinetics of birch leaves under different treatments were measured using a Multifunctional Plant Efficiency Analyzer (M-PEA, Hansatech, UK). The curves were induced by a saturating red light pulse (1000 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;). Fluorescence signals were recorded starting at 0.01 ms and ending at 2 s. On the OJIP curve, the O, J, I and P steps correspond to time points of 0.01, 2, 30, and 1000 ms, respectively, with fluorescence intensities denoted as F\u003csub\u003e0\u003c/sub\u003e, F\u003csub\u003eJ\u003c/sub\u003e, F\u003csub\u003eI\u003c/sub\u003e and F\u003csub\u003em\u003c/sub\u003e. The following parameters were derived: the maximum quantum yield of PSII photochemistry, \u003cem\u003eFv\u003c/em\u003e/\u003cem\u003eFm\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1-(\u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/F\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e), and Δ\u003cem\u003eI/I₀\u003c/em\u003e, reflecting PSI activity. Here, I₀ represents the maximum value of the \u003cem\u003eMR820\u003c/em\u003e curve, and ΔI is the difference between the maximum and minimum values of the \u003cem\u003eMR820\u003c/em\u003e curve. To analyze the changes in relative variable fluorescence at specific characteristic points, the segments of the OJIP transients between O-P, O-J, and O-K were normalized as follows. The normalized curves from the drought-treated plants of the three genotypes were then subtracted from those of their respective controls. The calculation methods are detailed below:\u003c/p\u003e \u003cp\u003eO-P segment normalization: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;P\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e), Δ\u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;J\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Δ[(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)], The relative variable fluorescence at the J step (2 ms) is obtained as: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eO-J segment normalization: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;J\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e), Δ\u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;J\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Δ[(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)], The relative variable fluorescence at the K step (0.3 ms) is obtained as: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eK\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eK\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eO-K segment normalization: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;k\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e), Δ\u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;k\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Δ[(\u003cem\u003eF\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)], The relative variable fluorescence at the L step (0.15 ms) is obtained as: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e=(\u003cem\u003eF\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/(\u003cem\u003eF\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e-\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of ROS content and physiological parameter quantification\u003c/h3\u003e\n\u003cp\u003eLeaves from the same position of each plant were selected for physiological measurements. First, the fresh weight (M1) of the leaves was recorded. The leaves were then immersed in distilled water for 4\u0026ndash;6 hours until fully saturated, blotted dry on the surface, and weighed to obtain the turgid weight (M2). Subsequently, the leaves were placed in an oven and dried at 65\u0026deg;C to a constant weight to obtain the dry weight (M3). The leaf relative water content (RWC) was calculated using the formula: RWC (%) = [(M1 \u0026ndash; M3) / (M2 \u0026ndash; M3)] \u0026times; 100%.\u003c/p\u003e \u003cp\u003eOther physiological and biochemical indices were measured using commercial assay kits (Griess Biotechnology Co, Ltd, Nanjing, China) according to the manufacturer's instructions. Specifically, the activities of peroxidase (POD), superoxide dismutase (SOD), and glutathione S-transferase (GST), as well as the contents of superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA), were determined using the corresponding kits. All measurements were performed with three independent biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003ebp-miR156c\u003c/b\u003e \u003cb\u003etargets and cleaves\u003c/b\u003e \u003cb\u003eBpSPL2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo validate the targeting and cleavage of \u003cem\u003eBpSPL2\u003c/em\u003e by \u003cem\u003ebp-miR156c\u003c/em\u003e in birch, we utilized the online prediction tool psRNATarget (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plantgrn.noble.org/psRNATarget/?function=3\u003c/span\u003e\u003cspan address=\"http://plantgrn.noble.org/psRNATarget/?function=3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict \u003cem\u003eBpSPL\u003c/em\u003e genes targeted by miR156 (see Supplementary Material). Constructs 35S::BpSPL2-GUS, 35S::bp-miR156c-GUS and 35S::bp-miR156c were generated (primer sequences and schematic diagrams of the constructs are provided in the Supplementary Material). Transient transformation in 4\u0026ndash;6 weeks old tobacco leaves was performed following the protocol described by Lu et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)(Lu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Agrobacterium tumefaciens strain GV3101 harboring the respective constructs was cultured in 20 ml of fresh LB liquid medium supplemented with 100 \u0026micro;g/mL kanamycin and 50 \u0026micro;g/mL rifampicin at 28\u0026deg;C with shaking at 200 rpm until the OD600 reached 0.6\u0026ndash;0.8. For co-infiltration, Agrobacterium cultures containing GV3101-pBI121-bp-miR156c and GV3101-pBI121-BpSPL2-GUS were mixed in equal volumes and injected into tobacco leaves. The infiltrated plants were kept in the dark at room temperature for 48 hours, followed by GUS staining.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of\u003c/b\u003e \u003cb\u003eBpSPL2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor each treatment, leaves from the same position of three biological replicate birch seedlings were collected, flash-frozen in liquid nitrogen, and stored at -80\u0026deg;C. RNA sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. on the Illumina NovaSeq 6000 platform. After quality control of the raw data, clean data were obtained for subsequent analysis. Based on the alignment results from HISAT2, transcripts were reconstructed using StringTie, and gene expression levels for each sample were calculated using RSEM. Genes satisfying the criteria of |log₂FC| \u0026ge; 1 and adjusted \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as significantly differentially expressed genes. Functional annotation and enrichment analysis of the differentially expressed genes were then conducted using GO and KEGG databases.\u003c/p\u003e\n\u003ch3\u003eYeast one-hybrid assay\u003c/h3\u003e\n\u003cp\u003ePromoter sequences containing the predicted GTAC-box motif (approximately 200 bp) from the target genes were cloned into the pAbAi vector to generate bait reporter constructs (pAbAi-proBpGSTF3, pAbAi-proBpASA1). The coding sequence of \u003cem\u003eBpSPL2\u003c/em\u003e (without the stop codon) was cloned into pGADT7-Rec to produce the effector plasmid pGADT7\u003cem\u003e-BpSPL2\u003c/em\u003e. Subsequently, the minimum inhibitory concentration of Aureobasidin A (AbA) for each bait vector was determined. Yeast one-hybrid assays were performed following the manufacturer's instructions (BD Matchmaker\u0026trade; Library Construction and Screening Kit User Manual). Yeast cells were plated onto selective dropout medium (SD/-Ura/-Leu) containing the corresponding concentration of AbA to screen for positive clones. Primers used are listed in the Supplementary Materials.\u003c/p\u003e\n\u003ch3\u003eDual-LUC transient expression assays\u003c/h3\u003e\n\u003cp\u003eTo analyze the regulatory role of \u003cem\u003eBpSPL2\u003c/em\u003e on its target genes, we constructed reporters by cloning the promoters of the target genes to drive the Luc gene in the pGreenII-0800-LUC vector (pGreenII-proBpGSTF3-LUC and pGreenII-proBpASA1-LUC), and used pGreen-62-SK to drive \u003cem\u003eBpSPL2\u003c/em\u003e as the effector (pGreen-BpSPL2-SK). The effector and reporter plasmids were individually transformed into Agrobacterium tumefaciens strain GV3101 harboring the pSoup helper plasmid. Following the method described by Zang et al.(Zang et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), bacterial cultures carrying the effector and reporter vectors were mixed in the required ratio to prepare the infiltration solution. Healthy, pre-flowering soil-grown tobacco plants at a vigorous growth stage were selected. The prepared bacterial suspension was infiltrated into tobacco leaves using a 1 mL needleless syringe. Each experimental group included at least three biological replicates. After infiltration, plants were kept in the dark for 12 hours and then transferred to normal growth conditions for 48 hours prior to fluorescence observation. Subsequently, the activities of REN and LUC were measured using a Dual-Luciferase Reporter Assay Kit (Beyotime, Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eChIP-PCR assay\u003c/h3\u003e\n\u003cp\u003eChromatin immunoprecipitation (ChIP) was performed following the method described by Zhao et al.(Zhao et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)Proteins and DNA were cross-linked using 3% (w/v) formaldehyde. After nuclei isolation and purification, chromatin was sheared by sonication into fragments of 200\u0026ndash;500 bp. Following sonication, chromatin was immunoprecipitated using an anti-GFP antibody (ChIP+). Sonicated chromatin immunoprecipitated with a nonspecific antibody (IgG) served as the negative control (ChIP\u0026ndash;). Cross-linking was reversed by digestion with proteinase K, and the ChIP products were purified using the TIANgel Maxi Purification Kit (Qiagen, Hilden, Germany). The ChIP-PCR program was as follows: 94\u0026deg;C for 3 min; 35 cycles of 94\u0026deg;C for 30 s, 58\u0026deg;C for 30 s and 72\u0026deg;C for 20 s; followed by a final extension at 72\u0026deg;C for 5 min and holding at 4\u0026deg;C. All primers used for ChIP-PCR are listed in the Supplementary Materials.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSignificant differences were analyzed using Student's t-test, one-way ANOVA combined with Tukey\u0026rsquo;s multiple comparison test in GraphPad Prism 9.0. An asterisk \u0026ldquo; * \u0026rdquo; indicates a significant difference at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and identical letters denote no significant difference. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ebp-miR156c\u003c/b\u003e \u003cb\u003enegatively regulates plant drought tolerance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder normal conditions, the \u003cem\u003ebp-miR156c\u003c/em\u003e-OE lines exhibited superior growth compared to the WT, the plant height significantly greater than that of the wild-type (by approximately 20%, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The leaf size (measured as the area of the third mature leaf) was also significantly larger than in wild-type plants (by about 52.3%, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). However, there were no significant differences in physiological indicators such as leaf water content, MDA, H₂O₂ and O₂⁻ levels, or in the activities of SOD and POD. After 7 days of drought stress, the leaves of the \u003cem\u003ebp-miR156c\u003c/em\u003e-OE lines showed obvious wilting and water loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The leaf water content of WT plants was 35.1% higher than that of the \u003cem\u003ebp-miR156c\u003c/em\u003e-OE lines (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the plants remained upright (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Measurement of oxidative stress-related parameters under drought conditions revealed that the \u003cem\u003ebp-miR156c\u003c/em\u003e-OE lines had significantly higher levels of MDA, H₂O₂ and O₂⁻ (increased by approximately 29.4%, 70.1% and 39.3%, respectively) compared to the WT lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, E). Furthermore, the activities of the antioxidant enzymes POD and SOD were significantly lower in the transgenic birch plants after drought stress compared to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). These results indicate that overexpression of \u003cem\u003ebp-miR156c\u003c/em\u003e in birch increases sensitivity to drought and reduces drought tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003eis a direct target of\u003c/b\u003e \u003cb\u003ebp\u003c/b\u003e\u003cb\u003e-\u003c/b\u003e\u003cb\u003emiR156c\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhile the canonical mechanism of miR156-targeted cleavage of SPL transcripts has been widely validated, the efficiency of cleavage by different miR156 family members on specific \u003cem\u003eSPL\u003c/em\u003e genes varies considerably across species. In this study, wild-type (WT) tissue-cultured plantlets were subjected to drought stress simulated by 20% PEG treatment. The results showed that with prolonged stress duration, the expression of \u003cem\u003ebp-miR156c\u003c/em\u003e was significantly down-regulated, whereas the expression pattern of \u003cem\u003eBpSPL2\u003c/em\u003e exhibited an opposite trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, we predicted the target cleavage site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and experimentally validated the \u003cem\u003ebp-miR156c\u003c/em\u003e mediated cleavage of \u003cem\u003eBpSPL2\u003c/em\u003e using GUS staining assays. The constructs 35S::bp-miR156c-GUS, 35S::BpSPL2-GUS and 35S::bp-miR156c (without GUS tag) were generated. Following agrobacterium mediated transient expression in tobacco leaves and GUS staining, positive controls (35S::bp-miR156c-GUS and 35S::BpSPL2-GUS fused with the GUS tag) showed blue coloration, while the negative control (35S::bp-miR156c without GUS tag) remained colorless. In the experimental group, due to the cleavage of \u003cem\u003eBpSPL2\u003c/em\u003e by \u003cem\u003ebp-miR156c\u003c/em\u003e, GUS activity was suppressed, resulting in significantly lighter or nearly undetectable staining compared to the positive controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results demonstrate that \u003cem\u003ebp-miR156c\u003c/em\u003e can specifically target and cleave \u003cem\u003eBpSPL2\u003c/em\u003e transcripts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003epositively regulates drought tolerance in birch\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the drought tolerance phenotype conferred by \u003cem\u003eBpSPL2\u003c/em\u003e, we compared the physiological performance of two independent \u003cem\u003e35S::BpSPL2-OE\u003c/em\u003e lines (OE3 and OE5) with that of WT plants under both normal and drought conditions. Under normal growth conditions, all lines exhibited similar growth phenotypes with no significant difference. However, under drought stress, leaves of WT plants lost water more severely and showed wilting earlier than those of the overexpressing lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Measurement of leaf relative water content revealed that, after drought stress, both OE lines maintained significantly higher water content (approximately 2-fold) compared to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSubsequently, we observed and statistically analyzed the belowground root phenotypes of these three lines before and after drought treatment. The results showed that under both normal and drought conditions, the root length and fresh weight of the two OE lines were significantly greater than WT line. Specifically under drought stress, the root lengths of \u003cem\u003eBpSPL2-OE\u003c/em\u003e3 and OE5 lines were 45.5% and 49.5% longer than that of the WT, respectively, while their root fresh weights were 30.5% and 54.6% higher than the WT, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003eAs lateral roots are a major component of the root system, we examined the root architecture of tissue-cultured seedlings (where the root structure is simpler and easier to observe) of \u003cem\u003eBpSPL2-OE\u003c/em\u003e3, OE5 and WT lines. The length and density of lateral roots were significantly higher in both OE lines compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, G). This explains why the root systems of the two \u003cem\u003eBpSPL2-OE\u003c/em\u003e lines were more developed than that of the WT during soil-based drought treatment. Collectively, these results indicate that \u003cem\u003eBpSPL2\u003c/em\u003e positively regulates drought tolerance in birch by promoting lateral root growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003eregulates glutathione metabolism in response to drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRNA-seq analysis of leaves from \u003cem\u003eBpSPL2\u003c/em\u003e transgenic birch plants before and after drought treatment yielded approximately 70 Gb of clean data. Each sample produced over 5.81 Gb clean data, with Q30 base percentages exceeding 95.86%. Comparative analysis between the drought stressed \u003cem\u003eBpSPL2-OE\u003c/em\u003e and WT lines identified 5597 differentially expressed genes (DEGs), comprising 2286 upregulated and 2771 downregulated genes (Fig. S2). When comparing the same genotype before and after stress, the WT line exhibited 2946 upregulated and 3324 downregulated DEGs, whereas the \u003cem\u003eBpSPL2-OE\u003c/em\u003e line showed only 1086 upregulated and 1148 downregulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The substantially less number of DEGs in the OE line indicates it was less perturbed by drought, consistent with its enhanced tolerance.\u003c/p\u003e \u003cp\u003eGO and KEGG enrichment analyses were performed on the DEGs identified between the OE and WT lines under drought conditions. GO enrichment revealed that these DEGs were primarily associated with processes such as photosynthesis (GO:0009765), light harvesting (GO:0009765), and oxidoreductase activity (GO:0016491) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). KEGG pathway analysis showed significant enrichment for pathways including MAPK signaling (map04016), photosynthesis - antenna proteins (map00196), photosynthesis (map00195), tryptophan metabolism (map00380), glutathione metabolism (map00480), and biosynthesis of secondary metabolites (map00999) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that \u003cem\u003eBpSPL2\u003c/em\u003e modulates drought resistance in birch mainly by influencing photosynthesis, hormone signal transduction, and cellular redox regulation. Therefore, we subsequently focused our analysis on key processes: photosynthesis (including light harvesting and electron transport), ROS scavenging related to glutathione metabolism, and the tryptophan metabolism pathway along with its downstream IAA signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003estabilizes photosynthetic electron transport and the expression of related genes under drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDrought stress leads to water loss and wilting in birch leaves, which significantly impacts the plant's photosystems. We investigated the effects of drought stress on PSII and PSI activity in birch leaves using parameters derived from prompt chlorophyll fluorescence transients (OJIP) and 820-nm reflection (\u003cem\u003eMR820\u003c/em\u003e). Under normal conditions, the overall shapes of the OJIP fluorescence induction curves and \u003cem\u003eMR820\u003c/em\u003e kinetics curves showed no significant differences among the WT, \u003cem\u003eBpSPL2-OE\u003c/em\u003e3 and \u003cem\u003eBpSPL2-OE\u003c/em\u003e5 lines. However, drought stress altered the curve morphology, primarily manifested as an increase in the relative fluorescence intensity at the O step, a decrease at the P step of the OJIP curve, and a reduction in the amplitude of the \u003cem\u003eMR820\u003c/em\u003e curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). Analysis of the chlorophyll fluorescence kinetics (OJIP) curve via the JIP-test revealed that the maximum quantum yield of PSII photochemistry (\u003cem\u003eFv/Fm\u003c/em\u003e) under drought stress was significantly higher in \u003cem\u003eBpSPL2\u003c/em\u003e transgenic plants compared to WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The \u003cem\u003eMR820\u003c/em\u003e curve reflects PSI activity. Under drought stress, the curve for WT plants was overall significantly lower than that for \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants. Quantitative data for Δ\u003cem\u003eI/I₀\u003c/em\u003e showed a significant decrease under drought, but overexpression of \u003cem\u003eBpSPL2\u003c/em\u003e partially alleviated this suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Under drought conditions, the OJIP curves of birch leaves exhibited significant distortion, mainly characterized by a marked decrease in fluorescence intensity at the P phase. Compared to WT plants, \u003cem\u003eBpSPL2-OE\u003c/em\u003e transgenic plants partially restored the OJIP curve morphology under drought stress, particularly improving performance at the P phase. Based on the normalized curves (\u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;P\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;J\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eO\u0026minus;K\u003c/sub\u003e) and double-normalized plots derived from the OJIP transients, \u003cem\u003eBpSPL2\u003c/em\u003e transgenic plants displayed distinct negative peaks, especially at the J (2 ms), K (0.3 ms) and L (0.15 ms) steps. The relative variable fluorescence values \u003cem\u003eV\u003c/em\u003e\u003csub\u003eJ\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eK\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e were also significantly lower in \u003cem\u003eBpSPL2\u003c/em\u003e transgenic plants than in WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-J). These results indicate that overexpression of the \u003cem\u003eBpSPL2\u003c/em\u003e gene can mitigate the drought-induced inhibition of both PSII and PSI, particularly by facilitating photosynthetic electron transport in PSII.\u003c/p\u003e \u003cp\u003eBased on the analysis of the drought transcriptome, we identified significant changes in a total of 18 genes encoding photosynthesis‑antenna proteins, including 6 Lhca and 12 Lhcb genes. Under drought stress, all these genes were down‑regulated; however, in the \u003cem\u003eBpSPL2\u003c/em\u003e‑OE5 line, the extent of down‑regulation for most antenna‑protein genes was less pronounced compared to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Moreover, three Lhca‑encoding genes (BpChr06G30386, BpChr06G30442, BpChr08G02997) showed significantly lower expression in WT than in OE‑5 under drought conditions, while Lhcb‑encoding genes overall exhibited the highest expression in OE‑5. Additionally, 25 differentially expressed genes related to the photosynthetic photosystem pathway (map00195) were identified in both OE‑5 and WT birch after drought treatment. Under non‑stress conditions, the expression levels of all PSII and PSI reaction‑center genes showed no significant differences. After stress, these genes were expressed significantly higher in OE‑5 plants than in WT, although compared to the pre‑drought levels, their expression was down‑regulated in both lines\u0026mdash;yet the down‑regulation was less severe in OE‑5 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). These results indicate that drought stress suppresses the expression of genes associated with PSI and PSII proteins. The transcriptome data are consistent with the corresponding phenotypic observations, demonstrating that overexpression of \u003cem\u003eBpSPL2\u003c/em\u003e reduces drought‑induced damage to photosynthetic antenna proteins and thereby alleviates photosynthetic inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003egene enhances ROS scavenging under drought stress and up-regulates the expression of genes involved in the glutathione metabolism pathway.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDrought stress severely impacts the plant's photosynthetic system, disrupting electron transport balance and leading to excessive accumulation of ROS. In situ staining of O₂⁻ and H₂O₂ with NBT and DAB, respectively, in birch leaves before and after drought stress showed no difference in staining intensity between \u003cem\u003eBpSPL2-OE\u003c/em\u003e and WT lines under normal conditions. After stress, however, the blue and brown staining was significantly deeper in WT leaves compared to \u003cem\u003eBpSPL2-OE\u003c/em\u003e leaves, indicating that overexpression of \u003cem\u003eBpSPL2\u003c/em\u003e reduced the accumulation of O₂⁻ and H₂O₂ under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Physiological measurements aligned with the staining results: under drought stress, the contents of O₂⁻ and H₂O₂ were significantly lower in both \u003cem\u003eBpSPL2-OE\u003c/em\u003e lines than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C), resulting in MDA content being 45.07% and 45.58% lower in the \u003cem\u003eBpSPL2-OE\u003c/em\u003e3 and \u003cem\u003eBpSPL2-OE\u003c/em\u003e5 lines, respectively, compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAs indicated by the earlier RNA‑seq results, \u003cem\u003eBpSPL2\u003c/em\u003e likely regulates ROS metabolism in birch leaves under drought stress mainly by affecting cellular redox processes, with the glutathione metabolism pathway being significantly enriched in the KEGG analysis. We performed a heatmap analysis of the transcriptome expression for genes significantly enriched in the glutathione metabolism process. The results revealed that among the 28 differentially expressed genes (DEGs), 24 were \u003cem\u003eGST\u003c/em\u003e genes, while the remaining included 1 \u003cem\u003eGPX\u003c/em\u003e, 2 \u003cem\u003eDHARs\u003c/em\u003e, and 1 \u003cem\u003eGR\u003c/em\u003e. In the \u003cem\u003eBpSPL2-OE\u003c/em\u003e5 transgenic plants, 10 GST members were significantly up‑regulated compared to the WT before and after drought stress, with three genes\u0026mdash;BpChr14G12602, BpChr05G19178, and BpChr05G19184\u0026mdash;showing particularly prominent induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). After screening, we selected BpChr05G19178 (\u003cem\u003eBpGSTF3\u003c/em\u003e), whose expression was markedly induced by \u003cem\u003eBpSPL2\u003c/em\u003e and whose promoter region contains multiple GTAC binding motifs for SPL transcription factors, for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBpSPL2\u003c/b\u003e \u003cb\u003epositively activates\u003c/b\u003e \u003cb\u003eBpGSTF3\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eBpASA1\u003c/b\u003e, \u003cb\u003eenhancing GST enzyme activity and promoting IAA accumulation.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEarlier, transcriptome data analysis predicted potential downstream regulatory pathways of the \u003cem\u003eBpSPL2\u003c/em\u003e gene under drought stress. We performed expression pattern analysis on key genes within these pathways, including \u003cem\u003eBpGSTF3\u003c/em\u003e, BpChr14G12602 and BpChr05G19184 from the GST family, as well as \u003cem\u003eBpASA1\u003c/em\u003e, a key rate-limiting enzyme gene in the tryptophan synthesis pathway. RT-qPCR results showed tissue-specific expression: \u003cem\u003eBpGSTF3\u003c/em\u003e and BpChr05G19184 were significantly induced by \u003cem\u003eBpSPL2\u003c/em\u003e mainly in leaves, while BpChr14G12602 expression was significantly higher in roots than in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Fig S3. A, B). In contrast, \u003cem\u003eBpASA1\u003c/em\u003e was primarily and significantly induced in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Promoter element analysis revealed that their promoters all contain the GTAC-box, a binding motif for \u003cem\u003eSPL\u003c/em\u003e transcription factors. To determine whether \u003cem\u003eBpSPL2\u003c/em\u003e can bind these DNA motifs to regulate gene expression, we performed ChIP-PCR assays on fragments of proBpGSTF3 and proBpASA1 containing the GTAC-box. The results showed that BpSPL2 binds to the P4 site of proBpASA1 and the P3 site of proBpGSTF3, confirming its ability to regulate gene expression by binding these DNA motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Subsequently, the promoter binding site fragments (200\u0026ndash;300 bp) identified by ChIP-PCR were cloned into the pAbAi vector for yeast one-hybrid (Y1H) validation. The results indicated that sequences containing the GTAC-box can be bound by BpSPL2, enabling growth on selective minimal medium containing Aureobasidin A (AbA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Furthermore, dual-luciferase (LUC) reporter assays confirmed that BpSPL2 binds to the proBpGSTF3 and proBpASA1 promoters and activates their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G, H).\u003c/p\u003e \u003cp\u003eDrought stress leads to excessive ROS production in plants, triggering severe oxidative stress. Previous experiments verified that birch \u003cem\u003eBpSPL2\u003c/em\u003e can target and regulate \u003cem\u003eBpGSTF3\u003c/em\u003e, activating its expression and increasing the activity of the antioxidant enzyme GST under drought stress (by approximately 36.7% and 29.6%, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). This enhances ROS scavenging capacity, reduces oxidative damage, and thereby improves drought tolerance in birch. We also measured the activities of other antioxidant enzymes involved in ROS scavenging (POD and SOD). The results showed no significant differences and generally low activity among the lines under normal conditions. However, after drought stress treatment, SOD (by 55%) and POD (by approximately 80%) activities were significantly higher in \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants compared to WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK, L). These results indicate that \u003cem\u003eBpSPL2\u003c/em\u003e can increase the antioxidant capacity in birch, reducing the degree of oxidative damage under drought stress, thereby enhancing drought tolerance. On the other hand, we also confirmed that \u003cem\u003eBpSPL2\u003c/em\u003e regulates \u003cem\u003eBpASA1\u003c/em\u003e, activating its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, H) and inducing IAA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI), which in turn promotes lateral root development in birch and enhances its drought tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDrought stress severely restricts plant growth and development, leading to premature leaf drop, delayed growth, and impaired nutrient accumulation, thereby causing significant economic losses(Luo \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Plants have evolved complex transcriptional regulatory networks to respond to drought stress, such as the miR156/SPL transcriptional regulatory module, which can be significantly induced under stress conditions to regulate a series of downstream stress responses. This study is the first to demonstrate that in birch, the miR156c/SPL2 module enhances drought tolerance by activating the key rate-limiting enzyme for auxin biosynthesis, \u003cem\u003eBpASA1\u003c/em\u003e and glutathione-S-transferase, \u003cem\u003eBpGSTF3\u003c/em\u003e, thereby promoting lateral root development and mediating the scavenging of ROS. While studies in herbaceous plants have largely shown the miR156/SPL module to act as a negative regulator of stress resistance, our findings reveal that \u003cem\u003eBpSPL2\u003c/em\u003e, targeted by \u003cem\u003ebp-miR156c\u003c/em\u003e, functions as a transcriptional activator playing a crucial role in the drought stress response of birch. This novel discovery provides a fresh perspective on the drought resistance mechanisms in birch and potentially other perennial woody plants.\u003c/p\u003e \u003cp\u003eThe miR156/SPL module is a key regulator that imparts plasticity to plant growth in response to light and other environmental factors(Sang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For instance, in tomato and Arabidopsis seedlings, overexpression of miR156 and suppression of SPL lead to elongated hypocotyls seeking light and activate light-responsive mechanisms(Sang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In response to drought and other environmental stresses, the miR156/SPL module can regulate plant stress tolerance by modulating anthocyanin production(Cui et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), auxin content(Feng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), antioxidant enzyme activity(Zhao et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), \u003cem\u003eFv/Fm\u003c/em\u003e values, chlorophyll content(Wen et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), ABA accumulation, and stomatal conductance(Visentin et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Drought stress affects both the morphology and physiology of birch. In this study, under PEG-simulated drought stress, the \u003cem\u003eBpSPL2\u003c/em\u003e gene was significantly induced, while \u003cem\u003ebp-miR156c\u003c/em\u003e was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This aligns with findings on the miR156/SPL module in apple under stress, where adversity leads to downregulation of miR156, releasing its inhibition of the downstream gene \u003cem\u003eMdSPL13\u003c/em\u003e, ultimately enhancing the expression of the stress-responsive gene \u003cem\u003eMdWRKY100\u003c/em\u003e and improving stress resistance in apple(Ma et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, however, \u003cem\u003ebp-miR156c-OE\u003c/em\u003e plants exhibited reduced drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), whereas \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants showed enhanced drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This phenotypic observation contrasts with conclusions in herbaceous plants (such as alfalfa and rice), where miR156 often enhances stress resistance by silencing \u003cem\u003eSPLs\u003c/em\u003e. For example, heterologous expression of \u003cem\u003eOsamiR156b/c\u003c/em\u003e simultaneously increases both biomass and stress tolerance in alfalfa(Arshad, Feyissa, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In cassava, a perennial woody starch crop often grown as an annual, \u003cem\u003eMeSPL9\u003c/em\u003e also negatively regulates drought tolerance(Li et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, in the study by Ning (Ning et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), birch \u003cem\u003eBpSPL9\u003c/em\u003e was described to improve ROS scavenging ability under salt and drought stress, although the molecular mechanisms were not investigated in depth. We speculate that this may reflect functional diversity of \u003cem\u003eSPL\u003c/em\u003e family transcription factors across different species.\u003c/p\u003e \u003cp\u003eTo further explore the molecular regulatory mechanisms underlying these morphological and physiological changes, we analyzed the transcriptomes before and after drought stress. A total of 5597 differentially expressed genes (DEGs) were identified between the \u003cem\u003eBpSPL2-OE\u003c/em\u003e and WT lines after stress. GO and KEGG enrichment analyses of these DEGs revealed that they are primarily involved in pathways such as photosynthesis, oxidoreductase activity, MAPK signaling, tryptophan metabolism, glutathione metabolism, and biosynthesis of secondary metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Drought stress led to dry and wilted birch leaves, significantly reduced water content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), and inhibited photosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). When photosynthesis is suppressed, excess electrons in the photosynthetic electron transport chain result in overproduction of ROS in chloroplasts, which in turn exacerbates photosynthetic inhibition, forming a vicious cycle. Moreover, excessive ROS induces lipid peroxidation and damages membranes, proteins, chlorophyll, nucleic acids, ultimately leading to cell death(Scandalios \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Smirnoff \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). By examining the effects of drought on the photosystems and several ROS-related indicators in \u003cem\u003eBpSPL2-OE\u003c/em\u003e and WT lines, we found that the \u003cem\u003eBpSPL2-OE\u003c/em\u003e lines better maintained ROS homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), thereby protecting birch plants and reducing oxidative damage caused by drought stress.\u003c/p\u003e \u003cp\u003eCombining phenotypic and transcriptomic analyses, we identified glutathione metabolism as a key pathway through which \u003cem\u003eBpSPL2\u003c/em\u003e regulates ROS balance in birch. Expression analysis of related genes in this pathway showed that most DEGs belonged to the \u003cem\u003eBpGSTs\u003c/em\u003e family, with \u003cem\u003eBpGSTF3\u003c/em\u003e being particularly prominent. \u003cem\u003eGSTs\u003c/em\u003e detoxify ROS via glutathione conjugation, and their induced expression under drought stress can reduce peroxide production during oxidation(Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Edwards and Dixon \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), thereby alleviating photoinhibition. Previous studies have reported that \u003cem\u003eSPL\u003c/em\u003e genes regulate downstream gene expression by recognizing and binding to GTAC motifs(Ma et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, Y1H, ChIP and LUC assays confirmed that \u003cem\u003eBpSPL2\u003c/em\u003e directly binds to the GTAC motif in the \u003cem\u003eBpGSTF3\u003c/em\u003e promoter, activating its expression and enhancing GST enzyme activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants, the activities of GST, SOD, and POD were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), effectively reducing H₂O₂ and O₂⁻ levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C) and thereby mitigating membrane lipid peroxidation (reduced MDA content, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This aligns with reports that overexpression of pear \u003cem\u003eGST\u003c/em\u003e or tamarisk \u003cem\u003eThGSTZ1\u003c/em\u003e enhances ROS scavenging capacity(Liu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants maintained higher PSII efficiency (\u003cem\u003eFv/Fm\u003c/em\u003e) and expression of light-harvesting protein genes under drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that GST-mediated ROS scavenging alleviated drought-induced photoinhibition, consistent with the protective role of the chloroplastic ROS scavenging system on photosynthetic machinery(Sun et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, studies have shown that \u003cem\u003eSPL\u003c/em\u003e genes can regulate the accumulation of osmoregulatory substances in plants, suggesting that \u003cem\u003eBpSPL2\u003c/em\u003e may also enhance drought tolerance in birch through osmotic adjustment. miR156-targeted SPLs regulate anthocyanin synthesis(Gou et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which acts as both an osmoregulant and a ROS scavenger(Landi, Tattini, and Gould \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In cassava, \u003cem\u003eSPL9\u003c/em\u003e regulates proline and anthocyanin synthesis via JA signaling(Li et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), implying that \u003cem\u003eBpSPL2\u003c/em\u003e might participate in osmoprotection through similar pathways, although this requires further experimental validation.\u003c/p\u003e \u003cp\u003eFurthermore, under drought stress, plants finely modulate their root system architecture to enhance water uptake capacity, among which the formation and development of lateral roots (LRs) is a key adaptive response. LR formation is a major determinant of root system architecture, significantly influencing water absorption efficiency, nutrient acquisition, and plant anchorage(P\u0026eacute;ret et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The initiation and development of LRs are inextricably linked to hormonal regulation, with auxin (IAA) being one of the primary regulatory hormones. Anthranilate synthase α-subunit 1 (\u003cem\u003eASA1\u003c/em\u003e), a key rate-limiting enzyme in the tryptophan-dependent IAA biosynthetic pathway, plays a central role in IAA biosynthesis(Niyogi and Fink \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Zhao and Last \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). \u003cem\u003eASA1\u003c/em\u003e catalyzes the conversion of chorismate to anthranilate, a precursor for tryptophan synthesis, which in turn leads to auxin production via the tryptophan-dependent IPA pathway. This pathway is widespread in plants and plays a crucial role in local auxin accumulation in root tips(Miles \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Zhao \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Teale, Paponov, and Palme \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Under drought stress, auxin synthesis and distribution in roots undergo significant alterations. The upregulation of \u003cem\u003eASA1\u003c/em\u003e expression may promote auxin synthesis, thereby stimulating the initiation and development of lateral root primordia, increasing lateral root density and root surface area, and consequently enhancing water absorption capacity. Additionally, auxin synthesized locally in roots also plays a vital role in responding to environmental stress, coordinating plastic root development in concert with shoot-derived auxin(Chen \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zhao \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The expression of \u003cem\u003eASA1\u003c/em\u003e is regulated by upstream transcription factors. For instance, in leaf explants, jasmonic acid (JA) biosynthesis occurs within two hours after wounding and transcriptionally activates \u003cem\u003eERF109\u003c/em\u003e, which encodes a protein that upregulates \u003cem\u003eASA1\u003c/em\u003e expression to promote auxin biosynthesis and facilitate de novo root organogenesis(Lee et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SPL transcription factor family, as target genes of miR156, is widely involved in regulating lateral root development. Niu et al(Yu et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), found that miR156 is differentially expressed in specific cells and tissues of lateral roots in Arabidopsis and regulates \u003cem\u003eSPL3\u003c/em\u003e, \u003cem\u003eSPL9\u003c/em\u003e, and \u003cem\u003eSPL10\u003c/em\u003e genes in response to hormone signaling pathways to suppress lateral root growth. In transgenic potatoes overexpressing \u003cem\u003eStu-miR156\u003c/em\u003e, the expression levels of the target gene \u003cem\u003eStSPL9\u003c/em\u003e were downregulated. Compared to controls, transgenic plants showed significantly reduced plant height and root length but a significant increase in lateral root number(Luo et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Apple \u003cem\u003eMdSPL23\u003c/em\u003e binds to the promoter of \u003cem\u003eMdNLP7\u003c/em\u003e to inhibit its expression, thereby negatively regulating nitrogen uptake and suppressing lateral root development in apple(Xu et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Rice \u003cem\u003eOsSPL14\u003c/em\u003e activates the expression of \u003cem\u003eOsSHR1\u003c/em\u003e; the expressed \u003cem\u003eOsSHR1\u003c/em\u003e further promotes the transcription of \u003cem\u003eOsSWEET16\u003c/em\u003e, reducing the accumulation of glucose and fructose in tiller buds and decreasing fructose and sucrose in roots, ultimately leading to fewer tillers and crown roots but increased root length(Feng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In reported results, \u003cem\u003eSPLs\u003c/em\u003e often function as negative regulators of lateral root development. However, we observed that birch \u003cem\u003eBpSPL2\u003c/em\u003e positively regulates lateral root development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), acting as a positive regulator to activate auxin synthesis and promote lateral root formation. Subsequent results also confirmed that in birch, \u003cem\u003eBpSPL2\u003c/em\u003e can positively activate the expression of the \u003cem\u003eBpASA1\u003c/em\u003e gene. The direct activation of \u003cem\u003eBpASA1\u003c/em\u003e by \u003cem\u003eBpSPL2\u003c/em\u003e significantly promoted auxin biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), thereby stimulating lateral root formation and enhancing birch's adaptive capacity under drought conditions. This finding not only provides new evidence for the functional diversity of the SPL protein family but also reveals the critical role of the SPL2-ASA1-auxin signaling pathway in birch lateral root development under drought stress. Furthermore, extensive crosstalk exists among multiple hormone signaling pathways in drought stress responses. ABA, a key hormone in drought response, interacts with the auxin signaling pathway through its receptors PYL8 and PYL9 to co-regulate lateral root growth and recovery(Zhao et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xing et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Transcription factors such as \u003cem\u003eMYB96\u003c/em\u003e and \u003cem\u003eWRKY46\u003c/em\u003e also integrate ABA and auxin signals to regulate lateral root initiation under drought conditions(Seo et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The miR156-SPL2-ASA1 regulatory module identified in this study may also interact with ABA signaling to coordinate root system plasticity in response to drought, but this requires further investigation. In summary, our study further reveals that in birch, \u003cem\u003eSPL2\u003c/em\u003e can directly activate \u003cem\u003eASA1\u003c/em\u003e expression to promote auxin synthesis and lateral root development, thereby enhancing drought tolerance. This finding expands the understanding of SPL family functions and provides a novel perspective on their role in drought resistance mechanisms in tree species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study systematically elucidates the molecular mechanism by which the \u003cem\u003ebp-miR156c\u003c/em\u003e/\u003cem\u003eBpSPL2\u003c/em\u003e module enhances drought tolerance in birch by promoting lateral root development and activating the antioxidant pathway. Drought stress significantly suppresses the expression of \u003cem\u003ebp-miR156c\u003c/em\u003e, thereby releasing its inhibition on the target gene \u003cem\u003eBpSPL2\u003c/em\u003e. Overexpression of \u003cem\u003ebp-miR156c\u003c/em\u003e reduces drought tolerance in birch, while its target gene \u003cem\u003eBpSPL2\u003c/em\u003e directly binds to the GTAC motifs in the promoters of the target genes \u003cem\u003eBpASA1\u003c/em\u003e and \u003cem\u003eBpGSTF3\u003c/em\u003e, activating their transcription. On the one hand, it induces IAA accumulation to promote lateral root development, thereby enhancing drought resistance. On the other hand, it increases GST enzyme activity to reduce ROS-induced oxidative damage and alleviate photosynthetic inhibition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Key Research and Development Program of China (No2021YFD2200304); Doctoral Student Independent Innovation Fund (2572025AW49); the Fundamental Research Funds for the Central Universities (No2572022DQ08); the National Natural Science Foundation of China (No32171738).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eH.P. performed most of the experiments, analysed the data and drafted the manuscript. Z.H. carried out the measurement of photosynthetic electron transport related indicators and data analysis. A.J. conducted phenotypic measurements of the growth of birch seedlings, including plant height and leaf area. Y.Z and D.H measured the relevant data of the lateral roots of the birch. Z.H. advised on all the experiments and thoroughly revised the manuscript. L.X. conceived, designed and directed the project, as well as revised and finalised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eData will be made available on request\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eAll\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eauthors have no conflict of interest for the publication of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArshad, M., Feyissa, B. 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'UV-B responsive microRNA genes in', Molecular Systems Biology, 3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9102551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9102551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDrought is one of the major abiotic stress factors affecting plant growth and productivity. The miR156/SPL module plays a crucial role in plant growth, development, and responses to abiotic stress; however, its regulatory mechanism in mediating drought adaptation in woody plants such as birch remains incompletely understood. In this study, we used transgenic plants overexpressing \u003cem\u003ebp-miR156c\u003c/em\u003e and \u003cem\u003eBpSPL2\u003c/em\u003e as experimental materials and employed GUS staining, RNA-seq, yeast one-hybrid assay, ChIP-PCR and dual-luciferase reporter assays to investigate the mechanism by which the miR156/SPL module regulates drought tolerance in birch. GUS staining results indicated that \u003cem\u003eBpSPL2\u003c/em\u003e is a target gene of \u003cem\u003ebp-miR156c\u003c/em\u003e and subject to its cleavage. Compared with wild-type plants, \u003cem\u003ebp-miR156c\u003c/em\u003e-overexpressing transgenic plants exhibited reduced drought tolerance under drought stress, whereas plants overexpressing its target gene \u003cem\u003eBpSPL2\u003c/em\u003e showed enhanced drought resistance. Specifically, \u003cem\u003eBpSPL2-OE\u003c/em\u003e lines under drought stress displayed alleviated photodamage in both PSII and PSI, along with reduced oxidative damage. Moreover, overexpression of \u003cem\u003eBpSPL2\u003c/em\u003e significantly promoted root system development, particularly lateral root growth, in birch. RNA-seq analysis revealed that, compared with the wild type, differentially expressed genes (DEGs) in \u003cem\u003eBpSPL2-OE\u003c/em\u003e plants under drought stress were significantly enriched not only in photosynthesis-related pathways but also in tryptophan metabolism, redox processes, and glutathione metabolism. We speculate that the alleviation of photosynthetic inhibition and oxidative damage in birch leaves under drought stress by \u003cem\u003eBpSPL2\u003c/em\u003e may be related to its regulation of ROS metabolism, while the promotion of lateral root development may be associated with activation of the tryptophan metabolic pathway and subsequent accumulation of IAA. Further studies demonstrated that the \u003cem\u003eBpSPL2\u003c/em\u003e transcription factor recognizes the GTAC motif and binds to the promoters of glutathione-S-transferase \u003cem\u003eBpGSTF3\u003c/em\u003e and the key rate-limiting enzyme in tryptophan synthesis gene \u003cem\u003eBpASA1\u003c/em\u003e, thereby enhancing their transcription. On one hand, this upregulates GST and antioxidant enzyme activities, mitigating drought-induced photodamage and oxidative injury; on the other hand, it promotes IAA accumulation, stimulating lateral root formation and ultimately improving drought tolerance in birch. In summary, our findings demonstrate that the miR156/SPL module enhances drought tolerance in birch by modulating ROS homeostasis and lateral root development, providing novel molecular insights and a theoretical foundation for drought resistance research in birch trees.\u003c/p\u003e","manuscriptTitle":"The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 14:01:02","doi":"10.21203/rs.3.rs-9102551/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-25T11:04:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-21T10:18:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130293505937771667488925783711948538986","date":"2026-04-13T08:25:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T01:57:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20990816826114637148288882456776107147","date":"2026-03-18T07:19:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T11:05:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T14:26:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-14T14:25:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-03-12T08:44:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3e6aa34f-b7a9-4a96-8a4e-193830ed4b49","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-25T11:09:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 14:01:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9102551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9102551","identity":"rs-9102551","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}