BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance in Betula platyphylla

Summary Glycine‐rich RNA‐binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, but the function of GRP genes involved in salt stress and the underlying mechanism remain unclear. In this study, we identified BpGRP1 (glycine‐rich RNA‐binding protein), a Betula platyphylla gene that is induced under salt stress. The physiological and molecular responses to salt tolerance were investigated in both BpGRP1‐overexpressing and suppressed conditions. BpGRF3 (growth‐regulating factor 3) was identified as a regulatory factor upstream of BpGRP1. We demonstrated that overexpression of BpGRF3 significantly increased the salt tolerance of birch, whereas the grf3‐1 mutant exhibited the opposite effect. Further analysis revealed that BpGRF3 and its interaction partner, BpSHMT, function upstream of BpGRP1. We demonstrated that BpmiR396c, as an upstream regulator of BpGRF3, could negatively regulate salt tolerance in birch. Furthermore, we uncovered evidence showing that the BpmiR396c/BpGRF3 regulatory module functions in mediating the salt response by regulating the associated physiological pathways. Our results indicate that BpmiR396c regulates the expression of BpGRF3, which plays a role in salt tolerance by targeting BpGRP1.


Introduction
Plant growth and productivity are under constant threat due to environmental challenges.Salt stress is a significant limiting factor that has a negative impact on various aspects of plant growth and development and on the quality and productivity of agroforestry (Safdar et al., 2019;Vishal et al., 2019;Yaldiz and Camlica, 2021).With the rapid development of a global society and economy and the acceleration of industrialization, poorly planned land utilization and development will further lead to continuous expansion of global saline and alkali land areas.Soil salinization makes it difficult to utilize a large portion of soil resources, which has become one of the serious factors affecting regional economic development and ecological restoration.It is therefore important to invest in the selection and development of salt-tolerant plants.
Posttranscriptional modification is important for regulating the expression of defence-related genes (Gough and Sadanandom, 2021).Posttranscriptional gene regulation involves several processes, including precursor mRNA (pre-mRNA) splicing, nucleocytoplasmic transport of mRNA and mRNA stability and decay, and translation is mainly achieved either directly by ribonucleic acid (RNA)-binding proteins (RBPs) or indirectly by RBPs modulating the function of other regulatory factors (Aguilar-Garrido et al., 2022;Hajieghrari and Farrokhi, 2022;Shi and Grifone, 2021).Plants contain numerous RBPs, including glycine-rich RNA-binding proteins (GRPs) containing an RNArecognition motif (RRM) at the N-terminus and a glycine-rich region at the C-terminus (Lu et al., 2019;Ma et al., 2021).
The first GRP was found in maize (Zea mays, Gomez et al., 1988), and since then, genes encoding homologous proteins have been consecutively isolated from a variety of plant species, such as Arabidopsis thaliana (Carpenter et al., 1994), Medicago sativa (Ferullo et al., 1997) and Nicotiana tabacum (Hirose et al., 1993).The expression of GRPs is regulated by a variety of external stimuli, including cold, drought stress and wounding stress (Ma et al., 2021;Shim et al., 2021;Xu et al., 2022).For instance, GRPs function as RNA chaperones during cold adaptation processes in rice and Arabidopsis plants.OsGRP1 and OsGRP4 rescue the growth defect of cold-sensitive Arabidopsis grp7 mutant plants under cold and freezing stress, whereas OsGRP6 enhances the frost resistance of grp7 (Kim et al., 2010).In addition, AtGRP2-or AtGRP7-expressing transgenic rice plants show higher recovery rates and grain yields under drought stress than wild-type plants (Yang et al., 2014).The overexpression of OsGRP3 alleviates reactive oxygen species (ROS) accumulation by regulating ROSrelated gene mRNA stability under drought stress, which confers drought tolerance (Shim et al., 2021).However, less is known about the regulatory mechanisms of GRPs in defence responses (including salt stress) than about their physiological functions.Therefore, it is necessary to identify salt tolerance-related GRPs.
Growth-regulating factors (GRFs) are plant-specific transcription factors that regulate stress-related physiological and metabolic pathways by binding to the promoter sequences of multiple downstream stress-related functional genes to activate or repress the expression of genes in response to stress (Omidbakhshfard et al., 2018).AtGRF7 inhibits AtDREB2A expression by binding to the TGTCAGG element of the AtDREB2A promoter to maintain the balance between plant growth and resistance to drought and abscisic acid (ABA) stress, which enables plants to survive in an adversarial environment (Kim et al., 2012).PdbGRF1 regulates the expression of PdbPOD17 and PdbAKT1 by binding to the DRE ('A/ GCCGAC') in their respective promoters to increase ROS scavenging, reduce the extent of damage to the plasma membrane and ultimately enhance the salt stress response in Populus davidiana 9 P. bolleana (Liu et al., 2022a).In addition, GRF can regulate the expression of different target genes by interacting with different proteins and thus participate in different physiological and metabolic pathways.For example, in Arabidopsis, AtGRF5, in addition to forming a macromolecular complex with AtGIF1, positively regulates the leaf size by promoting and/or maintaining the cell proliferation activity of the leaf primordia (Horiguchi et al., 2005); this protein also interacts with DELLA to regulate leaf growth and root elongation and development through the gibberellic acid (GA) pathway and thus participates in the cold stimulus response (Ourania et al., 2020).
Various studies have indicated that the regulation of GRFs by miR396 plays an important role in plant growth and development and is involved in a variety of stress responses (Li et al., 2019;Pegler et al., 2021).In Arabidopsis, one of the miR396 target genes, AtGRF7, functions as a repressor of stress-responsive genes in the plant's abiotic stress response (Kim et al., 2012).Sp-miR396a-5p plays a positive regulatory role in the abiotic stress response by targeting the NtGRF7-regulated expression of osmotic stress-responsive genes (Chen et al., 2015).Hpo-miR396b and HpGRF6 may exert an antagonistic effect to negatively regulate the response of pitaya (Selenicereus monacanthus) to various abiotic stresses, such as low temperature, high temperature, NaCl and ABA (Li et al., 2019).
In this study, we cloned and functionally characterized a glycine-rich RNA-binding protein, BpGRP1, from Betula platyphylla, an important timber and landscaping tree species that has been widely used in plate processing, appliance manufacturing and plant medicine development (Guo et al., 2017).Our results showed that the overexpression of BpGRP1 improved the tolerance of transgenic birch to salt stress.We further demonstrated that BpGRF3, an upstream regulator of BpGRP1, was regulated by BpmiR396c and was thus involved in the regulation of salt stress in birch.Based on the current data, we established a direct molecular link among BpGRP1, BpGRF3 and BpmiR396c for improving the salt tolerance of birch.

BpGRP1 improves salt stress tolerance in transgenic birch
Due to its preferred growth conditions, birch usually exhibits distinctive and obvious symptoms under salt stress.To identify the critical genes that respond to salt stress, we analysed RNA-Seq data under 200 mM NaCl treatment and identified many differentially expressed genes (Table S2), including the BpGRP1 gene.RT-qPCR was employed to analyse the expression patterns of BpGRP1 (GenBank accession number: BPChr06G07927) in different tissues at various time points under salt stress.The results showed that BpGRP1 was significantly upregulated in stems and leaves after salt stress (Figure 1a).
To further determine the functional roles of BpGRP1 in salt stress responses, BpGRP1 transgenic lines were obtained (Figure S1).Soil-grown transgenic birch plants, including OE, WT and SE lines, were exposed to salt to evaluate their stress responses.No substantial difference in phenotype was found among the OE, WT and SE lines under control conditions (Figure 1b), suggesting that BpGRP1 does not affect the growth phenotype or growth rate of the plants.Under salt stress, the OE lines displayed significantly higher growth rates, had greener foliage and exhibited less wilting compared with WT plants.In contrast, the SE lines exhibited more severe leaf rolling and wilting (Figure 1b).Moreover, the expression of stress-associated genes, including BpSODs (BpSOD1; BpSOD2), BpPODs (BpPOD4; BpPOD7), BpAPXs (BpAPX1; BpAPX2) and BpP5CS1, was significantly higher in the OE lines, whereas the expression levels of these genes were significantly lower in the SE lines (Figure 1c).
(i) The expression levels of BpSODs, BpPODs, BpAPXs and BpP5CS1 were increased in the OE lines and decreased in the SE lines and (ii) the balance between ROS production and ROS removal is closely related to plant resistance to stress.Therefore, in this study, the activity of SOD was measured as scavenging capability, and the analysis showed that SOD activity was significantly higher in the OE plants than in the WT and SE plants following salt treatment but remained unaltered under normal conditions (Figure 1d).Moreover, the H 2 O 2 and O 2 • ˉcontents under unstressed conditions were similar among all the studied lines.Under salt stress, the trend of the changes in the H 2 O 2 and O 2 • ˉcontents was opposite to that found for SOD activity, i.e., the SE lines exhibited the highest H 2 O 2 and O 2 • ˉcontents, followed by the WT plants and then the OE lines (Figure 1e,f).Malondialdehyde (MDA) is one of the end products of lipid peroxidation.To further analyse membrane lipid peroxidation in the studied lines, MDA levels were determined.Under normal conditions, the MDA content of the BpGRP1 transgenic seedlings did not differ from that of the WT seedlings.However, after salt stress, the MDA content of the WT plants was significantly lower than that of the SE plants but significantly higher than that of the OE plants (Figure 1g).Furthermore, to investigate the role of BpGRP1 in the salt stress response through the regulation of osmotic substances, the proline content was quantified and compared among the OE, WT and SE lines.Under normal conditions, no significant difference in the proline content was found among the studied plants, but after exposure to salt stress, the OE plants had the highest proline content, followed by the WT plants, and the SE plants had the lowest proline content (Figure 1h).
Taken together, these results demonstrated that BpGRP1 positively regulates the salt stress response by affecting salt stress-related gene expression, enhancing ROS scavenging capability, increasing proline levels and reducing MDA levels.Zhongyuan Liu et al.

BpGRF3 is a regulatory factor upstream of BpGRP1
To investigate the role played by BpGRP1 in the salt tolerance mechanism of birch, we analysed its promoter.A 2262-bp sequence upstream of the BpGRP1 translation start site was cloned from the birch genome sequence.A bioinformatics analysis of the BpGRP1 promoter revealed numerous stress-related consensus cisacting elements, including abiotic stress-related elements and hormone stress-related elements.The ABA-responsive element (ABRE) with the highest frequency of 10 times (Figure S2) was selected as the target for identifying the possible upstream genes of BpGRP1 by Y1H experiments.The results indicated that BpGRF3 (growth-regulating factor 3, GenBank accession number: BPChr01G18064) binds to the ABRE motif (Figure 2a).
We isolated the BpGRF3 gene with two highly conserved structural domains (QLQ, Glu-Leu-Glu; WRC and Trp-Arg-Cys) at the N-terminus in birch (Figure S3).Further subcellular localization analysis showed that BpGRF3-GFP preferentially localized to the nucleus, whereas control GFP was distributed in both the cytoplasm and the nucleus (Figure 2b).We further investigated whether BpGRF3 was the upstream regulator of BpGRP1.An electrophoretic mobility shift assay (EMSA), transient expression analysis and chromatin immunoprecipitation (ChIP) were performed.We found reproducible binding of the BpGRF3-maltose-binding protein specifically to the ABRE motif (Figure 2c).The effector construct was transformed into calli of birch together with the ABRE motif reporter construct pCAM-ABRE (Figure 2d).GUS activity determination showed that BpGRF3 highly activated the expression of the reporter gene in the presence of the ABRE motif (Figure 2e,f).The ChIP-qPCR results were consistent with the EMSA results; that is, BpGRF3 bound to the promoters of BpGRP1 (Figure 2g,h).Furthermore, under salt stress, the expression pattern of BpGRF3 showed an expression trend consistent with that found for BpGRP1 (Figure 2i).Based on our experimental evidence, we confirmed that BpGRP1 was also a direct target gene of BpGRF3.

The overexpression of BpGRF3 in birch improves tolerance to salt stress
To investigate the roles of BpGRF3 in salt tolerance, we upregulated BpGRF3 expression and assessed the success of the upregulation by RT-qPCR analysis.BpGRF3 was significantly overexpressed in the OE lines (the expression levels in the OE1, OE7 and OE11 lines were 135.61, 23.70 and 79.71 times higher, respectively, than those in the WT line), indicating that the OE lines had been successfully established (Figure S4).We then compared the phenotypes of the BpGRF3 (OE) lines with those of the WT cultured under both normal growth and salt stress conditions for 14 days.The BpGRF3-OE lines displayed growth phenotypes similar to those of the BpGRP1-OE lines.No significant difference in growth under normal growth conditions was found among the studied plants.However, under salt stress, the WT plants showed severely inhibited growth, with leaves that were withered, yellowed and curled and many leaves falling off the plants, whereas most of the OE lines showed normal growth with green and fully expanded leaves (Figure 3a).
To determine the salt tolerance mechanism mediated by BpGRF3 at the physiological level, the  3b-e).To investigate whether the reductions in ROS and lipid peroxidation were caused by altered antioxidant activity, the activities of superoxide dismutase (SOD) and peroxidase (POD) were studied.Under non-saline conditions, no difference in SOD or POD activity was found between transgenic birch and WT plants.However, under salt stress, SOD and POD activities increased in all the studied lines.The activities of SOD and POD were significantly higher in the OE plants than in the WT plants (Figure 3f,g).In addition, under salt stress, the trend for the change in the proline content in the BpGRF3-OE lines was similar to that found in the BpGRP1-OE lines (Figure 3h).
To further verify the function of BpGRF3 in birch salt tolerance, a BpGRF3-knockout mutant (grf3-1) was generated using the CRISPR-Cas9 system (Figure 4a).Under salt stress conditions, the activity of SOD was significantly lower in the grf3-1 mutant than in the WT plants (Figure 4b).Accordingly, the trend of the changes in the O 2 • ˉand MDA contents was the opposite of that found for SOD activity.Specifically, the grf3-1 mutant had higher O 2 • ˉand MDA contents than the WT (Figure 4d,e).Moreover, genes involved in the salt stress response, including BpSOD1, BpSOD2, BpPOD4, BpPOD7, BpAPX1 and BpAPX2, were studied.Consistent with the physiological indicators related to stress resistance, the expression of BpSODs, BpPODs and BpAPXs was lowest in the grf3-1 mutant, moderate in the WT plants and highest in the BpGRF3-OE lines (Figure 4f).Furthermore, the expression pattern of BpGRP1 exhibited opposite trends in the BpGRF3-OE lines and the grf3-1 mutant lines (Figure 4g).Collectively, these findings suggest that BpGRF3 enhanced salt stress tolerance in birch by regulating BpGRP1 gene expression to activate stress-associated physiological changes, such as enhancing the ROS scavenging capability, increasing the proline content and decreasing lipid peroxidation in cell membranes.

BpGRF3 and BpSHMT coregulate the expression of BpGRP1
To investigate whether BpGRF3 functions as a transcriptional activator, full-length BpGRF3 was fused to the GAL4 DNAbinding domain in the yeast expression vector pGBKT7.As shown in Figure 5a, little growth was observed in yeast transformed with the pGBKT7-BpGRF3 constructs.These results indicated that BpGRF3 is a nuclear localization protein but has no transcriptional activation activity.
To determine how BpGRF3 mediates BpGRP1 and then participates in the regulation of salt tolerance, we conducted further investigation of the proteins that interacted with BpGRF3 Figure 2 BpGRF3 regulates BpGRP1 by directly binding to ABREs in vitro and in vivo.(a) A yeast one-hybrid (Y1H) assay was used to verify the binding of BpGRF3 to ABREs.p53-HIS2/pGADT7-Rec2-p53 was used as a positive control.pGADT7-Rec-BpGRF3/P53-HIS2 was used as a negative control.(b) BpGRF3 was fused with the green fluorescence protein (GFP) gene under control of the 35S promoter, and 35S::GFP (control) was transiently expressed in onion epidermal cells via the particle bombardment method and visualized under a confocal microscope at 488 nm for the excitation of GFP and 507 nm longpass for emission.GFP, GFP fluorescence; Bright, bright field; Merge, merged bright field and fluorescence images.A nucleus-localized MYB transcription factor, ThMYB8, was used as a positive control (Liu et al., 2020).(c) The binding of BpGRF3 to the ABRE in BpGRP1 promoters was evaluated by EMSA.The biotinlabelled probe was used as a negative control; the biotin-labelled probe incubated with BpGRF3 protein was tested; and competitive probes were used at 10-fold and 100-fold (lack of biotin label).( d by utilizing the Y2H system and proximity labelling (PL) approach in conjunction with mass spectrometry (MS)-based quantitative proteomics.By screening the cDNA library of birch, a protein called BpSHMT (a serine hydroxymethyl transferase, GenBank accession number: BPChr12G11376) was identified as potentially interacting with BpGRF3.Additionally, the same BpSHMT protein was found through MS-based quantitative proteomics (data not published; some data shown in Table S3).Based on the results obtained, it can be inferred that the BpSHMT protein, which responds to salt stress, has a high probability of interacting with BpGRF3 (Figure S5).
We tested this interaction by regenerating a full-length BpSHMT-pGADT7-AD construct.Transformants harbouring BpGRF3-pGBDT7-BD and BpSHMT-pGADT7-AD exhibited a positive interaction on QDO/-X/-A solid medium, as indicated by a deep blue colour (Figure 5b).We then tested whether the BpGRF3-BpSHMT interaction occurs in planta by bimolecular fluorescence complementation (BiFC) in onion epidermal cells.The assays confirmed the interaction, as indicated by the detection of YFP signals from the interacting pairs, which colocalized with mCherry in the nucleus (Figure 5c).
We next explored the interaction of BpGRF3 and BpSHMT in the regulation of BpGRP1.The activation of BpGRP1 promoter activity by both proteins was tested using an in vivo luciferase expression system.Both BpGRF3 and BpSHMT enhanced BpGRP1 promoter activity, and stronger enhancement was obtained when the two proteins were expressed simultaneously (Figure 5d,e).In the LUC assay, coexpression of BpSHMT with BpproGRP1:LUC significantly increased LUC activity.The coexpression of BpGRF3 with the BpSHMT and BpproGRP1:LUC constructs further increased the LUC activity compared with that observed with only BpGRF3 or BpSHMT expression (Figure 5f).
Taken together, these results provide direct evidence showing that BpGRF3 and BpSHMT coregulate BpGRP1 and that the presence of both BpGRF3 and BpSHMT enhances BpGRP1 expression levels.

The suppression of BpGRF3 by BpmiR396c participates in the regulation of salt stress in birch
Previous studies have shown that the target gene of miR396 is GRF, which has been confirmed by RNA ligase-mediated 5'-rapid amplification of cDNA ends tests (5' RLM-RACE) (Peng et al., 2022).In this study, the BpGRF3 transcript levels exhibited a pattern opposite to that found for BpmiR396c in response to salt (Figures 2i and 6a).To further validate the target site of BpmiR396c, we localized the BpmiR396c-targeted cleavage sites in BpGRF3 using 5' RLM-RACE.The results showed that the mRNAs of BpGRF3 were cleaved by BpmiR396c between base pairs 11 and 12 (Figure 6b).Collectively, these findings support the notion that BpmiR396c directly cleaves BpGRF3 transcripts to decrease their abundance.
To investigate the function of BpmiR396c in salt tolerance, the growth phenotype under salt stress was observed.No difference in the growth phenotype was found among the BpmiR396c-OE lines (OE2, OE7 and OE9) and the WT plants under normal conditions.Under 0.2 M NaCl stress for 14 d, the leaves of the OE lines were withered, yellowed and curled, and many fell off the plants (Figure 6c).Moreover, the SOD activity and proline content were significantly lower in the OE plants than in the WT plants, whereas the H 2 O 2 , O 2 • ˉand MDA levels of the OE plants were significantly higher than those of the WT plants (Figure 6d-h).
To investigate the potential function of the BpmiR396c-BpGRF3 module, we isolated three independent transgenic lines (BpmiR396c-OE2, 7, 9) with high expression levels (Figure S6): the BpmiR396c transcripts were increased by 56.49, 5.40 and 13.21 times in the OE2, OE7 and OE9 lines, respectively.However, the expression level of BpGRF3 in the Bp-miR396-OE plants was significantly lower than that of the WT plants and was only 8.8%, 10.4% and 5.9%, respectively, of that in the WT plants (Figure 6i).Moreover, the phenotypes of the BpmiR396c-OE and BpGRF3-OE lines were opposite under salt stress.The BpmiR396c-OE lines were more sensitive to salt stress, whereas the BpGRF3-OE lines showed greater tolerance against these stresses (Figures 3a and 6c).Collectively, these results suggest that birch resistance to salt stress was negatively and positively modulated by BpmiR396c and BpGRF3, respectively.

Discussion
The stress-inducible gene BpGRP1 plays a positive role in salt stress tolerance Marked changes in physiology, metabolism and, most notably, gene expression always occur when plants respond to adversity (Gupta et al., 2020;Wang et al., 2022a).Prior research has demonstrated that stress-inducible genes are expressed at higher levels in plants accustomed to coping with salt stress (Li et al., 2022;Xing et al., 2020), and these genes include GRP genes (Tada et al., 2019).Furthermore, earlier studies have shown that the GRP protein exerts an influence on the growth and stress resilience of Arabidopsis plants under conditions of elevated salt levels and dehydration stress while also conferring tolerance to freezing (Kim et al., 2008).For example, AtGRP2 promotes Arabidopsis seed germination and growth under salt and dehydration stress treatments (Su et al., 2009).SvGRP1 enhances salt tolerance by increasing 3-aminopropanoic acid, citramalic acid and isocitric acid levels (Tada et al., 2019).
In the present investigation, we successfully isolated glycinerich RNA-binding protein 1 (BpGRP1) and subsequently identified it as a positive regulator in the salt stress response of birch plants (Figure 1a).Additionally, the modulation of BpGRP1 expression in response to salt stress plays a crucial functional role in birch, as demonstrated in our investigation of transgenic birch plants.Notably, the phenotypic analysis of the BpGRP1-OE and BpGRP1-SE lines under salt stress conditions yielded distinct outcomes (Figure 1b), which was consistent with previous studies showing that genes with positive effects on salt resistance increase the salt tolerance of plants (Ma et al., 2021), indicating that BpGRP1 positively regulates salt stress tolerance.Additional studies of the expression patterns of stress-related genes in the BpGRP1-OE and BpGRP1-SE lines, including BpSODs, BpPODs, BpAPXs and BpP5CS1, were performed.The findings revealed that BpGRP1 had a positive impact on the salt stress response by influencing the expression of salt stress-related genes (Figure 1c).
To elucidate the salt tolerance mechanism mediated by BpGRP1 at the physiological level, we conducted an analysis of various physiological indicators.Our analysis of physiological indicators showed that the BpGRP1-OE lines exhibited significantly improved SOD activity and proline contents and reduced levels of H 2 O 2 , O 2 • ˉand MDA, which enhanced their salt tolerance (Figure 1d-h).These results are in accordance with the capacity of LbGST1 to confer salt tolerance in Limonium bicolor (Bunge) Kuntze through modulation of some physiological pathways (Wang et al., 2012).Interestingly, our findings regarding the analysis of physiological indicators of BpGRP1-OE lines in response to salt stress contradict a previous report by Kim et al. (2007), who argued that (first) the H 2 O 2 content is higher in transgenic plants than in wild-type plants under salt stress and that (second) seed germination and seedling growth are hypersensitive to salt stress in atRZ-1a (glycine-rich RNA-binding proteins) OE Arabidopsis.
The above-described results suggest that homologous genes of GRP have different functions in different species and that their mechanisms of action are quite different, which is central to the significance of our investigation of the function of BpGRP1 in birch.
BpGRF3, as a regulatory factor upstream of BpGRP1, positively regulates salt stress in birch Numerous studies have demonstrated the critical role of cis-acting elements in regulating the expression of stress-responsive genes under challenging environmental conditions, which enables plants to withstand and mitigate stress-induced damage (Bo et al., 2022;Janiak et al., 2016;Wu et al., 2022).In this study, to investigate the role of BpGRP1 in the salt tolerance mechanism of birch, its promoter was analysed.A bioinformatics analysis of the BpGRP1 promoter revealed a significant number of stress-related consensus cis-acting elements, including those related to abiotic stress and hormone stress.Notably, the ABRE was found at the highest frequency, occurring 10 times (Figure S2).The results from a previous study revealed that ABRE is an essential binding site for the ABRE-binding bZIP factor (BnaABF2) to activate RD29B, RAB18 and KIN2 transcription under drought and salt stress (Zhao et al., 2016).ANAC096 functions cooperatively with ABF2, and ABF4 regulates the expression of RD29A by targeting the ABRE cis-acting element to confer both dehydration and osmotic tolerance (Xu et al., 2013).
Therefore, in this study, we focused on the ABRE and utilized it for cDNA library screening.Through Y1H experiments, the growth-regulating factor BpGRF3 was identified (Figure 2a).EMSA and transient expression analysis were then performed to determine whether BpGRF3 could directly bind to ABREs in vitro/ in vivo.BpGRF3 was able to bind to the ABRE, and the signal representing the complex was reduced or even disappeared in the presence of the competitor (Figure 2c).Similar binding was found by transient expression analysis, in which determination of GUS activity showed that BpGRF3 greatly activated the expression of the reporter gene in the presence of the ABRE motif (Figure 2d-f).The ChIP-qPCR results were consistent with the EMSA and transient expression analysis results: BpGRF3 bound to the promoters of BpGRP1 (Figure 2g,h).Furthermore, under salt stress, the expression pattern of BpGRF3 showed a consistent expression trend with that found for BpGRP1 (Figure 2i).Collectively, these results suggest that BpGRF3 is a regulatory factor upstream of BpGRP1.
A previous study showed that PdbGRF1 acts as a key positive regulator of plant salt tolerance by regulating the expression of PdbPOD17 and PdbAKT1 (Liu et al., 2022a).To investigate the role of BpGRF3 as a BpGRP1 upstream regulatory factor in salt tolerance, the phenotype and physiological parameters of the BpGRF3-OE line under salt stress were analysed.Similar to that found in the BpGRP1-OE lines, overexpression of BpGRF3 resulted in higher antioxidant enzyme activities and proline content and lower ROS and MDA levels under salt stress (Figure 3).However, the sensitivity of the grf3-1 mutant to salt stress may be attributed to its reduced SOD activity and elevated O 2 • ˉand MDA levels.Similarly, an analysis of the expression patterns of the stress-related genes BpSODs, BpPODs and BpAPXs in BpGRF3 transgenic plants was performed to explain the physiological differences between the BpGRF3-OE lines and grf3-1 mutant lines.Furthermore, the expression pattern of BpGRP1 exhibited opposite trends in the BpGRF3-OE lines and grf3-1 mutant lines (Figure 4).Overall, the results show that BpGRF3 enhances the salt tolerance of birch by combining with the ABRE of the promoter region to regulate the expression of the downstream gene BpGRP1.
Previous studies have revealed that GRF genes have the ability to recognize and bind specific elements, including ACTCGAC, CTTCTTC, CTGACA and TGTCAGG (Kim et al., 2012;Piya et al., 2020).In 84K poplar, PpnGRF5 enhances the expression of PagCKX1p-1 and PagCKX1p-2 by binding to the TGTCAG cisacting element, which results in enhancement of cell division and cell expansion in apical buds and young leaves and then enhancement of the growth and expansion of poplar leaves (Wu et al., 2021).The results from our previous study revealed that PdbGRF1 enhances salt stress tolerance by regulating the expression of genes, including PdbPOD17 and PdbAKT1, by binding to the dehydration-responsive element (DRE) element ('A/ GCCGAC') (Liu et al., 2022a).To date, few studies have investigated whether GRF-regulating target genes participate in the abiotic stress response by targeting ABREs.Interestingly, the present study showed that BpGRF3 regulated BpGRP1 to participate in salt stress regulation by directly binding to ABREs, which may be a previously unknown salt stress response pathway.
Moreover, ABA has been demonstrated to mediate numerous physiological and adaptive responses resulting from adverse environmental conditions (Long et al., 2013).Our results showed that ABRE cis-acting elements were highly enriched in the promoter region of BpGRP1.BpGRP1 expression may be induced by ABA and may participate in salt stress regulation.Hence, elucidating the specific role of ABA in the regulation of birch salt tolerance is our next area of research interest and will be the focus of a study that will be conducted soon.

BpGRF3 interacts with BpSHMT to positively regulate the expression of BpGRP1
Protein-protein interactions have been found to be useful for investigating complex biological activities and for understanding the ways in which external signals are perceived and transduced to trigger specific plant responses (Pazhamala et al., 2021;Struk et al., 2019).In Arabidopsis, AtGRF5 not only interacts with AN3 to regulate cell proliferation in leaves but also interacts with DELLA through the GA pathway to regulate leaf growth, root elongation and plant development and then participates in the response to cold stimuli (Horiguchi et al., 2005;Shahan, 2020).In this study, the fusion protein BpGRF3-pGBDT7-BD did not activate the yeast reporter gene (Figure 5a).Whether BpGRF3 mediates BpGRP1 and then participates in the regulation of salt tolerance remains unclear.We speculated that BpGRF3 interacts with other proteins to form functional complexes and thus regulates the involvement of BpGRP1 in salt stress.To verify this conjecture, the Y2H system and the PL approach combined with MS-based quantitative proteomics were used to investigate the proteins interacting with BpGRF3.Fortunately, through the abovementioned two pathways, we identified a common potential BpGRF3-interacting protein, BpSHMT.We then demonstrated the BpGRF3-BpSHMT interaction through a Y2H assay and BiFC in vitro/in vivo (Figure 5b,c).Previous studies have not found an interaction between GRF and SHMT.Thus, in this study, we were able to provide the first demonstration of the interaction between BpGRF3 and BpSHMT, and our subsequent resolution of the salt stress mechanism provides novel insight.
A previous study demonstrated that the OsSHMT3 gene could be significantly induced by salt stress in rice and that overexpression of the OsSHMT3 gene can significantly enhance the tolerance of Arabidopsis to salt stress (Mishra et al., 2019).The overexpression of ApSHMT in the freshwater cyanobacterium Synechococcus elongatus PCC7942 results in increased enzyme activities in serine biosynthetic pathways and enhanced salinity tolerance (Waditee-Sirisattha et al., 2017).Similarly, in our study, BpSHMT exhibited the highest expression level under salt stress (Figure S5), implying that BpSHMT may play an important role in salt stress.Importantly, our results provide direct evidence showing that BpGRF3 and BpSHMT coregulated BpGRP1 and that the presence of both BpGRF3 and BpSHMT enhanced BpGRP1 expression (Figure 5d,f).BpSHMT is generally a mitochondrial protein (Jamai et al., 2009;Wu et al., 2015); however, in birch, BpGRF3 interacts with BpSHMT in the nucleus to regulate BpGRP1 expression and then participates in the salt stress response.The pathway and mechanism by which BpSHMT enters the nucleus remain unclear and need further exploration.
The overexpression of BpmiR396c inhibits the expression of BpGRF3, which is involved in the response of birch to salt stress In plants, miR396 was previously recognized as a pivotal regulator governing various aspects of growth and development (Yu et al., 2020;Zhang et al., 2015).However, recent studies have also highlighted the significant role of miR396 in plant stress resistance (He et al., 2022;Pegler et al., 2021).For example, the Sp-miR396a-5p transcript levels are upregulated under salt and drought stresses (Chen et al., 2015); however, in Sporobolus alterniflorus, miR396 is downregulated under salinity stress (Qin et al., 2014).It is thus clear that the highly conserved miR396 has different functions in different species.The present study showed that BpmiR396c was downregulated in response to salinity stress (Figure 6a).Notably, the phenotypic and physiological parameters indicated that the expression of BpmiR396c can lead to increased sensitivity toward salt stress (Figure 6c-h), which suggests that BpmiR396c plays a negative role in regulating salt tolerance.
Prior research has indicated that the GRF gene is the target of miR396, a finding that has been corroborated through 5' RLM-RACE analysis in various species, including Oryza sativa (Duan et al., 2016), Panicum virgatum (Liu et al., 2021b) and Arabidopsis thaliana (Beltramino et al., 2021).Similarly, in the present study, we sequenced a degradome library with 5' RLM-RACE to confirm the cleavage sites.The BpGRF3 transcripts were cleaved at the site complementary to BpmiR396c between base pairs 11 and 12 from the 5' end of the miRNA (Figure 6b).Notably, the expression pattern of BpGRF3 transcripts in response to salt stress was opposite to that of BpmiR396c (Figures 2i and 6a).The expression level of BpGRF3 in the plants of the Bp-miR396-OE lines (OE2, OE7 and OE9) was significantly lower than that in the WT plants and was only 8.8%, 10.4% and 5.9%, respectively, of that in the WT plants (Figure 6h).Furthermore, in contrast to the suppressive effect of msi-miR164g on drought stress, the overexpression of MsNAC022 has been shown to enhance drought tolerance in transgenic Arabidopsis and apple (Peng et al., 2022).BpGRF3 was cloned into the pGBKT7 vector to examine its role in gene activation.pGBKT7-AtHSFA7b was used as a positive control (Zang et al., 2019).(b) A yeast two-hybrid (Y2H) assay was used to identify the interaction of BpGRF3 with BpSHMT in yeast.QDO: SD/-Leu/-Trp-His-Ade, QDO/-X/-A: SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA with 40 mg mL À1 X-a-Gal and 100 ng mL À1 AbA.pGBKT7-53/pGADT7-T was used as a positive control.(c) BiFC demonstrated that BpGRF3 interacted with BpSHMT in vivo.H2A-1:m Cherry served as a nuclear marker.All experiments were performed with at least three independent biological replicates.(d) Schematic diagrams of effector and reporter constructs used for the dual luciferase assay.(e) Firefly luciferase complementation imaging assays of the interaction of BpGRF3 with BpSHMT in tobacco leaves.A. tumefaciens GV3101 strains harbouring BpproGRP1-LUC and pROKII vectors were transfected into tobacco leaves.Luciferase imaging was performed 24 h after injection.(f) A dual-luciferase reporter assay showed that BpGRF3 and BpSHMT positively regulated the expression of BpGRP1.Similarly, we observed contrasting phenotypes between the BpmiR396c-OE lines and the BpGRF3-OE lines under salt stress (Figures 3a and 6c).Collectively, these results suggest that the resistance of birch to salt stress is negatively and positively modulated by BpmiR396c and BpGRF3, respectively.Based on the above-described findings, we propose a putative model of salt tolerance in birch mediated by the BpmiR396c-BpGRF3-BpGRP1 module, which may provide a new perspective for understanding the mechanism of birch salt tolerance (Figure 7).Under salt stress, a rapid decrease in BpmiR396c levels alleviated the cleavage of BpGRF3 transcripts, and the encoded transcription factor interacts with BpSHMT to form functional complexes that in turn activate BpGRP1 transcription.Through the interaction between BpGRF3 and BpSHMT, BpGRP1 is activated to regulate the associated physiological pathways, which increases the ROS scavenging ability, reduces the degree of damage to the plasma membrane and ultimately enhances the salt stress response in birch.

Plant materials and growth conditions
Birch seedlings (transgenic plants and wild-type plants were used in this study) were grown in a greenhouse under a 16 h light/8 h dark photoperiod under 400 lmol photons m À2 s À1 irradiation at 25 °C (Tan et al., 2020).Three-month-old plants were watered with a solution of 0.2 M NaCl, and tissues were collected at 6, 12, 24, 36 and 72 h post-watering.Seedlings watered with fresh water were harvested at the corresponding time points as controls.

Plasmid construction and birch transformation
BpGRP1, BpGRF3 and Bp-miR396c were amplified by PCR with KOD DNA polymerase (TOYOBO, Osaka, Japan), gene-specific primers and birch cDNAs.The full-length coding sequences of BpGRP1 and BpGRF3 were cloned into the pROKII vector under control of the cauliflower mosaic virus 35S promoter (35S) for overexpression.The Bp-miR396c precursor was introduced into  BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance 143 the PCAMBIA1302 vector under control of the 35S CaMV promoter to overexpress Bp-miR396c (Sahito et al., 2017).In addition, a truncated inverted-repeat cDNA of BpGRP1 (200 bp) was ligated into pFGC5941, an RNAi vector flanking the intron of Chsa (encoding chalcone synthase A), which yielded the pFGC5941::BpGRP1 construct to silence the expression of BpGRP1.
All plasmids were introduced into Agrobacterium tumefaciens strain EHA105 for birch transformation as described previously (Guo et al., 2017).All transgenic lines were verified by PCR and RT-qPCR analysis.The primers are listed in Table S1.

Assessment of the salt tolerance of transgenic birch
NaCl treatment was conducted to mimic salt stress conditions and to elucidate the function of BpGRP1, BpGRF3 and Bp-miR396c in the salt-tolerance response of birch.To observe the growth of birch plants, plants of similar size (height of approximately 5 cm) from the wild-type (WT) and all transgenic birch lines (BpGRP1, BpGRF3 and Bp-miR396c lines) were transplanted into 6-inch pots containing a perlite/vermiculite/soil mixture at a ratio of 1:1:4 (v/v) and were grown in a greenhouse.After two months, the roots of the plants were watered with 0.2 M NaCl, and plants watered with well water served as controls.After treatment for 14 d, the plant phenotype was observed and photographed.
The plants of WT and transgenic (BpGRP1, BpGRF3 and Bp-miR396c) birch lines that had been grown in soil for two months were watered with a solution of 0.2 M NaCl for 7 d.Well-watered plants were used as controls.The leaves of different lines were collected for the determination of physiological indices.The ROS, SOD, POD, MDA, H 2 O 2 , O 2 • ˉand proline levels were determined using a biological engineering kit from Nanjing Jiancheng Institute.

Subcellular localization analysis of transiently expressed fusion proteins
The coding sequence of BpGRF3 was fused in frame to the amino terminus of the enhanced green fluorescent protein (eGFP) coding sequence under control of the CaMV35S promoter, as previously described (Liu et al., 2020(Liu et al., , 2022a)).As a control, GFP transcribed using the CaMV 35S promoter (35S::GFP) was used.
Particle bombardment (Bio-Rad, Hercules, CA, USA) was used to introduce the constructs into onion epidermal cells.Confocal laser-scanning microscopy (LSM700, Zeiss, Jena, Germany) was then used to analyse the transformed cells.The primers are listed in Table S1.

Protein interaction
The full-length coding region of BpGRF3 was fused into the pGBDT7-BD vector, and yeast two-hybrid (Y2H) assays were performed as previously described (Liu et al., 2022b;Wang et al., 2022b).In brief, the BpGRF3-pGBDT7-BD recombinant plasmids were transformed into Y2HGold yeast competent cells, and the transformed yeast competent cells were then placed on synthetic dropout SD/-Trp and SD/-Trp/-His with 40 mg mL À1 X-a-Gal media to detect their growth status at 30 °C.The specific procedure used for PL combined with MS-based quantitative proteomics was described by Yang et al. (2021).

Yeast one-hybrid (Y1H) assay
The coding sequence of BpGRF3 was cloned into the pGADT7-Rec2 vector (Clontech) to generate BpGRF3-pGADT7-Rec2 constructs.The ABRE in the BpGRP1 promoter was inserted into the pAbAi vector (Clontech) immediately upstream of the AUR1-C gene.These constructs were cotransformed into the Saccharomyces cerevisiae strain Y187, and the Y1H assay was conducted as previously described (Liu et al., 2020).The primers are listed in Table S1.

EMSA
The full-length coding sequence of BpGRF3 was cloned into the pMAL-c5X vector and transformed into Escherichia coli strain ER2523 (NEB Express, Ipswich, MA, USA).An oligonucleotide probe containing the ABRE (ACGTG) motif derived from the BpGRP1 promoter was labelled with biotin at its 5' end by using an electrophoretic mobility shift assay (EMSA) Probe Biotin Labeling Kit (Beyotime, China).EMSAs were carried out with a chemiluminescence-based EMSA kit (Beyotime, China).The primers are listed in Table S1.

Detection of beta-glucuronidase (GUS) activity
For the construction of reporter vectors, the ABRE motif sequence with three tandem copies was fused with a 46-bp minimal 35S promoter to drive a GUS reporter gene.The effector, namely, the BpGRF3 overexpression vector (35S::BpGRF3), was transformed into birch calli together with each reporter.To normalize the transformation efficiency, the 35S::Luc vector was also cotransformed.GUS activity was assessed as described by Jefferson (1987).For each GUS activity assay, three biological replicates were analysed, and three technical replicates were performed for each biological replicate.The primer sequences used for vector construction are shown in Table S1.

ChIP-qPCR analysis
The 35S::BpGRF3-GFP construct was transiently transformed into birch for the ChIP assay, which was performed as described previously, with some modifications (Zhang et al., 2016;Zhao et al., 2020).Briefly, proteins and DNA were cross-linked using formaldehyde (3% v/v) for 5 min.The purified DNA-protein crosslinks (DPCs) were sonicated and immunoprecipitated with an anti-GFP antibody.The purified DPCs were also immunoprecipitated with an anti-human influenza haemagglutinin (HA) antibody, which served as the negative control.Chromatin before immunoprecipitation was used as the input control.ChIP-qPCR was performed to study the enrichment of the target DNA sequence.The fold enrichment of ChIP-qPCR was calculated according to the method described by Haring et al. (2007).For each transfection, three biological replicates were conducted, and three technical replicates were conducted for each biological replicate.The ChIP-qPCR primers are shown in Table S1.

Validation of cleavage sites of BpGRF3
The 5' RLM-RACE of the birch total RNA was performed using the GeneRacer kit (Invitrogen, Carlsbad, CA, USA), as described previously (Liu et al., 2021a).The amplification products were gel-purified and cloned into the pCloneEZ vector (CloneSmarter).Ten independent clones were picked for sequencing.

Statistical analysis
The data were analysed by one-way ANOVA with Statistical Product and Service Solutions (SPSS) 16.0 (IBM Corp., Armonk, NY, USA) to determine significance.Statistical significance was defined as follows: *P ≤ 0.05 and **P ≤ 0.01.

Figure 1
Figure 1 BpGRP1 improves the salt stress tolerance of transgenic birch.(a) RT-qPCR analysis of BpGRP1 expression during exposure to salt stress.(b) The growth of plants of the transgenic lines (OE lines, SE lines) and WT plants was compared under normal or salt stress conditions.Normal condition: normal growth conditions.Salt stress: treatment with 0.2 M NaCl for 14 d.(c) Analysis of the expression of salt-related genes (BpSODs, BpPODs, BpAPXs and BpP5CS1) in WT, BpGRP1-OE and BpGRP1-SE plants.All expression values were log 2 -transformed.(d-h) SOD activity, H 2 O 2 content, O 2 • ˉcontent, MDA content and proline content.Control: plants grown under normal growth conditions.NaCl: treatment with 0.2 M NaCl for 7 d.The error bars indicate the standard deviations (SDs) of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 22, 131-147 Zhongyuan Liu et al.
Figure2BpGRF3 regulates BpGRP1 by directly binding to ABREs in vitro and in vivo.(a) A yeast one-hybrid (Y1H) assay was used to verify the binding of BpGRF3 to ABREs.p53-HIS2/pGADT7-Rec2-p53 was used as a positive control.pGADT7-Rec-BpGRF3/P53-HIS2 was used as a negative control.(b) BpGRF3 was fused with the green fluorescence protein (GFP) gene under control of the 35S promoter, and 35S::GFP (control) was transiently expressed in onion epidermal cells via the particle bombardment method and visualized under a confocal microscope at 488 nm for the excitation of GFP and 507 nm longpass for emission.GFP, GFP fluorescence; Bright, bright field; Merge, merged bright field and fluorescence images.A nucleus-localized MYB transcription factor, ThMYB8, was used as a positive control(Liu et al., 2020).(c) The binding of BpGRF3 to the ABRE in BpGRP1 promoters was evaluated by EMSA.The biotinlabelled probe was used as a negative control; the biotin-labelled probe incubated with BpGRF3 protein was tested; and competitive probes were used at 10-fold and 100-fold (lack of biotin label).(d) Schematic diagram of the effector and reporter constructs used for coexpression in birch calli.(e) GUS staining of birch calli.(A) Transient expression of CAM-ABRE alone; (B) transient expression of the cotransformed effector and reporter under normal conditions; (C) transient expression of the cotransformed effector and reporter under salt stress.(f) GUS activity assay showing the binding of the BpGRF3 protein to the ABRE in vivo.The values represent the means AE SDs of three biological replicates.(g) Distribution of the BpGRF3-binding ABRE in the promoter of BpGRP1.(h) ChIP-qPCR analysis of the association of BpGRF3 with BpGRP1 promoters in vivo using an anti-GFP tag antibody.The relative abundance of BpGRF3targeted promoter fragments in chromatin isolated under normal conditions or salt treatment conditions (0.2 M NaCl) for 24 h.The error bars indicate the standard deviations (SDs) of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.(i) RT-qPCR analysis of BpGRF3 expression under salt stress.The error bars indicate the standard deviations (SDs) of three biological replicates.

Figure 3
Figure 3 The overexpression of BpGRF3 in birch improves tolerance to salt stress.(a) The growth of plants of the transgenic lines (OE lines) and WT plants was compared under normal or salt stress conditions.Control: plants grown under normal growth conditions.NaCl: treatment with 0.2 M NaCl for 14 d.(b-h) ROS content, H 2 O 2 content, O 2 • ˉcontent, MDA content, SOD activity, POD activity and proline content.Control: plants grown under normal growth conditions.NaCl: treatment with 0.2 M NaCl for 7 days.The error bars indicate the standard deviations of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.

Figure 4
Figure 4 BpGRF3 positively regulates salt tolerance in birch.(a) Gene structures of BpGRF3 with a CRISPR/Cas9 target site designed in the exon.The black lines, orange strips and blue strips indicate introns, untranslated regions (UTRs) and exons, respectively.The nucleotide sequences indicate regions targeted by the gRNA designed in this study, and the nucleotides in red indicate proto-spacer adjacent motifs (PAMs).(b-e) SOD activity, H 2 O 2 content, O 2 • ˉcontent and MDA content.Con: plants grown under normal growth conditions.NaCl: treatment with 0.2 M NaCl for 2 d.The error bars indicate the standard deviations of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.(f) Analysis of the expression of salt-related genes (BpSODs, BpPODs and BpAPXs) in WT, BpGRF3-OE and grf3-1 mutant plants.All expression values were log 2 -transformed.(g) Analysis of the expression of BpGRP1 in WT, BpGRF3-OE and grf3-1 mutant plants.All expression values were log 2 -transformed.

Figure 5
Figure5BpGRF3 interacts with BpSHMT.(a) Transcriptional activation activity of BpGRF3 in yeast.BpGRF3 was cloned into the pGBKT7 vector to examine its role in gene activation.pGBKT7-AtHSFA7b was used as a positive control(Zang et al., 2019).(b) A yeast two-hybrid (Y2H) assay was used to identify the interaction of BpGRF3 with BpSHMT in yeast.QDO: SD/-Leu/-Trp-His-Ade, QDO/-X/-A: SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA with 40 mg mL À1 X-a-Gal and 100 ng mL À1 AbA.pGBKT7-53/pGADT7-T was used as a positive control.(c) BiFC demonstrated that BpGRF3 interacted with BpSHMT in vivo.H2A-1:m Cherry served as a nuclear marker.All experiments were performed with at least three independent biological replicates.(d) Schematic diagrams of effector and reporter constructs used for the dual luciferase assay.(e) Firefly luciferase complementation imaging assays of the interaction of BpGRF3 with BpSHMT in tobacco leaves.A. tumefaciens GV3101 strains harbouring BpproGRP1-LUC and pROKII vectors were transfected into tobacco leaves.Luciferase imaging was performed 24 h after injection.(f) A dual-luciferase reporter assay showed that BpGRF3 and BpSHMT positively regulated the expression of BpGRP1.Each value represents the mean AE SD of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.
Figure5BpGRF3 interacts with BpSHMT.(a) Transcriptional activation activity of BpGRF3 in yeast.BpGRF3 was cloned into the pGBKT7 vector to examine its role in gene activation.pGBKT7-AtHSFA7b was used as a positive control(Zang et al., 2019).(b) A yeast two-hybrid (Y2H) assay was used to identify the interaction of BpGRF3 with BpSHMT in yeast.QDO: SD/-Leu/-Trp-His-Ade, QDO/-X/-A: SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA with 40 mg mL À1 X-a-Gal and 100 ng mL À1 AbA.pGBKT7-53/pGADT7-T was used as a positive control.(c) BiFC demonstrated that BpGRF3 interacted with BpSHMT in vivo.H2A-1:m Cherry served as a nuclear marker.All experiments were performed with at least three independent biological replicates.(d) Schematic diagrams of effector and reporter constructs used for the dual luciferase assay.(e) Firefly luciferase complementation imaging assays of the interaction of BpGRF3 with BpSHMT in tobacco leaves.A. tumefaciens GV3101 strains harbouring BpproGRP1-LUC and pROKII vectors were transfected into tobacco leaves.Luciferase imaging was performed 24 h after injection.(f) A dual-luciferase reporter assay showed that BpGRF3 and BpSHMT positively regulated the expression of BpGRP1.Each value represents the mean AE SD of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.

Figure 6
Figure 6 The overexpression of BpmiR396c inhibits the expression of BpGRF3, which enhances the salt tolerance sensitivity of transgenic birch plants.(a) RT-qPCR analysis of BpmiR396c expression under salt stress.All expression values were log 2 -transformed.The error bars represent the standard deviations (SDs) of three biological replicates.(b) BpmiR396c cleavage sites in BpGRF3.The positions corresponding to the 5 0 ends of the cleaved BpGRF3 mRNAs determined by 5 0 RACE and the frequency of 5 0 RACE clones corresponding to each site are shown by arrows.(c) The growth of OE and WT plants under salt stress was compared.(d-h) SOD activity, proline content, H 2 O 2 content, O 2 ˉcontent and MDA content.NaCl: treatment with 0.2 M NaCl for 7 days.The error bars represent the standard deviations (SDs) of three biological replicates.* indicates P ≤ 0.05, and ** indicates P ≤ 0.01.(i) Relative expression of BpmiR396c and BpGRF3 in plants of the WT and BpmiR396c-OE lines.All expression values were log 2 -transformed.The error bars represent the standard deviations (SDs) of three biological replicates.

Figure 7
Figure 7 A putative model for the mediation of the transcriptional regulation of BpGRP1 by the BpmiR396c-BpGRF3 module.This model may provide a new perspective for understanding the mechanism of birch salt tolerance.Under salt stress, a decrease in BpmiR396c diminishes cleavage of the downstream BpGRF3 transcripts, and the encoded protein activates transcription of the downstream gene BpGRP1.With the interaction between BpGRF3 and BpSHMT, BpGRP1 is activated to regulate the associated physiological pathways, which increases the ROS scavenging ability, reduces the degree of damage to the plasma membrane and ultimately enhances the salt stress response in birch.

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 22, 131-147 Zhongyuan Liu et al.
ROS, H 2 O 2 , O 2 • ˉand MDA contents were measured.The trend for the changes in the ROS, H 2 O 2 , O 2 • ˉand MDA contents in the BpGRF3-OE lines was consistent with that found in the BpGRP1-OE lines.Under salt stress conditions, the levels of ROS, H 2 O 2 , O 2 • ˉand MDA were significantly higher in the WT plants than in the OE plants (Figure