Long noncoding RNA from Betula platyphylla, BplncSIR1, confers salt tolerance by regulating BpNAC2 to mediate reactive oxygen species scavenging and stomatal movement

Summary

Long noncoding RNAs (lncRNAs) play an important role in abiotic stress tolerance. However, their function in conferring abiotic stress tolerance is still unclear. Herein, we characterized the function of a salt‐responsive nuclear lncRNA (BplncSIR1) from Betula platyphylla (birch). Birch plants overexpressing and knocking out for BplncSIR1 were generated. BplncSIR1 was found to improve salt tolerance by inducing antioxidant activity and stomatal closure, and also accelerate plant growth. Chromatin isolation by RNA purification (ChIRP) combined with RNA sequencing indicated that BplncSIR1 binds to the promoter of BpNAC2 (encoding NAC domain‐containing protein 2) to activate its expression. Plants overexpressing and knocking out for BpNAC2 were generated. Consistent with that of BplncSIR1, overexpression of BpNAC2 also accelerated plant growth and conferred salt tolerance. In addition, BpNAC2 binds to different cis‐acting elements, such as G‐box and ‘CCAAT’ sequences, to regulate the genes involved in salt tolerance, resulting in reduced ROS accumulation and decreased water loss rate by stomatal closure. Taken together, BplncSIR1 serves as the regulator of BpNAC2 to induce its expression in response to salt stress, and activated BpNAC2 accelerates plant growth and improves salt tolerance. Therefore, BplncSIR1 might be a candidate gene for molecular breeding to cultivate plants with both a high growth rate and improved salt tolerance.

Introduction

Plants, as sessile organisms, are frequently affected by various stress factors, such as salinity, cold, viral infection and high temperatures (Zhu, 2016). Salinity is one of the main abiotic stress factors that seriously affects plant growth and distribution. Excessive salt usually causes hyperosmotic stress, ion imbalance and even oxidative damage to plant cells (Zhu, 2001). In addition, plants activate multiple complex gene regulatory mechanisms to restore and re‐establish cellular homeostasis under salt stress to prevent damage and ensure survival (Atkinson and Urwin, 2012). One of the earliest plant responses to adverse stresses is the accumulation of reactive oxygen species (ROS; Baxter et al., 2014). As signalling components, ROS play important roles in plant signal transduction, such as osmotic regulation of cell membranes and regulating stomatal closure. To maintain ROS homeostasis, plants have evolved a variety of antioxidant mechanisms to eliminate excessive ROS. Therefore, dynamic redox homeostasis is quite important in the plant stress response.

A long noncoding RNA (lncRNA) is a transcript with a length of more than 200 nucleotides (nt) with no obvious protein‐coding ability or that lacks an open reading frame (ORF) encoding more than 100 amino acids (Hao et al., 2015; Wang et al., 2017; Yu et al., 2019). Based on their location in protein‐coding genes, lncRNAs can be classified into four types: sense, antisense, intronic and intergenic (Ponting et al., 2009). LncRNAs have lower sequence conservation and lower expression levels than protein‐coding mRNAs; therefore, they were initially considered as transcriptional ‘noise’ (Liu et al., 2015a,b; Chekanova, 2015). Recently, it was proposed that lncRNAs' functions are mainly related to their secondary structures (Pang et al., 2009). Although lncRNAs have little ability to encode proteins, they can act as key regulatory molecules that directly interact with DNA, proteins or other RNAs to regulate gene expression (Lucero et al., 2020). Plant lncRNAs are transcribed by plant‐specific RNA polymerases, Pol IV and Pol V, and Pol II and Pol III are considered potential regulators of plant responses to the environment (Dinger et al., 2009; Wierzbicki et al., 2008). With the continuous progress and development of sequencing technology, many lncRNAs from various eukaryotes, including plants, have been identified (An et al., 2018; Liu et al., 2012; Tian et al., 2016; Zhang et al., 2014). LncRNAs are important regulators of gene expression and plant growth and development. However, the functions of lncRNAs in plants are still poorly understood, and only a few lncRNAs have been characterized. For example, in tomatoes, transcription factor (TF) WRKY1 can activate lncRNA33732 to induce respiratory burst oxidase homolog protein (RBOH) expression, thereby increasing H₂O₂ accumulation and participating in the regulation of resistance to Phytophthora infestans in tomato (Cui et al., 2019). Cold‐induced lncRNA‐mediated chromatin modifications (lncR2Epi) in Arabidopsis repress the expression of FLOWERING LOCUS C (FLC) and inhibit its flowering (Csorba et al., 2014; Wang et al., 2016). Furthermore, evidence suggests that lncRNAs are important regulators of multiple biological processes and function through multiple mechanisms (Wang et al., 2017). Recent studies have found that lncRNAs play important roles in regulating gene expression in response to environmental stress (Chen et al., 2016; Di et al., 2014; Lv et al., 2016; Ye et al., 2022).

The clustered regularly interspersed short palindromic repeat (CRISPR) system is a revolutionary genomic editing technique that uses guide RNAs (gRNAs) to direct facile and efficient Cas‐mediated DNA editing (Jansen et al., 2002; Lino et al., 2018). The CRISPR system is a powerful technology for genetic manipulation, especially of lncRNAs (Zibitt et al., 2021), with merits of versatility and flexibility. CRISPR can target cytoplasmic or nuclear lncRNAs, induce or repress transcription and also disarrange the genome by selectively avoiding (or interacting with) adjacent genetic material (Zibitt et al., 2021).

Betula platyphylla (birch) is a fast‐growing woody plant that is widely distributed in the northern temperate zone of the northern hemisphere and can adapt to harsh environments, such as barren soil and cold temperatures. Birch is one of the main broadleaf tree species, with important applications in the pulp and biofuel industries (Borrega et al., 2013; Li et al., 2005). To date, many lncRNAs have been identified to be involved in biotic and abiotic stresses (Wen et al., 2020; Zhang et al., 2022). In our earlier study, we found that the expression of a birch lncRNA, BplncSIR1, was induced by salt stress (Jia et al., 2023). However, the function of BplncSIR1 in birch adaptation to salt stress remains unclear. In the present study, we generated the full‐length cDNA of BplncSIR1. In addition, BplncSIR1 CRISPR‐edited (knockout) and overexpressing plants were generated, and the function of BplncSIR1 in response to salt stress was characterized. Our study provides new insights into lncRNA‐mediated salt stress tolerance in birch plants.

Results

Cloning of the full‐length cDNA of BplncSIR1

Previously deposited public transcriptome or gene expression data (PRJNA790472) from the NCBI showed that the lncRNA BplncSIR1 was highly induced by salt stress in birch. Therefore, we first cloned the full‐length cDNA of BplncSIR1 using rapid amplification of cDNA ends (RACE). Four primers (P1–P4) were designed separately for RACE. The amplified 3′ and 5′ cDNA sequences of BplncSIR1 were 269 and 562 bp in length, respectively (Figure 1a). These two amplified sequences, together with the truncated BplncSIR1 sequence, were assembled to get the full‐length cDNA of BplncSIR1 (1139 bp; Figure S1a). Further study showed that BplncSIR1 is located in the first and second exons of BpDOM1 (encoding Domino 1) and is an intronic lncRNA.

Figure 1.

Cloning and characterizing the expression and subcellular location of BplncSIR1. (a) Cloning the full‐length cDNA of BplncSIR1 using 5′ and 3′ RACE, respectively. 5′RACE/3′RACE: the truncated region of BplncSIR1 is amplified by 5′RACE‐PCR (5′RACE) or 3′RACE‐PCR (3′RACE). Khaki and green region, respectively, indicate the amplified 5′ region and 3′ region of BplncSIR1 after the full cDNA length of the BplncSIR1 sequence is assembled. P1–P4: the regions amplified by qRT‐PCR in Figure 1e; four paired PCR primers were used. (b) Characterization of the expression of BplncSIR1 in response to salt stress using qRT‐PCR. (c) Analysis of the expression of BplncSIR1 using GUS staining. (d) Analysis of the expression of BplncSIR1 using GUS activity determination. (e) Subcellular location analysis of BplncSIR1. The nuclear and cytoplasmic mRNAs were isolated, respectively, and qRT‐PCR was performed to analyse the transcript of BplncSIR1.

We predicted the potential protein‐coding capability of BplncSIR1 using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/), and 11 potential peptides were predicted, with lengths of 27–84 amino acid residues (Figure S1b). However, no coding sequences were found for these ORFs when analysed using the Coding Potential Calculator (CPC) and Coding‐Potential Assessment Tool (CPAT) (Figure S1c).

BplncSIR1 is induced by salt stress in both leaves and roots

The expression of BplncSIR1 increased more in leaves than in roots after 6 h of 200 mm NaCl treatment, peaking at 48 h of salt treatment. The transcript of BplncSIR1 was induced by salt stress treatment for 6 h and kept increasing until 48 h in both roots and leaves (Figure 1b). To further study the expression of BplncSIR1, birch plants expressing a GUS gene under the control of the BplncSIR1 promoter were generated (proBplncSIR1::GUS). Consistently, GUS activity and staining assays of proBplncSIR1::GUS plants showed that the expression of BplncSIR1 was significantly induced in apical buds, tender leaves and roots when exposed to NaCl treatment (Figure 1c,d). Taken together, these results suggest that BplncSIR1 is involved in the birch salt stress response.

BplncSIR1 is located in the nucleus

The function of a lncRNA is closely related to its location; therefore, we studied the subcellular localization of BplncSIR1. The RNAs from the nucleus and cytoplasm were isolated separately and subjected to qRT‐PCR analysis. The U6 gene is expressed in the nucleus, which is used to determine the purity of isolated nuclear and cytoplasmic fractions. The qRT‐PCR results showed that U6 was mainly expressed in the nucleus and actin was mainly expressed in the cytoplasm (Figure 1e), indicating relatively low cross‐contamination in the nuclear and cytoplasmic fractions. Meanwhile, BplncSIR1 was highly enriched in the nucleus rather than in the cytoplasm (Figure 1e), suggesting that it is located in the nucleus.

Generation of birch plants overexpressing BplncSIR1 and CRISPR‐mediated knockout of BplncSIR1

To investigate the function of BplncSIR1, we generated transgenic birch overexpressing BplncSIR1 (Figure S2a). A total of 9 lines were generated, and qRT‐PCR showed that BplncSIR1 was successfully expressed in each transgenic line. Three lines (BplncSIR1‐OE2; BplncSIR1‐OE7; BplncSIR1‐OE8) with relatively higher expression levels of BplncSIR1 were selected for further study (Figure 2a). At the same time, a total of nine transgenic lines were generated using the CRISPR/Cas9 transformation system (Figure 2b). The primers were designed to respectively amplify the hygromycin and Cas9 regions, and the PCR products were analysed using agarose gel electrophoresis (Figure S2b, Cas9 panel). The results suggested that the transformation had been performed successfully. In addition, paired primers to amplify the truncated region of BplncSIR1 were designed, PCR amplification was performed and the amplicons were sequenced. The results showed that the DNA sequences were mutated with deletions, insertions, or substitutions (Figure 2b). Then, each PCR product from the CRISPR lines was mixed with that from WT plants, denatured and then renatured. The renatured DNA was digested with T7 endonuclease I (T7EI) to determine whether the mutation was heterozygous or homozygous. After T7 endonuclease digestion, the PCR products of some lines displayed more than one band (Figure S2b, T7E1 panels, two CRISPR editing sites were detected and were shown as two panels), indicating that these renatured DNAs had formed a loop and were cut by T7EI. Therefore, these lines should be mutated by CRISPR editing and were heterozygous. We further predicted the secondary structure of these mutated lines. Among them, only the lines of bplncsir1 ^# ‐1, bplncsir1 ^# ‐4 and bplncsir1 ^# ‐9 had changed their secondary structure compared with natural BplncSIR1, suggesting that their function might be lost because of their altered secondary structure (Figure S2c). Therefore, these three lines were selected for further study.

Figure 2.

BplncSIR1 confers salt tolerance. (a) Expression analysis of BplncSIR1 in the overexpressed lines. (b) Analysis of the CRISPR editing formats using Sanger DNA sequencing. (c) The growth phenotype of the BplncSIR1 overexpressing plants, BplncSIR1 knockout plants and WT plants. Bars, 5 cm. (d) Plant height analysis. (e) Comparison of fresh weight. (f) Comparison of chlorophyll content. (g) Comparison of root length. (h) Comparison of root weight. BplncSIR1‐OE2, 7 and 8: plant lines 2, 7 and 8 overexpressing BplncSIR1; bplncsir1 ^# ‐1, 4 and 9: plant lines 1, 4 and 9 with CRISPR‐mediated knockout of BplncSIR1; WT: wild‐type plant. The error bar indicates the standard deviation (SD) of the biological replicates. *Indicates statistically significant differences at P < 0.05; **indicates statistically significant differences at P < 0.01.

BplncSIR1 confers salt tolerance and facilitates plant growth in birch

The growth phenotypes among the WT, BplncSIR1‐OE and bplncsir1 ^# lines were compared. Under normal conditions, the height, fresh weight, root length and root weight of the plants overexpressing BplncSIR1 were all significantly higher than those of the WT and bplncsir1 ^# , while those in the bplncsir1 ^# lines were the lowest among all the lines (Figure 2c–e,g,h). These results suggested that BplncSIR1 facilitates plant growth. Under salt‐stress conditions for 21 days, the bplncsir1 ^# lines suffered obvious and heavy damage; all the leaves were wilted. However, the WT plants suffered lower levels of damage than the bplncsir1 ^# lines, and the BplncSIR1‐OE lines displayed the lowest amount of damage (Figure 2c). Simultaneously, the height, fresh weight, root length and root weight of BplncSIR1‐OE lines were all the highest, followed by the WT plants, with the bplncsir1 ^# lines having the lowest values (Figure 2c–e,g,h). Under normal conditions, there was no significant difference in the chlorophyll content among the studied plants (Figure 2f). The BplncSIR1‐OE lines showed the highest chlorophyll content, followed by the WT and lastly by the bplncsir1 ^# lines, after 150 mm salt stress for 21 days (Figure 2f). These results suggested that in birch, the expression of BplncSIR1 not only conferred salt tolerance but also facilitated plant growth.

To investigate whether BplncSIR1 exerts its function through its encoded short peptide, we generated a mutation in the longest peptide segment (ORF5) of BplncSIR1 (BplncSIR1‐MU). In this mutation, a single ‘A’ nucleotide after the start codon (ATG) was deleted, which will induce early termination of translation of the encoded peptide. To overexpress BplncSIR1‐MU, it was cloned into a plant expression vector driven by the 35S promoter (35S:BplncSIR1‐MU; Figure S1a,b) and stably transformed into birch. Three lines of birch overexpressing BplncSIR1‐MU were used for the study (Figure S3a). The BplncSIR1‐MU overexpressing plants displayed enhanced salt tolerance compared with the WT under salt stress conditions. However, the salt tolerance phenotype of plants overexpressing BplncSIR1‐MU was consistent with that of the BplncSIR1‐OE lines. These results indicated that mutation of its peptide coding regions did not alter the salt tolerance capability of BplncSIR1; therefore, BplncSIR1 does not exert its role dependent on its encoded short peptide (Figure S3b–f).

BplncSIR1 induces ROS scavenging capability and stomatal closure

To understand how BplncSIR1 enhances salt tolerance in birch, we measured physiological indicators involved in abiotic stress. Under salt conditions, the electrolyte leakage rate and MDA content were both significantly reduced in the BplncSIR1‐OE lines compared with WT plants. However, both the electrolyte leakage rate and MDA content in the bplncsir1^# lines were significantly increased relative to WT plants (Figure 3a,b).

Figure 3.

Determination of salt tolerance by physiological and histochemical analysis. (a) Electrolyte leakage analysis. (b) MDA analysis. (c) NBT and DAB staining analysis. Bars, 1 cm. (d) Determination of H₂O₂ contents. (e) POD activity analysis. (f) APX activity analysis. (g) CAT activity analysis. (h) Water loss rate analysis. (i) Comparison of stomatal apertures. (j) Analysis of the width–length ratio of stomatal apertures. BplncSIR1‐OE2, 7 and 8: plant lines 2, 7 and 8 overexpressing BplncSIR1; bplncsir1 ^# ‐1, 4 and 9: plant lines 1, 4 and 9 with CRISPR‐mediated knockout of BplncSIR1; WT: wild‐type plant. Error bar indicates the standard deviation (SD) of the biological replicates. *Indicates statistically significant differences at P < 0.05; **indicates statistically significant differences at P < 0.01.

Staining with DAB and NBT was performed to determine the accumulation of H₂O₂ and ${\mathrm{O}}_2^{-}$, respectively (Figure 3c). All the plants had similar levels of accumulation of H₂O₂ and ${\mathrm{O}}_2^{-}$ under normal conditions. When exposed to salt stress conditions, the bplncsir1 ^# lines had the highest accumulation of H₂O₂, and the BplncSIR1‐OE lines had the lowest accumulation of H₂O₂ compared with that in the WT plants (Figure 3c). The staining result was consistent with the measurement of H₂O₂ (Figure 3d). The overexpression of BplncSIR1 displayed reduced H₂O₂ and ${\mathrm{O}}_2^{-}$ accumulation. Therefore, we further studied whether the ROS scavenging capability was enhanced by overexpression of BplncSIR1. The activities of APX, POD and CAT were determined, which were all significantly increased in the BplncSIR1‐OE lines but significantly decreased in the bplncsir1 ^# lines compared with those in the WT plants under salt stress for 21 days (Figure 3e–g). However, there was no difference in APX, POD and CAT activities among the studied lines under normal conditions (Figure 3e–g). These results indicated that BplncSIR1 could increase the antioxidant capacity to decrease oxidative damage under salt stress, leading to improved salt tolerance.

The expression of BplncSIR1 confers salt tolerance; therefore, we further studied whether the water loss rate was altered. Overexpression of BplncSIR1 (BplncSIR1‐OE) significantly reduced the water loss rate. However, knockout of BplncSIR1 (bplncsir1 ^# ) significantly increased the water loss rate. These results indicated that BplncSIR1 could improve salt tolerance by controlling water loss (Figure 3h). We further studied whether the changes in water loss rates were caused by an altered stomatal aperture. The results showed that BplncSIR1‐OE lines displayed a reduced width–length ratio of the stomatal aperture. By contrast, the bplncsir1 ^# lines showed a significantly increased width–length ratio of the stomatal aperture. These findings suggest that BplncSIR1 has the capability to control the movement of the stomatal aperture. The regulation of stomatal aperture leads to a decrease in water loss rate, which ultimately improves salt tolerance (Figure 3i,j).

BpNAC2 is a direct transcriptional target of BplncSIR1

To identify the target genes of BplncSIR1, RNA‐seq was performed to compare gene expression between the WT and BplncSIR1‐OE birch plants under salt stress conditions. The quality and size of the RNA‐seq data are shown in Table S2. In total, 3223 DEGs were identified with an adjusted fold change >2 and a false discovery rate (FDR) < 0.01, including 1610 upregulated genes and 1613 downregulated genes (Table S3). Among the DEGs, 138 were TFs, including 24 induced TFs that were potentially involved in abiotic stress tolerance according to their functional annotation. These 24 TFs were further analysed using qRT‐PCR, and nine of them were observed to be significantly induced by BplncSIR1 (Figure 4a,b).

Figure 4.

Determination of the regulation of target genes by BplncSIR1. (a) The number of TFs among the DEGs. (b) Comparison of the target genes regulated by BplncSIR1 using qRT‐PCR and RNA‐seq. TFs potentially involved in salt tolerance were selected for this analysis. (c) Determination of the binding of BplncSIR1 to the promoters of genes using chromatin isolation by RNA purification (ChIRP). The upstream sequence of the translation initiation site of the gene (including the 5′UTR and promoter) was evenly divided into five regions (I–V); each region is 400 bp in length. (d) Analysis of the activation of BplncSIR1 on BpNAC2 using dual luciferase system. 35S::empty: empty pGreenII 62‐SK vector, 35S::LUC: empty pGreenII 0800‐Luc vector. 35S::BplncSIR1: BplncSIR1 was cloned into pGreenII 62‐SK under the control of 35S CaMV promoter. NAC2‐promoter‐LUC: the promoter of BpNAC2 was cloned into the pGreenII 0800‐Luc vector to drive the expression of LUC. The 35S::Ren vector was transformed together to normalize the transient transformation efficiency.

To study whether these nine TFs could be regulated by BplncSIR1, ChIRP was performed. The promoter regions (2000 bp upstream of the ATG translation initiation site) were divided into five equal fragments for ChIRP‐qPCR amplification. ChIRP‐qPCR identified one TF (BpNAC2) whose truncated promoter fragment (BpNAC2‐II) was significantly enriched using either ‘Even’ or ‘Odd’ probes, suggesting that this promoter can interact with BplncSIR1 (Figure 4c).

To further investigate the interaction between BplncSIR1 and the truncated promoter fragment (BpNAC2‐II), the effector (BplncSIR1‐OE) and reporter (LUC controlled by the BpNAC2‐II) vectors were constructed and transiently co‐transformed into birch, followed by dual luciferase activity determination. The LUC activity analysis showed that BplncSIR1 could bind to the BpNAC2‐II promoter fragment to activate gene expression (Figure 4d). These results suggested that BplncSIR1 could interact with the promoter of BpNAC2.

BpNAC2 enhances salt tolerance and growth in birch

BpNAC2 is directly regulated by BplncSIR1 according to the ChIRP results; therefore, we further characterized the function of BpNAC2. The expression of BpNAC2 was significantly increased in BplncSIR1‐OE lines and decreased in bplncsir1 ^# lines compared with WT plants according to qRT‐PCR (Figure S4a). These results indicated that BplncSIR1 binds to the promoter of BpNAC2 to activate its expression (Figure 4c,d; Figure S4a). Consistent with the response of BplncSIR1 to NaCl treatment, the transcript level of BpNAC2 was also significantly induced by NaCl treatment (Figure S4b). The BpNAC2 protein was localized in the nucleus (Figure S4c).

Eight lines overexpressing BpNAC2 (BpNAC2‐OE) were generated, and three lines with the highest expression of BpNAC2 (BpNAC2‐OE1, BpNAC2‐OE5 and BpNAC2‐OE10) were selected for further study (Figure 5a; Figure S4f). In addition, seven independent CRISPR‐edited lines with two editing target sites were generated. T7EI digestion was performed to determine whether the mutation was heterozygous or homozygous. After digestion, some lines generated PCR products with more than one band (Figure S4d, T7E1 panel), indicating that these renatured DNA had formed a loop and were cut by T7EI. Therefore, these lines were heterozygous mutations (Figure 5b; Figure S4d–e). Among them, we selected three lines, bpnac2 ^# ‐1, bpnac2 ^# ‐2 and bpnac2 ^# ‐4, for further study (Figure 5b; Figure S4f).

Figure 5.

BpNAC2 confers salt tolerance. (a) Expression analysis of the expression of BpNAC2 in overexpression lines. (b) Analysis of the CRISPR editing formats using Sanger DNA sequencing. (c) The growth phenotype of the plants overexpressing BpNAC2, plants with CRISPR‐mediated knocking out of BpNAC2 and WT plants. Bars, 5 cm. (d) Comparison of plant height. (e) Comparison of fresh weight. (f) Comparison of chlorophyll content. (g) Comparison of root length. (h) Comparison of root weight. BpNAC2‐OE1, 5 and 10: plant lines 1, 5 and 10 overexpressing BpNAC2; bpnac2 ^# ‐1, 4 and 9: plant lines 1, 2 and 4 with CRISPR knocking out BpNAC2; WT: wild‐type plant. The error bar indicates the standard deviation (SD) of the biological replicates. *indicates statistically significant differences at P < 0.05; **indicates statistically significant differences at P < 0.01.

The growth phenotypes were compared among BpNAC2‐OE, WT and bpnac2 ^# lines. Under normal conditions, overexpression of BpNAC2 could increase the growth rate, including plant height, fresh weight, root length and weight, whereas the bpnac2 ^# lines displayed reduced values for these growth characteristics when compared with those of the WT plants (Figure 5c–e,g,h). Under salt stress conditions, the BpNAC2‐OE lines showed the highest plant height, fresh weight, root length and weight, followed by the WT and lastly by the bpnac2 ^# lines (Figure 5c–e,g,h). In addition, the chloroplast contents of bpnac2 ^# lines and WT plants were significantly reduced compared with BpNAC2‐OE lines after salt stress (Figure 5f). These results showed that BpNAC2 could accelerate growth rates and confer salt tolerance simultaneously.

Identification of the target genes regulated by BpNAC2

To identify target genes for BpNAC2, gene expression between WT and BpNAC2‐OE was compared using RNA‐seq. The RNA‐seq data quality score 30 (Q30) of each sample was higher than 95.32%, and more than 7.7 GB of clean data were generated (Table S4). There were 2237 DEGs identified with an adjusted fold change>2; FDR < 0.01, including 584 upregulated and downregulated 1653 genes by BpNAC2 (Figure S5a; Table S5). GO enrichment analysis showed that DEGs were involved in abiotic stress and stomatal‐related processes, such as “response to abscisic acid” (GO:0009737), “response to water deprivation” (GO:0009414), “stomatal complex patterning” (GO:0010375), “response to osmotic stress” (GO:0006970), “stomatal complex development” (GO:0010374), “oxidoreductase activity” (GO:0016491) and “peroxidase activity” (GO:0004601; Figure S5b). In addition, many ROS‐scavenging and stomatal‐related genes were induced by BpNAC2 (Table S5).

BpNAC2 enhances ROS removal under salt stress

We further studied the physiological changes mediated by BpNAC2. Compared with WT plants, MDA content and electrolyte leakage rate were both significantly increased in the bpnac2 ^# lines but significantly reduced in the BpNAC2‐OE lines under salt stress conditions; however, these traits were similar under normal conditions (Figure 6a,b).

Figure 6.

Determination of salt tolerance by physiological and histochemical analysis. (a) Electrolyte leakage analysis. (b) MDA analysis. (c) NBT and DAB staining analysis. Bars, 1 cm. (d) Determination of H₂O₂ contents. (e) POD activity analysis. (f) APX activity analysis. (g) CAT activity analysis. (h) Water loss rate analysis. (i) Comparison of stomatal apertures. (j) Analysis of the width–length ratio of stomatal apertures. BpNAC2‐OE1, 5 and 10: lines 1, 5 and 10 for plants overexpressing BpNAC2; bpnac2 ^# ‐1, 2 and 4: lines 1, 2 and 4 of plants knocking out for BpNAC2; WT: wild‐type plant. The error bar indicates the standard deviation (SD) of the biological replicates. *indicates statistically significant differences at P < 0.05; **indicates statistically significant differences at P < 0.01.

Many ROS scavenging genes were induced by BpNAC2 according to RNA‐seq (Table S5); therefore, we further studied whether ROS scavenging was altered by the expression of BpNAC2. Under salt stress conditions, the bpnac2 ^# lines and BpNAC2‐OE lines had the highest and lowest H₂O₂ content, respectively (Figure 6c). Consistently, DAB and NBT staining indicated that the BpNAC2 ^# lines and BpNAC2‐OE lines accumulated the highest and lowest H₂O₂ and ${\mathrm{O}}_2^{-}$ levels, respectively, among the studied plants when exposed to salt stress (Figure 6c,d). In addition, to further understand how BpNAC2 reduces the accumulation of ROS, we studied the activities of POD, APX and CAT. Their activities were similar in all the studied lines under normal conditions. After salt stress, the BpNAC2‐OE and bpnac2 ^# lines showed the highest and lowest activities of POD, APX and CAT, respectively, among the studied plants (Figure 6e–g). These results showed that overexpression of BpNAC2 could improve the activities of antioxidant enzymes to reduce ROS levels, leading to increased salt tolerance.

We then determined whether the reduction of the stomatal aperture could affect the water loss rate. Our results showed that overexpression of BpNAC2 significantly reduced water loss; conversely, bpnac2 ^# plants showed an increased water loss rate (Figure 6h). The water loss rate is closely related to stomatal opening, and BpNAC2 regulates the stomatal‐related genes (Table S5). Therefore, we further determined whether BpNAC2 could mediate stomatal movement. Overexpression of BpNAC2 reduced the stomatal width–length ratio. Conversely, the bpnac2 ^# lines showed a significant increase in the stomatal width–length ratio (Figure 6i,j). These results suggested that BpNAC2 could regulate stomata‐related genes to facilitate stomatal closure. Furthermore, BpNAC2 might regulate genes involved in stomatal movement to decrease the stomatal aperture, leading to reduced water loss and thus improving salt tolerance.

Identification of the genes directly regulated by BpNAC2

To identify the genes directly regulated by BpNAC2, plants overexpressing BpNAC2‐eGFP were generated, and anti‐GFP antibodies were used for ChIP. In total, the clean bases of IP and input were 4.86 and 5.67 Gb respectively. The Raw Reads Q30 were 99% (Table S6), and IP and input had a low correlation coefficient (<0.8; Figure S6a). The distribution of reads of IP before and after peak calling was both substantially enriched (30.28% after peak calling) in the regions near Transcription Start Sites (TSS; Figure S6b–d), indicating that BpNAC2 binds to the regions near the TSS.

Joint analysis indicated that 1245 genes were identified simultaneously by both ChIP‐Seq and RNA‐seq (P < 0.05), which we tentatively called directly regulated genes (DRGs; Table S7; Figure 7a). GO term enrichment analysis showed that the induced DRGs were enriched in many GO terms involved in abiotic stress, such as “response to abscisic acid” (GO:0009737), “response to water deprivation” (GO:0009414), “heme binding” (GO:0020037), “response to hydrogen peroxide” (GO:0042542) and “peroxidase activity” (GO:0004601; Figure S6e).

Figure 7.

ChIP‐seq and RNA‐seq analysis. ChIP‐seq and RNA‐seq were performed with the plants overexpressing BpNAC2. (a) Distribution of genes that were potentially directly regulated by BpNAC2 using joint analysis of RNA‐seq and ChIP‐seq data. mRNA_down, up: The genes are significantly down‐ or up‐regulated by BpNAC2 according to the RNA‐seq results. ChIP: The genes whose promoters were bound by BpNAC2 according to the ChIP‐seq data. (b) The motifs are potentially bound by BpNAC2. These motifs were determined by analysing peak sequences of ChIP‐seq using MEME.

Identification of the cis‐acting element bound by BpNAC2

We further identified the cis‐acting elements that were potentially bound by BpNAC2, and MEME analysis was performed to analyse the peak‐enriched motifs. In total, 10 conserved motifs were identified (Figure 7b). According to the analysis of GO enrichment, it was found that the two key enzymes of stomatal regulation, abscisic acid‐deficient 2 (ABA2) and open stomata 1 (OST1), were highly enriched. At the same time, these two genes were found to be induced by BpNAC2 (Figure 8a).

Figure 8.

Analysis of the binding of BpNAC2 to different cis‐acting elements. Four genes whose promoter regions contain G‐box and ‘CCAAT’ sequences were studied. (a) Analysis of the expression of the studied genes regulated by BpNAC2; qRT‐PCR was performed using the cDNA from birch plant lines overexpressing BpNAC2. (b) ChIP‐qPCR analysis of the binding of BpNAC2 to G‐box and ‘CCAAT’ sequences in the promoter regions of the genes. (c) Yeast one‐hybrid analysis of the binding of BpNAC2 to the truncated promoters of these four genes containing G‐box and ‘CCAAT’ sequences. (d) EMSA analysis of the binding of BpNAC2 to G‐box and ‘CCAAT’ sequences. (e) Analysis of whether BpNAC2 can activate the expression of the studied genes using Dual‐Luciferase Reporter on Nicotiana benthamiana. (f) The LUC/REN ratio was determined to study whether BpNAC2 binds to the promoter of the gene to activate its expression. 35S::empty: empty pGreenII 62‐SK vector; 35S::LUC: empty pGreenII 0800‐Luc vector. 35S::BpNAC2: BpNAC2 was cloned into pGreenII 62‐SK under the control of 35S CaMV promoter. Promoter‐LUC: the promoter of the studied gene was cloned into pGreenII 0800‐Luc vector to drive the expression of LUC. 35S::Ren vector was transformed together to normalize the transient transformation efficiency.

In addition, because ROS accumulation was reduced by BpNAC2, the genes involved in ROS scavenging were identified from the DRGs and induced by BpNAC2 (Figure 8a), including ascorbate peroxidase 1 (APX1) and peroxidase 52 (PRX52). The promoters of these four genes were screened, and two motifs were identified using ChIP‐seq (Figure 7b), including “CACGTG” (G‐box) and ‘CCAAT’ motifs, which were present in the identified motif and potentially bound by BpNAC2.

The binding of BpNAC2 to these promoter regions with ‘CCAAT’ and ‘CACGTG’ motifs was first analysed using ChIP‐qPCR. The results showed that BpNAC2 could directly bind to the promoters of these four genes (Figure 8b). The qRT‐PCR results also showed that BpABA2, BpOST1, BpAPX1 and BpPRX52 were significantly and highly induced in BpNAC2‐overexpressing plants after salt treatment (Figure 8a). Furthermore, yeast one‐hybrid assays showed that the promoters of the four genes were specifically bound by BpNAC2 (Figure 8c). EMSA assays further confirmed that BpNAC2 could specifically bind to ‘CACGTG’ and ‘CCAAT’ motifs but failed to bind to the mutated probes (Figure 8d). Moreover, a dual luciferase (LUC) assay showed that expression of BpNAC2 in Nicotiana benthamiana leaves significantly activated the expression of BpABA2, BpOST1, BpAPX1 and BpPRX52. (Figure 8e,f). Taken together, these results suggested that BpNAC2 could directly bind to ‘CACGTG’ and ‘CCAAT’ motifs present in the promoters of genes to activate their expression.

Discussion

In the present study, we identified a lncRNA, BplncSIR1, that responds to salt stress and can accelerate plant growth and confer salt tolerance. The regulatory network of BplncSIR1 was further investigated, revealing that BplncSIR1 serves as a regulator of BpNAC2 to mediate salt tolerance.

BplncSIR1 does not exert its role by encoding a short peptide

Previous reports have shown that lncRNAs might have the capability to encode short peptides (Lin et al., 2020). Therefore, we first analysed the coding capability of BplncSIR1. Our results indicated that mutation of the short peptide of BplncSIR1 did not alter its capability of conferring salt tolerance, demonstrating that BplncSIR1's functions result only from its activity as an lncRNA and not from its coding short peptide (Figure S3).

Generation of BplncSIR1 knockout lines by CRISPR editing

CRISPR/Cas9 is an efficient technology to edit DNA sequences, which avoids the impact of low expression caused by RNAi technology (Li et al., 2018). The function of lncRNAs is dependent on their secondary structure, and mutation of a lncRNA might lead to an altered secondary structure causing loss of function. Therefore, lncRNAs can be edited by CRISPR/Cas9 to generate mutations by altering their secondary structure. In the present study, we generated and predicted the secondary structure of CRISPR‐edited BplncSIR1. The results showed that the secondary structures of BplncSIR1 in lines bplncsir1 ^# ‐1, bplncsir1 ^# ‐4 and bplncsir1 ^# ‐9 were changed compared with those of WT plants (Figure S2c). Further study showed that the function of BplncSIR1 in these lines was altered (Figure 2c–h), suggesting that mutations of BplncSIR1 had been successfully generated using the CRISPR‐Cas method.

BplncSIR1 accelerates plant growth and facilitates salt tolerance in birch

In the present study, BplncSIR1 was identified as a nucleus‐localized lncRNA that was induced by salt stress and mainly expressed in roots and leaves (Figure 1), suggesting that it might play a role in gene expression regulation and the salt stress response.

Our results indicated that the expression level of BplncSIR1 correlated positively with the height, fresh weight, root length and weight of plants (Figure 2c–e,g,h), suggesting that BplncSIR1 can accelerate plant growth. Plants exposed to high salinity and drought environments will lose water through gas exchange via the stomata on the leaves. Stomatal movement is influenced by many environmental factors, including hormone levels, light and biotic and abiotic stresses (Buckley, 2019). Our results showed that overexpression of BplncSIR1 could promote stomatal closure in leaves and reduce water loss to maintain a higher water content in birch leaves (Figure 3h–j), which leads to improved salt tolerance.

When plants are exposed to abiotic stress, there is a significant accumulation of ROS, such as H₂O₂ and ${\mathrm{O}}_2^{-}$, which disrupts the balance between ROS production and scavenging in cells, leading to cellular toxicity (Gould and Jain, 2015). The enzymatic antioxidant system scavenges accumulated ROS by promoting the activity of POD, CAT and APX. The expression of BplncSIR1 enhanced POD, CAT and APX activities, which scavenged ROS and reduced the oxidative damage to birch under salt stress conditions (Figure 3e–g), leading to improved salt tolerance. Taken together, these results suggested that BplncSIR1 regulates the ROS scavenging capability and reduces water loss in birch to confer salt tolerance.

BplncSIR1 regulates BpNAC2 to accelerate plant growth and confer salt tolerance

LncRNAs usually function by regulating transcription factors or structural genes and cannot function directly. For instance, salt‐induced Ptlinc‐NAC72 in poplar could directly upregulate PtNAC72.A/B expression, conferring resilience to salt stress in plants (Ye et al., 2022). Therefore, we further investigated the target genes of BplncSIR1. ChIRP combined with qRT‐PCR analysis showed that BplncSIR1 regulates the expression of BpNAC2 by binding to its promoter (Figure 4b–d).

The transcript level of BpNAC2 correlated positively with the height, fresh weight, root length and weight of plants (Figure 5c–e,g,h), suggesting that BpNAC2 can also accelerate plant growth, which is consistent with the performance of BplncSIR1 (Figure 2c–e,g,h). In addition, as a direct target gene of BplncSIR1, the functions of BpNAC2 in salt tolerance were consistent with those of BplncSIR1, including enhanced oxidative stress tolerance and reduced stomatal closure to reduce water loss (Figure 6). Together, these strongly indicate that BplncSIR1 facilitates plant growth and confers salt tolerance by regulating BpNAC2 expression.

BpNAC2 binds to different cis‐acting elements to regulate gene expression, thus mediating salt tolerance

Previous studies showed that NAC proteins can bind to different cis‐acting elements, such as the NAC recognition sequence (NACRS; Yuan et al., 2019), the ‘TTNCGT[G/A]’ sequence (Olsen et al., 2005), motifs with core sequences of ‘CACG’ or ‘CGT[G/A]’, NRS1 (‘TAGTT’) and NRS2 (‘GAATC’; He et al., 2018). To further study the function of BpNAC2, ChIP‐seq and RNA‐seq were performed, which indicated that BpNAC2 could bind to many cis‐acting elements to regulate genes expression when exposed to salt stress (Figure 7b). For instance, our results indicated that BpNAC2 binds to ‘CACGTG’ (G‐box) and ‘CCAAT’ motifs to directly regulate the expression of BpABA2, BpOST1, BpAPX1 and BpPRX52 (Figure 8). Xanthoxin dehydrogenase (ABA2) is involved in ABA biosynthesis (Cheng et al., 2002; Endo et al., 2014), and open stomata1 (OST1) is a key kinase for the ABA‐mediated regulation of stomata (Nakashima et al., 2009; Sun et al., 2022). These results indicated that the regulation of BpABA2 and BpOST1 by BpNAC2 suggested that BpNAC2 plays a role in the ABA regulatory pathway to regulate stomata movement. Both peroxidase (PRX52) and APX1 are key enzymes in the plants' response to oxidative stress and scavenging ROS during abiotic stress (Hong et al., 2023; Torres, 2010). BpNAC2 induces the expression of BpAPX1 and BpPRX52 (Figure 8), suggesting that BpNAC2 is involved in the ROS scavenging pathway.

Conclusion

In conclusion, a salt‐responsive lncRNA, BplncSIR1, was identified, which is mainly located in the nucleus and activates the expression of BpNAC2 by binding to its promoter. BpNAC2 then accelerates growth and confers salt tolerance. We further found that BpNAC2 binds to different cis‐acting elements, such as G‐box and ‘CCAAT’ motifs, to regulate the expression of genes responding to salt stress, including those involved in ROS scavenging and stomatal aperture movement. Regulation of these genes will lead to reduced ROS accumulation and a decreased water loss rate (Figure 9). Taken together, BplncSIR1 serves as an upstream regulator to induce the expression of BpNAC2. BpNAC2 regulates the expression of its target genes to form a regulatory cascade that mediates ROS scavenging and stomatal aperture movement. This regulatory cascade finally improves salt tolerance in birch.

Figure 9.

The regulatory network of BplncSIR1 in response to salt stress. Salt stress induces the expression of BplncSIR1, which in turn binds to the promoter of BpNAC2 to activate its expression. The BpNAC2 protein then binds to cis‐acting elements such as G‐box and ‘CCAAT’ sequences to regulate gene expression in response to salt stress, such as BpABA2, BpOST1, BpAPX1 and BpPRX52. The expression of these genes leads to increased ROS scavenging capability and reduced stomatal aperture to decrease the water loss rate, which finally improves salt tolerance.

Materials and methods

Plant growth conditions and treatments

Birch plants were grown in a mixture of turf soil and sand (3:1, v/v) in pots under greenhouse conditions of 400 μmol/m²/s light intensity, a photoperiod of 16/8 h light/dark and a relative humidity of 75%. To determine gene expression, 10‐week‐old birch seedlings were watered with a 200 mm NaCl solution for 0, 1, 3, 6, 12, 24 and 48 h, and the leaves and roots were harvested for further study. To analyse the phenotype and physiological traits, birch plantlets were grown in a mixture of turf soil and sand (3:1, v/v) in pots for 70 days. The plantlets were watered with 100 mL of a 150 mm NaCl solution every 3 days. After treatment for 21 days, the plants were subjected to physiological analyses, and their growth phenotypes were compared. Three biological replicates were analysed.

Rapid amplification of cDNA ends (RACE)

The expression of a truncated lncRNA (termed BplncSIR1) in response to salt stress was identified using RNA sequencing (RNA‐seq), and we used a SMARTer® RACE 5/3′ Kit (Takara, Dalian, China) to amplify the 5′ and 3′ sequences of BplncSIR1 according to the protocol of the kit. The 5′ and 3′ RACE products were assembled, together with the truncated BplncSIR1, to form the full‐length BplncSIR1 sequence. To investigate the accuracy of the assembled BplncSIR1, primers were designed to amplify the full‐length cDNA of BplncSIR1, and the PCR product was sequenced and compared with the assembled sequence. All the primers used are shown in Table S1.

Nuclear–cytoplasmic fractionation

Wild‐type birch was used to conduct nucleocytoplasmic separation experiments according to the method of Wang et al. (2011). After isolating the nucleus and cytoplasm of birch, RNA was extracted from the nucleus and cytoplasm, respectively, and reverse transcribed into cDNA as templates for quantitative real‐time PCR (qPCR) to determine the content of BplncSIR1. U6 and Actin were used as the internal controls for the nuclear and cytoplasmic extracts, respectively. Then, we designed four paired primers for BplncSIR1 to detect its subcellular localization using quantitative real‐time reverse transcription PCR (qRT‐PCR).

Construction of vectors and genetic transformation

The promoter sequence of BplncSIR1 (2000 bp in length) was used to replace the cauliflower mosaic virus (CaMV) 35S promoter to drive a GUS gene in vector pCAMBIA1301. To construct the overexpression vector, the full cDNA length sequence of BplncSIR1 or the coding sequence (CDS) of genes was cloned separately into pROK2 or pBI121‐eGFP under the control of the 35S CaMV promoter. The effective gRNA target sites were selected to construct CRISPR vectors using CRISPOR(http://crispor.tefor.net/; Haeussler et al., 2016). Cas9/gRNA vector construction was performed according to the method of Xing et al. (2014). The double‐target PCR fragment was amplified using pCBC‐DT1T2 as the template. The obtained target fragment was then ligated to the pG3H‐U6SC plasmid using Bsa I and T4 ligase, according to the Golden Gate reaction method (Smedley et al., 2021) to generate vector pG3H‐U6SC‐2gRNA, which was transferred into Agrobacterium strain EHA105. Genetic transformation into birch was performed using Agrobacterium tumefaciens‐mediated transformation (Tan et al., 2020). All the primers used are shown in Table S1.

Determination of physiological changes

The procedures for nitroblue tetrazolium (NBT) and 3′‐diaminobenzidine (DAB) staining followed the description by Zhang et al. (2011). The electrolyte leakage rate, malondialdehyde (MDA) content and peroxidase (POD) activity were determined as described by Zang et al. (2017). The chlorophyll content was determined according to the method of Brennan and Frenkel (1977). H₂O₂ levels were measured using a Hydrogen Peroxide (H₂O₂) Content Assay Kit (Jiancheng Biotech, Nanjing, China). Ascorbate peroxidase (APX) and catalase (CAT) activity were measured using an APX Activity Assay Kit and Catalase (CAT) Activity Assay Kit (Micro method; Sangon Biotech Co., Ltd., Shanghai, China), respectively. Each sample contained at least 10 seedlings, and each experiment had three biological replicates.

Analysis of stomatal aperture opening and the water loss rate

The lower epidermal peels of birch leaves were incubated in MES‐KCl buffer (10 mm MES‐KOH, pH 6.15, 50 mm KCl) under light for 2.5 h to induce stomatal aperture opening, and were then transferred to a MES‐KCl buffer supplied with 150 mm NaCl for 3 h. Stomatal apertures were visualized under a light microscope (BX43; Olympus, Tokyo, Japan) (Watkins et al., 2014). For the water loss rate analysis, detached leaves were weighed immediately (fresh weight, FW), dehydrated at room temperature (humidity, 45–50%; 20–22 °C) and weighed at designated time intervals (desiccated weights). The leaves were dried to a constant dry weight (DW). The water content (WC) was calculated according to the formula WC (%) = (desiccated weight − DW)/(FW − DW) × 100. Each sample contained at least 10 plantlets, and three biological replicates were performed.

qRT‐PCR

After reverse transcription (RT) to generate cDNA, qPCR was carried out with the following reaction system: 50 ng of cDNA, 0.5 μm each primer and 10 μL of SYBR Premix Ex Taq™ (Toyobo) in a total volume of 20 μL. qTower 2.2 (Analytik, Jena, Germany) was used to perform qPCR with the following thermal profile: 94 °C for 30 s; 40 cycles of 94 °C for 12 s, 58 °C for 30 s and 72 °C for 45 s. The genes encoding tubulin and ubiquitin were used as internal controls. Relative gene expression was calculated using the $2^{-\mathrm{\Delta \Delta }{C}_{\mathrm{T}}}$ method (Livak and Schmittgen, 2001). The primer sequences and the GenBank numbers of the genes are shown in Table S1.

Chromatin immunoprecipitation sequencing (ChIP‐seq), ChIP‐qPCR and RNA‐seq

The transgenic plants overexpressing 35S:BpNAC2‐eGFP (encoding NAC domain‐containing protein 2 fused to the enhanced green fluorescent protein) were used for ChIP‐seq. ChIP‐seq and ChIP‐qPCR were performed according to the methods of Zhao et al. (2020), and anti‐GFP antibodies were used for immunoprecipitation. For the RNA‐seq analysis, differentially expressed genes (DEGs) were displayed using a Venn diagram and heatmap. The functions of the DEGs were annotated by searching the Gene Ontology (GO) terms database and then visualized after enrichment analysis.

Chromatin isolation by RNA purification (ChIRP)

ChIRP was performed according to the method of Chu et al. (2012) with minor modifications. In brief, a set of probes was designed based on 100‐bp intervals in the sequence of BplncSIR1. The antisense DNA probes with biotin‐triethyleneglycol (TEG) at the 3' end were synthesized by Tsingke Biotechnology Co., Ltd (Beijing, China). The 10 probes were divided into two groups according to their positions. The ‘odd’ group (matching the forward DNA strand) contained probes numbered 1, 3, 5, 7 and 9. The ‘even’ group (matching the reverse DNA strand) contained probes numbered 2, 4, 6, 8 and 10. Birch samples were treated with 1% (v/v) glutaraldehyde for cross‐linking protein–DNA–RNA and then the nuclei of the sample were isolated. The purified nuclei were broken with 1% SDS, and then the cross‐linked chromatin was sonicated into 0.2–1.0 kb in length. Take 1/100 volume of sonicated chromatin as input. The remaining sonicated product was divided into two and respectively add with ‘odd’ and ‘even’ probes for hybridizing with the aim of lncRNA on chromatin. The target lncRNA hybridized with probes was isolated with streptavidin magnetic beads, and the separated product was eluted with the elution buffer containing RNase A and RNase H. The elution is treated with proteinase K for reverse cross‐linking and then purified with chloroform extraction. Three biological replicates were performed. The LacZ cDNA probe was employed as the negative control, having no homology with any RNA sequence from birch, and all the probes are shown in Table S1.

Yeast one‐hybrid (Y1H) assay

The promoter sequence of the target gene was cloned into the bait vector pHIS2, and the full‐length coding sequence (CDS) of BpNAC2 was cloned into the pGADT7‐rec2 prey vector (pGADT7‐BpNAC2). The constructs of bait and prey were co‐transformed into Y1H yeast cells following the user manual of the MatchMaker One‐Hybrid System (Clontech, CA, USA). pGAD‐p53 transformed together with p53HIS2 was used as a positive control, and p53HIS2 transformed together with pGADT7‐BpNAC2 served as a negative control. The positive clones were grown on SD/‐Leu/‐Trp (DDO) (as a positive growth control condition) and SD/‐His/‐Leu/‐Trp (TDO) medium supplied with 20 mm 3‐AT (3‐amino‐1,2,4‐triazole). The primers used for constructing the vectors are listed in Table S1.

Detection of the dual luciferase reporter gene

To analyse the binding of the lncRNA to the promoters of its target gene, the target gene promoter was cloned into pGreenII0800‐LUC as a reporter vector, and BplncSIR1 was cloned into the pGreenII 62‐SK (35S:BplncSIR1) as the effector vector. The reporter and effector vectors were transferred into Agrobacterium strain GV3101 (pSoup‐p19). The different plasmid combinations were co‐transformed into Nicotiana benthamiana leaves according to the method of Zang et al. (2017). After transformation for 48 h, the plants were moved to a culture medium supplied with 150 mm NaCl for 24 h. A dual luciferase assay kit (Sangon Biotech Co., Ltd.) was used to determine the dual luciferase (LUC) activity. Renilla LUC activity was used to normalize the luciferase activity, shown as a LUC/REN ratio. The primers used to construct the vector are listed in Table S1.

Electrophoretic mobility shift assay (EMSA)

The CDS of BpNAC2 was cloned into the vector pMAL‐c5X generate the NAC‐MBP (maltose‐binding protein) fusion protein (NEB, Ipswich, MA, USA) and transformed into Escherichia coli strain ER2523 for expression. The fusion protein was purified following the instructions supplied by the pMAL Protein Fusion & Purification System (NEB). Probes were labelled with biotin using the EMSA Probe Biotin Labelling Kit (Beyotime, Shanghai, China). Mutated probes were labelled with biotin and served as negative controls. The binding of proteins and probes was assessed using polyacrylamide gel electrophoresis and detected using a chemiluminescent EMSA kit (Beyotime).

Statistical analysis

A Student's t‐test was used to analyse the data, and the statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 22; IBM Corp., Armonk, NY, USA); statistical significance was set at P < 0.05 and high significance was set at P < 0.01.

Author contribution

YCW and YQJ designed and conceived this work. YQJ and HMZ performed the main experiments and the overall data analysis; YNN cloned the gene. YQJ and YCW wrote and edited the article; all authors revised the manuscript.

Supporting information

Figure S1 Characterization of the sequence of BplncSIR1.

Figure S2 Generation and characterization of birch plants overexpressing and CRISPR editing BplncSIR1.

Figure S3 Analysis of the function of short peptide coding by BplncSIR1.

Figure S4 Characterization of BpNAC2 and generation of plants overexpressing and CRISPR editing BpNAC2.

Figure S5 The RNA‐seq analysis of overexpressed BpNAC2 plants.

Figure S6 Analysis of the quality ChIP‐seq data.

Table S1 Primers used to construct plant expression vectors, qRT‐PCR and ChIP‐PCR.

Table S2 The characteristics of the high‐throughput sequencing results.

Table S3 The differentially expressed genes (DEGs) between the BplncSIR1‐OE and wild‐type plants under salt conditions.

Table S4 The characteristics of the high‐throughput sequencing results for BpNAC2 RNA‐seq.

Table S5 DEGs between the WT and BpNAC2‐OE plants under salt conditions.

Table S6 Statistics on the quality information of the clean data of BpNAC2 ChIP‐seq.

Table S7 Comprehensive analysis of ChIP‐seq and RNA‐seq data for BpNAC2.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.