BLH3 regulates the ABA pathway and lignin synthesis under salt stress in Lilium pumilum

BLH3 regulates the ABA pathway and lignin synthesis under salt stress in Lilium pumilum | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 28 February 2025 V1 Latest version Share on BLH3 regulates the ABA pathway and lignin synthesis under salt stress in Lilium pumilum Authors : Wenhao Wan , Huitao Cui , Lingshu Zhang , Miaoxin Shi , Hao Sun , Xingyu Liu , Wei Yang , Fengshan Yang , and Shumei Jin 0000-0003-1035-3486 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174071536.69426547/v1 Published Plants Version of record Peer review timeline 214 views 127 downloads Contents Abstract 3.7 BLH3 and KNAT3 synergistically activate the promoter Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract BLH3 (Bel1-like homeodomain gene3) plays a crucial role in plant development. However, its involvement in the salt stress response has not yet been studied.In this study, we investigated the molecular mechanism underlying the response of LpBLH3 to salt stress in Lilium pumilum ( L. pumilum ) using various techniques, including quantitative PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), dual-luciferase reporter assay (LUC), yeast two-hybrid (Y2H), and luciferase complementation imaging (LCI).RT-qPCR analysis revealed that LpBLH3 is most highly expressed in the leaves of L. pumilum . The expression of LpBLH3 peaks at 24 or 36 hours in the leaves under saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling, higher accumulation of proline and chlorophyll, lower levels of reactive oxygen species, and increased activity of antioxidant enzymes.The roots and stems of LpBLH3 overexpression lines exhibited significantly higher lignin content compared to those of the wild type (WT). EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 . Additionally, yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) analyses demonstrated that LpBLH3 interacts with LpKNAT3. LpBLH3 enhances the plant’s salt tolerance through the ABA pathway and lignin synthesis. not-yet-known not-yet-known not-yet-known unknown BLH3 regulates the ABA pathway and lignin synthesis under salt stress in Lilium pumilum Wenhao Wana, Huitao Cuia, Lingshu Zhanga, Miaoxin Shia, Hao Suna, Xingyu Liua, Wei Yangb, Fengshan Yangc,d,e, Shumei Jina# a Key Laboratory of Saline-Alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin 150028, Heilongjiang, China b Heilongjiang Agricultural Technology Extension Station, Harbin 150000, Heilongjiang, China c Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150080, Heilongjiang, China not-yet-known not-yet-known not-yet-known unknown d Heilongjiang Provincial Key Laboratory of Ecological Restoration and Resource Utilization for Cold Region, Heilongjiang University, Harbin 150080, Heilongjiang, China e Key Laboratory of Molecular Biology, College of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin 150080, Heilongjiang, China # Corresponding author not-yet-known not-yet-known not-yet-known unknown Abstract BLH3 (Bel1-like homeodomain gene3) plays a crucial role in plant development. However, its involvement in the salt stress response has not yet been studied.In this study, we investigated the molecular mechanism underlying the response of LpBLH3 to salt stress in Lilium pumilum (L. pumilum ) using various techniques, including quantitative PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), dual-luciferase reporter assay (LUC), yeast two-hybrid (Y2H), and luciferase complementation imaging (LCI).RT-qPCR analysis revealed that LpBLH3 is most highly expressed in the leaves of L. pumilum . The expression of LpBLH3 peaks at 24 or 36 hours in the leaves under saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling, higher accumulation of proline and chlorophyll, lower levels of reactive oxygen species, and increased activity of antioxidant enzymes.The roots and stems of LpBLH3 overexpression lines exhibited significantly higher lignin content compared to those of the wild type (WT). EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 . Additionally, yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) analyses demonstrated that LpBLH3 interacts with LpKNAT3. LpBLH3 enhances the plant’s salt tolerance through the ABA pathway and lignin synthesis. Key words: Lilium pumilum, BLH3 transcription factors, salt tolerance, ABA pathway, lignin content. Introduction Soil salinization turns out to be a vital abiotic stress, impeding the growth and progress of plants and consequently their productivity. In 2020, the Songnen Plain in Northeast China boasted an area of 2,702.39 km² of saline-alkali land. (lv, X. 2012). During previous laboratory studies, an in-depth analysis was carried out on the transcriptome of L. pumilum under alkaline stress conditions. This led to the discovery of the upregulation of the LpBLH3 gene. Thereafter, the LpBLH3 gene was cloned and a meticulous analysis was performed on it.The TALE (Three Amino Acid Loop Extension) homeobox genes can fall into two subfamilies: KNOTTED - like homeodomain (KNOX) and BEL1 - like homeodomain (BELL).(Tao, Y. et al. 2018). BLH3, as a member belonging to the BELL subfamily within the TALE homeobox gene group, is highly significant in both the advancement of plant growth and the reaction to stressors(Modrusan, Z. et al. 1994; Bürglin, T. R. 1997; Bellaoui, M. et al. 2001; Hay, A. andTsiantis, M. 2010). It features a conserved N-terminal SKY domain and a BELL domain positioned in front of the homeobox domain(Hamant, O. andPautot, V. 2010). The SKY along with the BELL domain jointly constitute the POX domain. This POX domain has the capacity to engage with the MEINOX domain present in KNOX family proteins, thereby giving rise to homodimers or heterodimers that are involved in governing plant growth as well as stress responses(Lee, J.-H. et al. 2008; Hamant, O. andPautot, V. 2010). The fact that these three functional domains are highly conserved is of great significance for ensuring the proper functioning of plant BLH family proteins(Niu, X. andFu, D. 2022). BLH family constituents play a pivotal part in enabling plants to withstand salt stress. In the case of cotton, GhBLH5-A05 functions as a facilitator for combating drought stress. When GhBLH5-A05 is overexpressed, it imparts enhanced drought resistance. Moreover, it teams up with GhKNAT6-A03 to trigger the expression of genes responsive to drought stress, namely GhRD20-A09 and GhDREB2C-D05, consequently bolstering cotton’s capacity to endure drought conditions(Zhang, J. 2021). In Arabidopsis thaliana, in comparison with the wild type (WT), the plants with overexpressed GmBLH4 exhibit a remarkably improved tolerance to the stress caused by high temperature and high humidity(Tao, Y., et al. 2018). Research indicates that transcription factors of the BLH family are capable of strengthening the structure of plant cell walls mainly through facilitating lignin synthesis and augmenting lignin accumulation. Lignin, being a vital constituent of the cell wall, holds a significant position in governing the biosynthesis of secondary cell walls(Liu, Y. 2015). In Arabidopsis thaliana, BLH2, BLH4 and BLH6 play a part in the generation and progression of the cell wall(Niu, X. andFu, D. 2022). In cotton, the GhBLH6-A13 gene participates in the regulation of secondary cell wall development. When GhBLH6-A13 is heterologously overexpressed in Arabidopsis thaliana, it markedly suppresses the synthesis of lignocellulose in the bundle sheath fibers(Ma, Q. et al. 2019). AtMYB46 has the capacity to directly govern 17 transcription factors associated with cell wall synthesis as well as 12 cell wall constituents, among which are AtBLH2, AtBLH3, AtBLH6, and AtBLH10 (Chen, H. et al. 2019). In camellia, CcBLH6 functions as a positive regulator in lignin biosynthesis throughout the lignification stage of camellia fruits. Meanwhile, in tomatoes, SlBLH4 is capable of directly suppressing the expression of SlPE (polygalacturonase). As a result, the texture of tomato fruits is reduced and the cell wall becomes thinner(Yan, F. et al. 2020). KNAT3 has the ability to interact via the MEINOX domain with the POX domain of the BELL family, giving rise to homodimers or heterodimers. These dimers, in unison, are responsible for governing plant growth and orchestrating stress responses(Shafi, A. et al. 2015). Take Arabidopsis thaliana as an illustration. BLH1 collaborated with KNAT3 and augmented the retention of KNAT3 within the nucleus. In this plant, AtKNAT3 plays a regulatory role in lignin biosynthesis, spurs the production of secondary cell walls in vessels, and furnishes mechanical support for the stems(Qin, W. et al. 2020; Wang, S. et al. 2020). Thus, we presume that LpKNAT3 communicates with LpBLH3 to modulate lignin synthesis, consequently assisting the plant in withstanding saline-alkali stress. The BLH family is closely related to the ABA pathway(Hoth, S. et al. 2002). ABI5, which belongs to the basic leucine zipper (bZIP) transcription factor family, participates in ABA signaling throughout seed maturation and germination. Moreover, it assumes a regulatory function under drought and high salinity circumstances by governing the expression of genes that harbor abscisic acid response elements (ABREs) within their promoter regions(Uno, Y. et al. 2000; Yan, F. et al. 2012; Skubacz, A. et al. 2016; Chang, H.-C. et al. 2019). For instance, GhBLH1 potentially contributes to the development of cotton fibers and might also take part in the plant’s reaction to environmental pressures, such as its response to ABA(Liu, C. et al. 2020). In Arabidopsis thaliana, BLH1 and KNAT3 work together to boost ABA responses. They achieve this by attaching to and then triggering the activation of the ABI3 promoter via the TGGA motif.(Kim, D. et al. 2013). In cotton, GhBLH1 can identify and fasten onto the TGGA motif within the promoters of its target genes, like ABI3(Jia, T. et al. 2024). In this study, we cloned LpBLH3, clarified the specific expression pattern of the gene, and eventually revealed the molecular mechanism of how LpBLH3 protein affects the saline tolerance in L. pumilum. Materials and Methods 2.1. Plant materials and growth conditions In Northeast China’s Daqing (46°58′N, 125°3′E), L. pumilum specimens were gathered from saline-alkaline terrains. Meanwhile, Nicotiana benthamiana plants were kept in the laboratory setting. All of these plants were cultivated under regulated circumstances, where the temperature was maintained at 25 ± 2°C. The light intensity reached 2000 lux, following a 16-hour light and 8-hour dark photoperiod, and the relative humidity ranged from 75% to 80%. 2.2. Cloning and bioinformatics analysis of LpBLH3 gene Total RNA was isolated from the leaves of L. pumilum by means of the OminiPlant RNA Kit (manufactured by CWBIO, Beijing, China). Subsequently, cDNA was synthesized with the aid of a reverse transcription kit (produced by Takara, Tokyo, Japan). The forward primer LpBLH3 -F and the reverse primer LpBLH3 -R were meticulously designed in accordance with the open reading frame (ORF) of LpBLH3, which was derived from the L. pumilum transcriptome. (It should be noted that all primer sequences are detailed in Supplementary Table S1.) The PCR products were then purified using the MolPure Gel Extraction Kit (developed by Co Win Biosciences, Beijing, China), ligated into the pMD18-T vector (produced by Takara, Tokyo, Japan), and finally transformed into Escherichia coli DH5α for sequencing. The sequences of homologous amino acids corresponding to the LpBLH3 protein within L. pumilum were analyzed and contrasted via DNAMAN software. Subsequently, a phylogenetic tree was built with the utilization of MEGA7 to investigate the associations among the BLH3 proteins present in L. pumilum . 2.3. Expression and subcellular localization of LpBLH3 The LpBLH3 gene was inserted into the pBI121 - GFP vector by means of specific primers, namely LpBLH3 - BamHI - F and LpBLH3 - SalI - R, and then transferred into Agrobacterium tumefaciens strain EHA105 . The sequences of these primers can be found in Supplementary Table S1. A comprehensive account of the subcellular localization procedures is provided in Methods S1. 2.4. Real-time quantitative PCR (RT-qPCR) analysis for LpBLH3 expression For the RT - qPCR analysis of LpBLH3, the primers LpBLH3 - qPCR - F and LpBLH3 - qPCR - R were employed, taking F - box family protein (FP) (Zhang, J. et al. 2017) and actin (ACT) (Liang, S. et al. 2013) as internal control genes. RNA was harvested from diverse anatomical parts, namely the root, bulb, leaf, flower, and seed of L. pumilum, and then transcribed in reverse to form cDNA. Subsequently, RT-qPCR was utilized to gauge the expression intensities of LpBLH3 within these distinct organs.(Zhang, L. et al. 2024). The in - depth analysis is elaborated in Methods S2. Every experiment was carried out three times. The sequences of the primers are presented in Supplementary Table S1. 2.5. Acquisition of LpBLH3 overexpressed transgenic lines The LpBLH3 gene was integrated into the plant expression vector pCXSN through the utilization of the XcmI restriction enzyme. Subsequently, the verified pCXSN - LpBLH3 plasmid was introduced into the Agrobacterium tumefaciens strain EHA105 .(Takara, Tokyo, Japan).Subsequently, transgenic plants were acquired by means of the Agrobacterium-mediated genetic transformation approach.(Wang, Z. et al. 2024).The specific procedures for obtaining and analyzing the transgenic lines are elaborated in Methods S3. Every sample was composed of three biological replicates. The sequences of the primers are presented in Supplementary Table S1. 2.6. Resistance analysis of LpBLH3 overexpressed and LpBLH3 mutant lines Wild-type (WT) and LpBLH3 -overexpressing plants with comparable sizes were cultivated in pots under non-stress conditions. Subsequently, stress inductions were carried out by irrigating them with 11 mM H2O2, 200 μM NaCl, 20 mM Na2CO3, or 20 mM NaHCO3 for a period of 7 days. The photosynthetic parameters, namely stomatal conductance, transpiration efficiency, net photosynthetic rate, and intercellular CO2 concentration, were gauged employing an LI-6400 photosynthesis apparatus. Meanwhile, the chlorophyll content was quantified using a CHLOROPHYLL METER SPAD-502PLUS (KONICA MINOLTA, Japan). The determination of Malondialdehyde (MDA) content was achieved using the thiobarbituric acid (TBA) approach.(Senthilkumar, M. et al. 2021). The activity of Peroxidase (POD) was gauged by means of the lignum sanctum vitae approach(Wang, X. 2006). The determination of Catalase (CAT) activity was carried out with the application of ultraviolet spectrophotometry(Shi, J. et al. 2016). The determination of free proline content was accomplished using the ninhydrin protocol(De, B. et al. 1996). In parallel, the measurement of superoxide dismutase (SOD) anion content was carried out by means of the hydroxylamine oxidation methodology(Yang, J. et al. 2020). Lignin staining was applied to the roots and stems of WT and LpBLH3 overexpressing lines by means of the Wiesner technique(Xu, L. et al. 2011). The lignin content determination was carried out with the application of ultraviolet spectrophotometry(Xie, X.-M. et al. 2011). 2.7. RT-qPCR analysis of stress-related gene expression In order to monitor the alterations in the expression of salt - related genes within LpBLH3 overexpressing lines, RT - qPCR was employed to gauge the expression magnitudes of LpSOS1, LpNHX1, LpABI5, and LpMYB4 genes. (It should be noted that all primer sequences are detailed in Supplementary Table S1.) Specifically, after subjecting both the LpBLH3 overexpressing lines and WT lines to a treatment with 500 mM NaHCO3 for 24 hours, the quantification was carried out in accordance with the previously described approach. 2.8. Cloning of the LpABI5 promoter and Analysis With the aid of a Genome Walking Kit, the promoter sequence of LpABI5 was replicated(Takara, Tokyo, Japan). The primers utilized for cloning the LpABI5 promoter, namely LpABI5 - SP1, SP2, and SP3, can be found in Supplementary Table S1. Once the LpABI5 promoter sequence was cloned, it was subjected to an analysis procedure with the assistance of the PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Subsequently, the screened cis - acting elements were visualized and mapped by means of TBtools software V1.0. The complete sequence of the LpABI5 promoter is presented in Supplementary Table S3. 2.9. BLH3 Regulation of the LpABI5 Promoter Validated by Dual-Luciferase Reporter The LpABI5 promoter was inserted into the pGreenII 0800 - LUC vector. Meanwhile, the LpBLH3 sequence was incorporated into the pGreenII 62 - SK vector with the application of primers LpBLH3 - BamHI - F and LpBLH3 - XhoI - R. Subsequently, the resultant construct plasmid vectors were transferred into Agrobacterium tumefaciens strain GV3101 via the freeze - thaw method(Ge, Q. 2005) . The primers employed for amplification can be found in Supplementary Table S1. The comprehensive procedure of the dual - luciferase reporter assay is elaborated in Methods S4. 2.10. DNA Electrophoretic mobility shift assay (EMSA) An electrophoretic mobility shift assay (EMSA) was carried out to confirm whether LpBLH3 can bind to the LpABI5 promoter in an in - vitro setting. This assay was carried out with the use of the BeyoGold™ Chemiluminescent EMSA Kit (Beyotime, Shanghai, China), following the guidelines provided by the manufacturer. Probes were designed for the LpBLH3 binding sites in the LpABI5 promoter region. (The primers LpABI5 - TGAC - F and LpABI5 - TGAC - R are presented in Supplementary Table S1.) The 5’ ends of these probes were labeled with biotin and synthesized by Comate BioScience (Changchun, China). The in - depth process of the electrophoretic mobility shift assay (EMSA) is detailed in Methods S5. 2.11. Screening of LpBLH3 interacting protein The construction of the L. pumilum cDNA library was accomplished by OE Biotechnology (Shanghai, China). After that, with the assistance of the EasyGeno Fast Recombination Cloning Kit (TIANGEN, Beijing, China), the LpBLH3 gene was inserted into the pGBKT7 vector by means of LpBLH3 - BamHI - F and LpBLH3 - BamHI - R primers. Subsequently, the resultant construct was transformed into the Y2H Gold yeast strain. The screening of the yeast library was carried out in accordance with the guidance provided by Clontech(www.clontech.com). The positive blue colonies were picked out, and PCR was carried out employing the pGADT7 universal primers T7 and 3’-AD. Subsequently, the PCR products were sent to Kumei Biotechnology (Changchun, China) for sequencing analysis. The sequences of the primers can be found in Supplementary Table S1. 2.12. Validation of the interaction between LpBLH3 and LpKNAT3 Yeast two-hybrid (Y2H) assay was employed to confirm the interaction between LpBLH3 and LpKNAT3. The coding sequence of the candidate interacting protein LpKNAT3 was inserted into the pGADT7 vector with the utilization of LpKNAT3 -EcoRI-F and LpKNAT3 -BamHI-R primers. Subsequently, additional interaction experiments were conducted on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade + X-α-gal + ABA solid media. The sequences of the primers are presented in Supplementary Table S1. The comprehensive procedure of the Y2H experiment is elaborated in Methods S6. In order to provide further verification for the interaction occurring between LpBLH3 and LpKNAT3, the Luciferase Complementation Imaging (LCI) experiment was implemented. The plasmids, namely LpBLH3 -pCAMBIA1300-Cluc (the corresponding primers being LpBLH3 -KpnI-F and LpBLH3 -SalI-R) and LpKNAT3 -pCAMBIA1300-Nluc (coupled with primers LpKNAT3 -BamHI-F and LpKNAT3 -SalI-R), were transferred into Agrobacterium tumefaciens strain GV3101 by means of the freeze-thaw procedure. You can refer to Supplementary Table S1 for the specific sequences of the primers. The elaborate procedure of the LCI experiment has been described in Methods S7. 2.13. Data statistical analysis All experimental procedures were carried out following a randomized design, with each treatment replicated three times. For the data processing of qPCR, MxPro - QPCR software v4.1 was employed. This software can be located on the relevant website (last accessed on 10 October 2024)( https://www.manualslib.com/manual/1418060/Agilent-Technologies-Mx3000p.html). The data underwent statistical analysis with the utilization of SPSS 23.0 software. Statistical significance was regarded as achieved when P < 0.05 and P < 0.01, which were denoted by * and ** correspondingly. Results Cloning and bioinformatic analysis of the LpBLH3 gene Sequencing results revealed that the open reading frame (ORF) of the LpBLH3 gene is 1800 bp in length, encoding a protein of 599 amino acids. Amino acid sequence alignment showed a high degree of homology to BLH3 proteins from other plants (Fig. S1). The LpBLH3 protein contains two conserved domains: a POX domain, located between amino acids 306-459, and a HOX (HD) domain, located between amino acids 502-566(Fig. S2). The phylogenetic analysis indicated that LpBLH3 is most closely related to the BLH3 protein from Asparagus officinalis (Fig. S3). Expression and subcellular localization of LpBLH3 To investigate the expression levels of LpBLH3 in different organs, RT-qPCR analysis revealed that LpBLH3 was expressed at the highest levels in the leaves and at the lowest levels in the roots (Fig. 1A). Under stress conditions with 200 mM NaCl, 20 mM Na2CO3, 20 mM NaHCO3, or 11 mM H2O2, LpBLH3 expression in L. pumilum leaves significantly increased, peaking at 24 hours, and gradually decreased with longer treatment times (Fig. 1B-E). These results indicate that LpBLH3 is highly responsive to saline stresses. Subcellular localization analysis using a Carl Zeiss fluorescence microscope detected green fluorescence in the nucleus (Fig. 1F), suggesting that LpBLH3 protein is localized in the nucleus, consistent with its role as a transcription factor. 2.9. BLH3 Regulation of the LpABI5 Promoter Validated by Dual-Luciferase Reporter not-yet-known not-yet-known not-yet-known unknown Fig. 1 . Expression and subcellular localization of LpBLH3. (A): Expression of LpBLH3 in the root, bulb, leaf, flower, and seed of wild-type L. pumilum plants. cDNA was obtained from the roots, stems, leaves, flowers, and seeds of L. pumilum, and the expression levels of LpBLH3 were quantified using Real-Time Quantitative PCR. (B): Real-Time Quantitative PCR analysis of LpBLH3 expression in L. pumilum under 11 mM H2O2 treatment for 6 h, 12 h, 24 h, 36 h, and 48 h. (C): Real-Time Quantitative PCR analysis of LpBLH3 expression in L. pumilum under 200 mM NaCl treatment for 6 h, 12 h, 24 h, 36 h, and 48 h. (D): Real-Time Quantitative PCR analysis of LpBLH3 expression in L. pumilum under 20 mM Na2CO3 treatment for 6 h, 12 h, 24 h, 36 h, and 48 h. (E): Real-Time Quantitative PCR analysis of LpBLH3 expression in L. pumilum under 20 mM NaHCO3 treatment for 6 h, 12 h, 24 h, 36 h, and 48 h. CK (No treatment) was used as a control. Asterisks (*) and (**) indicate statistically significant differences at P < 0.05 and P < 0.01, respectively. Data are presented as mean ± SD from three replicates. (F): Subcellular localization of LpBLH3 protein. Green fluorescence indicates GFP expression. pBI121-GFP and pBI121-LpBLH3-GFP constructs were transiently transformed into N. benthamiana cells using a biolistic transformation method. Samples were examined under a microscope equipped with a fluorescence module. Scale bar = 100 μm. Generation of LpBLH3 overexpressing and determinnation of physiological parameter RT-qPCR was used to measure the expression levels of LpBLH3 in L. pumilum . The lines pCXSN-BLH3 #1-#8 exhibited higher expression levels of LpBLH3 compared to the wild-type (WT). The three L. pumilum lines with the highest expression levels (#6, #7, #8) were selected for further experiments (Fig. S4). To further assess the tolerance of LpBLH3 overexpressing lines to saline stress, these lines were subjected to stress treatments with 1 M NaCl, 20 mM Na₂CO₃, 20 mM NaHCO₃, or 11 mM H₂O₂. Under normal conditions, there were no significant differences in growth between the wild-type (WT) and LpBLH3 overexpressing lines. However, under 11 mM H₂O₂, 1 M NaCl, 500 mM Na₂CO₃, or 500 mM NaHCO₃ stress, the leaves of LpBLH3 overexpressing lines remained mostly green with minimal wilting and yellowing. In contrast, the wild-type plants showed more severe wilting. The majority of the overexpressing lines remained upright with a low leaf lodging rate, while the wild-type exhibited significant wilting (Fig. 2A-D). Fig. 2. Phenotypes of transgenic Lilium pumilum under saline-alkali stress ( A-D ): Plant growth phenotype and leaf lodging rate (%) of wild-type (WT) and LpBLH3 overexpressing L. pumilum lines were evaluated. Plants were grown on the same medium supplemented with 11 mM H₂O₂, 200 mM NaCl, 20 mM Na₂CO₃, and 20 mM NaHCO₃ for 0 and 7 days. WT: wild-type; #6, #7, #8: LpBLH3 overexpressing lines. Bar, 10 cm. Photosynthesis is crucial for nutrient accumulation in plants. Measurements of chlorophyll content, stomatal conductance, transpiration rate, net photosynthetic rate, and intercellular CO₂ concentration revealed a decrease in these parameters to varying extents in the wild-type (WT) plants under stress. However, in the three LpBLH3 overexpressing lines, no significant changes were observed in chlorophyll content, stomatal conductance, or intercellular CO₂ concentration. While photosynthetic and transpiration rates still decreased, the reduction was less pronounced in the overexpressing lines compared to the WT. After exposure to the four stress treatments, the photosynthetic parameters in the LpBLH3 overexpressing lines were superior to those in the WT (Fig. S5A-D). Compared to the wild-type (WT), LpBLH3 overexpressing lines exhibited higher proline content and lower MDA levels (Fig. 4B-C), which enhanced cell water retention and enzyme activity. Saline-alkaline stress disrupts reactive oxygen species (ROS) homeostasis, primarily involving O₂•⁻ and H₂O₂, leading to ROS-induced damage. To further investigate the role of LpBLH3 overexpression in regulating ROS scavenging capacity, the accumulation of ROS was measured using an H₂O₂ assay kit (JianCheng, Nanjing, China) (Fig. 4A) and the hydroxylamine oxidation method (which detects O₂•⁻) (Fig. 4D). Additionally, superoxide dismutase (SOD) catalyzes the conversion of O₂•⁻ to H₂O₂, while catalase (CAT) and peroxidase (POD) decompose H₂O₂. These enzymes work together to regulate ROS homeostasis in plants. Under stress, CAT and POD levels were significantly higher in LpBLH3 overexpressing L. pumilum compared to WT (Fig. 3E-F). Fig. 3. Determination of physiological indexes of LpBLH3 overexperssing L pumilum under saline stress. ( A ): H 2 O 2 content. ( B ): MDA content. ( C ): Proline content. ( D ): superoxide anion. ( E ): CAT content. ( F ): POD activity. WT: wild type. #6; #7, #8: LpBLH3 overexpressing lines. Three selected LpBLH3 overexperssing lines with high expression level. *P＜0.05, **P＜0.01, ***P＜0.001 standard error of three biological replicates. Determinnation of lignin content Phloroglucinol staining of the stems and roots of wild-type (WT) and LpBLH3 overexpressing lines revealed that the lignin-rich areas in the stem cross-sections of the overexpressing lines were significantly wider and darker compared to those in WT plants (Fig. 4A). In the root cross-sections, there was a significant difference in lignin accumulation between the LpBLH3 overexpressing lines and WT, with the overexpressing lines showing higher and more intensely stained lignin. Ultraviolet spectrophotometry analysis further indicated that LpBLH3 promotes lignin accumulation over time, thereby enhancing the plants’ tolerance to saline-alkaline stress.(Fig.4B-C). Fig.4 . Determinnation of lignin content of LpBLH3 overexperssing L. pumilum. ( A ): Phloroglucinol staining of L. pumilum from detached stem and the root. WT: wild type. #6, #7 and #8: LpBLH3 overexpressing lines. The depth of staining reflects the amount of lignin accumulation in the cells. Scale bar =100 μm. ( B ): lignin content detached stems of 2 weeks old. WT: wild type. #6, #7, #8: LpBLH3 overexpressing lines. ( C ): lignin content detached root of ten weeks old. WT: wild type. #6, #7 and #8: LpBLH3 overexpressing lines. *P＜0.05, **P＜0.01, ***P＜0.001 standard error of three biological replicates. 3.5. RT-qPCR analysis of stress-related gene expression After 24 hours of treatment with 500 mM NaHCO₃, the expression levels of stress-related genes ABI5, SOS1, NHX1, and MYB4 were found to be higher in the three LpBLH3 overexpressing lines compared to the wild-type (WT) (Fig. S6). These results indicate that LpBLH3 overexpression upregulates the expression of SOS1, NHX1, ABI5, and MYB4 under 500 mM NaHCO₃ stress. Analysis of LpABI5 promoter A 852 bp upstream promoter sequence of LpABI5 was obtained through chromosome walking PCR . The cis-acting elements in the LpABI5 promoter were analyzed using PlantCARE, with the results presented in Table S4. A visual analysis of the promoter sequence was conducted using TBtools software (Fig. 5A). Sequence analysis revealed multiple recognition sites (TGGA) for the BLH3 transcription factor. In vitro, a dual-luciferase reporter assay confirmed that the LpBLH3 transcription factor binds to the LpABI5 promoter. The results of the Dual-luciferase reporter assay showed that the LUC signal was significantly stronger in leaves co-infiltrated with LpBLH3 -62SK and LpABI5Pro -LUC constructs compared to the empty vector control (Fig 5C), indicating that LpBLH3 can mediate the transcriptional activation of LpABI5 . Fig. 5 . Analysis of LpBLH3 bind to the LpABI5 promoter. ( A ): Visual analysis of the LpABI5 promoter. The cis-acting element analysis of the LpABI5 promoter sequence was carried out using the PlantCARE website. The obtained promoter sequences were visualized and analyzed using TBtools software. ( B ): Schematic diagram of the ABI5 promoter showing BLH3-binding TGGA motifs.Binding affinity of LpBLH3 to the promoter was evaluated using electrophoretic mobility shift assays. The first, second and third track represents control, normal and competitive respectively. ( C ): Dual-luciferase reporter assay. ( 1 ): LpBLH3 -62SK + LpABI5 -pro-LUC. ( 2 ): LpABI5 -pro-LUC +62SK. ( 3 ): LpBLH3 -62SK+LUC. Empty vector 62SK + LUC was used as a negative control. Scale bar = 1 cm. The optimal conditions for inducing LpBLH3 protein expression were determined to be an OD₆₀₀ of 0.6, with the addition of 0.5 mM IPTG, followed by incubation at 37 °C with shaking at 240 rpm for 4 hours. Induced protein expression was confirmed by the presence of distinct bands around 72 kDa at 1, 2, 3, and 4 hours in SDS-PAGE analysis (Fig. S7A). The LpBLH3 protein was then purified using BeyoGold™ His-tag Purification Resin Ni-NTA affinity chromatography (Fig. S7B). Further validation of LpBLH3 binding to the LpABI5 promoter was performed using electrophoretic mobility shift assay (EMSA), where the interaction between LpBLH3 protein and a biotin-labeled probe resulted in a shifted band, which was inhibited by the addition of a competitive probe (Fig. 5B). These results demonstrate that LpBLH3 directly upregulates LpABI5 expression by binding to its promoter. Analysis of LpBLH3-Interacting Proteins The plasmids of LpBLH3 -pGBKT7 was transformed into Y2H Gold yeast strains, and a yeast two-hybrid screen identified ten different full-length or partial interacting protein sequences, which were further analyzed by BLAST against the NCBI database (Table. S5). Prediction of LpBLH3 interacting proteins revealed that the candidate proteins included the class-II KNOX protein KNAT3 (Homeobox protein knotted-1-like 3) and OFP, among others (Fig. S8). Previous studies have shown that KNAT3 is involved in responses to abiotic stress and lignin bioaccumulation. Based on this, we hypothesized that LpBLH3 may regulate lignin deposition and contribute to the plant’s response to saline stress. Both the control and experimental groups grew normally on SD/-Trp-Leu medium. Co-transformation of LpBLH3 and LpKNAT3 into Y2H Gold yeast strains enabled growth on SD/QDO+X-α-gal+AbA medium, and the colonies turned blue, similar to the positive control (Fig. 6A). This result confirmed that LpBLH3 and LpKNAT3 proteins interact in the yeast two-hybrid system. Additionally, LCI analysis further validated the interaction between LpBLH3 and LpKNAT3 proteins, as fluorescence signals were observed only in tobacco leaves co-infiltrated with pBS-35S: LpBLH3 -VN154 and pBS-35S: LpKNAT3 -VC80 constructs (Fig. 6B). Fig. 6. Validation of the interaction between LpBLH3 and LpKNAT3. ( A ): Yeast two-hybrid assay to verify the relationship between LpBLH3 and LpKNAT3. The cotransformation of pGADT7 + pGBKT7, pGADT7 + pGBKT7- LpBLH3 , and pGBKT7 + pGADT7- LpKNT3 were used as controls. Only pGADT7- LpKNT3 and pGBKT7- LpBLH3 co-transformed colonies turned blue on SD/-Trp/-Leu/-His/-Ade + X-α-gal medium. ( B ): LCI assay to discover the relationship between LpBLH3 and LpKNAT3 were co-injected into N. benthamiana cells . NLUC + CLUC, LpKNAT3 -CLUC + NLUC, and LpBLH3 -NLUC + LpKNAT3 -CLUC were used as controls. Scale bar = 1 cm. 3.7 BLH3 and KNAT3 synergistically activate the promoter To investigate whether the interaction between LpBLH3 and LpKNAT3 affects the expression of ABI5 , the LUC signal was significantly stronger in leaves co-infiltrated with LpBLH3 -62SK, LpKNAT3 -62SK, and LpABI5Pro -LUC constructs compared to leaves co-infiltrated with LpBLH3 -62SK and LpABI5Pro -LUC constructs (Fig. 7A-B). These results suggest that KNAT3 enhances the binding affinity of LpBLH3 to the LpABI5 promoter. Co-transformation of LpBLH3 pro-LUC, LpBLH3 -62SK, and LpKNAT3 -62SK resulted in significantly higher luciferase activity, indicating that LpKNAT3 promotes LpBLH3 in regulating LpABI5 expression (Fig. 7C). Collectively, these findings suggest that LpBLH3 modulates LpABI5 expression by binding to its promoter, and the interaction between LpBLH3 and LpKNAT3 increases the binding affinity. Fig. 7 . BLH3 and KNAT3 synergistically activate the ABI5 promoter. ( A ): Dual-luciferase reporter assay. 62SK + LUC, LpABI5 -pro-LUC + 62SK, and LpBLH3 -62SK + LUC were used as a negative control. Scale bar = 1 cm. ( B ): Dual-luciferase reporter assay. 62SK + LUC, LpABI5 -pro-LUC + 62SK, and LpBLH3 -62SK + LpKNAT3 -62SK + LUC were used as a negative control. Scale bar = 1 cm. ( C ): Quantification was performed by normalizing firefly luciferase (LUC) activity to the activity of Renilla luciferase (REN), and 35S:REN was used as the internal control. Relative luciferase activities were determined using LpBLH3 -pGreenII62-SK and LpKNAT3 -pGreenII62-SK as the effector compared with the control effector (pGreenII62-SK empty vector). Values are means SD. Asterisks denote significant differences using a two-tailed t-test: **, P < 0.01; ***, P < 0.001. Discussion In previous laboratory studies, gene expression changes in L. pumilum under alkaline stress were analyzed, revealing that the LpBLH3 gene was upregulated. Consequently, the LpBLH3 gene was cloned and analyzed in detail. The BLH3 protein contains a HOX domain located between amino acids 502 and 566(Niu, X. andFu, D. 2022). The amino acid sequence alignment of LpBLH3 with BLH3 proteins from other species revealed a conserved region shared across all species. While the homology in these conserved regions is high, the homology in other regions is relatively low, suggesting that BLH3 proteins from different plant species may have distinct biological functions. The expression of LpBLH3 in L. pumilum was assessed using RT-qPCR, which revealed an increasing trend after short-term exposure to H₂O₂, NaCl, Na₂CO₃, and NaHCO₃ (Fig. 1B-E). This suggests that LpBLH3 may play a role in the stress resistance mechanisms of L. pumilum under saline stress. Saline stress can cause irreversible damage to photosynthetic organs at any stage of plant development, highlighting the importance of mitigating its impact on plant photosynthesis(Niu, X. andFu, D. 2022). The photosynthetic index measurements showed that LpBLH3 -overexpressing L. pumilum outperformed the wild type, with higher chlorophyll content. This suggests that LpBLH3 helps mitigate damage to photosynthesis in L. pumilum under stress (Fig. S5). POD and CAT are key antioxidant enzymes in plants, responsible for scavenging reactive oxygen species (ROS) during abiotic stress(Zhongming, Z. et al. 2004). In LpBLH3 overexpressing lines, the levels of POD and CAT were higher than in the wild type (Fig. 3E-F), indicating that LpBLH3 enhances the plant’s antioxidant capacity and its ability to scavenge ROS. Lignin accumulation is a key factor in plant resistance to salt stress(Hu, P. et al. 2019). Compared to normal plant lines, salt-tolerant lines often show increased lignin content and thickened cell walls, suggesting that the strengthening of cell wall physical properties is a key adaptation for plants to cope with salt stress(Chun, H. J. et al. 2019). Lignin staining and content determination were performed on LpBLH3 overexpressing and wild-type lines under 500 mM NaHCO₃ stress. The results showed that the lignin content in LpBLH3 overexpressing L. pumilum was significantly higher than in the wild type (Fig. 4A-C). These data suggest that the LpBLH3 overexpressing lines promote lignin accumulation and enhance the salt tolerance of the plants. Through yeast two-hybrid screening, proteins interacting with LpBLH3 were identified, including LpKNAT3. In plant cells, BLH proteins commonly interact with KNOX proteins. The binding of these two proteins forms a heterodimer, which is then translocated to the nucleus to perform its functions. Within the nucleus, their HD domains specifically bind to their target sequences, thereby regulating the expression of downstream genes(Yang, Q. et al. 2022). KNAT3 can interact with the POX domain of the BELL family through its MEINOX domain to form either homodimers or heterodimers, which together regulate plant growth and stress responses(Zhao, H. 2020). LpBLH3 can interact with LpKNAT3 both in vivo and in vitro, as demonstrated by yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) analyses (Fig. 6A-C). Previous studies have shown that class II KNOX genes, including KNAT3 , KNAT4 , KNAT5 , and KNAT7 , are expressed during the secondary cell wall (SCW) deposition process (Nookaraju, A. et al. 2022), KNAT3 and KNAT7 act synergistically to enhance the deposition of secondary cell walls in plants(Wang, S., et al. 2020). AAdditionally, KNAT3 can interact with the key transcription factors NST1 and NST2 during secondary cell wall formation, forming a heterodimer complex that regulates F5H to promote lignin synthesis(Qin, W., et al. 2020). Therefore, LpBLH3 and LpKNAT3 proteins may interact to collaboratively contribute to plant lignin synthesis and enhance salt tolerance. We investigated whether LpBLH3 enhances plant salt tolerance by regulating downstream genes. RT-qPCR results showed that the expression level of ABI5 increased in LpBLH3 overexpressing lines subjected to NaHCO₃ stress (Fig. S6). ABI5 encodes a member of the basic leucine zipper (bZIP) transcription factor family, which is involved in ABA signaling during seed maturation and germination, and plays a key role in abiotic stress responses(Collin, A. et al. 2021). ABI5 directly binds to the CAT1 promoter, activating CAT1 expression and regulating ROS homeostasis in Arabidopsis (Bi, C. et al. 2017). EMSA and dual-luciferase reporter assays confirmed that the LpBLH3 transcription factor can specifically bind to the promoter region of LpABI5 (Fig. 5A-C). In Arabidopsis , ABA promotes the interaction between BLH1 and KNAT3, leading to the formation of a dimer. This dimer then binds to the ABI3 promoter via the TGGA motif, thereby enhancing the expression of ABI3 (Kim, D., et al. 2013). We confirmed that LpBLH3 can interact with KNAT3 and promote the positive regulation of ABI5 gene expression (Fig. 7A-C). Therefore, we speculate that LpBLH3 may enhance the plant’s tolerance to saline stress by regulating the key gene LpABI5 within the ABA signaling pathway. Fig. 8. Model for the Action of LpBLH3 in Conferring Tolerance to Saline Stress in L. pumilum : Under saline conditions, LpBLH3 expression increases. The model includes three main pathways through which LpBLH3 enhances salt tolerance: ( 1 ): ROS Scavenging Pathway: LpBLH3 enhances the activity of antioxidant enzymes, including CAT, POD, and SOD, leading to reduced ROS levels and enhanced plant salt tolerance. ( 2 ) : ABA Pathway: LpBLH3 promotes the expression of LpABI5 by binding to the TGGA motif in the LpABI5 promoter, activating the ABA signaling pathway to further enhance the plant’s tolerance to salt stress. ( 3 ) : Lignin Pathway: LpBLH3 may interact with LpKNAT3 to increase lignin content, which strengthens the secondary cell wall, thereby improving the plant’s structural integrity and salt tolerance. Arrows indicate increased expression or activity in each pathway. In summary, we have identified three pathways through which LpBLH3 enhances plant salt tolerance: the ROS pathway, the ABA pathway, and the increased lignin content (Fig. 8). The expression levels of LpBLH3 were elevated when L. pumilum was subjected to saline stress. This increase in LpBLH3 expression led to enhanced activity of ROS-related enzymes (SOD, CAT, and POD), thereby improving the plant’s ability to scavenge ROS. As a transcription factor, LpBLH3 initiates the expression of LpABI5 , and its increased expression further upregulates LpABI5, contributing to improved salt tolerance through the ABA pathway. Under salt stress, both LpKNAT3 and LpBLH3 levels increase, and both contribute to elevated lignin content. It is likely that LpKNAT3 and LpBLH3 interact with each other, enhancing lignin accumulation, which in turn increases the thickness of the secondary cell wall and improves the plant’s salt tolerance. Funding This work was supported by the Heilongjiang Province Agriculture Research System-Ecological Agriculture ([2023] 1197) and Heilongjiang Province “Double First Class” Discipline Collaborative Innovation Achievement Project [LJGXCG2023-036]. Acknowledgements This work was supported by College of Life Sciences in Northeast Forestry University. Author contributions Shumei Jin conceived and designed the experiments; Wenhao Wan, Huitao Cui, Shangwei Ji, Lingshu Zhang, Miaoxin Sun,Xingyu Zhang and Hao Sun performed the experiments and experimental data analysis; Wenhao Wan wrote the manuscript; Shumei Jin revised and edited the article. All authors read and approved the paper. ORCID ShuMei Jin, https://orcid.org/0000-0002-5258-789X Competing interests The authors declare no competing interests. Additional files Supporting Information Additional Supporting Information may be found online in the Supporting Information section at the end of the article. Fig. S1. Analysis of the conserved domains of LpBLH3. Fig. S2 .Amino acid sequence alignment of LpBLH3 protein. Fig. S3. LpBLH3 evolutionary tree analysis. Fig. S4. Relative expression content of LpBLH3 in the WT and overexpressing 1-8 lines. Fig. S5 . Photosynthetic analysis of LpBLH3 overexperssing lines under saline-alkali stress. Fig. S6 . Determination of relative expression of genes related to salt-alkali stress ( LpSOS1 / LpNHX1 / LpABI5 / LpMYB4 ). Fig. S7 . Verification of LpBLH3 bind to the LpABI5 promoter by EMSA assay. Fig. S8 . Prediction of interaction protein of LpBLH3 based on STRING. Table. S1. All primer sequences in this research. Table. S2. The sequence of LpBLH3. Table. S3. LpABI5 promoter. Table. S4. Analysis of cisacting elements of LpABI5promoter. Table. S5. Candidate interacting proteins obtained by yeast two-hybrid screening of LpBLH3. Methods. S1. Subcellular localization Methods. S2. RT-qPCR analysis for LpBLH3 expression Methods. S3. Acquisition and analysis of transgenic lines Methods. S4. Dual luciferase reporter assay Methods. S5. DNA electrophoretic mobility shift assay (DNA-EMSA) Methods. S6. Yeast two-hybrid assay Methods. S7. Luciferase Complementation Imaging (LCI) Data availability All data generated or analyzed during this study are included within the article and its additional files. Protein sequences of different species are obtained from the NCBI (https://www.ncbi.nlm.n-ih.gov/). not-yet-known not-yet-known not-yet-known unknown 1 The sequence of LpBLH3 were deposited in NCBl GenBank:SUB15040377. 1 The sequence of LpBLH3 were deposited in NCBl GenBank:SUB15040377. References Bellaoui, M., Pidkowich, M. S., Samach, A., et al. (2001). The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. The Plant Cell, 13 (11), 2455-2470. doi:https://doi.org/10.1105/tpc.010161 Bi, C., Ma, Y., Wu, Z., et al. (2017). 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Reactive oxygen gene network of plants. 9 , 490-498. doi:https://doi.org/10.1016/j.tplants.2004.08.009 Information & Authors Information Version history V1 Version 1 28 February 2025 Peer review timeline Published Plants Version of Record 17 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords lilium pumilum aba pathway blh3 transcription factors lignin content signaling Authors Affiliations Wenhao Wan Northeast Forestry University View all articles by this author Huitao Cui Northeast Forestry University View all articles by this author Lingshu Zhang Northeast Forestry University View all articles by this author Miaoxin Shi Northeast Forestry University View all articles by this author Hao Sun Northeast Forestry University View all articles by this author Xingyu Liu Northeast Forestry University View all articles by this author Wei Yang Heilongjiang Academy of Agricultural Sciences Maize research institute View all articles by this author Fengshan Yang Heilongjiang University View all articles by this author Shumei Jin 0000-0003-1035-3486 [email protected] Northeast Forestry University View all articles by this author Metrics & Citations Metrics Article Usage 214 views 127 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wenhao Wan, Huitao Cui, Lingshu Zhang, et al. BLH3 regulates the ABA pathway and lignin synthesis under salt stress in Lilium pumilum. Authorea . 28 February 2025. DOI: https://doi.org/10.22541/au.174071536.69426547/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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