BLH3 Regulates the ABA Pathway and Lignin Synthesis Under Salt Stress in Lilium pumilum

Abstract

BEL1-like homeodomain protein 3 (BLH3) plays a crucial role in plant development. However, its involvement in the salt stress response has not 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), determination of physiological indices of plant after Saline-Alkali stress, yeast two-hybrid screening, luciferase complementation imaging (LCI), and chromosome walking to obtain the promoter sequence, analyzed by PlantCARE, electrophoretic mobility shift assay (EMSA), and then dual-luciferase reporter assay(LUC). 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 h in the leaves under different saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling and stronger photosynthesis. The overexpression of LpBLH3 can enhance lignin accumulation in root and stem by positively modulating the expression of crucial genes within the lignin biosynthesis pathway. Y2H and LCI analyses demonstrated that LpBLH3 interacts with LpKNAT3. Additionally, EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 and upregulate the expression of ABI5 downstream genes (LpCAT1/LpATEM/LpRD29B). In summary, LpBLH3 enhances the plant's salt tolerance through the ABA pathway and lignin synthesis. This study can enrich the functional network of the BLH transcription factor family, obtain Lilium pumilum lines with good saline-alkali resistance, expand the planting area of Lilium pumilum, and improve its medicinal and ornamental values. Additionally, the functional analysis of the BLH transcription factor family provides new insights into how crops adapt to the extreme growth environment of saline-alkali soils.

Keywords: ABA pathway; BLH3; Lilium pumilum; lignin content; salt stress.

Figure 1

Expression and subcellular localization of LpBLH3. (A) Expression of LpBLH3 in the root, bulb, leaf, flower, and seed of wild-type Lilium pumilum plants. cDNA was obtained from the root, bulbus, leaf, flower, and seed of L. pumilum, and the expression levels of LpBLH3 were quantified using Real-Time Quantitative PCR. CK (No treatment) was used as a control. Asterisks (**) and (***) indicate statistically significant differences at p < 0.01 and p < 0.001, respectively. Data are presented as mean ± SD from three replicates. (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. 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. (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. CK (No treatment) was used as a control. Asterisks (*) (**) and (***) indicate statistically significant differences at p < 0.05, p < 0.01 and p < 0.001, respectively. Data are presented as mean ± SD from three replicates. (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. 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. (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, p < 0.01 and p < 0.001, 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.

Figure 2

Phenotypes of transgenic L. pumilum under saline-alkaline 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. Scale bar = 10 cm. (E–H) Determination of relative expression of genes related to salt-alkali stress (LpSOS1/LpNHX1/LpABI5/LpMYB4). WT: wild type. #6, #7 and #8: LpBLH3 overexpressing lines. Note: *** p < 0.001, standard error of three biological replicates.

Figure 3

Determination of physiological indexes of LpBLH3 over-experssing L. pumilum under saline stress. (A) Chlorophyll content. (B) Intercellular CO₂ concentration. (C) Transpiration rate. (D) Photosynthetic rate. (E) Stomatal conductance. WT: wild type. #6; #7; #8: three selected LpBLH3 overexpressing lines with high expression level. ** p < 0.01 and *** p < 0.001 standard error of three biological replicates.

Figure 4

Analysis of gene expression in lignin synthesis pathway. (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. Standard error of three biological replicates. Scale bar = 100 μm. (B,C) Lignin content of stem and root. WT: wild type. #6, #7, #8: LpBLH3 overexpressing lines. Note: ** p < 0.01 and *** p < 0.001, standard error of three biological replicates. (D) Analysis of gene expression in lignin synthesis pathway. (LpPAL/Lp4CL/LpC3H/LpC4H/LpCCoAOMT/LpF5H). WT: wild type. #6, #7, #8: LpBLH3 overexpressing lines. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, standard error of three biological replicates.

Figure 5

Validation of the interaction between LpBLH3 and LpKNAT3. (A) Prediction of interaction protein of LpBLH3 based on STRING. (B) Yeast two-hybrid assay to verify the relationship between LpBLH3 and LpKNAT3. The cotransformation of pGADT7 + pGBKT7, pGADT7 + pGBKT7-LpBLH3, and pGBKT7 + pGADT7-LpKNTA3 were used as controls. Only pGADT7-LpKNTA3 and pGBKT7-LpBLH3 co-transformed colonies turned blue on SD/-Trp/-Leu/-His/-Ade + X-α-gal medium. (C) LCI assay to discover the relationship between LpBLH3 and LpKNAT3 were co-injected into N. benthamiana cells. (1) NLUC + CLUC; (2) LpKNAT3-CLUC + NLUC; (3) LpBLH3-NLUC + LpKNAT3-CLUC were used as controls. Scale bar = 1 cm.

Figure 6

BLH3 regulates the ABI5 expression. (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 ABI5 promoter was evaluated using EMSA. The first, second and third track represents control, normal, and competitive, respectively. (C) Dual-luciferase reporter assay. (a) LUC +62SK, (b) LpABI5-pro-LUC + 62SK, (c) LpBLH3-62SK + LUC, (d) LpBLH3-62SK + LpABI5-pro-LUC. Empty vector 62SK + LUC was used as a negative control. Scale bar = 1 cm. (D) Dual-luciferase reporter assay. (a) LUC +62SK, (b) LpABI5-pro-LUC + 62SK, (c) LpBLH3-62SK + LpKNAT3-62SK + LUC, (d) LpBLH3-62SK + LpKNAT3-62SK + LpABI5-pro-LUC. Empty vector 62SK + LUC were used as a negative control. Scale bar = 1 cm. (E) 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. (F) Analysis the expression levels of the LpABI5 downstream genes (LpCAT1/LpATEM/LpRD29B). WT: wild type. #6, #7, #8: LpBLH3 overexpressing lines. Note: ** p < 0.01 and *** p < 0.001, standard error of three biological replicates.

Figure 7

Model for the action of LpBLH3 in conferring tolerance to saline stress in L. pumilum: Under saline conditions, LpBLH3 expression increases. The model includes two main pathways through which enhances salt tolerance: ABA Pathway: LpNAT3 promotes LpBLH3 to regulate 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. Lignin Pathway: LpBLH3 may interact with LpKNAT3 to increase lignin content by positively modulating the expression of crucial genes within the lignin biosynthesis pathway, which strengthens the secondary cell wall, thereby improving the plant’s structural integrity and salt.