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Literature Sharing |The SBP transcription factor MdSPL13B positively regulates salt tolerance in apple
Release time:
2025-04-08
Salt stress has a major effect on the quality and yield of crops, and many transcription factors (TFs), such as WRKY, NAC, and ERF TFs have been shown to participate in the regulation of salt stress. Squamosa promoter binding protein-like (SPL) TFs play a role in plant floral organ development, metal ion responses and disease resistance. However, the precise function of SPL TFs in regulating the salt stress in plants remains unclear. In this study, we investigated the mechanism by which SPL TFs regulate salt stress in apple (Malus domestica). Overexpression of MdSPL13B in apple calli revealed that it positively regulated salt stress. The heterologous transformation of MdSPL3B also resulted in significant salt tolerance in Arabidopsis and tomato. Subsequently, a yeast two-hybrid assay showed that MdSPL13B could interact with MdNAC29 and MdPYL4, which was further confirmed by the bimolecular fluorescence complementation assay. Additionally, overexpression of MdNAC29 and MdPYL4 in apple calli, Arabidopsis and Nicotiana benthamiana plants indicated that MdNAC29 negatively regulated salt tolerance and that MdPYL4 positively regulated salt tolerance. Furthermore, the yeast one-hybrid, electrophoretic mobility shift assay and dual-luciferase assay showed that MdSPL13B could enhance salt tolerance through activation of MdWRKY100 in apple. In conclusion, our findings enhance the understanding of how MdSPL3B and its target gene regulate salt tolerance in apple, and have implications for the breeding of salt-tolerant apple.
To determine the homology of SPL13B proteins in different species, sequence alignment was performed on SPL13B proteins from multiple species. The results demonstrated that all the proteins contained typical SBP domains and exhibited high conservation (Fig.1, A). Phylogenetic tree displayed that MdSPL13B protein was closely related to PpSPL13B (Prunus persica SPL13B) protein, followed by FaSPL13B (Fragaria ananassa SPL13B) protein (Fig.1, B). We also further explored whether the MdSPL13B responds to salt stress, 'GL-3' self-rooted apple seedlings were subjected to 150 mmol · L-1 NaCl, and the whole plant material was collected after 0, 1, 3, 6, 12, and 24 h for expression analysis. As shown in Fig.1, C, the expression of MdSPL13B was upregulated under salt stress and peaked after 6 h, followed by gradual downregulation, which indicates that MdSPL13B may respond to salt stress. To further validate the prediction results, we transformed the MdSPL13B-GFP vector into onion cells for subcellular localization analysis, and the empty pRI101 vector without MdSPL13B (35S:GFP) served as the control. As a result, MdSPL13B was localized to the nucleus (Fig.1 D).
Fig. 1. Expression analysis and subcellular localization of MdSPL13B
To study the role of MdSPL13B, we obtained transgenic apple calli (MdSPL13B-OE1, MdSPL13B-OE2, MdSPL13B-OE3) through Agrobacterium-mediated transformation (Fig.2, A) and validated by qRT-PCR (Fig.2, B). Under salt treatment, the growth of WT plants was significantly inhibited and their fresh weight was significantly reduced compared with that of MdSPL13B-OE apple calli (Fig.2, C). Meanwhile, the MDA content and REC were higher in WT plants than in MdSPL13B-OE plants (Fig.2,D–E). This indicates that MdSPL13B positively regulates salt tolerance in apple calli.
Fig. 2. MdSPL13B enhances salt tolerance in apple calli
To further explore the impact of MdSPL13B on plants under salt treatment, we obtained transgenic Arabidopsis and tomato plants (Fig.3) and validated them by qRT-PCR (Fig. S1). A showed that there were no notable growth differences between Col-0 and MdSPL13B-OE Arabidopsis plants in the absence of salt. Nevertheless, the growth of Col-0 plants was significantly inhibited compared with that of MdSPL13B-OE plants after salt treatment, and their leaves were curled and wilted (Fig.3, A). Plants can generate reactive oxygen species (ROS) when exposed to abiotic stress, and DAB and NBT staining are frequently used to determine the contents of H2O2 and O2-.The degree of staining of MdSPL13B-OE Arabidopsis leaves was lower than that of Col-0 plants under NaCl treatment (Fig.3, B–C). As shown in Fig.3, D–G, the MDA content and REC were lower and the carotenoid and chlorophyll contents were higher in MdSPL13B-OE plants than in Col-0 plants. Moreover, changes in MdSPL13B-OE tomato plants were consistent with those observed in Arabidopsis after salt treatment (Fig.3, H–K). In summary, MdSPL13B is a positive regulatory factor of salt stress.
Fig. 3. MdSPL13B enhances salt tolerance in Arabidopsis and tomato plants
To explore how MdSPL13B regulates salt tolerance, we used Y2H assays to screen apple libraries for potential proteins interacting with MdSPL13B. As shown in Fig.4, A, the MdSPL13B protein encodes 415 aa (amino acids) and contains a typical SBP domain (97–171 aa). MdSPL13B has autonomous activation (Fig.4, B). This was further verified by segmentation (71–200 and 201–415 aa), and no autonomous activation was observed for 71–200 aa (which contained the SBP domain), indicating that this region could be used for the Y2H assay. Based on the results of the Y2H screening library (Table S4), we found that MdNAC29 (NAC TF) and MdPYL4 (ABA receptor protein) were capable of interacting with MdSPL13B. We then conducted a Y2H assay using pGBKT7-MdSPL13B (71–200 aa) as bait, pGADT7-MdNAC29/MdPYL4 as prey, and the empty vector as control. The Y2H assay results showed that only yeast strains cotransformed with MdSPL13B71–200aa + MdNAC29/MdPYL4 could perform QDO/X-α-Gal growth on medium (Fig.4, C). Interactions between the MdSPL13B and MdNAC29/MdPYL4 were further validated using BiFC. MdSPL13B-YFPN + MdNAC29/MdPYL4-YFPC vectors were transformed into onion epidermal cells for fluorescence observation (490–540 nm). The results revealed that only the combination of MdSPL13B + MdNAC29/MdPYL4 displayed yellow YFP fluorescence (Fig.4, D). Overall, the MdSPL13B protein can interact with the MdNAC29 and MdPYL4 proteins.
Fig. 4. Physical interaction assays for MdSPL13B and MdNAC29 / MdPYL4
Previously, we obtained MdNAC29-OE transgenic lines and found that MdNAC29 negatively regulated drought and cold tolerance in apple. Here, we selected MdNAC29-OE apple calli and Nicotiana benthamiana plants to examine the effects of MdNAC29 during salt treatment. The 14-d-old MdNAC29-OE and WT apple calli were treated for 10 d. MdNAC29-OE apple calli showed a more pronounced salt-sensitive phenotype after salt treatment (Fig.5, A), with less fresh weight, higher MDA and REC than WT (Fig.5,B–D). In addition, the growth of MdNAC29-OE Nicotiana benthamiana plants was reduced compared with that of WT plants, and their leaf curling and wilting phenotypes were more pronounced (Fig.5, E). Specifically, chlorophyll content significantly decreased, while the REC and MDA content significantly increased in MdNAC29-OE Nicotiana benthamiana plants than in WT plants (Fig.5, G–H). This indicates that MdNAC29 negatively regulates salt tolerance.
Fig. 5. Overexpression of MdNAC29 increases sensitivity to salt stress
PYL proteins are important regulators of responses to abiotic stress. Similar to MdSPL13B, the expression of MdPYL4 also peaked after 6 h and gradually decreased thereafter in salt treatment (Fig.S2). Subsequently, we obtained MdPYL4 transgenic Arabidopsis and apple calli by Agrobacterium-mediated transformation and validated by qRT-PCR (Fig.S3). Overexpression of MdPYL4 in apple calli resulted in higher salt tolerance (Fig.6, A). Compared to WT, MdPYL4 transgenic apple calli has a higher fresh weight, less MDA and REC after salt treatment. Consistent with the above results, the salt resistance of MdPYL4-OE Arabidopsis seedlings was much greater than that of Col-0 plants. The effect of salt treatment on MdPYL4-OE Arabidopsis seedlings was relatively weak. Compared with Col-0 plants, MdPYL4-OE Arabidopsis seedlings grew better and had more lateral roots (Fig.6, E–F). Furthermore, MdPYL4-OE Arabidopsis seedlings had a higher fresh weight, lower MDA content and REC . These findings indicate that MdPYL4 exerts a positive regulatory role in enhancing salt tolerance.
Fig. 6. Overexpression of MdPYL4 increases salt tolerance in apple calli and Arabidopsis seedlings
The MdSPL13B gene contains the SBP domain, which can bind to the promoter containing a cis-element (TNCGTACAA) to regulate the expression of downstream genes, with GTAC being the core binding sequence. In previous studies, MdWRKY100 (MD03G1057400) displays a active action in improving salt resistance and gloeosporioide resistance in apple. However, whether MdSPL13B can bind MdWRKY100 to regulate salt tolerance in apple remains unknown. Here, we detected two GTAC motifs in the MdWRKY100 promoter (Fig.7 ,A). Next, we selected two fragments with a length of 200 bp containing the GTAC motif, denoted as S1 and S2, ligated into the pAbAi vector. Subsequently, they were co-transformed into yeast cells with MdSPL13B-pGADT7. Only MdSPL13B-pGADT7 + MdWRKY100-S1/S2-pAbAi could grow on SD/-Leu /-Ura + 200 ng · mL-1 AbA medium (Fig.7, B), which indicates that MdSPL13B can bind to the promoter of MdWRKY100. EMSA also showed that MdSPL13B could bind to GTAC motifs (S1/S2) on the promoter of MdWRKY100, and the binding strength diminished when a gradient of cold probes was added (Fig.7, C–D). Meantime, DLR assays were performed to clarify how MdSPL13B regulates the expression of MdWRKY100. As shown in Fig.7, E, the luminescence activity of MdSPL13B-62SK + pMdWRKY100-S1/S2-LUC was significantly higher than that of the control, which indicates that MdSPL13B can activate the expression of MdWRKY100, thereby increasing salt tolerance.
Fig. 7. MdSPL13B can activate MdWRKY100 transcription
Meanwhile, how the interaction of MdNAC29 or MdPYL4 with MdSPL13B affects the ability of MdSPL13B to activate the MdWRKY100 promoter remains to be probed. Hence, we performed a simple validation using the DLR assay. As shown in Figs. S4 and A, the ability of MdSPL13B to activate the promoter of MdWRKY100 was decreased by the addition of 35S::MdNAC29-GFP compared to the MdSPL13B-62SK + pMdWRKY100-LUC. Similarly, the ability of MdSPL13B to activate the promoter of MdWRKY100 was enhanced after the addition of 35S::MdPYL4-GFP in Figs. S4 and B. These results initially suggested that MdNAC29 and MdPYL4 possibly regulate salt tolerance by weakening or enhancing the ability of MdSPL13B to activate the promoter of MdWRKY100. However, the molecular mechanism by which MdNAC29, MdPYL4, MdSPL13B, and MdWRKY100 jointly regulate salt resistance requires verification by subsequent studies.
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