This study identified a new bHLHm1 transcription factor MdSAT1 which functioned in mediating tolerance to salt and drought resistance.
Changes in the expression of stress-related genes play crucial roles in response to environmental stress. Basic helix-loop-helix (bHLH) proteins are the largest superfamily of transcription factors and a large number of bHLH proteins function in plant responses to abiotic stresses. We identified a new bHLHm1 transcription factor from apple and named it MdSAT1. β-Glucuronidase (GUS) staining showed that MdSAT1 expressed in various tissues with highly expressed in leaves. Promoter analysis revealed that MdSAT1 contained multiple response elements and its transcription was induced by several environmental cues, particularly salt and drought stresses. Overexpression of MdSAT1 in apple calli and Arabidopsis resulted in a phenotype of increased tolerance to salt and drought. Altering abscisic acid (ABA) treatment increased the sensitivity of MdSAT1-OE Arabidopsis to ABA, and heavy metal stress, osmotic stress, and ethylene did not participate in MdSAT1 mediated plant development. These findings reveal the abiotic stress functions of MdSAT1 and pave the way for further functional investigation.
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Calcium-dependent protein kinase
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Salt overly sensitive pathway
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This work was supported by Agricultural Variety Improvement Project of Shandong Province (2019LZGC007), Natural Science Foundation of China (Grants 31972378), Major Program of Shandong Provincial Natural Science Foundation (ZR2017ZC0328, ZR2018MC021), and Ministry of Agriculture of China (CARS-27).
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Fig. S1 Tissue expression pattern of MdSATs. a Tissue (stems, roots, leaves, flowers, and fruits) expression analysis of MdSAT1 using MdUBQ as control gene by qRT-PCR in Gala. Take the expression of MdSATs in the bud as the relative value, and analyze the significant difference of the expression of MdSAT1 in other tissues. b Tissue expression analysis of MdSAT1 by GUS staining in ProMdSAT1::GUS transgenic Arabidopsis. c Tissue (stems, roots, leaves, flowers, and fruits) expression analysis of MdSAT1-2, MdSAT1-3, and MdSAT1-4 using MdACT as control gene by qRT-PCR in Gala. d Tissue (stems, roots, leaves, flowers, and fruits) expression analysis of MdSAT1-2, MdSAT1-3, and sMdSAT1-4 using MdUBQ as control gene by qRT-PCR in Gala. Take the expression of MdSAT1-2 in various tissues as the relative value, and analyze the significant difference of the expression of MdSAT1-3 and MdSAT1-4 in various tissues. Each set of experiments was repeated three times. Error bars represent standard deviations and * significant values at P < 0.05, ** at P < 0.01 (JPG 837 KB)
Fig. S2 The abiotic stress response of MdSAT1. The expression of MdSAT1 with MdUBQ as the control gene by qRT-PCR in Gala treated with 100 mM NaCl (a), 6% PEG (b), 250 mM DL mannitol (c), 4 °C (d), 50 μM Cd (e), 50 μM Cu (f), 200 μM ABA (g) and 100 μM ETH (h), respectively. Take the expression of MdSAT1 in each time period under the control treatment as a relative value, and analyze the significant difference of the expression of MdSAT1 in each time period under other treatments. Each set of experiments was repeated three times. Error bars represent standard deviations and * significant values at P < 0.05, ** at P < 0.01 (JPG 1748 KB)
Fig. S3 GUS staining in ProMdSAT1::GUS transgenic Arabidopsis. a The ProMdSAT1::GUS transgenic Arabidopsis treated with for 48 h. b The GUS activity of MdSAT1 of a. Take the GUS activity of MdSAT1 under 23 °C treatment for 0 h as the relative value, and analyze the significant difference of the CUS activity of MdSAT1 under other treatment times. Each set of experiments was repeated three times. Error bars represent standard deviations and * significant values at P < 0.05 (JPG 768 KB)
Fig. S4 The expression of MdSAT1 in transgenic plants. a The expression of MdSAT1 with MdACT as the control gene by qRT-PCR in transgenic calli. b The expression of MdSAT1 using MdACT as the control gene by qRT-PCR in transgenic Arabidopsis plants. c The expression of MdSAT1 using MdUBQ as the control gene by qRT-PCR in transgenic calli. d The expression of MdSAT1 with MdUBQ as the control gene by qRT-PCR in transgenic Arabidopsis plants. Take the expression of MdSAT1 in control lines as the relative value, and analyze the significant difference of the expression of MdSAT1 under MdSAT1 transgenic plants. Each set of experiments was repeated three times. Error bars represent standard deviations and * significant values at P < 0.05, ** at P < 0.01 (JPG 835 KB)
Fig. S5 The expression of stress-related genes in Col, EV, and MdSAT1-OE plants. The expression of SOS1, SOS3, CDPK6, ABI1, ABI2, and PLC5 with AtUBC as the control gene by qRT-PCR in Col, EV, and MdSAT1-OE plants. b The expression of ZEP, NCED9, AAO3, P450, RD16, RD29A, KIN2, and DREB2 using AtUBC as the control gene by qRT-PCR in Col, EV, and MdSAT1-OE plants. The expression level of the measured gene in the control line was taken as the relative value, and the significant difference was analyzed about the expression level of the measured gene in the EV and MdSAT1 transgenic lines. Each set of experiments was repeated three times. Error bars represent standard deviations and * significant values at P < 0.05 (JPG 946 KB)
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Yang, YY., Zheng, PF., Ren, YR. et al. Apple MdSAT1 encodes a bHLHm1 transcription factor involved in salinity and drought responses. Planta 253, 46 (2021). https://doi.org/10.1007/s00425-020-03528-6
- Abiotic stress