GsMAS1 Encoding a MADS-box Transcription Factor Enhances the Tolerance to Aluminum Stress in Arabidopsis thaliana

Abstract

The MADS-box transcription factors (TFs) are essential in regulating plant growth and development, and conferring abiotic and metal stress resistance. This study aims to investigate GsMAS1 function in conferring tolerance to aluminum stress in Arabidopsis. The GsMAS1 from the wild soybean BW69 line encodes a MADS-box transcription factor in Glycine soja by bioinformatics analysis. The putative GsMAS1 protein was localized in the nucleus. The GsMAS1 gene was rich in soybean roots presenting a constitutive expression pattern and induced by aluminum stress with a concentration-time specific pattern. The analysis of phenotypic observation demonstrated that overexpression of GsMAS1 enhanced the tolerance of Arabidopsis plants to aluminum (Al) stress with larger values of relative root length and higher proline accumulation compared to those of wild type at the AlCl₃ treatments. The genes and/or pathways regulated by GsMAS1 were further investigated under Al stress by qRT-PCR. The results indicated that six genes resistant to Al stress were upregulated, whereas AtALMT1 and STOP2 were significantly activated by Al stress and GsMAS1 overexpression. After treatment of 50 μM AlCl₃, the RNA abundance of AtALMT1 and STOP2 went up to 17-fold and 37-fold than those in wild type, respectively. Whereas the RNA transcripts of AtALMT1 and STOP2 were much higher than those in wild type with over 82% and 67% of relative expression in GsMAS1 transgenic plants, respectively. In short, the results suggest that GsMAS1 may increase resistance to Al toxicity through certain pathways related to Al stress in Arabidopsis.

Keywords: Al stress; Arabidopsis thaliana; Glycine Soja; GsMAS1; MADS.

Figure 1

Homology analysis of GsMAS1 protein and other MADS-box transcription factors in plants. (a) Sequence alignment of GsMAS1 protein and MADS-box transcription factors. (b) Phylogenetic analysis of GsMAS1 and MADS-box transcription factors. The sequence alignment of amino acids was carried out by using the software DNAMAN6.0. The phylogenetic tree was constructed by the software MEGA (V6.0) with the neighbor-joining method. The amino acid sequences of MADS-box transcription factors are from the databases of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) and Phytozome (http://phytozome.net/). The information of accession number and species for MADS-box transcription factors is as follows. The proteins of AtAGL13 (AAC49081), AtAGL17 (OAP11731), AtAGL19 (AAG37901), AtAGL21 (NP_195507.1), AtAGL44 (NP_179033.1), AtAP1 (CAA78909), AtSHP1 (OAP06129), AtSEP2 (AAF61626), AtSHP2 (NP_850377), and AtSVP (OAP09056) are from Arabidopsis thaliana. The proteins of Dt2 (NP_001340272), Glyma.08g065300 (KRH42047), Glyma.08g065400 (XP_014634038), Glyma.15g059600 (KRH10635), GmAGL1 (NP_001304521), GmAGL15 (NP_001237033), GmAGL18 (XP_006575259), GmAP1 (XP_003547792), GmFULa (ahi43155), GmMADS28 (NP_001236390), GmMADSI1 (XP_014623536), GmMADSI2 (XP_025981482), GmNMH7 (NP_001236857), GmNMHC5 (XP_006593452), GmSEP1 (AAZ86071) and GmSOC1 (NP_001236377) are from Glycine max, whereas GsMADS23L1 (XP_028204324), GsMADS23L2 (XP_028187089), GsMADS23L4 (XP_028187090), GsMADS27 (RZB82838), and GsMAS1 are from wild soybean (Glycine soja). The proteins of OsMADS8 (Q9SAR1), OsMADS20 (Q2QQA3) and OsMADS27 (XP_015626695) are from Oryza sativa; MTR_2g009890 (AES63546) is from Medicago truncatula, BRADI_5g12437v3 (KQJ82998) is from Brachypodium distachyon.

Figure 2

Expression patterns of GsMAS1 in tissues and under acidic aluminum exposure. (a) Expression pattern analysis of GsMAS1 in soybean tissues. Samples of roots, stems, and leaves are from young seedlings; flowers and pods were taken at the period of blooming and 15 days after soybean flowering, respectively. (b) Expression pattern of GsMAS1 under the AlCl₃ treatments. After seed germination for two days, the seedlings of wild soybean (BW69 line) were transferred to AlCl₃ solutions, which were set as 0, 15, 30, 50, 75, and 100 µM (pH 4.3, 0.5 mM CaCl₂). After AlCl₃ treatments for 6 h, the 6-cm-long roots of seedlings were harvested to analyze the expression levels of GsMAS1. (c) Temporal expression pattern of GsMAS1 under acidic aluminum exposure. The 2-day seedlings after germination were cultured in a solution of 0.5 mM CaCl₂ (pH 4.3) for 24 h, and then they underwent the 50 µM AlCl₃ treatment (pH 4.3, 0.5 mM CaCl₂). The 6-cm-long roots were taken from the seedlings at the processing time nodes of 2, 4, 6, 8, 12, and 24 h. The qRT-PCR was carried out to assess the transcript abundance of GsMAS1 by the 2^−∆∆Ct method with ACT3 as an internal control [92]. Three independent biological experiments were carried out to calculate the relative expression value of GsMAS1. Data are means ± SE, and the asterisks (**) represent a significant difference (p = 0.01).

Figure 3

Subcellular localization of GsMAS1 protein.

Figure 4

Molecular identification for GsMAS1 transgenic lines in Arabidopsis. (a) PCR identification of GsMAS1 transgenic plants. (b) qRT-PCR identification of GsMAS1 transgenic lines. The 3-week-old seedlings of Arabidopsis were used to identify GsMAS1 transgenic plant and lines. M1: DNA maker DL5000; M2: DNA marker DL500; Lanes 1–15: PCR products with different DNA templates set as ddH₂O for lane 1, genomic DNA from wide type for lane 2, genomic DNA from GsMAS1 transgenic plants for lanes 3–15. WT: wild type of Arabidopsis Columbia-0; L4 to L13: six transgenic lines of GsMAS1 in T₃ generation. Data are means ± SE. Error bars represent the standard error of four replicates.

Figure 5

GsMAS1 enhanced the resistance of Arabidopsis plants to Al stress (a) The phenotypes of GsMAS1 transgenic lines tolerant to Al stress. (b) Statistical analysis of relative root elongation. (c) The determination of free proline content. WT: wild type of Arabidopsis (Col-0); L4, L9, L13: GsMAS1 overexpression transgenic lines of T₃ generations. The two-day seedlings after seed germination were transferred to culture medium containing AlCl₃ (pH 4.3, 0.5 mM CaCl₂) at 0, 50, 100, 150, and 200 µM, individually. After being cultured for seven days, phenotypic images and contents of free proline for the GsMAS1 transgenic lines were recorded and/or determined for statistical analysis. Data were represented as the means ± SE of three biological replicates (Student’s test, ** p = 0.05). The programs of Image J, SPSS20, and EXCEL2000 were used to measure and/or analyze the data.

Figure 6

Expression patterns of Al stress responsive genes regulated by GsMAS1. Arabidopsis seeds of WT and the GsMAS1 transgenic line L9 of T₃ generation were sown in 1/2 MS (Murashige-Skoog) culture media and cultured in the chamber room. The two-week-old seedlings were subjected to the treatment of solutions with or without 50 µM AlCl₃ (pH 4.3, 0.5 mM CaCl₂) for 24 h. The seedling samples from Arabidopsis plants were taken to extract total RNA. The transcription abundance of investigated genes was quantified by qRT-PCR using ACT3 as the inner reference gene. The quantitative variation between the examined replicates was determined by the 2^−∆∆Ct method. The details of specific primers for GsMAS1, ACT3 and measured genes are listed in Table S1 (Additional file 3). WT: wild type; GL9: GsMAS1 transgenic line L9 of T₃ generation. The data were represented as the mean ± SE of three biological replicates (t-test, ** p = 0.05).