GsMYB10 encoding a MYB-CC transcription factor enhances the tolerance to acidic aluminum stress in soybean

Abstract

Background: MYB transcription factors (TFs) play crucial roles in the response to diverse abiotic and biotic stress factors in plants. In this study, the GsMYB10 gene encoding a MYB-CC transcription factor was cloned from wild soybean BW69 line. However, there is less report on the aluminum (Al)-tolerant gene in this subfamily.

Results: The GsMYB10 gene was up-regulated by acidic aluminum stress and rich in the roots with a constitutive expression pattern in soybean. It was found that GsMYB10 protein contains the MYB and coiled-coil (CC) domains, localizes in the nucleus and holds transcriptional activity. The analysis of the transgenic phenotype revealed that the taproot length and root fresh weights of the GsMYB10-OE plants were greater than those of the wild type when subjected to AlCl₃ treatments. While the accumulation of Al³⁺ in root tip of GsMYB10 transgenic plants (59.37 ± 3.59 µg/g) significantly reduced compared with that of wild type (80.40 ± 3.16 µg/g) which were shallowly stained by hematoxylin under the treatments of AlCl₃. Physiological indexes showed that the proline content significantly increased 39-45% and the malondialdehyde content significantly reduced 37-42% in GsMYB10-OE plants compared with that of wild type. Transcriptomic analysis showed that overexpression of GsMYB10 induced a large number of differentially expressed genes (DEGs) with Al-treatment, which were related to wall modification related genes included PGs (such as Glyma.19g006200, Glyma.05g005800), XTHs (such as Glyma.12g080100, Glyma.12g101800, Glyma.08g093900 and Glyma.13g322500), NRAMPs and ABCs.

Conclusions: In summary, the data presented in this paper indicate that GsMYB10, as a new soybean MYB-CC TF, is a positive regulator and increases the adaptability of soybeans to acidic aluminum stress. The findings will contribute to the understanding of soybean response to acidic aluminum stress.

Keywords: Aluminum stress; MYB-CC family; Soybean; Transcription factor.

Fig. 1

The phylogenetic tree of MYB-CC family proteins from soybean, rice and Arabidopsis. (A) The phylogenetic tree was constructed using MEGA X with the Maximum likelihood (ML) method. The three groups corresponding to three branches are marked by numbers (I-III). Bootstrap values in percentages (1000 replicates) are indicated on the nodes. Different subgroups use different colors of clades to distinguish. B and C are comparative alignments of conserved domain sequences of known functional genes in the MYB-CC family. (B) The MYB domain and CC (coiled-coil) domain of GsMYB10 and other MYB-CC proteins. The Genbank accession numbers of proteins or genes loci for other species are as follows: AtAPL (At1g79430), GmPHR1 (Glyma.19g122700), GmPHR25 (Glyma.15g123100), AtMYR1 (At5g18240), AtMYR2 (At3g04030), OsMYBc (LOC_Os09g12770), OsPHR1 (LOC_Os03g21240), OsPHR2 (LOC_Os07g25710), OsPHR3 (LOC_Os02g04640), OsPHR4 (LOC_Os06g49040), AtPHR1 (At4g28610), AtUNE16 (At4g13640)

Fig. 2

Expression patterns analysis of GsMYB10 in wild soybean BW69 line. (A) Tissue expression pattern of GsMYB10. The samples of roots, stems, leaves, flowers, pods and tops (apical tissues) were harvested during the pod stage. (B) The three-day seedlings were cultured in 0.5 mM CaCl₂ solution containing 50 µM AlCl₃ (pH 4.5) for 0, 1, 2, 4, 8, 12 and 24 h. (C) Soybean seedlings were treated with 0, 25, 50, 75 and 100 µM AlCl₃ in 0.5 mM CaCl₂ solution (pH4.5) for 8 h. Total RNA was extracted from root apices (0–6 cm). The data were represented as the mean ± SD of three biologic replicates. Student’s t-test was used to calculate the p-values, *, P<0.05;**, P < 0.01

Fig. 3

Transcriptional activity assays and subcellular localization of GsMYB10 protein. (A) Transcriptional activation activity of GsMYB10 protein in yeast cells. (B) Subcellular localization of the 35S::GsMYB10-GFP fusion protein in leaf epidermal cells of Nicotiana benthamiana. Leaf epidermal cells transformed with 35S::GFP were used as a control. The RFP was nuclear localization protein marker (PJIT-mCherry-Nuc). Scale bars = 10 μm

Fig. 4

Physiological indexes related to aluminum stress change. The hematoxylin staining (A) root Al content (B) proline concentration (C) and MDA concentration (D) in transgenic plants and wild type under Al stress. Both WT and GsMYB10 transgenic lines were exposed to 0.5 mM CaCl₂ solution with 25 µM AlCl₃ (pH 4.5) for 8 h (in A and B) or 24 h (in C and D). Data were represented as mean ± SD of three biological replicates. Student’s t-test was used to calculate the p-values, ns = no significant difference

Fig. 5

Overexpression of GsMYB10 conferred enhanced aluminum tolerance in transgenic plants. (A) The phenotype of GsMYB10 and wild type soybean seedlings which were treated using the solution including 0 µM, 25 µM and 50 µM AlCl₃ in 0.5 mM CaCl₂ solution (pH4.5) for 7 days. The taproot length (B), the fresh weight (C) and the aluminum content (D) of the underground part of transgenic lines (L1, L2) and wild type (WT). Data were represented as mean ± SD of three biological replicates, Student’s t-test was used to calculate the p-values, ns = no significant difference

Fig. 6

Transcriptome analysis of differentially expressed genes regulated by GsMYB10. (A) Upset diagram showing number of differentially expressed genes (DEGs) in different groups. (B) GO terms which were statistically enriched in GsMYB10 regulated genes involved in aluminum treatment which were identified using the DEGs in A. (C) Heatmap of DEGs in the GO terms of xyloglucan metabolic process, extracellular region, plant-type vacuole, cellulose catabolic process, polygalacturonase activity. (D) Expression levels of the Al-responsive genes in GsMYB10 overexpression transgenic soybean plants. Data were represented as mean ± SD of three biological replicates. Student’s t-test was used to calculate the p-values, *, P < 0.05; **, P < 0.01