Functional Conservation and Divergence of Soybean GmSTOP1 Members in Proton and Aluminum Tolerance

Abstract

Proton (H⁺) and aluminum (Al) rhizotoxicity are two major factors limiting crop production in acid soils. Orthologs of the zinc-finger transcription factor, Sensitive To Proton Rhizotoxicity1 (STOP1), have been found to play an essential role in the tolerance to both stresses by regulating the transcription of multiple H⁺ and Al tolerant genes. In the present study, color three GmSTOP1 homologs were identified in the soybean genome. All three GmSTOP1 exhibited similar properties as reflected by the harboring of four potential zinc finger domains, localizing in the nucleus, and having transactivation activity. Expression profiling showed that H⁺ stress slightly modulated transcription of all three GmSTOP1s, while Al significantly up-regulated GmSTOP1-1 and GmSTOP1-3 in root apexes and GmSTOP1-3 in basal root regions. Furthermore, complementation assays in an Arabidopsis Atstop1 mutant line overexpressing these GmSTOP1s demonstrated that all three GmSTOP1s largely reverse the H⁺ sensitivity of the Atstop1 mutant and restore the expression of genes involved in H⁺ tolerance. In contrast, only GmSTOP1-1 and GmSTOP1-3 could partially recover Al tolerance in the Atstop1 mutant. These results suggest that the function of three GmSTOP1s is evolutionarily conserved in H⁺ tolerance, but not in Al tolerance.

Keywords: Al rhizotoxicity; GmSTOP1; proton rhizotoxicity; soybean; transcription factor.

FIGURE 1

Phylogenetic tree and amino acid alignments of predicted C₂H₂ zinc finger domains in plant STOP1s. (A) Phylogenetic tree was generated based on an amino-acid alignment with STOP1 orthologs from several plant species. (B) Alignment of the amino acid sequences of predicted C2H2 zinc finger domains in STOP1 proteins. Black background indicates identical residues. Asterisks indicate conserved Cys and His residues of C2H2 motifs. The plant STOP1 proteins aligned include representatives from Glycine max (GmSTOP1-1, XP_006588359.1; GmSTOP1-2, XP_006598713.1; GmSTOP1-3, XP_014628358.1), Arabidopsis thaliana (AtSTOP1, NP_174697.1), Nicotiana tabacum (NtSTOP1, AB811781), Lotus japonicus (LjSTOP1, BAN67817.1), Vigna umbellata (VuSTOP1, KP637172), Camellia sinensis (CsSTOP1, BAN67815.1), Populus nigra (PnSTOP1, BAN67813.1), Eucalyptus (EguSTOP1, BAO56822.1), Triticum aestivum (TaSTOP1-A, AGS15201.1; TaSTOP1-B, AGS15202.1; TaSTOP1-D, AGS15195.1), Oryza sativa (OsART1, AB379846), and Physcomitrella patens (PpSTOP1, BAN67814.1).

FIGURE 2

Subcellular localization and transactivation assay of GmSTOP1s. (A) 35S::GmSTOP1s-GFP and 35S::GFP (control) constructs were introduced individually into Nicotiana benthamiana leaves. Scale bars: 20 μm. (B) Transactivation assay of GmSTOP1-1 (I), GmSTOP1-2 (II), GmSTOP1-3 (III), and control (IV). β-galactosidase activity is indicated by blue color on the filter paper using X-gal as the substrate.

FIGURE 3

Expression patterns of GmSTOP1s in response to Al toxicity. (A) Relative expression of GmSTOP1s in root tips (0–2 cm), basal roots (>2 cm) and leaves after 4 h of –Al (0 μM) or +Al (50 μM) treatment. (B) Relative expression of GmSTOP1s in soybean root tips (0–2 cm) treated with different concentrations of Al for 4 h. (C) Relative expression of GmSTOP1s in soybean roots tips (0–2 cm) in response to Al (50 μM) for different treatment times. Asterisks indicate significant differences between the +Al treatment and –Al control (^∗0.01 < P < 0.05; ^∗∗0.001 < P < 0.01; ^∗∗∗P < 0.001).

FIGURE 4

Effects of low pH and Al stresses on the growth of Arabidopsis Atstop1 mutant plants overexpressing GmSTOP1s. Uniform seedlings with ∼1.5 cm root lengths were treated with low pH (pH 4.7) and Al stresses (2 μM Al and pH 5.0) for 7 days. The phenotypes of wild-type (WT), Atstop1 and GmSTOP1 overexpressing Atstop1 lines in response to H⁺ and Al were photographed (A). Root elongation for each line was measured under control (B), low pH (C), and Al (D) treatments. Each bar represents the mean of four biological replicates with standard error. Asterisks indicate significant differences in comparison to the Atstop1 mutant (^∗0.01 < P < 0.05; ^∗∗0.001 < P < 0.01; ^∗∗∗P < 0.001). Scale bars: 5 mm.

FIGURE 5

Transcriptional accumulation of genes regulated by GmSTOP1s under low pH and Al stresses. Wild-type (WT), Atstop1 mutant and complemented lines overexpressing GmSTOP1-1 (#5 and #6), GmSTOP1-2 (#12 and #15), and GmSTOP1-3 (#47 and #54) were exposed to low pH (pH 4.7) and Al treatments (AlCl₃: 2 μM; pH 5.0) for 24 h. Transcript abundances of GDH1, GDH2, GABA-T and AtNADP-ME2 were quantified from plants grown in the low pH treatment, while expression levels of PMI (At2g45220), AtTDT (At5g47560), AtNADP-ME2 (At5g11670), and AtMATE (At1g51340) were quantified in Al treated samples. UBQ1 transcript levels were used as the internal standard. Data are expressed as means of four replicates. Asterisks indicate significant differences in comparison to the Atstop1 mutant (^∗0.01 < P < 0.05; ^∗∗0.001 < P < 0.01; ^∗∗∗P < 0.001).