The Stylo Cysteine-Rich Peptide SgSnakin1 Is Involved in Aluminum Tolerance through Enhancing Reactive Oxygen Species Scavenging

Abstract

Stylo (Stylosanthes spp.) is an important pasture legume with strong aluminum (Al) resistance. However, the molecular mechanisms underlying its Al tolerance remain fragmentary. Due to the incomplete genome sequence information of stylo, we first conducted full-length transcriptome sequencing for stylo root tips treated with and without Al and identified three Snakin/GASA genes, namely, SgSnakin1, SgSnakin2, and SgSnakin3. Through quantitative RT-PCR, we found that only SgSnakin1 was significantly upregulated by Al treatments in stylo root tips. Histochemical localization assays further verified the Al-enhanced expression of SgSnakin1 in stylo root tips. Subcellular localization in both tobacco and onion epidermis cells showed that SgSnakin1 localized to the cell wall. Overexpression of SgSnakin1 conferred Al tolerance in transgenic Arabidopsis, as reflected by higher relative root growth and cell vitality, as well as lower Al concentration in the roots of transgenic plants. Additionally, overexpression of SgSnakin1 increased the activities of SOD and POD and decreased the levels of O₂^·- and H₂O₂ in transgenic Arabidopsis in response to Al stress. These findings indicate that SgSnakin1 may function in Al resistance by enhancing the scavenging of reactive oxygen species through the regulation of antioxidant enzyme activities.

Keywords: ROS; Snakin/GASA family; Stylosanthes; aluminum.

Figure 1

Transcriptome analysis in stylo roots treated with or without 10 µM AlCl₃, and phylogenetic tree of Snakins/GASAs in different plant species. (A) Gene ontology (GO) statistical sort. (B) Phylogenetic analysis of Snakins/GASAs in different plant species. The first two letters of each Snakin/GASA represent the abbreviated species name. The phylogenetic tree was constructed by the MEGA 5.0 program using the neighbor-joining method, with 1000 bootstrap replicates. Red arrows show the positions of SgSnakin1, SgSnakin2, and SgSnakin3. (C) Prediction of disulfide bonds in SgSnakin1. Different colors mean different amino acids, as indicated by different letters.

Figure 2

Effects of duration of Al stress on stylo root growth and the regulation of three SgSnakin genes. (A,B) Phenotype (A) and RRG (relative root growth) (B) of stylo seedlings in response to Al treatments. (C,D) Al–hematoxylin staining (C) and Al concentration (D) of stylo root tips. (E) Time-course analyses of SgSnakin genes transcript levels in root tips. Uniform seedlings were treated with 0 and 10 µM AlCl₃ in hydroponics for 3, 6, 9, and 12 h. Values are means ± SE (n = 4). Different letters represent significant differences (p < 0.05); asterisks indicate significant differences between −Al and +Al treatments. * p < 0.05, 0.001 < ** p < 0.01, *** p < 0.001.

Figure 3

Responses of stylo to different Al concentrations and the regulation of three SgSnakin genes. (A) Al–hematoxylin staining in root tips of stylo seedlings. (B) Al concentration in stylo root tips. Uniform seedlings were treated with 0, 10, 50, and 100 µM AlCl₃ in hydroponics for 12 h. Values are means ± SE (n = 4). Different letters represent significant differences (p < 0.05). (C) Relative expression of three SgSnakins in response to Al dosage. Values are means ± SE (n = 4). Asterisks indicate significant differences between the Al treatments and the 0 µM AlCl₃ control; *** p < 0.001.

Figure 4

Subcellular and histochemical localization of SgSnakin1. (A) Subcellular localization of SgSnakin1. Green fluorescence was observed in transgenic epidermal cells of tobacco leaves and onion expressing 35S::SgSnakin1-GFP and 35S::GFP by confocal laser scanning microscopy; (B) histochemical GUS staining of transgenic Arabidopsis expressing pSgSnakin1::GUS in different Al treatments. −Al: only 0.5 mM CaCl₂ solution; +Al: 0.5 mM CaCl₂ solution with 10 µM AlCl₃ for 48 h.

Figure 5

Effects of SgSnakin1 overexpression on Al tolerance of transgenic Arabidopsis. (A) Phenotype of wild-type (WT) and transgenic Arabidopsis (OX1, OX2, OX3); bar = 0.5 cm. (B) RRG of WT and three OX lines; asterisks indicate significant differences between WT and OX lines. 0.001 < ** p < 0.01. (C) Al concentration in roots of WT and OX lines. Values are means ± SE (n = 4). Asterisks indicate significant differences between WT and OX lines; 0.001 < ** p < 0.01. (D) Hematoxylin staining in root tips of WT and OX lines; bar = 0.5 mm. (E) Observation of dead cells of WT and OX lines; red fluorescence derived from propidium iodide (PI) staining was captured by confocal laser scanning microscopy. Bar = 100 µm. −Al: Onoy 0.5 mM CaCl₂ solution; +Al: 0.5 mM CaCl₂ solution with 10 µM AlCl₃ for 72 h.

Figure 6

Effects of SgSnakin1 overexpression on ROS accumulation and activities of SOD and POD. (A) Nitroblue tetrazolium (NBT) and (B) Diaminobenzidine (DAB) staining was used to represent O₂^·− and H₂O₂ levels in root tips of WT and transgenic Arabidopsis with SgSnakin1 overexpression (OX1, OX2, and OX3). (C,D) SOD (C) and POD (D) activity in roots of WT and three OX lines under different Al treatments. −Al: only 0.5 mM CaCl₂ solution; +Al: 0.5 mM CaCl₂ solution with 10 µM AlCl₃ for 72 h. Values are means ± SE (n = 4). Asterisks indicate significant differences between WT and OX lines. * p < 0.05, 0.001 < ** p < 0.01, *** p < 0.001.