Vegetative cell wall protein OsGP1 regulates cell wall mediated soda saline-alkali stress in rice

Abstract

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.

Keywords: Abiotic stress; Gene cloning; Genetic transformation; OsGP1; Rice.

Figure 1. Schematic diagram of the T-DNA region in pGWB5-OsGP1 plasmid.

LB, T-DNA left border; RB, T-DNA right border.

Figure 2. Electropherogram of OsGP1 DNA.

(A) Electropherogram of PCR amplification product from rice cDNA. M, Marker, DL2000; #1 and #2, PCR amplification products of OsGP1. (B) Recombinant pMD18-T-OsGP1 plasmid digested with SalI/BamHI. M: Marker, DL5000; #1, pMD18-T-OsGP1 plasmid; #2, pMD18-T-OsGP1 plasmids digested with SalI/BamHI.

Figure 3. Bioinformatics analysis of OsGP1.

(A), Nucleotide sequence and deduced amino acid sequence of OsGP1 coding region. (B) Secondary structure prediction. Blue, α-helix (h); green, β-turn (t); red, extended strand (e); purple, random coil (c). (C) Tertiary structure prediction. The model was constructed with Q851X5_ORYSJ as the template. The Global Model Quality Estimate (GMQE) score is 0.62 and the colours represent different model confidence scores. (D) Prediction of conserved domains of OsGP1 protein. Red, signal peptide; blue, transmembrane region; purple, low complexity region. (E) Prediction of the signal peptide. The S-score is higher in the signal peptide region, the C-score is highest at the cleavage site, the Y-score is a parameter that comprehensively considers the S-score and C-score, and the Y-max score is the putative cleavage site. (F) Protein interaction relationship. The red node is OsGP1 (OsJ_13402), and the nodes in other colors represent the ten proteins predicted to interact with OsGP1. Different coloured lines represent different interaction types. Black, co-expression; purple, experimentally determined; yellow, textmining. (G) Analysis of OsGP1 promoter. All cis-acting elements selected for display have a PlantCARE matrix score ≥5.

Figure 4. Phylogenetic tree and multiple sequence alignment of OsGP1 protein.

(A) Phylogenetic tree. (B) Multiple sequence alignment of the sequences in the same branch with OsGP1. Sequences were aligned using Clustal Omega with default settings. The phylogenetic tree was constructed in MEGA6.0 by using the neighbour-joining (NJ) method (bootstrap value, 1,000).

Figure 5. Subcellular localization of OsGP1-GFP fusion protein in onion epidermal cells.

GFP and OsGP1-GFP driven by 35S promoter under fluorescence, bright field, and merged views. Bar, 50 μm.

Figure 6. Identification of OsGP1 transgenic rice lines.

(A) Identification of T0 generation transgenic rice. The integration of OsGP1 was detected by PCR using specific and vector primers. M, Marker, DL2000. WT, Wild-type line, Lj11; #1-#6, OsGP1 transgenic lines. (B) The expression level of OsGP1 in T3 generation transgenic rice were detected by qRT–PCR. The expression level of Lj11 was set to 1, and the Os18sRNA gene was used as an internal reference control. Data represent the mean ± SD of three replicates. Statistical analyses were performed using Student’s t test: **P ≤ 0.01.

Figure 7. Tolerance analysis of rice overexpressing OsGP1 to soda saline-alkali stress.

The three-leaf stage overexpression lines T3-#2, #4, #5 and Lj11 seedlings were treated with different ratios of SAE for 7 days, and water as control. (A) Growth phenotypes. (B) Fresh weight of five seedlings. (C) Plant height. (D) MDA content. (E) SOD activity. (F) POD activity. Data show the mean ± SEM of three replicates. Statistical differences are labelled with different letters using Tukey test (P ≤ 0.05, one-way ANOVA).