Effect of Singlet Oxygen on the Stomatal and Cell Wall of Rice Seedling Under Different Stresses

Abstract

Singlet oxygen (¹O₂), a reactive oxygen species, can oxidize lipids, proteins, and DNA at high concentrations, leading to cell death. Despite its extremely short half-life (10^-5 s), ¹O₂ acts as a critical signaling molecule, triggering a retrograde pathway from chloroplasts to the nucleus to regulate nuclear gene expression. In this study, rice seeds were treated with 0, 5, 20 and 80 μM Rose Bengal (RB, a photosensitizer) under moderate light for 3 days to induce ¹O₂ generation. Treatment with 20 μM RB reduced stomatal density by approximately 25% in three-leaf-stage rice seedlings, while increasing the contents of pectin, hemicellulose, and cellulose in root cell walls by 30-40%. Under drought, salinity, or shading stress, 20 μM RB treatment significantly improved rice tolerance, as evidenced by higher relative water contents (49-58%) and chlorophyll contents (60-76%) and lower malondialdehyde (37-43%) and electrolyte leakage (29-37%) compared to the control. Moreover, RT-qPCR analysis revealed that the significant up-regulation of stomatal development genes (OsTMM and OsβCA1) and cell wall biosynthesis genes (OsF8H and OsLRX2) was associated with RB-induced ¹O₂ production. Thus, under controlled environmental conditions, ¹O₂ may regulate stomatal development and cell wall remodeling to enhance rice tolerance to multiple abiotic stresses. These results provide new perspectives for the improvement of rice stress tolerance.

Keywords: Rose Bengal; cell wall; rice; singlet oxygen; stomatal density.

Figure 1

¹O₂ levels of rice seeds under different RB concentration treatments. (A) Representative images of rice seeds stained with SOSG solution under various RB concentration treatments; Bar = 200 μm; ¹O₂ fluorescence signal quantification results (B), with the ¹O₂ fluorescence signal expression level of control normalized to 1; 0, 5, 20, and 80 μM represent the concentrations of RB. Error bars in the graphs indicate the mean ± standard deviation (SD) of ten biological replicates from independent rice plants, and different lowercase letters denote significant differences at the 0.05 (p < 0.05) level.

Figure 2

The effect of RB on stomatal traits in rice seedlings. (A) Stomatal images of 14-day rice seedling leaves under a microscope, with stomata pseudocolored in green for ease of identification. Bar = 20 μm. Statistics were obtained for (B) stomatal density and stomatal index (C). CK, control; 5, 20, and 80 μM represent the concentrations of RB. Error bars in the plots indicate the mean ± SD of ten biological replicates from independent rice plants, and different lowercase letters indicate significant differences at the 0.05 (p < 0.05) level.

Figure 3

The effect of RB on the cell wall of rice roots. (A) Propidium iodide (PI) staining of cell walls from 14-day-old rice roots. Bar = 100 μm. Quantification of PI fluorescence signals (B), pectin (C), hemicellulose (D), and cellulose (E) content in cell wall fractions of rice roots. CK, control; 5, 20, and 80 μM represent the concentrations of RB; PI fluorescence signals are expressed as a percentage of CK levels, which were normalized to 1. FW, fresh weight. DW, dry weight. Error bars in the graphs indicate the mean ± SD of three biological replicates, and different lowercase letters denote significant differences at the 0.05 (p < 0.05) level.

Figure 4

Moderate ¹O₂ enhanced rice tolerance to drought, salinity, and shade stress. The phenotype changes in three-week-old rice seedlings under no stress (A), drought stress (B), salt stress (C), and shade stress (D) were observed after 14 days. Bar = 5 cm. The fresh weight (E), dry weight (F), and moisture content (G), as well as the chlorophyll a, chlorophyll b, and carotenoid content (H–J) of the rice seedlings are also presented. 0, 5, 20, and 80 μM represent the concentrations of RB. FW, fresh weight. The error bars in the graph indicate the mean of three biological replicates ± SD, and different lowercase letters denote significant differences at the 0.05 (p < 0.05) level.

Figure 5

Oxidative damage parameters of rice seedlings under three stress conditions. (A) O₂⁻ and (B) H₂O₂ staining in leaves. Bar = 5 cm. Malondialdehyde content (C), relative electrical conductivity (D), and Proline content (E). CK, control; 5, 20, and 80 μM represent the concentrations of RB. FW, fresh weight. Error bars in the graphs indicate the mean ± SD of three biological replicates, and different lowercase letters denote significant differences at the 0.05 (p < 0.05) level.

Figure 6

Alterations in stomatal characteristics and cell walls of rice seedlings subjected to drought, salt, and shade stresses. (A) Microscopic images of stomata on rice leaves under normal conditions and those exposed to drought, salt, and shade stresses. Bar = 10 μm. Stomatal conductance (B) and stomatal density (C) of rice seedlings. Contents of cell wall components, pectin (D), hemicellulose (E), and cellulose (F), in rice seedlings under normal conditions and each stress condition. Concentrations of 0, 5, 20, and 80 μM represent the RB levels. FW, fresh weight. Error bars in the graphs denote the mean ± SD of three biological replicates, with different lowercase letters indicating significant differences at the 0.05 (p < 0.05) level.

Figure 7

The effects of RB on genes associated with stomatal development and cell wall synthesis in rice under various stress conditions were examined. Quantitative real-time PCR analysis was employed to assess the expression levels of representative genes OsTMM (A), OsβCA1 (B), OsF8H (C), and OsLRX2 (D). The expression levels of the control seedlings were normalized to 1. Error bars in the graphs represent the mean ± SD of three biological replicates, with different lowercase letters denoting significant differences at the 0.05 (p < 0.05) level.