Salicylic acid reverses pollen abortion of rice caused by heat stress

Abstract

Background: Extremely high temperatures are becoming an increasingly severe threat to crop yields. It is well documented that salicylic acid (SA) can enhance the stress tolerance of plants; however, its effect on the reproductive organs of rice plants has not been described before. To investigate the mechanism underlying the SA-mediated alleviation of the heat stress damage to rice pollen viability, a susceptible cultivar (Changyou1) was treated with SA at the pollen mother cell (PMC) meiosis stage and then subjected to heat stress of 40 °C for 10 d until 1d before flowering.

Results: Under control conditions, no significant difference was found in pollen viability and seed-setting rate in SA treatments. However, under heat stress conditions, SA decreased the accumulation of reactive oxygen species (ROS) in anthers to prevent tapetum programmed cell death (PCD) and degradation. The genes related to tapetum development, such as EAT1 (Eternal Tapetum 1), MIL2 (Microsporeless 2), and DTM1 (Defective Tapetum and Meiocytese 1), were found to be involved in this process. When rice plants were exogenously sprayed with SA or paclobutrazol (PAC, a SA inhibitor) + H₂O₂ under heat stress, a significantly higher pollen viability was found compared to plants sprayed with H₂O, PAC, or SA + dimethylthiourea (DMTU, an H₂O₂ and OH· scavenger). Additionally, a sharp increase in H₂O₂ was observed in the SA or PAC+ H₂O₂ treatment groups compared to other treatments.

Conclusion: We suggest that H₂O₂ may play an important role in mediating SA to prevent pollen abortion caused by heat stress through inhibiting the tapetum PCD.

Keywords: H2O2; Heat stress; Oryza sativa L.; Pollen viability; Salicylic acid; Tapetum.

Fig. 1

Effect of SA on the pollen viability and seed-setting rate of rice under heat stress at the pollen mother cell meiosis stage. a, the images of pollen grains in rice plants sprayed with salicylic acid under control and heat stress; a-f and g-i were the images of pollen grains of rice plants under control and heat stress, respectively. b, the images of rice plants with panicles sprayed with SA under control and heat stress. a and c, the images of rice plant sprayed with H₂O (SA0); b and d, the images of rice plants and panicles sprayed with 10 mM SA (SA10). c, the data in the figure (a) and (b) were shown as the mean of ten and three replicates, respectively. Vertical bars denote standard deviations (n = 10 and 3 in (a) and (b), respectively). Different letters indicate significant differences between the SA treatments under control and heat stress (P < 0.05)

Fig. 2

Transmission electron micrographs of cross-sections through anthers of rice plants at the vacuolization microspore stages sprayed with SA under control and heat stress. a-h, anthers under control pretreatment with different SA concentrations; i-p, anthers under heat stress pretreatment with different SA concentrations. Bar = 2μm. P, pollen grain; T, tapetum; M, middle layer; E, endothecium

Fig. 3

Detection of fragmented DNA via TUNEL assay in the tapetum of the anther under control and heat stress. TUNEL-positive signal was marked by a white arrow. a and b, control with 0 and 10 mM SA respectively; c and d, heat stress pretreatment with 0 and 10 mM SA respectively

Fig. 4

Effect of SA on the caspase 3 activity of anthers in rice plants in response to heat stress. Vertical bars denote standard deviations (n = 3). Different letters indicate significant differences between the SA treatments under control and heat stress (P < 0.05)

Fig. 5

Effect of SA on the reactive oxygen species of anthers under control and heat stress. The anthers were incubated with 5 μM DCFH-DA, and were measured after 30 min by a fluorescence microscope. a, the fluorescence images of the anthers; b, these data were obtained from the fluorescence images (n = 10). Vertical bars denote standard deviations (n = 10). Different letters indicate significant differences between SA treatments under control and heat stress (P < 0.05)

Fig. 6

Effect of SA on the MDA concentration of anthers in response to heat stress. Vertical bars denote standard deviations (n = 3). Different letters indicate significant differences between the SA treatments under control and heat stress (P < 0.05)

Fig. 7

Effect of SA on activities of the antioxidant enzyme including SOD (a), POD (b), CAT(c) and APX (d) in anthers of rice under control and heat stress. Vertical bars denote standard deviations (n = 3). Different letters indicate significant differences between the SA treatments under control and heat stress (P < 0.05)

Fig. 8

Effects of SA on the expression levels of tapetum development genes in rice anther in response to heat stress. a, EAT1 gene (Eternal Tapetum 1); b, MIL2 gene (Microsporeless 2); c, DTM1 gene (Defective Tapetum and Meiocytese 1). Vertical bars denote the standard deviation (n = 3). A t-test is conducted to compare difference between control and heat stress. * denotes P < 0.05, ** denotes P < 0.01

Fig. 9

The changes in pollen viability, MDA and H₂O₂ of rice plants sprayed with H₂O, SA, DMTU, H₂O₂, and PAC along or together under control and heat stress. A-(a-j), the photos of pollen grains was stained with 1% with KI/I₂ by a microscope (Leica, DM4000). A-(a-e), pollen grains under control condition. A-(f-j), pollen grains under heat stress condition. B-a and B-b, MDA and H₂O₂ at anther respectively. The data in the Fig. (A-k) were shown as the mean of ten replicates and Fig. (B-a and b) were shown for three replicates. DMTU, dimethylthiourea, an H₂O₂ and OH· scavenger. PAC, paclobutrazol, a SA inhibitor. Different letters indicate significant differences between treatments under control and heat stress (P < 0.05)

Fig. 10

Descriptive model of the salicylic acid functions toward preventing pollen abortion of rice in response to heat stress. Under heat stress, H₂O₂ is significantly increased in anthers induced by SA under heat stress, which in turn enhances the antioxidant capacity to scavenge excessive ROS. This can inhibit PCD in anthers, and thus prevent tapetum degradation caused by heat stress. Genes such as EAT1, MIL2, and DTM1 are involved in the process of SA-preventing tapetum degradation caused by heat stress, which may be independent of H₂O₂. H₂O₂, hydrogen peroxide; MDA, Malondialdehyde; Casp3, caspase 3 activity; MIL2, Microsporeless 2; EAT1, Eternal Tapetum; DTM1, Defective Tapetum and Meiocytese. The arrow mark “→” indicates induction, while “⊣” indicates inhibition