Influence of stripe rust infection on the photosynthetic characteristics and antioxidant system of susceptible and resistant wheat cultivars at the adult plant stage

Abstract

Wheat stripe rust (Puccinia striiformis f. sp. tritici, Pst), is one of the most serious diseases of wheat (Triticum aestivum L.) worldwide. To gain a better understanding of the protective mechanism against stripe rust at the adult plant stage, the differences in photosystem II and antioxidant enzymatic systems between susceptible and resistant wheat in response to stripe rust disease (P. striiformis) were investigated. We found that chlorophyll fluorescence and the activities of the antioxidant enzymes were higher in resistant wheat than in susceptible wheat after stripe rust infection. Compared with the susceptible wheat, the resistant wheat accumulated a higher level of D1 protein and a lower level of reactive oxygen species after infection. Furthermore, our results demonstrate that D1 and light-harvesting complex II (LHCII) phosphorylation are involved in the resistance to stripe rust in wheat. The CP29 protein was phosphorylated under stripe rust infection, like its phosphorylation in other monocots under environmental stresses. More extensive damages occur on the thylakoid membranes in the susceptible wheat compared with the resistant wheat. The findings provide evidence that thylakoid protein phosphorylation and antioxidant enzyme systems play important roles in plant responses and defense to biotic stress.

Keywords: Triticum aestivum L.; antioxidant enzyme; chlorophyll fluorescence; photosystem II; stripe rust.

FIGURE 1

Stripe rust disease symptoms at 14 days post inoculation (dpi) on the susceptible (Sy95-71) and resistant (CN19) wheat cultivars at the adult plant stage. CK, un-inoculated leaves.

FIGURE 2

Chlorophyll (A), net photosynthetic rate (B), relative water content (RWC) (C), and total protein content (D) in inoculated and un-inoculated leaves of Sy95-71 and CN19. Error bars represent the standard deviation based on three biological replicates. Different letters depict significant differences between the susceptible and resistant wheat cultivars (P < 0.05). Statistical analysis was performed using one-way ANOVA followed by Duncan’s multiple range test. CK, un-inoculated wheat plants.

FIGURE 3

Measurement of reactive oxygen species (ROS) after stripe rust infection. Histochemical assays for superoxide anion radicals (${\mathrm{O}}_{\mathrm{2}}^{\mathrm{\cdot –}}$) and hydrogen peroxide (H₂O₂) by nitro blue tetrazolium (NBT) (A) and 3,3-diaminobenzidine (DAB) (B) staining, respectively. Then, ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{\cdot –}}$ production (C) and the H₂O₂ content (D) were measured. Values are means ± SD from three independent biological replicates. Different letters depict significant differences between the susceptible (Sy95-71) and resistant (CN19) wheat cultivars (P < 0.05). CK, un-inoculated wheat plants.

FIGURE 4

Effects of stripe rust infection on the malondialdehyde (MDA) content (A) and electrolyte leakage (B) of the susceptible (Sy95-71) and resistant (CN19) wheat cultivars. Bars represent standard deviations of three independent biological replicates and values followed by different letters are significantly different at P < 0.05 according to Duncan’s multiple range test. CK, un-inoculated wheat plants.

FIGURE 5

Effects of stripe rust infection on the POD, Peroxidase (A); SOD, superoxide dismutase (B); catalase CAT, catalase; (C); APX, ascorbate peroxidase (D); GPX, glutathione peroxidase (E); and GR, glutathione reductase (F) in the susceptible (Sy95-71) and resistant (CN19) wheat cultivars. Bars represent standard deviations of three independent biological replicates and values followed by different letters are significantly different at P < 0.05 according to Duncan’s multiple range test. CK, un-inoculated wheat plants.

FIGURE 6

Effects of stripe rust infection on chlorophyll fluorescence parameters (F_v/F_m; qP, photochemical quenching; NPQ/4, non-photochemical quenching coefficient; and ΦPSII, quantum yield of PSII electron transport) in Sy95-71 and CN19. Quantitative values (±SD) are shown below the individual fluorescence images. CK, un-inoculated wheat plants.

FIGURE 7

Immunoblot analyses of thylakoid proteins in inoculated and un-inoculated wheat plants (Sy95-71 and CN19). Immunoblot analyses of thylakoid membrane proteins were performed using antibodies specific for representative PSI, photosystem I (A); PSII, photosystem II (B); and Rubisco proteins. One microgram of total chlorophyll was loaded into each electrophoretic lane. CK, un-inoculated wheat plants. Rubisco was used as the standard reference for western blotting.

FIGURE 8

Thylakoid protein phosphorylation after wheat stripe rust infection of the susceptible (Sy95-71) and resistant (CN19) wheat cultivars. Thylakoid proteins extracted from the inoculated and un-inoculated wheat plants were fractionated by SDS-PAGE in 12% acrylamide separation gel with 6 M urea. Immunoblot analysis of thylakoid membrane proteins was performed using anti-phosphothreonine antibodies (A). Loading was based on an equal amount of chlorophyll (1 μg chlorophyll). The SDS-PAGE results after Coomassie blue staining (CBS) are shown in the bottom panel (B). CK, un-inoculated wheat plants. (C) Quantification of immunoblot data. Results are presented relative to the amount of respective CK (100%). Asterisks indicates statistically significant differences at the P < 0.05 level. Values are means ± SD from three independent biological replicates.

FIGURE 9

Transmission electron microscope analysis of chloroplasts after stripe rust infection of the susceptible (Sy95-71) and resistant (CN19) wheat cultivars. CK, un-inoculated wheat plants.