Stress Sensitivity Is Associated with Differential Accumulation of Reactive Oxygen and Nitrogen Species in Maize Genotypes with Contrasting Levels of Drought Tolerance

Abstract

Drought stress decreases crop growth, yield, and can further exacerbate pre-harvest aflatoxin contamination. Tolerance and adaptation to drought stress is an important trait of agricultural crops like maize. However, maize genotypes with contrasting drought tolerances have been shown to possess both common and genotype-specific adaptations to cope with drought stress. In this research, the physiological and metabolic response patterns in the leaves of maize seedlings subjected to drought stress were investigated using six maize genotypes including: A638, B73, Grace-E5, Lo964, Lo1016, and Va35. During drought treatments, drought-sensitive maize seedlings displayed more severe symptoms such as chlorosis and wilting, exhibited significant decreases in photosynthetic parameters, and accumulated significantly more reactive oxygen species (ROS) and reactive nitrogen species (RNS) than tolerant genotypes. Sensitive genotypes also showed rapid increases in enzyme activities involved in ROS and RNS metabolism. However, the measured antioxidant enzyme activities were higher in the tolerant genotypes than in the sensitive genotypes in which increased rapidly following drought stress. The results suggest that drought stress causes differential responses to oxidative and nitrosative stress in maize genotypes with tolerant genotypes with slower reaction and less ROS and RNS production than sensitive ones. These differential patterns may be utilized as potential biological markers for use in marker assisted breeding.

Keywords: drought stress; maize seedlings; reactive nitrogen species; reactive oxygen species.

Figure 1

Photosynthetic parameters of seedling leaves from the sensitive genotypes, B73 and Lo1016, and the tolerant ones, Lo964 and Va35, under well-watered (WW) and drought stressed (D) conditions. Photosynthetic metrics were measured in sensitive and tolerant genotypes during drought stress and post-recovery including P_n (A), G_s (B), C_i (C), and T_r (D) measured from 9:00 to 11:00 in the morning for every collection time, at day zero (0), 3, 6, 9 or 12, on young leaves. Different letters indicate significant differences (p < 0.05) based on Tukey’s test between control and treatments and between different treatment times. Data represent the mean ± SD of three or more replicates.

Figure 2

ABA and IAA content in maize seedling leaves under well-watered (WW) and drought (D) conditions. Phytohormone levels were measured in sensitive and tolerant genotypes over drought stress and recovery including ABA (A); and IAA (B). Different letters indicate significant differences (p < 0.05) based on Tukey’s test between control and treatments and between different treatment times. Data represent the mean ± SD of three or more replicates.

Figure 3

Visualization of superoxide radical and hydrogen peroxide in the leaves of maize plants under well-watered (WW) and drought stressed (DT) conditions. Endogenous O₂^·− levels were monitored by staining O₂^·− using a Nitro blue tetrazolium (NBT) staining method (A); and the endogenous H₂O₂ level was monitored by staining H₂O₂ using 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) (B). Scale bar in (A,B), 5 mm.

Figure 4

Effects of drought treatments on SOD and CAT activities in maize seedling leaves. The activities of ROS-remediating enzymes were measured in sensitive and tolerant genotypes over drought stress and recovery including SOD (A) and CAT (B). Different letters indicate significant differences (p < 0.05) based on Tukey’s test between control and treatments and between different treatment times. Data represent the mean ± SD of three or more replicates.

Figure 5

Visualization of nitric oxide (NO) in maize leaf tissues subjected to drought stress. Endogenous NO levels was monitored in maize leaves by staining NO using DAF-FM diacetate, and displayed by green fluorescence; the red fluorescence represents the chlorophyll intensity. WW refers to well-watered leaves; DT refers to drought treated leaves of all tested maize genotypes. Scale bar, 5 mm.

Figure 6

The NOS and GSNOR activities in maize seedling leaves under well-watered (WW) and drought (D) conditions. Nitrosative stress-related enzyme activities were measured in sensitive and tolerant genotypes over drought stress and recovery including NOS (A); and GSNOR (B). Different letters indicate significant differences (p < 0.05) based on Tukey’s test between control and treatments and between different treatment times. Data represent the mean ± SD of three or more replicates.

Figure 7

Principal component analysis (PCA) of all physiological and biochemical data in six different lines under well-watered and drought treated conditions. The blue points represent the well-watered (W) plants, while the red points represent the drought-stressed (D) plants.

Figure 8

Correlation and variance analysis of all tested dataset in six tested lines under well-watered and drought treated conditions. (A) shows the correlation among each parameter determined; and (B) shows the variance among each trait and each sample during the period of drought treatments.

Figure 8

Correlation and variance analysis of all tested dataset in six tested lines under well-watered and drought treated conditions. (A) shows the correlation among each parameter determined; and (B) shows the variance among each trait and each sample during the period of drought treatments.