Multiple Abiotic Stresses Applied Simultaneously Elicit Distinct Responses in Two Contrasting Rice Cultivars

Abstract

Rice crops are often subject to multiple abiotic stresses simultaneously in both natural and cultivated environments, resulting in yield reductions beyond those expected from single stress. We report physiological changes after a 4 day exposure to combined drought, salt and extreme temperature treatments, following a 2 day salinity pre-treatment in two rice genotypes-Nipponbare (a paddy rice) and IAC1131 (an upland landrace). Stomata closed after two days of combined stresses, causing intercellular CO2 concentrations and assimilation rates to diminish rapidly. Abscisic acid (ABA) levels increased at least five-fold but did not differ significantly between the genotypes. Tandem Mass Tag isotopic labelling quantitative proteomics revealed 6215 reproducibly identified proteins in mature leaves across the two genotypes and three time points (0, 2 and 4 days of stress). Of these, 987 were differentially expressed due to stress (cf. control plants), including 41 proteins that changed significantly in abundance in all stressed plants. Heat shock proteins, late embryogenesis abundant proteins and photosynthesis-related proteins were consistently responsive to stress in both Nipponbare and IAC1131. Remarkably, even after 2 days of stress there were almost six times fewer proteins differentially expressed in IAC1131 than Nipponbare. This contrast in the translational response to multiple stresses is consistent with the known tolerance of IAC1131 to dryland conditions.

Keywords: TMT labeling; heat shock proteins; multiple abiotic stress; physiology; proteomics; rice.

Figure 1

Photosynthetic responses and ABA content of Nipponbare and IAC1131 in response to multiple abiotic stress at three time points (0d = C0, 2d stress = S2 and 4d stress = S4). (A) Assimilation (Ar), (B) stomatal conductance (gs) (C) ratio of intracellular to ambient CO₂ concentration (Ca/Ci) and (D) ABA concentration. Standard errors were obtained from three biological replicate measurements. An asterisk (*) indicates statistically significant difference between the control and stress conditions, according to Student’s t-test (p-value < 0.05).

Figure 2

Differential protein expression of two rice genotypes after multiple abiotic stress treatment. (A) Number of significantly differentially expressed proteins seen at each time point for each genotype. (B) Unique and common proteins significantly increased and decreased in abundance across IAC1131 and Nipponbare. (C) Range of fold-change of DEPs in both genotypes after stress treatments.

Figure 3

Differential protein expression of two rice genotypes after multiple abiotic stress treatment. (A) Polar plot of number of significantly differentially expressed proteins in each variety. (B) Unique and common differentially abundant proteins between IAC1131 and Nipponbare. (C) Fold change of DEPs in both genotypes after stress treatment.

Figure 4

Comparison of differentially expressed proteins at 2d and 4d of multiple abiotic stress. (A) Polar plot of number and percentage of significantly differentially expressed proteins at stress each time point. (B) Unique and common differentially abundant proteins between the two time points. (C) Scatter plots of fold-change values of the differentially expressed proteins found in both varieties at 2d and 4d of stress, measured against control and plotted against each other.

Figure 5

Visualization of differentially abundant proteins identified and quantified under two stress time points in IAC1131 and Nipponbare. (A) Venn diagram indicating the overlap in proteins uniquely and commonly identified and quantified in IAC1131 and Nipponbare after 2d and 4d of stress. (B) Expanded view of some important protein families represented in the commonly increased in abundance protein category.

Figure 6

Expression patterns of common DEPs in IAC1131 and Nipponbare after exposure to 2d and 4d multiple abiotic stress. The fold changes of 41 significantly changed proteins were log 2-transformed. and hierarchical clustering was performed using Euclidean as the distance metric and average as the linkage criterion.

Figure 7

Box and whisker plots showing the spread of relative fold change of 11 HSP family proteins overexpressed in all conditions in both IAC1131 and Nipponbare. The shaded box indicates the interquartile range, while the color boundary within the shaded box indicates the median. The horizontal bars indicating the range of the data show the lesser value of either the minima or maxima or 1.5 times the interquartile range.