Impact of Single and Combined Salinity and High-Temperature Stresses on Agro-Physiological, Biochemical, and Transcriptional Responses in Rice and Stress-Release

Abstract

Here, for the first time, we aimed to identify in rice the key mechanisms and processes underlying tolerance to high-temperature (HT) or salt stress (SS) alone, the co-occurrence of both stresses, and recovery using physiological and biochemical measurements and gene expression analysis. We also investigated whether recovery from the two stressors depended on the relative intensities/relief of each stressor. Wild type ('Yukinkomai') rice plants were found to be more susceptible to salinity or heat applied individually. SS leads to a depletion of cellular water content, higher accumulation of Na⁺, and alterations in photosynthetic pigments. The stress-tolerant cultivar 'YNU31-2-4' (YNU) displayed a lower Na⁺/K⁺ ratio, higher water content in cells and improved photosynthetic traits, antioxidant system, and expression of defence genes. Strikingly, the SS + HT combination provided a significant level of protection to rice plants from the effects of SS alone. The expression pattern of a selected set of genes showed a specific response and dedicated pathways in plants subjected to each of the different stresses, while other genes were explicitly activated when the stresses were combined. Aquaporin genes were activated by SS, while stress-related (P5CS, MSD1, HSPs, and ions transporters) genes were shaped by HT. Hierarchical clustering and principal component analyses showed that several traits exhibited a gradually aggravating effect as plants were exposed to the combined stresses and identified heat as a mitigating factor, clearly separating heat + salt-stressed from salt-non-heat-stressed plants. Furthermore, seedling recovery was far more dependent on the relative intensities of stressors and cultivars, demonstrating the influence of one stressor over another upon stress-release. Taken together, our data show the uniqueness and complexity of the physiological and molecular network modules used by rice plants to respond to single and combined stresses and recovery.

Keywords: coupled stress; heat; independent/interactive effects; individual stress; recovery; salt stress.

Figure 1

Morphological responses to salinity (SS), high-temperature (HT), and combined salinity and heat (SS + HT) of ‘Yukinkomai’ (WT) and ‘YNU31-2-4’ (YNU) rice seedlings; (A) Shoot (top panel) and root (lower panel) lengths, (B) shoot (top panel) and root (lower panel) fresh weight (FW), (C) shoot (top panel) and root (lower panel) dry matter (DM), (D) relative water content (RWT), and (E) salt tolerance index (STI) of WT and YNU plants under control normal-temperature (NT) (C: 26/23 °C, 0 mM NaCl), salt stress (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), combined stress (SS + HT: 30/26 °C, 75 mM NaCl), and stress-release (recovery) conditions. Bars = 10 cm. Values represent the means ± SE determined from three independent experiments using 4 plants in each experiment. Means within the same graph followed by different lowercase (WT) and uppercase (YNU) letters are significantly different at p < 0.05 according to the Tukey’s HSD test. Asterisks represent statistical significance of difference using Student’s t-statistic with *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant.

Figure 2

Chlorophyll content of rice seedlings under salinity (SS), high-temperature (HT), and combined salinity and heat (SS + HT) conditions; (A) Chlorophyll a (Chl a) and (B) chlorophyll b (Chl b) content of WT and YNU plants under control (C: 26/23 °C, 0 mM NaCl), salt stress (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), and combine stress (SS + HT: 30/26 °C, 75 mM NaCl) conditions. Values represent the means ± SE determined from three independent experiments using 4 plants in each experiment. Means within the same graph followed by different lowercase (WT) and uppercase (YNU) letters are significantly different at p < 0.05 according to the Tukey’s HSD test. Asterisks represent statistical significance of difference using Student’s t-statistic with *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant.

Figure 3

Na⁺ and K⁺ ion content of rice seedlings under salinity (SS), high-temperature (HT), and combined salinity and heat (SS + HT) conditions; (A) K⁺ content of shoot (top panel) and root (down panel), (B) Na⁺ content of shoot (Top panel) and root (down panel), and (C) Na⁺/K⁺ ratio of shoot (Top panel) and root (down panel) of WT and YNU seedlings under control (C: 26/23 °C, 0 mM NaCl), saline (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), and combined salinity and heat (SS + HT: 30/26 °C, 75 mM NaCl) conditions. Values represent the means ± SE determined from three independent experiments using four plants in each experiment. Means within the same graph followed by different lowercase (WT) and uppercase (YNU) letters are significantly different at p < 0.05 according to Tukey’s HSD test. Asterisks represent statistical significance of difference using Student’s t-statistic with *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant.

Figure 4

Biochemical responses to salinity (SS), high-temperature (HT), and combined salinity and heat (SS + HT) of rice seedlings; (A) Malondialdehyde (MDA), (B) proline, and (C) protein of WT and YNU plants under control (C: 26/23 °C, 0 mM NaCl), salt stress (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), and combined salinity and heat (SS + HT: 30/26 °C, 75 mM NaCl) conditions. Values represent the means ± SE determined from three independent experiments using four plants in each experiment. Means within the same graph followed by different lowercase (WT) and uppercase (YNU) letters are significantly different at p < 0.05 according to Tukey’s HSD test. Asterisks represent statistical significance of difference using Student’s t-statistic with *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant. nd: not determined.

Figure 5

Antioxidant enzyme responses to salinity (SS), high-temperature (HT), and combined salinity and heat (SS + HT) of rice seedlings; (A) catalase (CAT), (B) superoxide dismutase (SOD), and (C) ascorbate peroxidase (APX) of WT and YNU plants under control (C: 26/23 °C, 0 mM NaCl), salt stress (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), and combined salinity and heat (SS + HT: 30/26 °C, 75 mM NaCl) conditions. Values represent the means ± SE determined from three independent experiments using four plants in each experiment. Means within the same graph followed by different lowercase (WT) and uppercase (YNU) letters are significantly different at p < 0.05 according to Tukey’s HSD test. Asterisks represent statistical significance of difference using Student’s t-statistic with *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant. nd: not determined.

Figure 6

Hierarchical clustering analysis (HCA) of the relative transcript level of ion transporters, aquaporins, and stress-related genes in (A) shoot and (B) root tissues of WT and YNU seedlings under control (C: 26/23 °C, 0 mM NaCl), salt stress (SS: 26/23 °C, 75 mM NaCl), high-temperature (HT: 30/26 °C, 0 mM NaCl), and combined salinity and heat (SS + HT: 30/26 °C, 75 mM NaCl) conditions. Kendall’s Tau and Average linkage were used as distance measurement and clustering methods, respectively. ND: not detected.

Figure 7

Principal component analyses (PCA) of WT and YNU traits under (A) DAT5; 5 days of stress, (B) DAT10; 10 days of stress, (C) DAT15; 5-day stress-release recovery, and (D) DAT20; 10-day stress-release recovery.