Effects of β-sitosterol on plant growth and water status of rice seedlings exposed to prolonged UV-B stress
Plants are vulnerable to absorption of strong UV-B light, and this can affect different aspects of plant growth and development. According to the objectives of this study, rice plants not supplemented with βSito (Nβ) were grown in normal light under non-stress (ns) and under prolonged UV-B stress (uvs) conditions (termed as Nβns and Nβuvs, respectively). Similarly, rice plants supplemented with βSito (Sβ) were grown under non-stress and UV-B stress conditions (and termed as Sβns and Sβuvs, respectively) for comparative analysis. Initially, we have observed phenotypic changes which clearly show markedly enhanced tolerance of Sβuvs plants; however, Nβuvs plants were severely damaged, and all the leaves become twisted and crinkled under stress conditions (Fig. 1A). Under non-stress conditions, shoot and root lengths of Sβ plants were increased as compared to Nβ plants (Sβns vs. Nβns). Upon prolonged exposure to UV-B stress, the shoot and root length was not affected in Sβuvs plants (Sβuvs vs. Sβns); in contrast, a significant decrease in shoot (57.15% decrease in Nβuvs vs. Nβns, 64% decrease in Nβuvs vs. Sβuvs) and root (40% decrease in Nβuvs vs. Nβns, 42% decrease in Nβuvs vs. Sβuvs) length was noticed in Nβuvs plants (Fig. 1B, C). Another aspect of measuring plant growth under stress conditions is the evaluation of whole plant biomass. Under normal light conditions (non-stress), our results demonstrate that plant biomass for Sβ plants was increased up to 33% than Nβ plants (Sβns vs. Nβns). Notably, the plant biomass of Sβuvs plants was not affected even after UV-B stress. Contrastingly, we found a substantial decrease of plant biomass in Nβuvs plants, i.e., nearly 52% lower as compared with Sβuvs plants under prolonged UV-B irradiation stress (Fig. 1D). To check the water status of plants, we analyzed leaf relative water content both in Nβ and Sβ plants under non-stress and UV-B stress. No significant difference was observed between Nβns and Sβns plants under normal conditions, but when plants were subjected to prolonged UV-B stress, a marked decrease of 44% was noticed in Nβuvs plants as compared to Sβuvs plants (Fig. 1E). Overall, our result reveals the positive effect of βSito on the growth performance and water status of rice plants under prolonged UV-B stress conditions.
Supplementation of β-sitosterol affects photosynthetic pigments and ultrastructure of chloroplast in rice plants exposed to prolonged UV-B irradiation
Photosynthetic pigments Chl a, Chl b, and Caro are important for capturing light to perform efficient photosynthesis and are good indicators for plant adaptability to stress conditions. Therefore, we measured the content of these pigments in Nβ and Sβ plants under non-stress and UV-B stress. Under non-stress conditions, there were no significant differences for all the photosynthetic pigments between Nβns and Sβns plants except for Caro, contents of which were relatively increased in Sβns plants (Fig. 2D). Interestingly, there was a significant reduction in the levels of Chl a, Chl b, total chlorophyll, and Caro in Nβuvs plants after exposure to UV-B stress (Nβuvs vs. Nβns and Nβuvs vs. Sβuvs). Conversely, we have found that the contents of Chl a, total chlorophyll, and Caro in Sβuvs plants were significantly higher (Sβuvs vs. Sβns and Sβuvs vs. Nβuvs), whereas the content of Chl b was relatively constant (Sβuvs vs. Sβns) after stress (Fig. 2A–D). Moreover, we have also examined any effects on the ultrastructure of chloroplast in Nβ and Sβ plants subjected to prolonged UV-B stress. For this purpose, we make use of transmission electron microscopy which shows disorganization of grana stacks in the chloroplasts of leaves from Nβuvs plants after exposure to prolonged UV-B irradiation, while no significant effect was observed in Sβuvs as compared to non-stress plants (Fig. 2E). Collectively, the results demonstrated that βSito was involved in maintaining chloroplast development and chlorophyll synthesis under stress conditions thus showing superior performance of Sβ plants compared to Nβ plants.
Effects of β-sitosterol on net photosynthesis of rice plants exposed to prolonged UV-B stress
Of all the biological processes, photosynthesis is the central and most crucial process for measuring the physiological characteristics of plants under various environmental challenges. Any change in photosynthetic-related parameters affects the overall sensitivity of photosynthesis and subsequently changes the plant physiology. In this study, almost 27.78% increase in Pn was recorded in Sβuvs (vs. Sβns) plants exposed to UV-B stress; however, no change was seen in WUEi, but Tr was reduced slightly after stress (Fig. 3). A similar increasing trend was noted for Gs, Fv/Fm, and NPQ in Sβuvs vs. Sβns plants after stress (Fig. 3). Furthermore, under non-stress conditions, Pn, Fv/Fm, and WUEi were also enhanced in Sβns vs. Nβns. In contrast, photosynthesis was negatively affected in Nβuvs plants, and as we expected, the values for all these parameters were significantly reduced in Nβuvs plants after UV-B irradiation (Fig. 3). In case of Pn, almost 52.20% and 26.67% reduction (Nβuvs vs. Sβuvs and Nβuvs vs. Nβns, respectively) was observed in Nβuvs plants (Fig. 3A). Notably, this result was also consistent with reduced chlorophyll content and a decrease of plant biomass in Nβuvs plants. In conclusion, these results clearly indicate the positive effects of βSito application on net photosynthesis and associated gas exchange parameters in Sβ plants under prolonged UV-B irradiation.
Effects of β-sitosterol on oxidative stability of UV-B exposed rice plants
One of the many consequences in UV-B-stressed plants is the accumulation of high levels of ROS in cellular compartments that has harmful effects on cell homeostasis, structures, and functions and results in oxidative stress. In order to understand the effect of βSito on cell membrane integrity and oxidative stability of rice plants under prolonged UV-B irradiation, we have initially analyzed the amount of H2O2 and O2 in Nβ and Sβ plants under non-stress and after UV-B stress continuously until 5 days of treatment. Interestingly, H2O2 and O2 contents in Nβuvs vs. Sβuvs plants were not different under non-stress conditions, but the levels of these ROS were markedly abundant in Nβuvs plants as compared to Sβuvs plants during the entire period of stress (Fig. 4B, C). Moreover, when the leaves were visualized using the NBT staining method, significantly intense accumulation of intracellular ROS was detected in Nβuvs plants than in Sβuvs plants (Fig. 4A). Further, to assess membrane damage by lipid peroxidation, MDA contents were measured in plants under non-stress and stress conditions. Although MDA level was slightly increased in Sβuvs vs. Sβns plants, the magnitude of increase was much higher in Nβuvs plants (up to 12.78 μmol g−1 FW) that shows an elevated activity of lipid peroxidation in Nβ plants after stress (Fig. 4D).
Furthermore, abundant evidence supports the idea that the equilibrium between the production and detoxification of ROS is sustained by antioxidant enzyme activities thereby maintaining ROS at low concentrations in stressed plants and thus allowing them to perform normal functions. Therefore, we measured the activities of antioxidant enzymes (POD, SOD, CAT, and APX) in Nβ and Sβ plants under non-stress and stress conditions. Under non-stress conditions, the difference in activities of antioxidant enzymes was not changed between Nβns and Sβns plants, but we recorded a dramatic increase in SOD (446.32 U g−1 FW), POD (143.21 U g−1 FW), CAT (58.44 U g−1 FW), and APX (184.57 U g−1 FW) activities in Sβuvs plants (Fig. 5). In case of Nβuvs plants, we detect only a slight increase in SOD and POD activities as compared to non-stress conditions, whereas CAT and APX activities (27.81 U g−1 FW and 33.42 U g−1 FW, respectively) were reduced after exposure to prolonged UV-B light (Fig. 5). These results demonstrate an elevated level of oxidative stress tolerance in Sβ plants under stress through avoiding oxidative damage caused by UV-B. Maintaining a high level of antioxidant enzymes in Sβ plants eventually helps plants to immediately scavenge ROS in cells and thus restore redox homeostasis.
Effects of β-sitosterol on metabolic profile of rice plants during UV stress
To investigate the effect of βSito on the biochemical snapshot of rice plants subjected to UV-B stress, we utilized a metabolomics approach to quantify the changes in specific metabolites in Nβ and Sβ plants under non-stress and after UV-B stress. A total of 72 key metabolites were detected and quantified using GC-TOFMS in leaf samples from Nβ and Sβ plants, out of which 27 belongs to organic acids, 19 belongs to sugars, 15 were amino acids, and 11 others (Supplementary Table S1). Differences in metabolite contents are visualized as a heat map that shows the overall changes in 72 metabolites under non-stress and UV-B stress conditions (Fig. 6). Under UV-B stress conditions, a large number of metabolites were induced in Sβuvs vs. Nβuvs plants, whereas a significant decrease in accumulation of several metabolites was recorded in Nβuvs vs. Nβns plants (Fig. 6). However, Sβuvs vs. Nβuvs, we have also noticed a reduction in levels of some organic acids and sugars. This implies a significantly different mechanism of metabolite regulation in Sβ and Nβ plants subjected to UV-B stress. Under non-stress conditions, we have also observed different metabolite accumulation patterns (Sβns vs. Nβns), with the most obvious differences recorded for organic acids and sugars (Fig. 6). In case of Sβuvs vs. Nβns, sugars, amino acids, and other key metabolites were mainly induced, while no obvious differences were noted for organic acids (with regard to total organic acids) accumulation (Fig. 6). In total, these results thus demonstrate that exogenous application of βSito has significantly induced several metabolites belonging to organic acids, sugars, and amino acids as well some other key metabolites in Sβ plants compared with Nβ plants under stress. Contrastingly, we recorded a significant downregulation of several metabolites belonging to organic acids, sugars, and amino acids in Nβ plants under UV-B stress (Fig. 6). Importantly, these results also imply that application of βSito has mainly affected the accumulation of various metabolites in rice plants under UV-B stress (Sβuvs vs. Nβuvs) rather than unstressed plants (Sβns vs. Nβns).
Effects of β-sitosterol on accumulation of key metabolites belonging to different metabolic pathways
After prolonged UV-B stress, significantly reduced levels of few organic acids were observed especially fumaric acid, oxalic acid, malic acid, shikimic acid, palmic acid, ferulaic acid, and pyruvic acid in Nβ plants as compared to non-stress plants (Nβuvs vs. Nβns, Fig. 6). In contrast, several organic acids were accumulated in large amounts in Sβuvs vs. Nβuvs plants, such as 4.5-fold increase in oxalic acid, 6.2-foldsincrease in fumaric acid, 5-fold increase in salicylic acid, and 6.3-fold increase in benzoic acid followed by 2.6-fold increase in pyruvic acid (Figs. 6 and 7). In case of sugars, we found that glucose, fructose, sorbitol, and trehalose were reduced in Nβuvs vs. Nβns plants (Fig. 6 and Supplementary Table S1). Analysis of sugars in Sβuvs vs. Nβuvs plants reveals that trehalose, mannitol, glucose, fructose, and raffinose were increased significantly (up to 19-folds, 4.5-folds, 6-folds, 2.3-folds, and 5.1-folds, respectively) after stress is applied (Figs. 6 and 7). Conversely, the levels of these sugars were low in Nβuvs vs. Nβns plants (Fig. 6 and Supplementary Table S1). In case of amino acids, we found an obvious reduction in the levels of glycine and tryptophan in Nβ plants under UV-B stress as compared to non-stress (Nβuvs vs. Nβns, Fig. 6 and Supplementary Table S1). Interestingly, the relative abundance of amino acids such as tryptophan, isoleucine, and γ-aminobutyric acid (GABA) was increased up to 17-folds, 3.2-folds, and 2.2-folds, respectively, in Sβuvs vs. Nβuvs plants (Figs. 6 and 7). For proline, we noticed an increased accumulation in Nβuvs vs. Nβns and Sβuvs vs. Nβns, but the level of increase was higher in Sβuvs vs. Nβns plants (Fig. 6). Analyzing the other key metabolite profile in Sβuvs vs. Nβuvs plants, we found 4.9-folds and 2.7-fold increase in 1-hexadecanol and 2-aminoadipic acid, respectively (Fig. 7). Accumulation of different metabolites is crucial for the activation of various signaling and biochemical pathways in plants to overcome environmental challenges especially under high UV-B light. Differential accumulation of metabolites (including organic acids, sugars, amino acids, and others) between Nβ and Sβ plants suggest that various metabolic pathways are regulated in plants through β-sitosterol, which further explains the underlying mechanisms of UV-B stress tolerance in Sβ plants. Overall, these results imply that βSito is an essential part of stress signaling and participates in the tolerance mechanism against UV-B in rice. Importantly, differential accumulation of several key metabolites in Sβ plants is regulated via exogenous application of βSito and is associated with sugar and amino acid metabolism, GABA shunt, and tricarboxylic acid (TCA) cycle.