Augmenting Rice Defenses: Exogenous Calcium Elevates GABA Levels Against WBPH Infestation

Abstract

This study investigates the impact of exogenous calcium and gamma-aminobutyric acid (GABA) supplementation on rice growth and stress tolerance under white-backed planthopper (WBPH) infestation. We evaluated several phenotypic traits, including shoot/root length, leaf width, tiller number, panicle length, and relative water content, alongside physiological markers such as oxidative stress indicators, antioxidant enzymes activities, hormonal levels, and amino acids biosynthesis. Our results indicate that WBPH stress significantly reduces growth parameters but calcium and GABA supplementation markedly enhance shoot length (by 26% and 36%) and root length (by 38% and 64%), respectively, compared to WBPH-infested plants. Both supplementations also reduced oxidative stress, as evidenced by decreased H₂O₂ and O₂^•- levels and a lower electrolyte leakage. Notably, calcium and GABA treatments increased antioxidant enzyme activities, with GABA boosting catalase (CAT) activity by 800%, peroxidase (POD) by 144%, and superoxide dismutase (SOD) by 62% under WBPH stress. Additionally, calcium and GABA enhanced the accumulation of stress hormones (abscisic acid ABA) and salicylic acid (SA) and promoted stomatal closure, contributing to improved water conservation. This study reveals that calcium regulates the GABA shunt pathway, significantly increasing GABA and succinate levels in both root and shoot. Furthermore, calcium and GABA supplementation enhance the biosynthesis of key amino acids and improve ion homeostasis, particularly elevating calcium (Ca), iron (Fe), and magnesium (Mg) levels under WBPH stress. Overall, this study highlights the potential of exogenous calcium and GABA as effective strategies for enhancing rice plant tolerance to WBPH infestation by modulating various physiological and biochemical mechanisms. Further research is warranted to fully elucidate the underlying mechanisms of action.

Keywords: antioxidant activity; calcium; gamma-aminobutyric acid; hormones; oxidative stress; white-backed planthopper.

Figure 1

Calcium and GABA enhance rice growth parameters against WBPH stress. Panel (A) provides a pictorial representation of plant length and development. Panels (B–H) depict the effects of calcium and GABA supplementation on shoot length, root length, leaf width, tiller number, panicle length, seed number per panicle, and leaf relative water content, respectively. Data in panels (B–H) are derived from three independent biological replicates, presented as means ± standard deviation (SD), and statistically analyzed using Bonferroni post hoc tests. Statistical significance is denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).

Figure 2

Exogenous calcium and GABA mitigate WBPH-induced oxidative stress in rice plants. Panel (A) illustrates symptoms of WBPH infestation on rice stems at the seedling stage. Panels (B,C) depict the in situ detection of oxidative damage in rice leaves induced by the generation of reactive oxygen species, with (B) showing DAB staining and (C) showing trypan blue staining. Panel (D) shows H₂O₂ accumulation, while panel (E) displays O₂^•− accumulation in rice leaves under different treatments. Panel (F) represents electrolytic leakage from rice leaves under various treatments. Data presented in panels (D–F) were obtained from three independent biological replicates (± standard deviation, SD), and statistical analysis was conducted using Bonferroni post hoc tests. Significance levels are denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).

Figure 3

Calcium and GABA supplementation mitigate lipid peroxidation and enhance antioxidant enzymatic activity in rice plants under WBPH stress. Panel (A) illustrates lipid peroxidation, measured by MDA (malondialdehyde) contents accumulation under different treatments in response to WBPH stress. Panels (B–D) depict the activities of catalase, peroxidase, and superoxide dismutase enzymes, respectively. Data were derived from three independent biological replicates (± standard deviation, SD), and statistical significance was assessed using Bonferroni post hoc tests. Significance levels are denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).

Figure 4

Calcium and GABA induce the synthesis of ABA and SA, facilitating the regulation of stomatal dynamics in rice plants under WBPH stress. (A) shows stomata opening and closing patterns induced by calcium and GABA supplementation under WBPH stress. (B,C) shows accumulation of ABA and SA, respectively, in rice plants induced by calcium and GABA under WBPH stress. The data presented in (B,C) were analyzed in three independent biological replicates (± standard deviation, SD), and the means were compared using Bonferroni post hoc tests. ** indicates p < 0.01, and *** indicates p < 0.001. White arrow indicate stomata while scale bare shows 20 µm.

Figure 5

Exogenous calcium triggers the synthesis of GABA and succinate in rice plants, accompanied by the upregulation of their respective biosynthetic genes. Panels (A,B) show the increased levels of GABA in shoot and root, respectively, following calcium application. Panels (C–E) display the temporal expression patterns of key biosynthetic genes GAD, GABA-T, and SSADH, after calcium supplementation. Panels (F,G) depict GABA accumulation in shoot and root under WBPH stress, while panels (H,I) show succinate accumulation in shoot and root under the same stress condition. Panels (J–L) illustrate the expression levels of GAD, GABA-T, and SSADH in calcium- and GABA-supplemented rice plants under WBPH stress. Panel (M) outlines the GABA shunt pathway. Data represent means from three independent biological replicates (± standard deviation, SD), with statistical significance indicated by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) determined via Bonferroni post hoc tests.

Figure 6

Calcium and GABA supplementation enhance total amino acid content, promote photosynthesis, and regulate Ca, Fe, and Mg ion homeostasis in rice plants exposed to WBPH stress. Panel (A) illustrates the regulation of specific free amino acids, panel (B) shows chlorophyll content, while panels (C–E) depict the levels of Ca, Fe, and Mg ions, respectively, under WBPH stress. Data represent means from three independent biological replicates (± standard deviation, SD), with statistical significance indicated by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) determined via Bonferroni post hoc tests.

Figure 7

Schematic representation of calcium-induced GABA shunt and its associated pathways regulated during WBPH stress in rice plant. ABA, abscisic acid; Ca²⁺, calcium ion; CaM, calmodulin; CAT, catalyze; GABA, GAD, glutamate decarboxylase; H₂O₂, hydrogen peroxide; MDA, malondialdehyde; O₂^•−, superoxide radical; POD, peroxidase; PR, pathogenesis-related; SA, salicylic acid; SAR, systemic acquired resistance; SOD, superoxide dismutase.