Gamma-aminobutyric acid treatment promotes resistance against Sogatella furcifera in rice

Abstract

The Sogatella furcifera (Horváth) (Homoptera: Delphacidae) is a white-backed planthopper (WBPH) that causes "hopper burn" in rice, resulting in severe yield loss. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter that inhibits neurotransmission in insects by binding to specific receptors. In this study, we investigated the potential role of GABA in modulating rice resistance to WBPH and evaluated possible defense mechanisms. The experiment was conducted in green house in pots consist of four groups: control, GABA-treated, WBPH-infested, and WBPH-infested treated with GABA. Among the various tested concentration of GABA, 15 mM GABA was applied as a single treatment in water. The treatment was administered one week before WBPH infestation. The results revealed that 15 mM GABA treatment strongly increased WBPH resistance. A plate-based assay indicated that direct application of 15 mM GABA increased the mortality rate of WBPH and increased the damage recovery rate in rice plants. We found that GABA treatment increased the activation of antioxidant enzymes and reduced the reactive oxygen species content and malondialdehyde contents, and reduced the damage rate caused by WBPH. Interestingly, GABA-supplemented plants infested with WBPH exhibited increased phenylalanine ammonia-lyase and pathogenesis-related (PR) genes expression levels. GABA induced the accumulation of abscisic acid (ABA) and salicylic acid (SA) and enhanced the stomata closure and reduced leaf vessels to reduce water conductance during WBPH stress. Furthermore, we found that GABA application to the plant induced the expression of Jasmonic acid (JA) biosynthesis genes (LOX, AOS, AOC, and OPR) and melatonin biosynthesis-related genes (TDC, T5H, ASMT, and SNAT). Our study suggested that GABA increases resistance against WBPH infestation by regulating antioxidant defense system, TCA cycle regulation, phytohormonal signaling, and PR gene regulation.

Keywords: Sogatella furcifera; antioxidant; gamma-aminobutyric acid; melatonin; phytohormone; tricarboxylic acid cycle.

Figure 1

Application of GABA enhance rice plant growth against WBPH stress. The rice seedlings were first treated with GABA and after one week of treatment the plants were infested with WBPH for one month. After one month of infestation, the data presented in this figure was recorded. (A, B) shows pictorial and graphical representation of GABA effects on shoot length under WBPH stress. (C, D) shows pictorial and graphical representation of GABA effects on root under WBPH stress. (E, F) shows pictorial and graphical representation of GABA effects on leaf width under WBPH stress. (G, H) shows effect of GABA on shoot and root fresh weight respectively, under WBPH stress. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Different letters on the bars shows significant differences (p ¾ 0.05) as evaluated by DMRT test.

Figure 2

GABA reduces WBPH damagein rice stem and leaves. (A, B) shows original picture of leaf and stem respectively. (C, D) shows the ImageJ analyzed picture of leaf and stem respectively, indicating the damage induced by WBPH infestation. (E, F) shows the quantitative analysis of the leaf and stem damage induced by WBPH, respectively. ** indicates p < 0.01, and *** indicates p < 0.001.

Figure 3

GABA inhibit WBPH survival and reduces their population in rice plant. (A, B) shows pictorial and graphical representation of effect of direct application of GABA different concentration on WBPH survival. (A) represent dead insects while red arrow in (C) indicate presence of WBPH on plants. (C, D) shows effect of WBPH population percentage in plants supplemented with different concentrations of GABA. All the control plants, 5mM, 10mM, and 15mM GABA supplemented plants were grown in the same insectarium and were infested seven days after GABA supplementation. DATA in the graphs were presented in percentage. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

Figure 4

Application of GABA reduces oxidative stress, induced by WBPH infestation in rice plants and regulate ions homeostasis. (A, B) shows in situ detection of oxidative stress caused by generation of ROS during WBPH stress, using DAB and trypan blue staining respectively. (C-E) shows H₂O₂, O₂ ^.−, and electrolytic leakage. (F-H) shows Fe²⁺, Mg⁺, and Ca⁺ contents. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Different letters on the bars shows significant differences (p ¾ 0.05) as evaluated by DMRT test.

Figure 5

Exogenous application of GABA regulate TCA cycle in rice plant. (A, B) shows accumulation of GABA in rice shoot and roots respectively. (C, D) shows accumulation of succinate in leaf and root respectively. (E, F) shows accumulation of NADPH in shoot and root respectively. (G-I) shows the expression of GABA shunt genes i.e GAD, GABA-T, and SSADH respectively. (J) shows schematic representation of GABA shunt and TCA cycle pathway. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Different letters and on the bars shows significant differences (p ¾ 0.05) as evaluated by DMRT test. Asterisks on bars shown in (G-I) represent significant differences (p ¾ 0.05) as evaluated by Bonferroni post-hoc test.

Figure 6

GABA induces Phenylalanine ammonia-lyase, pathogenesis related genes and phytohormons in rice under WBPH stress. (A) represent PAL gene expression level. (B-G) represent gene expression of PR1, PR2, PR3, PR4A, PR8A, and PR8B respectively. (H, I) shows the accumulation of ABA and SA hormones respectively. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Asterisks on bars shown in (G-I) represent significant differences (p ¾ 0.05) as evaluated by Bonferroni post-hoc test.

Figure 7

Exogenous application of GABA regulates rice leaf stomata closure and leaf vessels reduction. (A) shows stomata opening and closure and (B) shows vessels dimeter variations. Red arrows shows stomata while yellow arrows shows vessels in leaf lamina.

Figure 8

GABA application induces antioxidant defense system in rice plant against WBPH stress. (A) shows MDA contents, (B) shows APX activity, (C) shows GPx activity, (D) shows CAT activit, (E) shows POD activity, (F) shows SOD activity, (G) shows ABTS activity, and (H) shows DPPH activity. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Asterisks on bars shown in (G–H) represent significant differences (p ¾ 0.05) as evaluated by Bonferroni post-hoc test.

Figure 9

GABA induces melatonin biosynthesis in response to WBPH stress. (A–D) shows the transcript level of melatonin biosynthesis pathway genes i.e. TDC, T5H, ASMT, and SNAT respectively. (E) shows the general pathway of melatonin and their genes. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Asterisks on bars shown in (A–D) represent significant differences (p ¾ 0.05) as evaluated by Bonferroni post-hoc test.

Figure 10

GABA induces JA biosynthesis pathway in response to WBPH stress. (A–D) shows the transcript level of JA biosynthesis pathway genes i.e. LOX, AOS, AOC, and OPR respectively. (E) shows the general pathway of JA and their genes. Data represented in graphs were analyzed as a mean of three independent biological replicates ± SD. Asterisks on bars shown in (G–I) represent significant differences (p ¾ 0.05) as evaluated by Bonferroni post-hoc test.

Figure 11

Schematic representation of GABA shunt and its associated pathways regulated during WBPH stress in rice plant. ABA, Adenosine triphosphate; ABTS, Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; ADP, Adenin di-phosphate; AOC, Allene oxide cyclase; AOS, Allene oxide synthase; APX, Ascorbate peroxidase; ASMT, Acetyl-serotonin methyltransferase; ATP, Adenin tri-phosphate; Ca2+, Calcium Ion; CaM, Calmodulin; CAT, Ahloramphenicol acetyltransferase; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EOT, Epoxyoctadecatrienoic acid; GABA, Gamma-aminobutyric acid; GABA-T, GABA transaminase; GAD, Glutamate decarboxylase; GHB, γ-Hydroxybutyric acid; GPx, Glutathione peroxidase; H₂O₂, Hydrogen peroxide; HPOT, Hydroperoxyoctadecatrienoic acid; JA, Jasmonic acid; LOX, Lipoxygenase; MDA, Malondialdehyde; NAD+, Nicotinamide adenine dinucleotide; NADP+, Nicotinamide adenine dinucleotide phosphate; NADH, Nicotinamide adenine dinucleotide+hydrogen; NADK, NAD+ kinase; NADPH, Nicotinamide adenine dinucleotide phosphate+hydrogen; NADPOX, NADPH oxidase; O₂ ^•−, Superoxide radical; OPC, Oxo-phytodienoic acid; OPDA, Oxophytodienoic acid; OPR, Oxo-phytodienoic acid reductases; PAL, Phenylalanine ammonia-lyase; POD, Peroxidase; PR, Pathogenesis related; ROS, Reactive oxygen species; SAR, Systemic acquired resistance; SNAT, Serotonin N-acetyl transferase; SOD, Superoxide dismutase; SSADH, Semialdehyde dehydrogenase; SSR, Succinic semialdehyde reductase; T5H, Tryptamine 5-hydroxylase; TDC, Tryptophan decarboxylase.