The phytohormone abscisic acid (ABA) is a sesquiterpenoid with a C15 backbone (Nambara and Marion-Poll, 2005). ABA was initially identified as a compound that accelerated abscission (abbreviated as “abscission II”) in cotton and induced dormancy (abbreviated as “dormin”) in the leaves of Firmiana simplex. Further analyses showed that dormin has the same structure as abscission II, and both compounds are considered analogues of ABA (Taishi et al., 2011). The physiological effects of ABA were first detected in the early 1950s (Addicott and Lyon, 1969) when researchers isolated acidic fractions from plant extracts that had growth inhibitory effects on oat germinal sheaths. These extracts were classified as members of the β-inhibitor complex, and their function was consistent with what we know about the physiological effects of ABA (Taishi et al., 2011). Abiotic stressors have huge impacts on agricultural productivity and induce the production of many compounds that function in stress tolerance. ABA has numerous functions at the cellular level including the induction of the dehydration-responsive element-binding (DREB) transcription factors. DREB proteins regulate the expression of stress-induced genes by binding to DRE/CRT cis-elements in the promoter region to improve drought and salinity tolerance (Li et al., 2014). In response to abiotic stressors, mutations in the gene encoding serine-threonine protein kinase (Open Stomata1, OST1) rendered Arabidopsis thaliana guard cells insensitive to ABA, which kept the stomata open (Assmann, 2003). ABA also plays an important role in temperature stress and can reduce the risk of frost damage in plants. In tobacco plants exposed to a short period of heat stress, increased ABA concentrations reduced the cellular damage caused by high temperatures (Itai et al., 1978). ABA can also increase tolerance to biotic stressors; for example, ABA improved the resistance of rice to fungi, bacteria and the brown planthopper (BPH; Nilaparvata lugens) (Dunn et al., 1990; Robert-Seilaniantz et al., 2007; Fan et al., 2009; De Vleesschauwer et al., 2010; Liu et al., 2014; AbuQamar et al., 2017; Liu et al., 2017; Camisón et al., 2019; Darma et al., 2019; Boba et al., 2020). ABA has important roles in regulating plant growth, inhibiting seed germination, and promoting leaf senescence (Hao et al., 2009; Liang et al., 2014). Furthermore, ABA reduces water consumption by regulating stomatal closure, decreasing transpiration (Christmann et al., 2007) and regulating the activity of water channel proteins (Parent et al., 2009). ABA increased hydraulic conductivity and promotes water uptake by roots when soil begins to harden and dry out (Hose et al., 2000), which influences root and shoot growth (Hao et al., 2009). Thus, ABA has crucial roles in plant growth, development and stress tolerance.

In higher plants, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis and is involved in the rate-limiting step of ABA biosynthesis. NCED is induced earlier than other ABA synthase genes and is considered a pivotal step in ABA synthesis (Qin and Zeevaart, 1999). In rice, the OsNCED gene family is comprised of five genes, OsNCED1–5 (Zhu et al., 2009), and these have different roles in plant growth and response to external stressors. OsNCED1 is primarily expressed in rice leaves but is significantly suppressed during water stress; whereas OsNCED2 is more highly expressed in rice seeds as compared to the other four OsNCED genes (Zhu et al., 2009; Ye et al., 2011). OsNCED3 is highly induced during water stress (Ye et al., 2011) and regulates the accumulation of the dehydrin and late embryogenesis abundant (LEA) proteins (Hundertmark and Hincha, 2008; Xiang et al., 2008). OsNCED3 and OsNCED4 exhibit an overlapping expression pattern in rice seeds, which suggests a level of functional redundancy and common control of ABA biosynthesis in rice seeds in response to salinity stress (Hwang et al., 2018). In response to rice black-stripe dwarf virus, the expression of OsNCED4 and OsNCED5 increased in rice as the duration of viral infection became more prolonged, which suggests a role for both genes in the regulation of ABA synthesis during viral infection (Ni et al., 2015).

ABA is synthesized via oxidative cleavage of epoxy-carotenoids. In maize, xanthophyll epoxygenase catalyzes the epoxidation of zeaxanthin and antioxidant xanthophylls to form purple xanthophylls and neoxanthophylls. These products are isomerized to produce 9-cis-isomers that are cleaved by NCED to form xanthotoxin; the latter is converted to ABA by short-chain dehydrogenase/reductase and aldehyde oxidase 3 (Wen, 2019).

OsNCED3 is systematically expressed in various tissues of rice and is induced by NaCl, PEG and H₂O₂, which supports a role for OsNCED3 in abiotic stress tolerance (Huang et al., 2018). When MhNCED3 from Malus hupehensis was expressed in Arabidopsis thaliana exposed to chlorine stress, the growth and development of transgenic A. thaliana improved, and plants exhibited an increase in ABA content and a decrease in transpiration (Zhang et al., 2015). Hwang et al. (2010) reported that heterologous expression of NCED3 in Arabidopsis increased ABA levels (Hwang et al., 2010). NtNCED3–2 is one of the NCED genes in tobacco, and NtNCED3–2 knockout plants had reduced levels of diterpenes, photosynthetic pigments, and phytohormones. Furthermore, knockdown of NtNCED3–2 resulted in decreased expression of genes in the isoprenoid metabolic pathway as compared to wild-type plants, resulting in reduced photosynthetic capacity (Yang et al., 2018). In rice plants, drought stress significantly induced OsNCED3 expression, which was down-regulated when watering was resumed. Transgenic lines overexpressing OsNCED3 after drought stress had higher ABA levels (Xu et al., 2018a). Southern hybridization experiments in Lycium barbarum showed that NCED was present in low copy numbers, and NCED expression gene was synchronized with the accumulation of endogenous ABA after salt and dehydration stress (Lu et al., 2013). Our previous work found that OsNCED3 had a positive role in defense against the brown planthopper through transcriptome profiling but more details not showed (Sun et al., 2022). In this study, we used OsNCED3 overexpression, RNA interference and wild-type (WT) rice to investigate the role of OsNCED3 in conferring resistance to the brown planthopper (BPH, Nilaparvata lugens). Resistance was evaluated by examining physiological and biochemical parameters, changes in hormone content, and defense gene expression. The results provide a foundation for analyzing ABA function in the regulation of BPH resistance.

Plant insect tolerance is the ability of plants to tolerate adversity and compensate for it by relying on factors such as their own growth as well as reproductive capacity when subjected to harsh biotic stresses similar to those of insect-sensitive species (Yang, 2005). Our findings suggest that BPH preferred to feed on osnced3-RNAi rice as compared to WT and OsNCED3-OE rice. The overexpression of OsNCED3 reduced BPH-induced damage and improved insect tolerance. These results suggest that OsNCED3 expression is induced by BPH feeding and correlates with improved insect tolerance in a very short amount of time.

The overexpression and silencing of OsNCED3 in rice resulted in multiple changes in the concentrations of flavonoids, soluble sugars, oxalic acid, and amino acids in the OsNCED3-OE and osnced3-RNAi rice lines. We observed a rapid increase in flavonoid content in OsNCED3-OE rice that was significantly higher than levels in the knockdown line, osnced3-RNAi. Plants produce a number of polyphenolic compounds including flavonoids (Ballester et al., 2010; Wang et al., 2011), which function as antioxidants that scavenge free radicals in plants. Furthermore, flavonoids have key roles in antimicrobial activity and stress tolerance (Heim et al., 2002; Chen et al., 2007). Soluble sugars provide energy for growth and development and have signaling functions in plants (Takahashi et al., 2003; Kato-Noguchi et al., 2010; Boriboonkaset et al., 2013). There are two opinions on the roles of soluble sugar in plant resistance to insects; for example, one view is that higher levels of soluble sugars improve plant resistance to insects. For example, Ji et al. (2006) studied oviposition and feeding selectivity of the tobacco whitefly, Bemisia tabaci, on different cucumber varieties. Higher concentrations of soluble sugars in cucumber reduced whitefly numbers and development (Ji, 2006). He et al. (2017) also reported that peppers with higher resistance would have higher resistance to whitefly (Jing et al., 2017). After Solanum lycopersicum was treated with exogenous MeJA, Zhang et al. (2009) analyzed the nutrients, secondary metabolites, and defense enzymes in tomato leaves infested with whiteflies. The content of soluble sugars and proteins were reduced and resistance to whiteflies improved, leading the authors to speculated that exogenous MeJA improved plant resistance by reducing nutrients such as soluble sugars (Zhang et al., 2020). Our data shows that soluble sugars weaken plant resistance to plant hopper. Oxalic acid is another defensive compound that has an important role in plant resistance to abiotic and biotic stressors (Tian et al., 2009). For example, kiwifruit sprayed with oxalic acid had increased levels of defense enzymes and were more resistant to Penicillium causativeum (Zhu et al., 2016). Melons (Cucumis melo) treated with oxalic acid were more resistant to pink mold rot caused by Trichothecium roseum, and oxalic acid treatment elevated the activity of defense enzymes such as POD and PPO (Deng et al., 2015). Free amino acids function in the metabolic homeostasis of plants and also play an important role in plant resistance (Gao, 2016). Zeng et al. (1992) reported that the free amino acid content in BPH-resistant varieties of rice was lower than in susceptible cultivars (Zeng et al., 1992).

SOD, POD and CAT are important antioxidant enzymes that scavenge free radicals in plants and protect cells from oxidative damage. SOD scavenges free radicals and converts them to O₂ and hydrogen peroxide, which reduces cellular damage. CAT protects plant cells by utilizing hydrogen peroxide as a substrate and converting free radicals to oxygen and water, thus eliminating the toxicity of H₂O₂ (Xing et al., 2015; Hu, 2020). In addition to metabolizing hydrogen peroxide, POD catalyzes the polymerization of phenolics into lignin, which strengthens the plant cell wall and provides a level of protection from invading insects and pathogens (Tian et al., 2001; Jia et al., 2004). PPO catalyzes the conversion of phenolic compounds into highly reactive quinones and provides precursors for defense compounds such as lignin and phytochelatins (Zhang et al., 2020). In this study, we observed higher levels of SOD, POD and CAT in the OsNCED3-OE rice line as compared to osnced3-RNAi and WT rice; this was especially true for CAT activity. PPO activity increased rapidly beginning at 12 h and levels were significantly higher than those in the osnced3-RNAi and WT lines.

Our results definitively show that defense compounds such as flavonoids and oxalic acid were highest in the OsNCED3-OE line, and the increase was more obvious beginning 12 h after BPH exposure. The soluble sugar content was comparable in all three rice lines. The levels of the antioxidant enzymes SOD, POD and CAT were higher in the OsNCED3-OE line than the WT beginning at 0 h. In contrast, PPO activity began to rise at 12 h after BPH exposure. The average level of damage and the index of functional loss were exclusively discussed in Sun’s article for overexpression lines, while no mention was made regarding RNAi interference line, this study demonstrates that the OsNCED3-OE line exhibits higher insect resistance compared to the osnced3-RNAi line (Sun et al., 2022). Sun’s EPG analysis reveals that the feeding of BPH is inhibited by the OsNCED3-OE line. The data presented by Sun aligns with our results, indicating the involvement of OsNCED3 in rice’s mechanism of insect resistance (Sun et al., 2022). Collectively, these results also indicate that levels of defense compounds were highest in the OsNCED3-OE line, which is also is more resistant to BPH than WT and osnced3-RNAi rice.

SA has been extensively studied in plants since its original isolation from willow bark in 1828. SA has an important role in tolerance to abiotic (e.g., cold and salt stress) and biotic stressors (diseases and insects) (Kang et al., 2007; Palma et al., 2009; Hussain et al., 2014; Ueda et al., 2019; Cheng et al., 2020). JA-Ile is the bioactive form of JA, and induces the expression of defense-related genes to protect the plant from biotic stress (Wang, 2018). Although no significant changes were observed in SA content in the three rice lines ( Figure 6A ), both JA and JA-Ile levels increased rapidly in the OsNCED3-OE line beginning at 6 h after BPH feeding. Beginning at 0 h, the ABA content in OsNCED3-OE rice was higher than the levels in osnced3-RNAi rice, suggesting a role for OsNCED3 in regulating ABA synthesis. At 6–12 h after BPH feeding, the ABA content in all three rice lines increased and then declined rapidly, which was similar to the increase and decrease in JA and JA-Ile levels ( Figure 6 ). At 24 h of BPH feeding, the ABA content was comparable in all three rice lines. Based on the existing research in our group, we hypothesized that there was a synergistic interaction between ABA and JA in plants after BPH feeding.

Allene oxide synthase (AOS1) is the second enzyme in the JA biosynthesis pathway, whereas MYC2 is a transcription factor that positively regulates JA biosynthesis. Vos et al. (2013) found that exogenous ABA induced MYC2 expression in Arabidopsis, which resulted in a JA-mediated defense response. The ABA synthesis loss-of-function mutant aba2–1 impeded the transcription of the cabbage worm Pieris rapae post-feeding expression of resistance genes regulated by the transcription factor MYC2 (Vos et al., 2013). JAZ (Jasmonate ZIM-domain) is a transcriptional repressor of JA signaling that inhibits JA-activated responses by repressing MYC2. JAZ proteins are dependent on the JA signaling pathway to negatively regulate plant defense against biotic and abiotic stressors (Browse, 2009; Fu et al., 2017). OsKSL4, a biosynthesis gene for the diterpene phytocannabinoid ryanodiolactone, acts as a positive regulator of the defense response in rice (Xu et al., 2007; Yoshida et al., 2017). OsABA8ox3 is an ABA catabolism-related gene in rice, and OsPYL9 (pyrabactin resistance 9-like) is an ABA receptor. OsPYL positively regulates the ABA response when seed germination occurs, whereas overexpression of OsPYL9 can significantly improve drought and cold tolerance in rice (Tian et al., 2015). OsbZIP23 belongs to the rice bZIP transcription factor family, which promotes in plant resistance (Mellacheruvu et al., 2016). Osbph6 (brown plant hopper resistance 6) confers broad-spectrum resistance to BPH (Guo et al., 2018). RT-qPCR after BPH feeding showed that the expression of 8 genes was higher in OsNCED3-OE rice than in the WT and osnced3-RNAi lines. The expression patterns of OsAOS1, OsMYC2, and OsJAZ1 correlated with changes in JA hormone content, indicating that JA biosynthesis was promoted during BPH feeding. Expression of the ABA catabolism gene OsABA8ox3 in OsNCED3-OE rice was not significantly different from expression in WT rice ( Figure 7 ). However, expression of OsPYL9 in OsNCED3-OE was significantly higher than the WT and osnced3-RNAi lines. Based on the results obtained for OsKSL4, OsbZIP23 and Osbph6, we concluded that the resistance of rice lines overexpressing OsNCED3 gene was enhanced and the expressed genes identified in Sun et al. (2022)’s article were found to align with the transcriptome data, and their expression patterns exhibited consistency, suggesting a positive impact on enhancing rice resistance against insects (Sun et al., 2022).

The resistance of three rice lines (OsNCED3-OE, osnced3-RNAi, and WT) to BPH and the role of OsNCED3 in BPH tolerance was examined by monitoring physiological and biochemical parameters, changes in hormone content, and defense gene expression. The results indicate that OsNCED3 expression is induced by BPH feeding and correlates with improved insect tolerance in a very short amount of time. The overexpression of OsNCED3 reduced BPH-induced damage and improved insect tolerance. BPH clearly preferred to feed on osnced3-RNAi vs. the OsNCED3-OE and WT lines, and BPH populations on the OsNCED3-OE line were significantly lower than those on the WT and osnced3-RNAi. BPH that fed on the osnced3-RNAi line also had lower larval survival rates and reduced numbers of eggs per plant as compared to osnced3-RNAi and WT rice. one graphical summary model showed in Figure 8 .

Our results indicate that levels of defense compounds (flavonoids and OA) were highest in the OsNCED3-OE line, which was more resistant to BPH than WT and osnced3-RNAi rice. Our findings also show that OsNCED3 activated rice defense mechanisms, which led to increases in the defense enzymes superoxide dismutase, peroxidase, and polyphenol oxidase. JA, JA-Ile and ABA concentrations rapidly accumulated in the OsNCED3-OE line after BPH infestation, and these levels declined as the infestation time increased. The findings outlined in this study indicate that modulation of endogenous genes in rice may be a valid management tactic for reducing yield loss, which is beneficial for the environment due to the reduced use of chemical agents.

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding authors.

JtL: Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. HL: Investigation, Formal analysis, Software, Writing – original draft. XL: Software, Supervision, Visualization, Writing – original draft. WW: Investigation, Formal analysis, Writing – original draft. XL: Investigation, Conceptualization, Formal analysis, Methodology, Writing – original draft. LC: Conceptualization, Writing – original draft. YW: Conceptualization, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JlL: Writing – review & editing, Conceptualization, Data curation, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization.