Plants, especially perennial fruit trees such as pear, are frequently subject to attack by a variety of insect pests. In response to insect herbivore attack, plants have evolved highly sophisticated strategies allowing them to perceive various herbivory-associated molecules from insects’ oral secretions (or regurgitant), oviposition fluids or larval frass and trigger complex defense systems (Mao et al., 2022; Zhang et al., 2022). After the recognition of herbivores, plant initiate a series of early signaling events such as Vm variation, Ca²⁺ elevation, production of reactive oxygen species and activation of MAP kinases, as well as phytohormone-centered regulatory networks to broad transcriptional reprogramming, ultimately leads to the synthesis and accumulation of defensive proteins and defense-associated secondary metabolites (Erb and Reymond, 2019). Cross-talk between different phytohormone signaling pathways help plants to fine-tune their responses to herbivore-specific cues. It is well known that the jasmonic acid (JA) pathway as a core route in plant defenses generally acts antagonistically toward the salicylic acid (SA) pathway (Erb and Reymond, 2019).

(E)-β-ocimene, a secondary metabolite commonly found in plants, plays important physiological and ecological roles in attracting pollinators and natural enemies of pests, as well as protecting plants directly against herbivores and pathogens (Shimoda et al., 2012; Su et al., 2022). This compound belongs to monoterpene which are derived from the methyl-erythritol-phosphate (MEP) pathway mainly in plastids and produced by terpene synthases (TPSs) using geranyl diphosphate (GPP) or neryl diphosphate (NPP) as the substrate. Several TPSs including (E)-β-ocimene synthase have been functionally characterized in many plants except for wild pear Pyrus betuleafolia (Chen et al., 2011; Jiang et al., 2019; Ding et al., 2020). According to phylogenetic relationships, plant TPS family fall into seven subfamilies including TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g and TPS-h, two of which, angiosperm TPS-b and TPS-g, consists mainly of monoterpenes (Karunanithi and Zerbe, 2019).

In modern pear production, cultivated species are generally used as scions, while wild species such as P. betuleafolia was commonly used as rootstocks (Dong et al., 2020). Native to China, P. betuleafolia is widely cultivated in pear orchards across the world due to its vigorous growth, high-affinity for different cultivars of Asian pears and European pears, and resistance or tolerance to several abiotic as well as biotic environmental stresses. It has thus been gaining more scientific attention, and its recently released genomic sequence (Dong et al., 2020) is an excellent resource for mining functional genes.

Pear trees emit more than 40 different volatile organic compounds, mostly composed of terpenes, esters, aldehydes and alcohols (Chen et al., 2020). Various terpenes are present in pear fruits, flowers and vegetative tissues. For instance, α-farnesene is a major terpenoid released from ripe fruits (Chen et al., 2006). Pear trees at the flowering and fruiting stages emit 14 terpenes including (E)-β-ocimene, caryophyllene, pinene, limonene and α-farnesene, which play crucial roles in the seasonal host shift of the lepidopteran pest Grapholita molesta from peach to pear trees (Najar-Rodriguez et al., 2013). (E)-β-ocimene and linalool emitted by flowers of Pyrus bretschneideri and P. communis were crucial for attracting honey bees (Su et al., 2022). Emissions of 17 terpenes including (E)-β-ocimene and α-farnesene are increased in leaves of pear trees after pear psylla infestation (Scutareanu et al., 2003), which is attractive to conspecifics of this insect (Horton and Landolt, 2007).

Transcriptional changes of P. betuleafolia in response to abiotic stresses such as drought and potassium deficiency have been well investigated (Li et al., 2016; Yang et al., 2020b). However, little is known about transcriptional changes in wild pears infested by herbivores. It was reported that PcAFS1, a sesquiterpene synthase in Pyrus communis, could catalyze the formation of α-farnesene attracting natural enemies of herbivores (Gapper et al., 2006). Yet, the biosynthesis of monoterpenoid including (E)-β-ocimene in pears, especially in P. betuleafolia has not been elucidated. Here, transcriptional changes in P. betuleafolia infested by lepidopteran Spodoptera litura, a notorious agricultural and horticultural pest, were investigated to figure out the molecular mechanisms underlying defense responses of pear plants to chewing insects and identify candidate TPSs responsible for terpene biosynthesis. Based on RNA-seq, PbeOCS, the (E)-β-ocimene synthase gene, was newly identified. Recombinant PbeOCS was then heterologously expressed in Escherichia coli, and its catalytic products were characterized using in vitro enzymatic assays. The impacts of (E)-β-ocimene on choice behavior and growth of S. litura larvae were also assessed. Our findings provide valuable insights in understanding of the molecular mechanisms underlying defense responses of pear plants against chewing insects and shed new light on the (E)-β-ocimene biosynthesis in pear plants.

In this study, transcriptional changes in P. betuleafolia in response to chewing S. litura were investigated. Phytohormone biosynthesis and signaling pathways have been reported to play a central role in mediating plant defense responses against herbivores attack through linking perception of herbivore-derived stimuli and early signaling networks to broad downstream transcriptional reprogramming and defense induction (Erb and Reymond, 2019). Different herbivores often trigger distinct transcriptional responses and signal-transduction pathways in plants. Generally, chewing herbivores such as caterpillars and cell-content feeders such as thrips and mites activate JA pathway, while phloem-feeding insects such as whiteflies and aphids trigger SA pathway (Stam et al., 2014). SA signaling and JA signaling pathways often act through negative crosstalk. So not surprisingly, our findings consistently showed that chewing S. litura larvae activated JA signaling pathway while SA-dependent defenses were suppressed ( Table S4 ). Moreover, other phytohormone pathways such as ethylene, brassinosteroids, cytokinin, gibberellic acid, auxin and abscisic acid were also involved in pear defenses against S. litura ( Table S4 ). These findings indicated that S. litura larvae infestation triggered complex phytohormone signaling networks in pear plants, and crosstalk between these phytohormones might allow the pear seedlings to fine-tune its induced defense response to specific herbivores.

TF families are known to play important roles in herbivore-induced direct and indirect defenses in plants, which regulate the transcript levels of the target genes and potentiate defense responses (Erb and Reymond, 2019). In the present study, 135 TFs, primarily bHLH (29 genes), AP2-EREBP (28 genes) and WRKY (23 genes), were differentially expressed in S. litura-infested P. betuleafolia leaves ( Table S6 ). Several TFs involved in the transcriptional regulation of insect-induced volatile terpenes, including (E)-β-caryophyllene in Arabidopsis thaliana (Hong et al., 2012), (E)-β-farneseneas and (E)-α-bergamotene in Zea mays (Li et al., 2015) linalool in Freesia hybrida (Yang et al., 2020a), and Dendrobium officinale (Yu et al., 2021), valencene and (E)-geraniol in Citrus sinensis (Shen et al., 2016; Li et al., 2017) and α-farnesene in M. domestica (Wang et al., 2020), have been identified and functionally characterized. However, the role of TFs in terpene biosynthesis in pear is still not clear and need further investigation.

Many genes involved in biosynthesis of defensive metabolites, such as CYP (64 genes) and UGTs (18 genes), were differentially expressed ( Table S8 ). In addition, 24 genes associated with the biosynthetic pathways of volatile terpenoids and HPL-derived green leaf volatiles were upregulated after S. litura infestation, highlighting their potential roles in S. litura-induced pear defenses. Unlike defense-related nonvolatile and volatile secondary metabolites, growth-related photosynthesis was suppressed in S. litura-infested P. betuleafolia ( Table S7 ), which is similar to the findings reported in the defense response of potato (Mao et al., 2022), cotton (Zhang et al., 2022), rice (Wang et al., 2020; Satturu et al., 2021), wheat (Zhang et al., 2020) and tomato (Ke et al., 2021) plants induced by herbivores. In herbivore-damaged plants, suppression of photosynthesis might be a general phenomenon as a result of a trade-off between growth and defense, which allow plants to reallocate its limited resources toward activation of anti‐herbivore defense responses and production of inducible secondary metabolites (Zhou et al., 2015).

In plants, (E)-β-ocimene is one of the most common volatiles that act as an attractant in flower-pollinator interactions, a defensive compound in leaf-pest interactions and/or a priming signal in plant-plant communication (Fernández-Milmanda, 2022; Onosato et al., 2022). (E)-β-ocimene in flower and leaves of pear has multiple ecological roles in attracting pollinators and plant-pest interactions (Scutareanu et al., 2003; Su et al., 2022). The TPS in catalyzing (E)-β-ocimene biosynthesis, namely (E)-β-ocimene synthase, has been functionally characterized in several plant species such as Antirrhinum majus (Dudareva et al., 2003), Arabidopsis thaliana (Fäldt et al., 2003), Camellia sinensis (Chen et al., 2021; Jian et al., 2021), Citrus unshiu (Shimada et al., 2005), Lotus japonicus (Arimura et al., 2004), Malus domestica (Nieuwenhuizen et al., 2013), Matricaria recutita (Irmisch et al., 2012), Medicago truncatula (Navia-Giné et al., 2009), Morus notabilis (Ding et al., 2020), Nicotiana attenuata (Xu et al., 2020), Osmanthus fragrans (Zeng et al., 2015), Phaseolus lunatus (Arimura et al., 2008), Populus trichocarpa (Irmisch et al., 2014) and Vitis vinifera (Martin et al., 2010). In the current study, the (E)-β-ocimene synthase gene PbeOCS was present in the RNA-seq data and functionally characterized in vitro ( Figure 5 ). The upregulation of PbeOCS after S. litura infestation suggested that PbeOCS and its enzymatic product (E)-β-ocimene are likely to be involved in insect-induced pear defenses. Interestingly, subsequent insect bioassay revealed that (E)-β-ocimene had a repellent effect on S. litura larvae ( Figure 6 ) and significantly increased larval mortality ( Figure 7 ). Additionally, candidate TPSs involved in the biosynthesis of α-pinene, limonene, linalool, germacrene D, β-caryophyllene (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) were identified in our transcriptomic data, which still need to be further functionally characterized.

In this study, transcriptomic analyses of wild pear P. betuleafolia after S. litura infestation were performed to elucidate the molecular mechanisms underlying pear-herbivore interactions and to mine new TPSs. Our results revealed that S. litura infestation triggered a drastic and extensive transcriptomic reprogramming in pear leaves, including activation of a series of early signaling events and induction of complex phytohormone signaling networks. Significantly, almost all DEGs in the JA pathway were significantly upregulated, indicating that the chewing S. litura mainly activated JA-dependent defense responses. Moreover, defense-related transcription factors and genes involved in biosynthetic pathway of green leaf volatiles and volatile terpenes were also activated, whereas growth-related photosynthesis and SA-dependent defenses were suppressed. In addition, a terpene synthase gene PbeOCS was present in the RNA-seq data and functionally characterized in vitro. Recombinant PbeOCS catalyzed GPP as substrate to produce (E)-β-ocimene and the compound had significant impacts on choice behavior and growth of S. litura larvae in insect bioassays. These findings should contribute to generating valuable markers for further breeding insect-resistant pear varieties and developing an environmentally friendly strategy for pest control.

The original contributions presented in the study are publicly available. This data can be found here: NCBI, PRJNA783361 and GenBank, OK635579.

WS and YZ conceived and designed the study. XZH, HZ, HL, MW, XG, and EL performed the experiments and analyzed the data. XQH, CZ, and LL contributed the resources. XZH, HZ, WS, and YZ wrote the manuscript. All authors read and approved the submitted manuscript.

This work was supported by the National Natural Science Foundation of China (32272638, 31972338, 31772176, 31701800), and the Open Fund Project of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201901).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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