Mechanism of Exogenous Jasmonic Acid-Induced Resistance to Thrips palmi in Hemerocallis citrina Baroni Revealed by Combined Physiological, Biochemical and Transcriptomic Analyses

Abstract

Jasmonic acid (JA) is a regulator of plant resistance to phytophagous insects, and exogenous JA treatment induces plant insect resistance. This study investigated the mechanism of exogenous JA-induced resistance of Hemerocallis citrina Baroni (daylily) to Thrips palmi at the biochemical and molecular levels. Daylily leaves sprayed with JA showed significantly higher levels of secondary metabolites—tannins, flavonoids, and total phenols, and activity of defense enzymes—peroxidase, phenylalanine ammonia lyase, polyphenol oxidase, and protease inhibitor (PI) than control leaves; the most significant effects were observed with 1 mmol L⁻¹ JA. Owing to an improved defense system, significantly fewer T. palmi were present on the JA-treated plants than control plants. The JA-treated leaves had a smoother wax layer and fewer stomata, which was unfavorable for insect egg attachment. The differentially expressed genes (DEGs) were significantly enriched in insect resistance pathways such as lignin and wax biosynthesis, cell wall thickening, antioxidant enzyme synthesis, PI synthesis, secondary metabolite synthesis, and defense hormone signaling. A total of 466 DEGs were predicted to be transcription factors, mainly bHLH and WRKY family members. Weighted gene co-expression network analysis identified 13 key genes; TRINITY_DN16412_c0_g1 and TRINITY_DN6953_c0_g1 are associated with stomatal regulation and lipid barrier polymer synthesis, TRINITY_DN7582_c0_g1 and TRINITY_DN11770_c0_g1 regulate alkaloid synthesis, and TRINITY_DN7597_c1_g3 and TRINITY_DN1899_c0_g1 regulate salicylic acid and ethylene biosynthesis. These results indicate that JA treatment of daylily improved its resistance to T. palmi. These findings provide a scientific basis for the utilization of JA as an antagonist to control T. palmi in daylily.

1. Introduction

Plants encounter numerous insect herbivores during their lifespan. Various compounds from the oral and oviposition secretions of insects—such as metabolites, peptides, and proteins—act as ligands for membrane receptors in plant cells, activating signaling cascades that lead to a jasmonic acid (JA) burst at the wound sites [1]. JA and its precursors and derivatives, referred to as jasmonates, are important molecules in the regulation of several physiological processes in plant growth and development, especially the mediation of plant responses to biotic and abiotic stresses [2]. JA synthesis occurs (Figure 1) [3] via the octadecanoid pathway, which has been extensively characterized in the model plant Arabidopsis [4]. JA biosynthesis begins in the chloroplast with the release of α-linolenic acid (18:3) from membranes and the sequential actions of lipoxygenase, allene oxide synthase, and allene oxide cyclase proteins, releasing 12-oxo-phytodienoic acid (OPDA) [5]. OPDA is then converted to JA in the peroxisome by OPDA reductase 3 (OPR3) via three cycles of beta oxidation [6]. In addition to the canonical JA biosynthetic pathway utilizing the peroxisomal OPR3, there is an alternative biosynthetic pathway dependent on the cytosolic OPR3 homologs OPR1 and OPR2; OPR1 and OPR2 catalyze the reduction of 4,5-didehydro-JA to JA in the cytosol independent of OPR3 [7,8]. There are multiple JA conjugates in the cytoplasm, of which jasmonoyl-L-isoleucine (JA-Ile) is the bioactive form. In the cell nucleus, the JA co-receptors CORONATINE-INSENSITIVE1 (COI1) and Jasmonate ZIM domain (JAZ) bind to JA-Ile, activating JA-responsive transcription factors (TFs) involved in the regulation of the biosynthesis of plant defense compounds [9].

The phytohormone JA is a key signal that induces the synthesis of insect-resistant compounds and the formation of insect-resistant tissues in plants, which adversely affects feeding, physiological activities, and host selection of pests, and produces a direct insect-resistant defense response [10,11]. Stomata and cuticular wax are structural features of plant leaves to defend against insect damage, and it was shown that the stomatal density of the leaf surface was significantly reduced [12] and the cuticular wax was significantly thickened after JA induction [13], which affects the egg-laying behavior of the insect as well as the growth and development of the larvae. Exogenous application of JA in cucumber induced leaf thickening, significantly increased trichome density and phenol content, enhanced the resistance to Liriomyza sativae, and significantly decreased the degree of pest damage [14]. Exogenous jasmonic acid induces plants to produce toxic secondary metabolic compounds (e.g., phenolic acids, tannins, etc.) or defensive enzymes (e.g., peroxidase (POD), phenylalanine ammonia-lyase (PAL), polyphenol oxidases (PPO), protease inhibitors (PI), etc.) that impede the digestion and utilization of plant nutrients by the pests, interfering with the normal physiology and behavior of the pests, and causing the plants to show a certain degree of resistance to the pests [15,16,17]. In rice, JA-induced accumulation of anti-herbivore compounds (such as phenolic amides and trypsin inhibitors) mediates resistance to the rice leaf folder Cnaphalocrocis medinalis [18]. In tomato plants, exogenous JA application increased the activity of PPO, PI, and POD and decreased feeding damage by Spodoptera exigua Hubner [19]. The activities of antioxidant enzymes such as POD, superoxide dismutase (SOD) and catalase (CAT) were increased and aphid feeding was reduced in wheat leaves after JA spraying; in particular, 1 mmol L⁻¹ was better than 0.1 mmol L⁻¹ [20]. Exogenous JA-induced expression of insect resistance genes is similar to pest induction, binding specifically to receptors on plant cells and undergoing a series of intracellular signaling, ultimately inducing the expression of specific genes in the nucleus and the synthesis of TFs. TFs are trans-regulatory factors that bind cis-elements on the promoters of resistance genes, inducing the expression of resistance genes and ultimately inducing plant insect resistance. Jasmonate-responsive TFs that regulate the jasmonate-induced accumulation of secondary metabolites belong to diverse families such as APETALA2/ethylene response factor (AP2/ERF), basic helix–loop–helix (bHLH), myeloblastosis-related factor (MYB), and WRKY [21]. Genes involved in cell wall synthesis, chlorophyll metabolism, calcium-transporting ATPase activity, protein kinase activity, flavonoid biosynthesis, and aromatic acid synthesis pathways in Arabidopsis were upregulated during JA stress [22].

Hemerocallis citrina Baroni (daylily) is a perennial liliaceous herbaceous plant. The genus is widely cultivated for food, medicinal value and ornamental interest [23]. Thrips palmi, an important pest of vegetables and ornamentals, transmits several tospoviruses [24]. T. palmi concentrates on the back of daylily leaves or the heart and leaves of flower shoots, filing and sucking the sap of young plant tissues, harming the leaves, heart and shoots, affecting the normal growth of stem shoots and flower buds, and seriously affecting daylily yields and commercial value [25]. Thrips, which is highly insidious, reproduces quickly and is prone to outbreaks, often causes serious disasters when it is detected, and is currently the only insect pest that can lead to the extinction of daylily. At present, thrips control mainly adopts chemical control, but the excessive use of pesticides has led to thrips resistance to a variety of pesticides, and a large amount of killing and maiming of natural enemies and environmental pollution [26]. Therefore, it is important to study the mechanism of thrips resistance and thrips green control measures for the sustainable development of the daylily industry. Treatment of plants with synthetic JA induces a defensive response similar to that observed during an herbivore attack and is a potential strategy for integrated pest management. However, there are no reports of exogenous JA inducing insect resistance in daylily. To this end, we used Hemerocallis citrina ‘Datong Huanghua’ (daylily) as the experimental material to study induced resistance to T. palmi after exogenous JA application. The present study aimed to examine whether exogenous application of JA (1) improves the resistance of daylily and reduces the T. palmi-induced damage in the field and (2) affects the expression of defense-related traits such as stomata, wax layer, secondary metabolite content, and defense enzyme activity in daylily leaves, with a view to providing a theoretical basis and application technology for the use of exogenous JA to induce resistance to insects of the daylily to realize the green prevention and control of T. palmi.

2. Materials and Methods

2.1. Daylily Varieties, Planting and Treatments

‘Datong Huanghua’ cultivated at the Horticultural Station of Shanxi Agricultural University was used as the test plant. The plots were set up in 4-row zones with 3.5 m row length and 45 cm row spacing, with a plot area of 14 m², and field management was carried out as in conventional daylily production fields. Experiments were conducted during the spring seedling growth period. When the seedlings reached the seven-leaf stage, pest- and disease-free seedlings from approximately the same growth conditions were selected and exogenously sprayed with three concentrations of JA (95% purity; cat no. 77026-92-7; Sigma Aldrich, Shanghai, China; 0.01, 0.1, and 1 mmol L⁻¹, named 0.01JA, 0.1JA, and 1JA). The selected JA concentrations were selected on the basis of the preliminary findings, as the three concentrations not only promoted the growth of daylily plants but also effectively reduced the population of T. palmi. A total of 0.1 g of JA was dissolved using 7 mL of anhydrous ethanol, and then the dissolved JA was prepared into 0.01, 0.1, and 1 mmol L⁻¹ solutions using distilled water. A 0 mmol/L JA solution was prepared by adding equal amounts of anhydrous ethanol and distilled water, and control check (CK) was sprayed with equal amounts of 0 mmol/L JA. One plot was selected for each treatment, and 20 daylily seedlings were selected from each plot for a uniform spray treatment using a small hand-held sprayer. The plants were sprayed evenly with the appropriate treatment solution (approximately 200 mL per plant) until the solution dripped off the leaves; then, the whole plant was covered with transparent plastic bags for 8 h to prevent evaporation of the treatment solution. Polysorbate 80 (cat no. T8360; Solarbio, Beijing, China) was used as an additive in all treatment solutions to facilitate adhesion of the solution to the leaves.

2.2. Determination of Plant Secondary Metabolite Content

The fifth to seventh leaves (new leaves) of each daylily seedling were selected for each analysis at 1, 3, 5, 7 and 9 days after spraying in the three JA treatment groups, and the fifth to seventh leaves collected at the same time points in the CK group were used as the control. To determine the leaves’ secondary metabolites, a pooled sample from five daylily seedlings was taken for each sample, and three biological replicates were taken from each sample. Tannins were extracted from the leaves of different treatments with dimethylformamide, centrifuged after extraction, and color development was carried out by adding chromogen, while a blank control was made, and then the absorbance value of the solution to be tested was measured at 525 nm with an enzyme marker, and the tannin content in the leaves was determined by using tannic acid as the standard curve (cat no. BC1395; Solarbio). After the leaves of different treatments were dried and crushed through a 40-mesh sieve, 60% ethanol was added to ultrasonic treatment (power of 300W, temperature of 60 °C) and centrifuged after extraction for 30 min, and color development was carried out by adding colorant, while a blank control was made, and then the absorbance value of the solution to be measured was determined by using an enzyme standard apparatus at a wavelength of 470 nm, and rutin was used as the standard curve to determine the content of flavonoids in the leaves (cat no. BC1335; Solarbio). After the leaves of different treatments were dried and crushed through a 40-mesh sieve, 60% ethanol was added to ultrasonic treatment (power 300 W, temperature 60 °C) and centrifuged after extraction for 30 min, and color development was carried out by adding colorant, while a blank control was made, then the absorbance value of the solution to be tested was measured by enzyme marker at 760 nm, and the total phenol content of leaves was determined by using gallic acid as the standard curve (cat no. BC1340; Solarbio).

2.3. Determination of Plant Defense Enzyme Activity

The fifth to seventh leaves (new leaves) of each daylily seedling were selected for each treatment at 1, 3, 5, 7, and 9 days after spraying in the three JA treatment groups, and the fifth to seventh leaves collected at the same time points in the CK group were used as the control. To determine the activity of defense enzymes in leaves, a mixed sample of five daylily seedlings was taken for each sample, and three biological replicates were taken from each sample. Leaves were homogenized in an ice bath by adding the extract (prepared in the kit) (cat no. BC0090; Solarbio), centrifuged, and then the reagents were added and mixed thoroughly according to the steps in the kit, the change in absorbance (OD) value at 470 nm was determined using a spectrophotometer, and one unit of enzyme activity was defined as a change in OD₄₇₀ of 0.01 per min per gram of tissue in the reaction system. Leaves were homogenized in an ice bath by adding the extract (prepared in the kit) (cat no. BC0215; Solarbio), centrifuged, and then the reagents were added and mixed thoroughly according to the steps in the kit, the change in OD value at 290 nm was determined using a spectrophotometer after 10 min of standing, and one unit of enzyme activity was defined as a change in OD₂₉₀ of 0.01 per min per gram of tissue in the reaction system. Leaves were homogenized in an ice bath by adding the extract solution (which was prepared in the kit) (cat no. BC0195; Solarbio), centrifuged, reagents were added according to the steps in the kit and mixed thoroughly, and a water bath at 25 °C for 10 min was followed immediately by a boiling water bath for 10 min, the supernatant was collected by centrifugation after cooling, the change in OD value at 290 nm was determined using a spectrophotometer, and one unit of enzyme activity was defined as a change in OD₄₁₀ of 0.01 per min per gram of tissue in the reaction system. Leaves were homogenized in an ice bath by adding PBS (PH 7.4), centrifuged and operated according to the kit steps (cat no. SP39933; Spbio, Wuhan, China), and the change in OD at 450 nm was determined using a spectrophotometer after termination of the reaction, and the concentration of PI in the samples was calculated on the basis of the standard curve.

2.4. Effect of Exogenous Jasmonic Acid on the Population of Thrips palmi in Daylily

Five sites were randomly selected in each plot, five daylilies were selected at each site, and each site was counted as one replicate. T. palmi adults were attached to the JA-treated daylily plants (20 heads/plant) with a fine soft brush and covered with an 80 µm insect net to prevent their escape. The population size of T. palmi on the plants from various treatment groups was visually surveyed at 1, 3, 5, 7 and 9 days after inoculation at 3 p.m. every day. During the population survey, the number of adult T. palmi on one upper, one middle, and one lower leaf on each plant was counted by gently turning the leaves.

2.5. Microscopic Observation of the Morphological Structure of Leaves’ Epidermal Stomata and Waxy Layer

To explore the effect of exogenous JA on the epidermal structure of daylily leaves, we used scanning electron microscopy to observe the structural differences in leaf stomata and wax layer. The concentration of JA that improved the physiological indexes of resistance and decreased the number of T. palmi most significantly was selected for subsequent experimentation. After treating leaves with this concentration of JA for 5 days, samples were taken from the fifth to seventh leaves (new leaves) for microscopic observation of stomata and the morphology and structure of the waxy layer; CK leaves at the same positions collected at the same time were used as controls. The surface of sampled leaves was rinsed clean with distilled water and immediately placed in 2.5% glutaraldehyde (0.1 mol L⁻¹, pH 7.2 phosphate buffer preparation), and the materials were fixed at 0–4 °C for 2 days after pumping the material sink by vacuum pump. The samples were rinsed thrice with the same phosphate buffer and dehydrated by treating with 30%, 50%, 70%, 80%, 90%, and 95% ethanol for 15 min each and in 100% ethanol twice for 20 min each. Then, the samples were subjected to tert-butanol replacement and freeze-drying (HITACHI ES-2030, Tokyo, Japan). After drying, the material was adhered to the sample stage with electrical tape, and platinum was sprayed on the samples using an ion sputter coater (HITACHI E-1010, Tokyo, Japan). The prepared samples were subjected to scanning electron microscopy (HITACHI U8010, Tokyo, Japan) for morphological observation and imaging [27,28].

2.6. Transcriptome Sequencing and Analysis

At 1, 3, 5, 7, and 9 days after spraying leaves with the optimal JA concentration, the fifth to seventh leaves of daylily plants were collected (named TG1–TG5), immediately dipped in liquid nitrogen, and then stored at −80 °C. CK leaves of the same part of the same period were used as control (named CK1–CK5). Three replicate samples were collected at each time point. Total RNA was extracted using the Trizol method [29]. RNA integrity was accurately detected by Agilent 2100 bioanalyzer (Agilent, Beijing, China), and cDNA libraries were constructed from total RNA that passed the quality control. The Illumina NovaSeq 6000 (Illumina, CA, USA) was used as the sequencing platform, and the raw reads obtained from sequencing were first filtered, and the sequencing error rate and GC content distribution were checked to obtain clean reads for subsequent analysis, and then the transcripts were spliced by Trinity (version 2.4.0), and hierarchical clustering of the transcripts was performed by the Corset (version 4.6) program to remove redundant transcripts. The transcripts were then spliced into transcripts using Trinity (version 2.4.0), and then hierarchically clustered using the Corset (version 4.6) program. After removing the redundant transcripts and using the clustered sequences as a reference, the longest transcripts (Unigene) in each gene were selected for subsequent functional annotation and analysis.

2.7. Screening and Analysis of Differentially Expressed Genes

Significantly DEGs were screened based on two-by-two sample comparisons with the following conditions: FDR < 0.05, |log₂FC| > log₂(2) [30]. All significantly DEGs in the samples were clustered into expression patterns using ShortTime-series Expression Miner, and the set of genes with the same expression pattern was analyzed for Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment, and the p-value was obtained by hypothesis test counting, and the q-value was corrected for FDR to define the significantly enriched KEGG/GO in the trend, with q-value KEGG/GO ≤ 0.05 defined as the significantly enriched KEGG/GO in the trend [31]. GO functional enrichment was used to understand the major biological functions exercised by differentially expressed genes, and pathway enrichment analysis was used to understand the major signaling pathways and biochemical metabolism pathways in which differentially expressed genes were involved. Finally, TFs were predicted using the Plant Transcription Factor Database.

2.8. Weighted Gene Co-Expression Network Analysis

Based on the transcriptome differential gene expression matrix, 30% of the low-expression genes were first filtered out using R language (version 3.4.4), and the remaining genes were used to construct the co-expression network and divide the gene modules with the WGCNA package (version 1.6.6), and subsequently, key gene modules were searched for through the analysis of the correlation with secondary metabolites and defense enzymes. Cytoscape (version 3.9.1) software was used to construct the secondary metabolite and defense enzyme-related structural gene interaction network, and the core structural genes in the interactions network were screened by the MCC algorithm of CytoHubba plug-in in this software.

2.9. qRT-PCR Validation

Six genes were selected for qRT-PCR to validate the RNA-seq results. The design of specific primers used Primer-BLAST from the NCBI website, and the actin gene was used as an internal control. Primer sequences are listed in Table S1. Analyses were performed using a Bio-Rad CFX96 RT-PCR detection system fluorescence quantitative PCR instrument according to the TB Green^® Premix Ex Taq^TM II (Takara, Beijing, China) instructions. Each qRT-PCR experiment (15 μL) consisted of 7.5 μL of 2× SG Fast qPCR Master Mix, 0.6 μL of each primer (10 μM), 40 ng of cDNA template, and ddH2O up to 15 μL. The amplification program was pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 56 °C for 30 s, and 40 cycles. Three replicates were set up for each treatment and the relative expression of genes was calculated by the 2^−ΔΔCT method.

2.10. Data Analysis

One-way ANOVA using SPSS v20.0 software was used to analyze the differences between the means (±standard error) of the treatment groups, and Tukey’s HSD and Duncan’s new complex polar deviation methods were used to perform a significant ANOVA for differences (p < 0.05). Plots were drawn using Microsoft Excel 2010 software.

3. Results

3.1. Effects of Exogenous Jasmonic Acid on Secondary Metabolite Content of Daylily Leaves

Daylily leaves treated with the three concentrations of JA showed a significantly higher content of secondary metabolites than the control leaves at all five time points. The levels of tannins, flavonoids, and total phenols were most significantly affected by the 1JA treatment, with the highest flavonoid content at day 3, which was four-fold higher than that in CK (Figure 2B), and the highest tannin and total phenols levels at day 5, which were 2.1 and 2.3 times higher than those in CK, respectively (Figure 2A,C).

3.2. Effects of Exogenous Jasmonic Acid on Defense Enzyme Activity in Daylily Leaves

Daylily leaves treated with the three concentrations of JA showed significantly higher POD, PAL, and PI activity than control daylily leaves at all five time points; however, PPO activity of JA-treated leaves was not significantly different from CK leaves at day 9. POD, PPO, PAL, and PI activity were most significantly affected by the 1JA treatment, with peak PPO activity at day 3, which was 2.2-fold higher than that in CK (Figure 3B), and peak POD and PAL activity at day 5, which was 10.1 and 1.8 times higher than that in CK, respectively (Figure 3A,C). PI activity peaked at day 1 and was 1.7 times higher than that in CK (Figure 3D).

3.3. Effect of Exogenous Jasmonic Acid on Thrips palmi Population

Application of exogenous JA increased daylily resistance, thereby adversely affecting T. palmi. The three concentrations of exogenous JA treatments were effective in suppressing the population of T. palmi, and the 1JA treatment group had the lowest number of T. palmi per plant (Figure 4). It indicated that exogenous JA affected the fecundity of T. palmi, and the number of T. palmi decreased gradually with the increase in JA concentration.

3.4. Effects of Exogenous Jasmonic Acid on Stomatal and Wax Layer Morphology in Daylily Leaves

It was observed that the stomata on the adaxial surface of 1JA-treated daylily leaves and CK leaves were arranged in the same manner, in a single linear row in the intercostal region of the leaf blade. The number of stomata on the adaxial surface of 1JA-treated leaves was lower than that in CK leaves, and the rows of stomata were more loosely packed (Figure 5A,B). The abaxial surface of 1JA-treated leaves was smoother with fewer stomata, and there was less material at the edges of and inside the stomata to facilitate insect egg attachment. In contrast, the abaxial surface of CK leaves was rougher, and the stomata were more conducive to egg attachment (Figure 5C,D). The wax layer on the abaxial surface of both 1JA-treated daylily leaves and CK leaves was distributed around the stomata. The wax layer on CK leaves was more raised, and it was waxy around and inside the stomata, whereas the abaxial surface of 1JA-treated leaves was smoother, and wax did not fill around and inside the stomata (Figure 5E,F).

3.5. Quality Assessment of Transcriptome Sequencing Results

In total, 141.94 GB of clean data was obtained from 10 samples, which were filtered using Illumina 2 × 150 bp double-end sequencing; clean data from each sample reached >6.03 Gb, and the average Q30 was >94.18%; the comparison efficiency of clean reads ranged between 80.59% and 82.82% (

Table 1. Transcriptome sequencing data statistics.

Sample	Raw Reads/bp	Clean Reads/bp	Q20 (%)	Q30 (%)	Mapped Reads/bp
CK1	48,597,229	43,722,323	96.95	94.35	17,805,077 (81.45%)
CK2	50,907,231	42,588,800	96.90	94.25	17,268,027 (81.10%)
CK3	46,682,755	41,929,344	96.94	94.36	17,363,380 (82.82%)
CK4	49,858,899	41,816,134	96.91	94.30	17,136,180 (81.96%)
CK5	52,020,018	42,876,465	96.87	94.18	17,551,857 (81.86%)
TG1	44,946,127	42,324,159	97.82	95.66	17,519,708 (82.79%)
TG2	46,777,333	43,246,189	97.69	95.43	17,512,535 (81.00%)
TG3	45,726,123	41,665,462	97.68	95.36	16,787,678 (80.59%)
TG4	45,821,658	43,084,535	97.74	95.50	17,607,582 (81.74%)
TG5	45,995,561	44,713,283	97.97	95.96	18,222,903 (81.48%)

). The results indicated that the quality of the sequencing output data was good, and the data could be used for further analysis.

3.6. Daylily Genes Differentially Expressed in Response to Jasmonic Acid Treatment

The transient expression of genes in daylily leaves in response to 1JA treatment was investigated. In total, 32,326 DEGs were identified at the five time points after 1JA treatment. The TG1 stage had the highest number of DEGs (10,253); the TG4 stage had the lowest number of DEGs (3391) (Figure 6A). Only 475 genes were differentially expressed in all TG1–TG5 stages due to the low number of DEGs during T4 (Figure 6B).

The GO enrichment analysis showed that the DEGs were more enriched in biological process (BP) subcategories, including secondary metabolic process (GO:0019748), L-phenylalanine biosynthetic process (GO:0009094), terpenoid biosynthetic process (GO:0016114), regulation of stomatal complex development (GO:2000038), cell wall macromolecule metabolic process (GO:0044036), hormone-mediated signaling pathway (GO:0009755), glutathione metabolic process (GO:0006749). Among cellular component (CC) subcategories, plastid organization (GO:0009657) and extracellular region (GO:0005576) were significantly enriched. Protein phosphatase inhibitor activity (GO:0004864) and peroxidase activity (GO:0004601) were significantly enriched among molecular function (MF) subcategories (Figure 6C).

In addition, amino acid metabolic pathways such as alpha-linolenic acid metabolism, cyanoamino acid metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis and plant insect resistance pathways such as isoquinoline alkaloid biosynthesis; flavonoid biosynthesis; anthocyanin biosynthesis; cutin, suberin, and wax biosynthesis; and ascorbate and aldarate metabolism were enriched significantly based on the results of KEGG enrichment analysis for the common DEGs (Figure 6D).

3.7. Expression Patterns of Genes Related to Jasmonic Acid-Mediated Improvement of Daylily Resistance

The expression levels of 475 genes associated with daylily resistance were analyzed over five periods of time (Figure 7). The results showed that these genes were divided into two clusters on the basis of their expression patterns. Cluster1 contained 281 genes. The functions of these genes include activation of POD, PAL, PPO, and PI activities; promotion of flavonoids, alkaloids, and terpenoids synthesis; and regulation of hormone signaling such as JA, salicylic acid (SA), abscisic acid (ABA), and ethylene (ET). These genes were mainly upregulated after 1 JA treatment and were more significantly upregulated in the TG1 and TG2 stages. Cluster2 contained 194 genes; their functions include polysaccharide metabolic process, stomatal complex development, sulfur compound catabolic process, carbohydrate metabolism, and photosynthesis, and they were mainly downregulated after 1 JA treatment.

3.8. Transcription Factor Prediction and Expression Patterns

To explore the TFs associated with 1 JA-induced insect resistance in daylily, 466 TFs were screened from 32,326 DEGs and clustered into 26 TF families, including MYB, AP2/ERF, bHLH, C2C2, WRKY, and NAC, which are closely related to plant resistance (Figure 8A, Table S1). These genes could be divided into six expression patterns (Figure 8B,C). The TFs in Cluster1, Cluster3, Cluster4, and Cluster6 were upregulated after 1 JA treatment. The TFs in Cluster1 and Cluster6 were mainly MYB and C2C2; the Cluster1 and Cluster6 TFs were significantly upregulated at the TG3 and TG5 stage, respectively. The TFs in Cluster3 were mainly AP2/ERF and bHLH and were significantly upregulated at the TG1 stage. The TFs in Cluster 4 were mainly AP2/ERF and WRKY and were significantly upregulated at the TG2 stage. The TFs in Cluster2 and Cluster5 were mainly NAC and B3, which were downregulated in comparison with CK. Cluster3 and Cluster4 included the highest numbers, with 70 and 110 upregulated TFs, respectively, indicating that they play an important role in the induction of insect resistance in daylilies by 1 JA.

3.9. WGCNA-Identified Hub Genes Responded to the Jasmonic Acid-Induced Resistance

To analyze gene expression profiles in transcriptomic data and to search for gene modules and co-expressed genes related to secondary metabolite synthesis and defense enzyme activities, the 32,326 differentially expressed genes in the transcriptome data were filtered out of the 30% low-expression genes to obtain 21,281 highly expressed genes, which were used for WGCNA analysis. The soft threshold β = 5 was determined when the fitting curve was close to 0.8 for the first time (Figure 9A), and a total of 23 co-expression modules were obtained using the dynamic shear tree method, among which the turquoise module was the largest, with 3901 genes, whereas the dark green module was the smallest, with only 53 genes (Figure 9B,C). The genes in the blue module are closely related to secondary metabolite synthesis and defense enzyme activities, and the correlation coefficients are all greater than 0.77 (Figure 9C). Four key modules (magenta, tan, midnight blue, and blue) that are highly correlated with the seven phenotypes were selected, and Cytoscape (version 3.9.1) software was used to construct the interactions networks of these structural genes, respectively, and the MCC algorithm of the CytoHubba plug-in of this software was used to screen the hub genes in the interaction networks (Figure 9D). A total of 13 network hub genes were identified as key genes and were annotated using Asparagus officinalis, Nymphaea colorata, Juglans regia, and Viscum album. Among them, TRINITY_DN16412_c0_g1 regulates stomatal development; TRINITY_DN6953_c0_g1 plays a crucial role in the synthesis of lipid barrier polymers, which constitute the first line of physical defense in the plant. TRINITY_DN7582_c0_g1 regulates anthocyanin synthesis and is annotated to the zinc-finger domain; TRINITY_DN7597_c1_g3 regulates the synthesis of phenolic acid secondary substances such as SA and protocatechuic acid and enhances the chemical defense of plants. TRINITY_DN1899_c0_g1 regulates ET synthesis and is annotated to bHLH; TRINITY_DN3105_c0_g1 initiates the plant MAPK pathway, regulates protein tyrosine and serine/threonine kinase, and is annotated to ABC 1. TRINITY_DN11770_c0_g1 regulates the synthesis of alkaloids and is annotated to the LRE family, which are involved in the regulation of plant growth and development and response to adversity and other biological processes (

Table 2. Hub genes and predicted functions.

Gene ID	Homologous Species/Gene	Gene Function
TRINITY_DN16412_c0_g1	Viscum album AEM46061.1	Actin-related protein; cytoskeleton
TRINITY_DN1021_c1_g1	Asparagus officinalis ONK62957.1	Mitochondrial outer membrane; protein turnover
TRINITY_DN7582_c0_g1	Asparagus officinalis XP_020250650.1	lysine-specific demethylase JMJ25; zinc-finger domain of monoamine-oxidase A repressor R1
TRINITY_DN11770_c0_g1	Juglans regia KAF5481697.1	strictosidine synthase activity; SMP-30/gluconolaconase/LRE domain protein
TRINITY_DN9399_c0_g1	Asparagus officinalis XP_020251516.1	Lysine-specific demethylase JMJ25
TRINITY_DN7597_c1_g3	Asparagus officinalis XP_020257495.1	Polyadenylate-binding protein RBP47
TRINITY_DN95913_c0_g1	Asparagus officinalis XP_020265081.1	Intramolecular lyase activity; plant transposase (Ptta/En/Spm family)
TRINITY_DN9965_c0_g1	Nymphaea colorata XP_031476351.1	Photosystem I reaction center subunit II; energy production and conversion
TRINITY_DN28770_c0_g1	Asparagus officinalis XP_020276044.1	Serine/threonine protein kinase
TRINITY_DN1899_c0_g1	Asparagus officinalis XP_020246802.1	Protein dimerization activity; ethylene-responsive protein; transcription factor bHLH113-like
TRINITY_DN6953_c0_g1	Asparagus officinalis XP_020264347.1	C2 and GRAM domain-containing protein; VAD1 analog of StAR-related lipid transfer domain
TRINITY_DN10849_c0_g3	Asparagus officinalis XP_020275006.1	Protein dephosphorylation; metal ion binding; myosin phosphatase activity; PP2C
TRINITY_DN3105_c0_g1	Asparagus officinalis XP_020273072.1	MAPK-Plant; protein kinase activity; protein tyrosine and serine/threonine kinase; ABC1 atypical kinase-like domain

).

3.10. Real-Time Quantitative PCR (qRT-PCR) Verification of Transcriptome Data

The expression trends of the six DEGs verified by qRT-PCR were consistent with the transcriptome sequencing results, and the correlation reached 0.8388 (Figure 10 and Figure S1), indicating that the RNA-Seq results were highly credible.

4. Discussion

JA is a naturally occurring hormone in plants that acts as a crucial signaling molecule in various developmental and defense responses, including insect resistance [32]. The JA phytohormone signaling pathway orchestrates multiple plant responses to herbivory [33]. Several studies have demonstrated that exogenously applied JA induces plant resistance against various insects in many crop plants [34,35,36,37,38]. Exogenous JA increases the content of the wax layer of plant leaves and decreases stomatal density in response to biotic stress [39,40]. Spraying exogenous JA on the whole plant rapidly stimulates the JA pathway, induces the expression of a series of defense genes, and produces numerous defense proteases and secondary metabolites, which inhibit or interfere with the normal growth and development of insects [41].

Numerous studies have shown that JA induces an increase in the content of metabolites with anthelmintic activity and the activity of defense enzymes in plants [42]. Treatment with 1000 µmol L⁻¹ JA for 6 days induced a significant increase in PI activity as well as total phenolic and tannin content in peanut leaves [43]. Similarly, 1.5 mmol L⁻¹ JA induced a significant increase in PPO activity in tomato and reduced the number of four types of insects (lepidoptera larvae, beetles, aphids, and thrips) infesting the plant [44]. In the present study, the content of secondary metabolites such as tannins, flavonoids, and total phenols as well as the activity of defense enzymes such as PPO, POD, PAL, and PI were significantly higher in daylily plants treated with different concentrations of JA than in control plants. Moreover, the higher the concentration of JA, the more significant the increase in the secondary metabolite content and enzyme activity, indicating a concentration effect of JA-induced insect resistance in daylily. In addition to JA concentration, the time of JA induction was also associated with the resistance of daylily. The content of secondary metabolites and activity of all enzymes except that of PI—which gradually decreased with time—showed a tendency to first increase and then decrease. These findings indicate that JA treatment enhanced the content of secondary metabolites and activity of defense enzymes with anti-insect activity in daylily, but the sensitivity and lag period of different substances to JA treatment differed. Insect resistance induction in plants through exogenous JA application to control pest-induced damage has been attempted in pest control of tomato, tobacco, and other crops with definite effects [45,46]. In the present study, the population size of T. palmi on JA-treated daylily plants was significantly lower than that in the control, indicating that the JA-induced resistance is crucial for reducing the population density of T. palmi. This induced resistance may be attributed to exogenous JA-induced stimulation of defense gene expression in daylily leaves, which leads to changes in secondary metabolic processes and production of compounds with toxic, antinutritional, or antidigestive effects on T. palmi. Stomatal density and waxy layer morphology are key indicators of plant resistance to insects. JA negatively regulates stomatal density and affects wax synthesis in daylily leaves, resulting in a smoother leaf surface, which is not conducive to T. palmi oviposition and attachment, which is similar to the previously reported methyl jasmonate-induced decrease in stomatal density and the thickening and smoothing of cuticle in eggplant leaves [47].

It has been demonstrated that JA affects the synthesis and accumulation of secondary metabolites by activating or repressing the activity of the corresponding TFs, which in turn regulate the expression of key enzyme genes related to plant secondary metabolism [48,49]. In Picea abies treated with exogenous JA, the transcriptional level of WRKY was upregulated, which promoted paclitaxel biosynthesis [50]. Upregulated expression of bHLH promoted tanshinone biosynthesis in Salvia miltiorrhiza after the application of exogenous MeJA [51]. Both bHLH and WRKY family TFs in Coptis chinensis are involved in regulating the biosynthesis of isoquinoline alkaloids [52]. Exogenous JA maintains Arabidopsis cellular homeostasis by repressing cytokinin oxidase/dehydrogenase1 gene expression through the MYC2 [53]. In the present study, JA was demonstrated to regulate the biosynthetic pathways of secondary metabolites such as tannins, flavonoids, and total phenols through the upregulation of TFs such as MYB, AP2/ERF, bHLH, C2C2, WRKY in daylily, which resulted in the production of secondary metabolites that are toxic to T. palmi. In addition, the expression of TFs such as NAC and B3 was downregulated, thereby regulating plant nutrient growth pathways such as stomatal opening and closing, photosynthetic efficiency, protein and lipid metabolism, etc., slowing down plant nutrient growth so that nutrients available to T. palmi were reduced, and changes in the physical structure of daylily leaves surfaces affected the attachment of T. palmi eggs, so as to enable daylily to achieve the purpose of insect resistance. JA serves as an important stress-responsive signal that interacts with other phytohormones such that plant resistance mediated through these signaling pathways is finely regulated [54]. We found that JA treatment of daylily resulted in DEGs enriched in defense hormone pathways such as JA, SA, ABA, and ET, indicating that JA interacts with various hormones to enhance plant resistance.

5. Conclusions

In conclusion, the present findings elucidate the potential mechanisms and hub genes involved in exogenous JA-induced resistance to T. palmi in daylily. The underlying mechanisms include the regulation of leaf stomatal and wax layer development as the first physical line of defense against T. palmi in daylily; catalytic production of secondary metabolites harmful to T. palmi by defense enzymes; and synergistic effects of JA with SA, ABA, and ET on the regulation of daylily resistance to reduce T. palmi damage. These findings provide a scientific basis for the utilization of JA as an antagonist to control T. palmi in daylily.