Introduction

Insect feeding is a major cause of biotic stress to plants. During their co-evolution with insects, plants have developed complex defense systems to resist insect feeding. Almost all plants can be harmed by certain molecules in the oral secretions (OS) of herbivorous insects such as fatty acid-amino acid conjugates (FACs)1,2. However, plants can also resist the attack of pests by activating a series of signaling events, including cell membrane depolarization, activation of mitogen-activated protein kinases, and accumulation of stress-related plant hormones3,4,5, which may alter the expression of resistance-related genes, to increase the levels of defensive metabolites such as plants of the Brassicales order6,7, agglutinin in tobacco (Nicotiana glauca)8 and benzoxazinoids in maize (Zea mays)9,10. Carbon dioxide (CO2), a basic substance required for plant photosynthesis, is essential for plant growth and development. The global average atmospheric concentration of CO2 hit 409 ppm in February 2017 (Mauna Loa Observatory), which was 47% higher than the 278 ppm in 175011 and is projected to reach 750 ppm by the end of this century12. An elevated carbon dioxide (ECO2) concentration is expected to have profound effects on many aspects of plant physiology, including increased photosynthetic rate, biomass and seed production13,14,15. For example, previous free-air CO2 enrichment (FACE) studies have shown that the yield of staple crops (such as sorghum, cotton, wheat and rice) can be increased by an average of 17% under ECO2 (700 ppm)14.

ECO2 is thought to enhance photosynthetic rate and affect plant-insect interactions of C3 plants16. According to the carbon-nitrogen balance theory, increasing C-based metabolites can enhance photosynthesis and thus reduce the relative content of proteins. Since nitrogen (N) is a limiting factor for the growth of many herbivores17,18, the compensatory feeding hypothesis suggests that insects may have to consume more foliage to obtain sufficient N-based nutrients (mainly proteins)19,20,21. ECO2 associated changes in plant nutrients, especially in protein content, are known to affect plant defense against insects. For example, ECO2 reduces the leaf nitrogen of peanut and ramie, resulting in increased food consumption, reduced growth rate and prolonged phlegm time in Spodoptera litura and Achaea janata22.

The phytohormone jasmonic acid (JA) is vital in plant defense against insects. For example, in wild-type tobacco, silencing JA biosynthetic gene lipoxygenase 3 (LOX3), or the JA signaling receptor coronatine insensitive 1 (COI1) will reduce plant resistance to Manduca sexta larvae, due to decreased defense-related metabolites23,24. Similar findings were also reported in tomato (Solanum lycopersicum), almond (Amygdalus communis), and rice (Oryza sativa) Arabidopsis thaliana plants harboring mutations in genes that are involved in JA biosynthesis or signaling25,26,27,28. ECO2 can alter plant defense against pests by regulating JA level. For example, ECO2 reduces tomato resistance to cotton bollworm by inhibiting JA accumulation29. Under ECO2 leaf JA and JA-Ile concentrations increase, and may induce the production of flavonoids30. ECO2 improved the feed intake and reproductive performance of Japanesebeetle (Popillia japonica Newman)31,32 and western corn rootworm (Diabrotica virgifeya virgifer)33,34 feeding on soybean (Glycine max) in FACE experiments, which is associated with downregulated expression of JA biosynthetic genes lipoxygenase 7 (LOX7), LOX8, allene oxide synthase (AOS) and allene oxide cyclase (AOC), and an ethylene (ET) biosynthetic gene 1-aminocyclopropanecarboxylic acid synthase (ACS)34,35.

There are many other factors that can affect plant and insect growth and their interactions. For example, temperature and climate can affect the growth of plants36 and insects37, and may also affect the way plants resist insects38. ECO2 will have an impact on the atmospheric temperature and climate environment, thus indirectly affecting the growth of plants and insects, as well as the resistance of plants to insects. In this study, we selected the more direct factors that affect the growth and interaction of plants and insects to elucidate the mechanism of ECO2 affects plants resistance insect through the jasmonic acid signaling pathway.

Tobacco and melon are both important C3 plants. In order to explore how ECO2 affects the defense of plants (especially C3 plants) against lepidopteran insects, we studied the interaction of tobacco-Spodoptera litura (Lepidoptera: Noctuidae), and melon-Diaphania indica (Lepidoptera: Coleoptera) to investigate molecular mechanisms of plant resistance to lepidopteran insects under ECO2. The results showed that under an ECO2 of 750 ± 20 mmol/mol, the resistance of tobacco to S. litura increased, while the resistance of melon to D. indica decreased, and the changes in plant resistance and JA level showed the same pattern. More importantly, we demonstrated that ECO2 alters plant-to-insect resistance by affecting herbivoryinduced JA level. Based on the correlations of plant total nitrogen, total protein, JA level and the growth of insects, we also found that plants are able to inhibit the growth of S. litura and D. indica mainly by regulating herbivory-induced JA level, while the changes in leaf total nitrogen or total protein have little effect on the growth of the two insects.

Results

Effects of ECO2 on photosynthesis of tobacco and melon plants

To determine whether ECO2 affects the photosynthesis in tobacco and melon plants, the light responsive curve and CO2 response curve were plotted. As the light intensity increased, tobacco and melon plants grown under ECO2 showed increased photosynthetic rates, compared with the plants grown under ambient CO2 (ACO2) (Fig. 1A,B). However, according to the CO2 response curves, the plants grown under ACO2 showed higher photosynthetic rate than the those grown under ECO2, with intercellular CO2 level increasing (Fig. 1C,D), indicating that ECO2 increased the photosynthetic rate but weakened the photosynthetic capacity of tobacco and melon plants, which may be due to the decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase level under ECO239,40,41.

Figure 1
figure 1

Photosynthetic rates and biomass of tobacco and melon plants under ACO2 and ECO2. (A) Light response curves of five-week-old tobacco plant; (B) Light response curves of five-week-old melon plant; (C) CO2 response curves of five-week-old tobacco plant; (D) CO2 response curves of five-week-old melon plant; (E) Fresh weight of above-ground part of tobacco plant; (F) Fresh weight of above-ground part of melon plant; (G) C:N ratio of tobacco plant; (H) C:N ratio of melon plants. Pn, photosynthetic rate; PAR, photosynthetically active radiation; Ci, intercellular CO2 concentration. Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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As the photosynthetic rate increased, the fresh weight of tobacco decreased by 49.6% (Fig. 1E), and that of melon increased by 22.3% under ECO2 (Fig. 1F). In addition, the C:N ratios of tobacco and melon under ECO2 increased by approximately 28.2% and 8.5%, respectively (Fig. 1G,H).

Resistance of tobacco and melon plants to lepidopteran insects under ACO2 and ECO2

In order to determine whether or not ECO2 affects the resistance of tobacco and melon to insects, the mass of insects gained was measured respectively under ECO2 and ACO2. The results showed that under ECO2, the average mass of S. litura feeding on tobacco plant decreased by 44%, 46% and 31% on day 4, day 6 and day 9, respectively (Fig. 2A). In contrast, under ECO2, the average mass of D. indica feeding on melon plant increased by 21%, 27%, and 43% on day 4, day 6 and day 11, respectively (Fig. 2B). The total dry matter of the two insects changed in similar patterns to their mass under ECO2 (Fig. 2A,B). These results indicate that ECO2 increases the resistance of tobacco to S. litura but reduces the resistance of melon to D. indica.

Figure 2
figure 2

Growth of larvae feeding on tobacco and melon plants under ACO2 and ECO2. Total mass of S. litura on tobacco (A) and D. indica on melon (B) under ACO2 and ECO2. Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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JA level in tobacco and melon under ECO2 and ACO2

Plant hormones, especially JA, play a crucial role in regulating plant defenses against insects. To uncover the mechanisms by which ECO2 affects plant resistance to insects, we determined the levels of JA and JA-isoleucine conjugate (JA-Ile) in tobacco and melon plants treated by different CO2 concentrations.

Since insect feeding is difficult to control, it was simulated by wounding the leaves with a fabric pattern wheel, following which the oral secretion (OS) of S. litura or D. indica was immediately applied to the wounds. The results showed that the peak value of JA in tobacco plants induced by S. litura OS (1 h after induction) under ECO2 was 51% higher than that under ACO2. However, the peak value of JA in melon plants induced by D. indica OS under ECO2 was 32% lower than that under ACO2 (Fig. 3A,B). JA-Ile conjugate is a JA derivative that binds to the COI1 receptor and thereby activates JA-induced responses28,42. We observed that simulated herbivory induced JA-Ile level changed in the same patterns of simulated herbivory induced JA in both plants (Fig. 3C,D).

Figure 3
figure 3

Changes in phytohormones in simulated herbivory treated tobacco and melon plants under ACO2 and ECO2. Changes in simulated herbivory-induced JA concentration in tobacco (A) and melon (B); Changes in simulated herbivory-induced JA-Ile concentration in tobacco (C) and melon (D). Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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Expression of the genes involved in the JA pathway under ECO2 and ACO2

LOX (lipoxygenase)22,43, AOS (allene oxide synthase)44, AOC (allene oxide cyclase)45, and JAR (jasmonic acid resistance)46,47 are closely associated to JA and JA-Ile biosynthesis. To assess the effects of ECO2 on simulated herbivory -induced JA and JA-Ile levels, the expression levels of these genes in tobacco and melon were determined.

In tobacco, the expression levels of NtLOX (1.5 h), NtAOC (0.5 h) and NtJAR (1.5 h) induced by S. litura feeding under ECO2 were increased by 49%, 88% and 35%, respectively, (Fig. 4A,C,D), compared with those under ACO2, while the expression levels of NtAOS changed little (Fig. 4B). In contrast, the peak expression levels of CmLOX, CmAOS, CmAOC and CmJAR in melon induced by D. indica feeding decreased by 15%, 35%, 23% and 36% (Fig. 4E–H). These data indicated that herbivory- induced JA in both tobacco and melon changes consistently with the expression levels of JA biosynthesis-involved genes.

Figure 4
figure 4

Changes in relative expression levels of defense-related genes in simulated herbivory-treated tobacco and melon plants under ACO2 and ECO2. Relative expression levels of NtLOX (A), NtAOS (B), NtAOC (C) and NtJAR (D) in tobacco; Relative expression levels of CmLOX (E), CmAOS (F), CmAOC (G) and CmJAR (H) in melon. Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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Effects of JA on plant resistance to insects under ECO2

After the JA level in tobacco plants under ACO2 was increased using exogenous JA to the same level as in the plants grown under ECO2, we found that the mass of S. litura feeding on exogenous JA treated tobacco plants was 43% lower than the mass of S. litura feeding on untreated tobacco plants under ACO2, but showed no significant difference from that under ECO2 (Fig. 5A). After the JA level in melon plants grown under ECO2 was increased using exogenous JA to the same level as in the plants grown under ACO2, we found that the mass of D. indica feeding on exogenous JA treated melon plants was 37% lower than the mass of D. indica feeding on untreated plants under ECO2, but showed no significant difference from that under ACO2 (Fig. 5B). The results proved that ECO2 changes the resistance of tobacco and melon plants to insects via the JA pathway.

Figure 5
figure 5

Effect of JA on the growth of S. litura feeding on tobacco plants and D. indica feeding on melon plants grown under ACO2 and ECO2, (A) Weight of S. litura that had fed on untreated tobacco plants, exogenous JA treated tobacco plants under ACO2, or untreated tobacco plants under ECO2 for 6 days; (B) Weight of D. indica that had fed on untreated melon plants, exogenous JA treated melon plants under ECO2, or untreated melon plants under ACO2 for 6 days Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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Total nitrogen and protein of tobacco and melon, and the effects on larval growth

Plant nutrients, especially protein, which is the limiting nitrogen source for many herbivores17, are critical for larval growth. The total nitrogen and protein contents of both tobacco and melon plants decreased under ECO2, which was possibly due to the increased C-based metabolites diluting the contents of proteins and N-based metabolites. Under ACO2, the total weight gain of S. litura feeding on exogenous JA-treated tobacco plants was 19% lower than that on untreated plants when the larvae ingested the same amount of nitrogen. The total nitrogen content of exogenous JA-treated tobacco plants under ACO2 was about 23% higher than that of untreated tobacco plants under ECO2, but the S. litura larvae that ate the same amount of leaf of the two treatments gained the same weight (Fig. 6A,B), which was similar to the findings in protein content and larval growth (Fig. 6C,D). Similar results were also obtained in melon plants (Fig. 6E–H). These results suggest that it is the JA level rather than leaf nitrogen content or protein content of plants that determines the weight gain of S. litura and D. indica.

Figure 6
figure 6

Total nitrogen and protein contents of exogenous JA-treated and untreated tobacco and melon plants under ACO2 and ECO2, and weight gain of insects in these treatments. (A) Total nitrogen of exogenous JA-treated and untreated tobacco plants under ACO2, and that of untreated tobacco plants under ECO2; (B) Total nitrogen and larval biomass of exogenous JA-treated and untreated tobacco plants under ACO2 and ECO2; (C) Total protein of exogenous JA-treated and untreated tobacco plants under ACO2, and that of untreated tobacco plants under ECO2; (D) Total protein and larval biomass of exogenous JA-treated and untreated tobacco plants under ACO2 and ECO2; (E) Total nitrogen of exogenous JA treated and untreated melon plants under ECO2, and that of untreated melon plant under ACO2; (F) Total nitrogen and larval biomass of exogenous JA-treated and untreated melon plants under ACO2 and ECO2; (G) Total protein of exogenous JA-treated and untreated melon plants under ECO2, and that of untreated melon plants under ACO2; (H) Total protein and larval biomass of exogenous JA treated and untreated melon plants under ACO2 and ECO2; Diferent letters for each species denote signifcant diferences (p ≤ 0.05).

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Discussion

ECO2 has a profound impact on plant physiology, especially in C3 plants. It enhanced the defense of tobacco against S. litura, but reduced the defense of melon against D. indica, suggesting that its effect on plant defense is species-specific. In addition, our study also showed that ECO2 alters plant defense against lepidopteran insects mainly by affecting the JA level of plants

ECO2 can promote photosynthesis and biomass accumulation of plants. In this study, we found that the photosynthesis rate and growth rate of tobacco and melon plants were both increased under ECO2. The study of Zhu et al.48 showed that the growth rate of soybean seedlings was increased from 6 to 22 μm·min-1 with CO2 concentration increasing from 400 to 800 ppm, but began to decrease when CO2 concentration exceeded 900 ppm.

The two plant species tomato and melon responded differently to insect feeding under ECO2. In detail, the JA content and JA biosynthesis-related gene expression in tobacco plants both increased, while those in melon plants decreased in response to insect OS29. It has also been reported that the JA level in tomato plants grown under ECO2 reduces in response to cotton bollworm feeding, which suggests that herbivory induced-JA level in plants grown under ECO2 changes in a species-specific manner. Our data also showed that the expression of JA and JA-Ile biosynthesis-involved genes in both tobacco and melon under ECO2 changed consistently with herbivory induced-JA level, but the mechanism by which ECO2 affects plant JA level needs to be further studied. The photosynthetic activity of both tobacco and melon plants also increased significantly under ECO2. Considering the key role of chloroplasts in JA biosynthesis and photosynthesis, we speculate that JA level is associated with plant photosynthesis activity. ECO2 may change JA level by regulating the content of polyunsaturated fatty acid, which is the substrate for the JA pathway49. In addition, environmental CO2 concentration may also play an important role in controlling plant defense through a JA-independent pathway. For example, when wounded Arabidopsis plants were grown under ECO2 they had a lower level of total glucosinolate than those grown under ACO2, while they had similar levels of JA and JA-Ile whether grown under ECO2 or ACO250.

Our results showed that amongst the three factors tested the JA level rather than the content of nitrogen or nutrients is the main factor influencing larval growth. The contents of both nitrogen and proteins in tobacco and melon plants were reduced under ECO2, which may be due to the increased C-based metabolites diluting the total nitrogen and proteins in plants. By assessing the effects of plant proteins and defensive metabolites on the defense of exogenous JA treated tobacco and melon plants against pests, we found that defensive metabolites are more influential than protein to plant defense under ECO2. Knepp et al.51 reported that although ECO2 had no significant effect on leaf nitrogen of black oak, the larvae feeding on black oak grown under ECO2 gained less weight than under ACO2. Further analysis revealed ECO2 decreased the approximate digestibility of black oak leaf by larvae, leading to a 29% reduction in leaf consumption, a 30% reduction in larval weight gain and a 20% increase in larval mortality under ECO2. This suggests the level of defensive metabolites is more influential than the level of proteins in black oak defense mechanisms against pests.

In summary, our data showed that ECO2 can affect plant defense against lepidopteran insects, by enhancing the rate of photosynthesis and increasing the plant’s biomass, and altering the level of plant JA induced by insect feeding. In both the crops and insects we studied, JA level played a more important role than leaf protein in determining larval growth under ECO2. However, the effect of ECO2 on plant defense against insects was shown to be species-specific. Therefore, we believe that it is important to study how the resistance of plants changes in the interaction between important agricultural crops and their major pests, and the changes in crop-pest interaction and in plant resistance to pests under ECO2 may provide a theoretical basis for predicting the impact of elevated atmospheric CO2 on future agro-ecosystems.

Materials and Methods

Plant cultivation

Tobacco ‘NC89’ and melon ‘Jiashi’ plants were grown separately in two climate chambers with the same temperature, light intensity and humidity regimes. Tobacco and melon were planted in each carbon dioxide concentration environment respectively, and the required number of plants were randomly selected during the experiment. The CO2 concentration was (400 ± 20 mmol/mol) in one chamber (ACO2), and 750 ± 20 mmol/mol in the other (ECO2). Tobacco (Nicotiana tabacum cv. Samsun) plants were grown in 10 L plastic pots. From seed germination, the plants were exposed to a photoperiod 16 hours of light (33 ± 0.05 klux, 28 ± 1 °C) and 8 hours of darkness (20 ± 1 °C) at a relative humidity of 60% ± 2%, watered once every 6 days and fertilized with 1 g/L nitrogen, phosphorus, potassium and trace elements at a ratio of 20:20:20:0.5, once every two weeks. The seeds of Jiashi melon were seeded in a 128-well plate containing a mixed matrix (meteorite: perlite = 1:1), After germination, 1/2 of the nutrient solution (N:P:K = 20:20:20) was watered every 2 days, when the seedlings grew to two leaves, they were transplanted into 20 L PVC barrels (1 plant per barrel), other cultivation environment conditions are the same as tobacco.

Determination of photosynthetic parameters

Photosynthetic rate was measured using a LI-COR 6400 portable photosynthesis system (LI-COR Biosciences). The photosynthetic parameters were determined using the fourth true leaf of tobacco plants and the third and fourth true leaves of melon plants. The CO2 concentration in the chambers was set to 400 mmol/mol, and the Pn (photosynthetic rate) and PAR (photosynthetically active radiation) values at 11 light intensities (0, 20, 50, 100, 200, 300, 400, 600, 1000, 1500 and 2000 mmol·m−2·s−1) were measured to plot the light response curves. To fit CO2 response curves, the Pn and Ci (intercellular CO2 concentration) were measured at 12 different CO2 concentrations (0, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1600, 2000 mmol/mol), and at a light intensity of 1000 mmol·m−2·s−1. During the measurements, the chamber temperature was controlled at 27 °C and relative humidity at 60%.

Larva feeding and simulated insect feeding

S. litura and D. indica eggs were purchased from Genralpest Biotech (http://genralpest.b2b.hc360.com/). Following emergence the larvae were reared on artificial diets for two days and then transferred onto five-week-old plants. To determine the effect of ECO2 on plant resistance to insects, 15 tobacco plants were infested. with 50 to 80 S. litura larvae (3 to 4 larvae per plant), and 15 melon plants were infested. with 50 to 80 D. indica larvae (3 to 4 larvae per plant). And their weights were measured on the indicated days.

The oral secretions (OS) of 4th- and 5th-instar S. litura and D. indica larvae were collected from the tobacco and melon plants respectively, and stored at −80 °C. To simulate insect feeding, the leaves were wounded with a fabric pattern wheel, before 20 μL of S. litura or D. indica OS was gently rubbed onto the freshly created wounds, and these experiments repeated 3 times.

Plant hormone analysis

Approximately 100 mg of leaf tissue of each sample was ground in liquid nitrogen, before 1 mL of ethyl acetate containing 20 ng of internal standards D4-SA, D5-JA and JA-13C6-Ile was added, thoroughly mixed, and centrifuged at 13 000 g for 10 minutes at 4 °C. The supernatant was transferred to a new tube and evaporated to dryness using a vacuum concentrator (Eppendorf, Hamburg, Germany) at 30 °C. Leaf samples were immersed in 0.6 mL of methanol- water mixture (70:30, v/v) and centrifuged at 13 000 g for 10 minutes at 4 °C. The supernatant was transferred to a glass vial and injected into the Ultra Performance Liquid Chromatography-Mass Spectrometry/MS system (LCMS-8040 system, Shimadzu). The peak areas of the internal standard and each standard compound were used to calculate phytohormone concentration.

Protein extraction and quantification of total C and N

Approximately 50 mg of leaf sample that had been ground was mixed with 0.3 mL of extraction buffer containing 0.1 M Tris-HCl (pH 7.6), 5% (m/v) polyvinylpolypyrrolidone, 2 mg/mL phenylthiourea, 5 mg/mL diethyldithiocarbamate, and 0.05 M Na2EDTA, and then immediately centrifuged at 16 100 g for 20 minutes at 4 °C. After that, 200 mL of the supernatant was transferred to a new tube. Protein concentration was determined photometrically (595 nm) in a 96-well plate using Bradford 1x Dye Reagent (BIO-RAD).

For the measurement of total C and total N, leaves samples were dried in an oven at 100 °C for 2 days, and pulverized, before total C and N contents were quantified using an Elemental Combustion System (Elementar, vario MICRO).

RNA extraction and Real-time quantitative RT-PCR expression analysis

Total RNA samples were extracted from leaf material with the EasyPure Plant RNA Kit (TRANS, China) following manufacturer protocol. To amplify the selected genes, cDNA was amplified by PCR using the following primerswere listed in (Table 1) and were synthesized with the EasyScript First-Strand cDNA Synthesis SuperMix (TRANS China), to be used as the template for RT-PCR52. Real-time quantification RT-PCR reactions were performed in Bio-RAD MyiQTM Real-time PCR Detection System (Bio-Rad, USA) using the TransStart Top Green qPCR SuperMix (TRANS, China) according to the manufacturer’s instructions and the tobacco elongation factor 2 and melon actin (CmActin) was employed as the internal control for RT-PCR analysis53. Amplifcation was carried out through initial denaturation at 94 °C for 2 min, followed by 38 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, and elongation at 72 °C for 2 min. The PCR products from each amplifcation reaction were separated on 2.5% (w/v) agarose gels.

Table 1 Primers used in this study.
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