Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Elevated CO2 Reduces the Resistance and Tolerance of Tomato Plants to Helicoverpa armigera by Suppressing the JA Signaling Pathway

  • Huijuan Guo ,

    Contributed equally to this work with: Huijuan Guo, Yucheng Sun

    Affiliations State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China, Graduate School, Chinese Academy of Sciences, Beijing, People's Republic of China

  • Yucheng Sun ,

    Contributed equally to this work with: Huijuan Guo, Yucheng Sun

    Affiliation State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China

  • Qin Ren,

    Affiliation Jining Normal College, Inner Mongolia Autonomous Region, Jining, People's Republic of China

  • Keyan Zhu-Salzman,

    Affiliation Department of Entomology, Texas A&M University, College Station, Texas, United States of America

  • Le Kang,

    Affiliation State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China

  • Chenzhu Wang,

    Affiliation State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China

  • Chuanyou Li,

    Affiliation State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China

  • Feng Ge

    gef@ioz.ac.cn

    Affiliation State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China

Abstract

Both resistance and tolerance, which are two strategies that plants use to limit biotic stress, are affected by the abiotic environment including atmospheric CO2 levels. We tested the hypothesis that elevated CO2 would reduce resistance (i.e., the ability to prevent damage) but enhance tolerance (i.e., the ability to regrow and compensate for damage after the damage has occurred) of tomato plants to the cotton bollworm, Helicoverpa armigera. The results showed that elevated CO2 reduced resistance by decreasing the jasmonic acid (JA) level and activities of lipoxygenase, proteinase inhibitors, and polyphenol oxidase in wild-type (WT) plants infested with H. armigera. Consequently, the activities of total protease, trypsin-like enzymes, and weak and active alkaline trypsin-like enzymes increased in the midgut of H. armigera when fed on WT plants grown under elevated CO2. Unexpectedly, the tolerance of the WT to H. armigera (in terms of photosynthetic rate, activity of sucrose phosphate synthases, flower number, and plant biomass and height) was also reduced by elevated CO2. Under ambient CO2, the expression of resistance and tolerance to H. armigera was much greater in wild type than in spr2 (a JA-deficient genotype) plants, but elevated CO2 reduced these differences of the resistance and tolerance between WT and spr2 plants. The results suggest that the JA signaling pathway contributes to both plant resistance and tolerance to herbivorous insects and that by suppressing the JA signaling pathway, elevated CO2 will simultaneously reduce the resistance and tolerance of tomato plants.

Introduction

In the last 250 years, atmospheric carbon dioxide (CO2) has risen from 280 ppm to greater than 390 ppm, and is anticipated to reach at least 550 ppm by year 2050 [1]. Because elevated CO2 increases the carbon to nitrogen (C∶N) ratio and reduces the N content in the tissue of most plant species, elevated CO2 is expected to alter plant synthesis of phenolics, terpenes, and other secondary metabolites [2], [3]. Such changes in C∶N and in the content of secondary metabolites will alter the nutritional quality and palatability of host plants for herbivores and could therefore affect the performance of herbivorous insects [4].

Plants have evolved a variety of mechanisms to reduce the negative impacts of herbivory [5], [6]. When damaged by herbivorous insects, plants can produce herbivore-deterrent metabolites or defensive proteins to limit the damage [7]. This kind of induced defense (i.e., resistance) is energy and resource costly, however, and cannot be maintained at high levels throughout the growing season [8]. An alternative to resistance is tolerance, which compensates for tissue loss after insect attack [9]. In expressing tolerance, plants reallocate energy and resources from undamaged to damaged tissues (for example, by increasing sucrose-transport enzymes in the damaged tissues) and increase photosynthetic rates and growth parameters [10], [11]. Although researchers generally assume that there is a trade-off between resistance and tolerance (i.e., plants with high resistance have low tolerance and vice versa), the relationship between plant resistance and tolerance to herbivores varies among studies and often depends on the plant species, soil resource, and environment [12], [13].

Elevated CO2 is likely to increase constitutive levels of defensive metabolites, including phenolics and tannins, in plant leaves [2], [14], and such increases in phenolics and tannins have an negative influence on the development and fitness of chewing herbivorous insects [15]. However, the induced phenolic compounds are decreased by elevated CO2 when responding to damage of insect [16]. Additionally, jasmonic acid (JA) signaling defense (JA is considered as the most important defense hormone involved in resistance against chewing insects) has been reported to be suppressed by elevated CO2 [17], and CO2-induced decreases in the expression of downstream genes of JA pathway (i.e., proteinase inhibitors) increased the consumption of soybean leaves by herbivorous insects [18].

Little is known about how CO2 affects plant tolerance to herbivores but the possible effects of resource availability on tolerance have been described by three classic models or hypotheses. The compensatory continuum hypothesis (CCH) predicts that plants growing in resource-rich or low-competition environments will be more tolerant to herbivores than those growing in resource-poor, stressful environments [19]. The main rival to the CCH is the growth rate model (GRM), which predicts that plants grow at a low relative growth rate will be more tolerant than plants grow at a high relative growth rate, because, unlike plants growing in stress-free environments, plants growing in stressful environments are not growing at their maximum rate and therefore have the potential to increase their growth rate [19]. The limiting resource model (LRM) predicts that tolerance will depend on the particular resource that is limiting plant fitness and how acquisition of that resource is affected by herbivory; according to the LRM, the relative effects of a stressful vs. a stress-free environment on tolerance will therefore depend on the nature of the resource [20].

Some researchers have reported that elevated CO2 increased plant susceptibility to herbivorous insects [21], [22], [23], while others found that elevated CO2 increased compensatory growth in response to artificial herbivory, i.e., in response to researcher removal of buds from cotton plants [24], [25]. Elevated CO2 could possibly affect the re-growth ability or tolerance by increasing C∶N and by decreasing the N concentration of plant tissues [26]. Although research has established that JA plays a crucial role in plant resistance to herbivorous insects [27] and that plant tolerance and resistance are not independent [13], it is still unclear how tolerance is affected by the JA signaling pathway and how the JA signaling pathway, and therefore resistance and tolerance, are affected by elevated CO2.

Using the cotton bollworm, Helicoverpa armigera, and tomato, the current study investigated the relationships between elevated CO2, insect herbivory, the JA pathway, and plant tolerance and resistance. Helicoverpa armigera is a leaf-chewing insect that causes great damage to cotton, tomato, and many other crops in Northern China [28]. To determine whether elevated CO2 influenced resistance and tolerance of tomato plants by affecting the JA pathway, we used the JA pathway-impaired mutant spr2. We tested the hypothesis that, by altering the JA signaling pathway, elevated CO2 would reduce tomato plant resistance (the ability to prevent damage) against H. armigera while enhancing plant tolerance (the ability to re-grow after H. armigera damage). Our specific aims were to determine (1) whether elevated CO2 affects the JA-dependent defense of tomato plants and the midgut enzyme activities of cotton bollworm associated with different tomato genotypes, and (2) whether elevated CO2 affects tolerance (in terms of re-growth ability, as indicated by photosynthetic rate, sucrose phosphate synthases, sucrose synthases, biomass, flower number, height, and branch length) of wild-type and spr2 plants after H. armigera damage.

Materials and Methods

Open-top chambers

This experiment was carried out using eight octagonal, open-top chambers (OTC), each 4.2 m in diameter, located at the Observation Station of the Global Change Biology Group, Institute of Zoology, Chinese Academy of Science (CAS) in Xiaotangshan County, Beijing, China (40°11′N, 116°24′E). The atmospheric CO2 concentration treatments were: (1) current atmospheric CO2 levels (375 µl/L) (“ambient CO2”), and (2) doubled ambient CO2 levels (750 µl/L) (“elevated CO2”). Four OTCs were used for each CO2 concentration treatment. During the period from seedling emergence to the harvesting of tomato plants, CO2 concentrations were monitored and adjusted with an infrared CO2 analyzer (Ventostat 8102; Telaire, Goleta, CA, USA) once every 20 min to maintain the CO2 concentrations. The automatic-control system for adjusting the levels of CO2 concentration, as well as specifications for the OTC, are detailed in Chen and Ge [29].

Tomato plants

Wild-type (WT) tomato plants (Lycopersicum esculentum cv. Castlemart) and jasmonate-deficient mutant plants (spr2) were kindly provided by Professor C. Li of the Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences. The JA-biosynthesis mutant, suppressor of prosystemin-mediated responses2 (spr2), reduces chloroplast ω3 fatty acid desaturase, which impairs the synthesis of JA [30]. WT tomato was the parent for the spr2 mutant. After they had grown in sterilized soil for 2 weeks, the tomato seedlings were individually transplanted into large plastic pots (20 cm diameter and 22 cm height) containing sterilized loamy field soil (organic carbon, 25 g/kg; N, 500 mg/kg; P, 200 mg/kg; K, 300 mg/kg) and placed in OTCs on 23 May 2010. Each OTC contained 40 plants. Pots were rearranged randomly within each OTC once every week. No chemical fertilizers and insecticides were used. Water was added to each pot once every 2 days.

Herbivore treatment

In its native habitat in China, Helicoverpa armigera is one of the most abundant of the lepidopteran herbivores on L. esculentum cv. Castlemart. H. armigera larvae tend to attack leaves and fruits. Thirty-six days after the tomato plants had been placed in the OTCs, 40 plants of each genotype at each CO2 level ( = 10 plants per OTC) were randomly selected and infested with H. armigera. Six 4th-stage H. armigera larvae were placed on each of two leaves at mid-plant height, and the leaves were caged (80 mesh gauze); corresponding leaves of control plants were caged in the same way. After 2 days, the larvae were removed and were weighed and analyzed for midgut proteases as described later in the Methods. The leaves of eight randomly selected plants infested with larvae and of eight noninfested plants were harvested and immediately stored in liquid nitrogen (Fig. 1).

thumbnail
Figure 1. Experimental design and replications.

A flow diagram of the design used for the plant treatments, tissue collection and replications. There were two CO2 levels, two plant genotypes, two insect treatments and four OTC replications for plant treatments.

https://doi.org/10.1371/journal.pone.0041426.g001

Plant photosynthesis

Net photosynthetic rate was determined in the presence or absence of herbivores on four 5-week-old plants for each genotype at each CO2 level. Gas exchange was measured on the distal portion of the leaf blade of undamaged leaves using a Li-6400 with a red/blue LED light source (6400-02B; Li-Cor, Lincoln, NE, USA). The CO2 concentration of the incoming air was adjusted to 400 µmol mol−1 CO2 or 750 µmol mol−1. Relative humidity corresponded to ambient conditions. Before gas exchange was measured, photosynthetic active radiation (PAR) for the leaf in the measuring cuvette was increased in steps to 1200 µmol m−2 s−1.When the CO2 assimilation rate was stable for at least 2 min, a light response curve was recorded. Measurements were carried out after herbivores had been allowed to feed on plants for 2 days.

JA measurement

A 0.5 g sample of fresh leaves was ground to a fine powder on ice. The powder was mixed with 4 ml of 80% methanol (80 methanol/20 water, V/V) and kept at −20°C for 12 h, and then added to 6 µl of [9], [10]-dihydro-JA (dihydro-JA) for use as an internal standard. The total extracted preparation was centrifuged at 8,000 g for 20 min. The condensed endogenous JA were extracted according to procedures described by ren et al., [31].

Endogenous JA and its internal standards (dihydro-JA) were analyzed using full GC/MS scans. Retention times were identified using Xcalibur 1.2, the NIST 2003 mass library. Endogenous JA was measured by GC-MS selected ion monitoring (SIM). The characteristic ions (m/z) were 151/224 for JA and 153/226 for the internal standard (dihydro- JA).

Plant enzyme activity (SPS, SS, LOX, PIs, PPO, POD, and PAL)

For analyses of sucrose synthases (SS) and sucrose phosphate synthases (SPS), 0.5 g of fresh tomato leaves was homogenized for 1.5 min at 4°C in 1∶10 (fresh weight/buffer volume ratio) 100 mMphosphate buffer, pH 7.4, containing 100 Mm KCl and 1 mM EDTA. Homogenates were centrifuged at 10 000 g for 10 min, and the supernatants were subjected to SS and SPS analysis. SS and SPS were analyzed according to the protocol of the reagent kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China).

About 0.1 g of frozen leaf tissue was used for determination of plant defensive enzyme activity, including the activities of lipoxygenase (LOX), proteinase inhibitors (PIs), polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia lyase (PAL). Extract was obtained from individual leaflets by grinding them in a 50 mM Tris HCl buffer (pH 7.8, 3 ml/g of leaf tissue) containing 7% polyvinylpolypyrrolidine, 1.67 mM phenylthiourea, 0.3 M KCl, and 0.4 mM ascorbic acid. This extract was immediately frozen for later use. For assays, the thawed extract was centrifuged at 13,000 g for 10 min, and enzyme activity was measured in the supernatant. LOX assays were performed in a 1-ml reaction mixture containing 20 µl of supernatant and 980 µl of reaction buffer. The reaction buffer consisted of 0.113 g of linoleic acid, which had been dissolved in 3 ml of methanol in 100 ml pH 7.8 Tris-HCl buffer (w/v, pH 7.0), and 800 µl of Tween-20. PIs were measured as in Thaler et al. [32]. PPO activity was measured as in Mahanta et al. [33]. POD activity was determined according to Tegelberg et al. [34]. Reaction mixtures for determination of PAL activity contained 950 µl of 0.02 mM L-phenylalanine (0.33 g of L-phenylalanine dissolved in 100 mL of Tris-HCl, pH 7.8) and 50 µl of supernatant. The changes in absorbance were detected with a spectrophotometer at 290 nm (SPEKTRAmax®Plus; Molecular Devices, Sunnyvale, California, USA).

Midgut proteases of H. armigera

After they had fed on plants for 48 h, 30 larvae of uniform size from each treatment were selected. Larvae were anaesthetized by chilling on ice, and their midguts were removed. The midgut tissue was cut longitudinally, and the external surface of the midgut tissue was cleaned by placing it in 0.15 mol/L NaCl. Finally, the tissues were stored at −80°C.

All enzyme substrates were obtained from Sigma-Aldrich. Weak alkaline trypsin-like enzyme (WATE) activity with Nα-tosil-L-arginin methyl ester (TAME) substrates was assayed spectrophotometrically at 248 nm according to the methods of Erlanger et al. [35]. The substrates were initially dissolved in dimethyl formamide (DMF) and further dissolved in 20 mM Tris–HCl buffer, pH 8.5.

Active alkaline trypsin-like enzyme (AATE) activity was measured according to Erlanger et al. [35] by using N-benzoyl-DL-arginin-ρ-nitroanilide (BApNA) and 7.5% (v/v) dimethyl sulfoxide (DMSO) dissolved in Glycine-NaOH buffer (0.1 mM, pH 10.5) at a final concentration of 1.4 mM. The mixtures were incubated for 20 min at 30°C, and the reactions were stopped by adding 0.5 ml of 30%(v/v) acetic acid. The enzyme activity was measured at 406 nm.

Chymotrypsin activity in extracts was determined spectrophotometrically according to Blackwood et al. [36], based on the hydrolysis of N-benzoyl-L-tyrosin ethyl ester (BTEE), which was determined at 256 nm (l M in 10% (v/v) methanol with 0.15 M NaC1). Tris-HCl buffer (0.2 M, pH 8.5) was used. One unit of enzyme activity was defined as 1 mol of BTEE hydrolyzed per min. The extinction coefficient of BTEE was 964.

Total protease was measured with azocasein as the substrate. Azocasein (20 mg ml−1) was dissolved in 0.15 M NaC1. The sample was then dissolved in 0.2 M glycine-NaOH buffer (pH 8.0). The reaction was run by adding 300 µl of reaction buffer to 0.3 ml of the azocasein solution and incubating the mixture for 24 h at 30°C. The reaction was stopped by adding 0.6 ml of 20% (W/V) trichloroacetic acid (TCA). Samples from each time point were centrifuged at 10000 g for 15 min at 4°C, and the absorbance of the supernatant was measured once at 366 nm. One absorbance unit from the mixture was defined as one unit of azocasein under the given assay conditions.

Mean relative growth rate of H. armigera

H. armigera larvae were weighed with an automatic electrobalance before and after they had fed on tomato plants. Mean relative growth rate (MRGR) was calculated following Chen et al. [37]: MRGR = (ln W2−ln W1)/t, where W1 is the initial weight; W2 is the final weight; and t is the time in days between weighings. For each combination of genotype and CO2 level, 85 larvae were weighed.

Resistance and tolerance analysis

The following variables were considered to be measures of resistance: the activities of LOX, PIs, PPO, POD, and PAL in plants; the activities of midgut enzymes in H. armigera; and the MRGR of H. armigera.

Tolerance was measured by comparing the parameters on plants that were or were not exposed to H. armigera and that were exposed to ambient or elevated CO2. In the short term, the following variables were considered to be measures of tolerance: photosynthetic rate, sucrose phosphate synthases and sucrose phosphate synthases. In the long term, the following variables were considered to be measures of tolerance (i.e. re-growth ability): total branch length per plant, plant height, cumulative number of flowers per plant, and final root and shoot mass. These data were obtained 60 days after the larvae had been added from five plants from each combination of genotype and herbivore treatment per OTC ( = 20 plants per OTC and 160 plants in total).

JA content was considered to be one measure of both tolerance and resistance.

Statistical analysis

A split-split plot design was used to analyze the univariate responses of the measured variables (i.e., plant traits, chemical components, phytohormones) (ANOVA, PASW, 2009). In the following ANOVA model, CO2 and block (a pair of OTCs with ambient and elevated CO2) were the main effects, tomato genotype was the subplot effect, and herbivore level was the sub-subplot effect:where C is the CO2 treatment (i = 2), B is the block (j = 4), G is the tomato genotype (k = 2), and H is the herbivore treatment (l = 2). Xijklm represents the error because of the smaller scale differences between samples and variability within blocks (ANOVA, SAS institute, 1996). Effects were considered significant if P<0.05. The effect of block and the interactive effects of block and other factors were not significant (P>0.45), and the effect of block and its interaction with other factors are not presented so as to simplify the presentation. Least significant difference (LSD) tests were used to separate means when ANOVAs were significant. For quantifying the midgut protease and weight of H. armigera on different tomato genotypes under two CO2 levels, a split-plot design was also applied, with CO2 and block as the main effects and tomato genotype as the subplot effect. R software (version 2.15.0, http://www.r-project.org/) was used to calculate the standard error of the difference value of parameters between herbivore infested and uninfested treatment.

Results

Photosynthesis and growth

Relative to ambient CO2 and in the absence of H. armigera, under elevated CO2, the photosynthetic rate were significantly increased by 87.9% and 43.8%, biomass by 31.6% and 19.8%, flower number by 51.1% and 53.0%, total branch length by 27.5% and 31.8%, and plant height by 56.7% and 44.5% for WT and spr2 plants, respectively. The root to shoot ratio (R∶S) were significantly decreased by 27.2% for WT plants and by 8.2% for spr2 plants under elevated CO2 (Fig. 2).

thumbnail
Figure 2. Growth parameters of two tomato genotypes grown under ambient and elevated CO2 without H. armigera infestation.

(A) Photosynthetic rate (A), (B) biomass, (C) root biomass: shoot biomass ratio (R∶S), (D) cumulative flower number, (E) plant height, and (F) total branch length. Each value represents the average (±SE) of 20 replicates. Different lowercase letters indicate significant differences between CO2 level within the same tomato genotype (LSD test: d.f. = 3.12; P<0.05). Different uppercase letters indicate significant differences between WT plants and spr2 plants within the same CO2 level (LSD test: d.f. = 2.9; P<0.05).

https://doi.org/10.1371/journal.pone.0041426.g002

SPS and SS activity

Relative to ambient CO2 and in the absence of H. armigera, SPS activity were significantly increased by 2.2-fold for WT plants and by 4.4-fold for spr2 plants under elevated CO2 (Fig. 3A) but SS activity of either WT or spr2 plants were not affected (Fig. 3B). SPS and SS activities were unaffected by genotype (Fig. 3).

thumbnail
Figure 3. Activity of sugar transport enzymes in leaves of two tomato genotypes grown under ambient and elevated CO2 without H. armigera.

(A) sucrose phosphate synthase (SPS) activity, and (B) sucrose synthase (SS) activity. Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between CO2 level within the same tomato genotype (LSD test: d.f. = 3.12; P<0.05). Different uppercase letters indicate significant differences between WT plants and spr2 plants within the same CO2 level (LSD test: d.f. = 2.9; P<0.05).

https://doi.org/10.1371/journal.pone.0041426.g003

JA level and defense enzyme activity

Genotype, H.armigera infestation, the interaction between CO2 and genotype, the interaction between genotype and H.armigera infestation, as well as the interaction among CO2 level, genotype and H.armigera infestation, significantly affected plant JA content (Table S1). JA levels and LOX, PI, and PPO activities of WT plants were decreased when damaged by H. Armigera under elevated CO2 (Fig. 4A–D). Elevated CO2 also decreased LOX and PI activity of undamaged WT plants. POD and PAL activity of undamaged plants in both genotypes were higher under elevated CO2 (Fig. 4E, F).

thumbnail
Figure 4. Chemical defensive components in two tomato genotypes grown under ambient (AM) and elevated CO2 (EL) without and with H. Armigera (+HA).

(A) JA content, and the activity of (B) lipoxygenase (LOX), (C) proteinase inhibitors (PIs), (D) polyphenol oxidase (PPO), (E) peroxidase (POD), and (F) phenylalanine ammonia lyase (PAL). Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences among combinations of H. armigera and CO2 level within the same tomato genotype (LSD test: d.f. = 3.12; P<0.05). Different uppercase letters indicate significant differences between WT plants and spr2 plants within the same CO2 (LSD test: d.f. = 2.9; P<0.05).

https://doi.org/10.1371/journal.pone.0041426.g004

H. armigera significantly increased the levels of JA and all defensive proteins in WT plants under ambient CO2 (Table S1, Fig. 4). Under elevated CO2, in contrast, H. armigera only increased JA levels and PI activity in WT plants.

Midgut proteases and weight of H. armigera

Total Proteolysis was affected by CO2 and the interaction between CO2 and genotype (Table S2). H. armigera that consumed leaves of WT plants grown under elevated CO2 had substantially higher gut protease activities than those that consumed leaves of WT plants grown under ambient CO2 (Table S2, Fig. 5). However, elevated CO2 did not affect gut protease activity in H. armigera that consumed spr2 foliage. Under ambient CO2, H. armigera that consumed leaves of WT plants had higher total protease and CTE activity than those that consumed spr2 foliage (Fig. 5A, B). Moreover, H. armigera that consumed leaves of WT plants had a lower MRGR than those that consumed spr2 foliage under ambient CO2 (Fig. 6).

thumbnail
Figure 5. Activities of digestive proteases in the guts of H. armigera that fed for 2 days on tomato genotypes grown under ambient and elevated CO2.

(A) total protease, (B) chymotrypsin (CTE) activity, (C) weak alkaline trypsin-like enzyme (WATE) activity, and (D) active alkaline trypsin-like enzyme (AATE) activity. Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between ambient CO2 and elevated CO2 treatment. Different uppercase letters indicate significant differences between feeding on the WT plants and spr2 plants within the same CO2.

https://doi.org/10.1371/journal.pone.0041426.g005

thumbnail
Figure 6. Mean relative growth rate (MRGR) of H. armigera that fed for 2 days on tomato genotypes grown under ambient and elevated CO2.

Each value represents the average (±SE) of 85 replicates. Different lowercase letters indicate significant differences between ambient CO2 and elevated CO2 treatment. Different uppercase letters indicate significant differences between feeding on WT plants and spr2 plants within the same CO2 treatment.

https://doi.org/10.1371/journal.pone.0041426.g006

Regrowth and sucrose transportation after H. armigera attack

For WT plants under ambient CO2, H. armigera increased the photosynthetic rate and SPS and SS activities but did not significantly affect biomass, flower number, height, and total branch length. For WT plants under elevated CO2, however, H. armigera not only reduced the photosynthetic rate but also decreased plant biomass by 13.7% and flower number by 24.9% (Fig. 7D, F). For spr2 plants under ambient CO2, H. armigera reduced plant biomass by 20.1%, flower number by 24.2%, and plant height by 11.6% (Fig. 7D, F, G). For spr2 plants under elevated CO2, H. armigera reduced plant biomass by 18.8%, flower number by 33.8%, and total branch length by 14.6% (Fig. 7D, F, H). In addition, H. armigera increased the R∶S of both genotypes under both CO2 levels (Fig. 7E).

thumbnail
Figure 7. Tolerance (as indicated by differences in values of growth traits or enzyme activities between damaged plants and undamaged plants) of two tomato genotypes grown under ambient and elevated CO2.

Damaged plants are those that were fed on by H. armigera. (A) photosynthetic rate (A), (B) sucrose phosphate synthase (SPS), (C) sucrose synthase (SS), (D) biomass, (E) root biomass: shoot biomass (R∶S), (F) cumulative flower number, (G) plant height, and (H) total branch length. Each value represents the average (±SE) of 20 replicates. Symbols above columns indicate levels of significant differences between variables of damaged and undamaged plants (*, P<0.05). Positive differences indicate that values were greater for damaged plants than for undamaged plants.

https://doi.org/10.1371/journal.pone.0041426.g007

Discussion

The effects of elevated CO2 on resistance and tolerance ability of tomato plants to H.armigera attack

Many studies have evaluated the relationship between plant resistance and tolerance to herbivores [38], [39], but little information is available regarding how the relationship between tolerance and resistance is affected by an abiotic stress such as global CO2 enrichment. Our results suggested that elevated CO2 decreased tomato plant resistance against H. armigera by suppressing the critical defensive signal molecule JA and JA-pathway-related defensive enzymes. Our results also indicated that tomato plants grown under elevated CO2 are less tolerant to H. armigera than plants grown under ambient CO2. Phenotypic plasticity is a principal means by which plants cope with biotic or abiotic stress [40], and the decreased resistance and tolerance to herbivores under elevated CO2 in this study suggests that elevated CO2 reduces the phenotypic plasticity of plant response to herbivorous insect attack.

Prior studies have revealed that elevated CO2 increases total nonstructural carbohydrates in plant tissues and that the excess C is probably allocated to the increased synthesis of secondary metabolites, such as terpenes and phenolics [41], [42], which in turn can reduce the development of chewing insects [43]. Furthermore, PAL is known to be a principle enzyme involved in a rate-limiting step of phenolic biosynthetic process [41]. Our results with both genotypes of tomato plants were consistent with the previous finding that elevated CO2 increases PAL activity in plants (Fig. 4F). Generally, attack by chewing insects induces a complex set of defense responses in plants [44]. In WT plants under ambient CO2, H. armigera attack increased anti-oxidant enzymes (in terms of POD), followed by triggered JA signaling pathway defense (in terms of JA and LOX) and caused plants to increase PIs and PPO activities (Fig. 4A–E). To the undamaged WT plants, elevated CO2 decreased LOX and PIs activity. Additionally, when damaged by H.armigera, elevated CO2 reduced LOX activity and JA level as well as PI and PPO activity in WT plants (Fig. 4A–D). It seems that elevated CO2 tends to impair the JA-dependent defense induced by H. armigera. Although responses to elevated CO2 by plants and insects are species-specific [14], the current results are not the first to show that elevated CO2 changes the plant–insect interaction by modifying the JA-dependent pathway. In soybean plants, elevated CO2 also suppressed the JA signaling pathway and increased susceptibility to the Japanese beetle, Popillia japonica [17], [18].

Proteinase inhibitors (PIs) of plants are able to reduce the feeding fitness of chewing insects by suppressing insect gut proteases [45]. WT plants grown under elevated CO2 had reduced PI activity, the reduced resistance resulted in increased gut protease activities for H. armigera (Fig. 5); these results may explain our previous finding that H. armigera consumed more wheat biomass when the wheat was grown under elevated vs. ambient CO2 conditions [46]. Although increases in gut protease activities may result in increased consumption under elevated CO2, the H. armigera MRGR did not change (Fig. 6). Perhaps the increased consumption only complemented feeding and enabled the insect to maintain development and growth when consuming leaves grown under elevated CO2, i.e., on leaves with a reduced N concentration.

Plant tolerance to herbivorous insects can depend on the availability of particular resources such as C resources [9]. Elevated CO2 increases C assimilation and causes re-allocation of C (especially sucrose) in plant tissue [47]. In the transport of sucrose from leaves to sink tissues via phloem, SPS and SS are key regulatory enzymes [48]. Because elevated CO2 significantly increases plant growth and C metabolism (Fig. 2), the CCH hypothesis would predict that plant tolerance to herbivores would be increased in the resource-rich, elevated-CO2 environment. The CCH hypothesis, however, was not supported by the current study. Under ambient CO2, WT plants expressed substantial tolerance to H. armigera attack in that herbivory increased C assimilation and sucrose synthesis (as indicated by SPS activity in leaves) and transportation (as indicated by SS activity in roots) such that plant biomass and other parameters measured at week 8 did not differ between plants with and without H. armigera infestation. This suggests that WT plants can completely compensate for H. armigera damage under ambient CO2. Under elevated CO2, in contrast, H. armigera consumption of WT plants reduced photosynthesis, biomass, flower number, and plant height and did not increase SPS and SS activity. We conclude that elevated CO2 reduces the tolerance of WT tomato plants to H. armigera. Furthermore, our results are consistent with the GRM hypothesis, which predicts that plants growing under conditions that promote a high growth rate will be less tolerant to herbivores than plant growing under conditions that reduce the growth rate [23].

The interaction between elevated CO2 and jasmonate signalling

In addition to the essential role of the JA pathway in resistance against herbivorous insects, JA has been found to regulate the interaction between tolerance and resistance in Nicotiana attenuata against Manduca sexta [49]. While WT plants in the current study exhibited substantial tolerance against H. armigera under ambient CO2, spr2 mutant plants did not, i.e., tolerance under ambient CO2 was much lower in spr2 than in the WT (Fig. 7). This is consistent with that finding that treatment of Populus with JA increased C transport to the roots, nutrient uptake, and regrowth capacity, and therefore increased tolerance [50]. Results concerning the effect of the JA pathway on tolerance, however, have been inconsistent. N. attenuata and its JA-deficient genotype (asLOX) did not differ in capsule production after simulated herbivore attack [9], and the tolerance to defoliation did not differ between WT Arabidopsis and the overexpressing-JA genotype JMT [51]. This indicates that tolerance to herbivorous insects may depend on some mechanisms other than the JA pathway. Under elevated CO2, the tolerance of spr2 plants to H. armigera was not lower than WT plants except the total branch length (Fig. 7A–G). This result confirmed that JA pathway plays an important role in tolerance of plants when attacked by H.armigera under elevated CO2. Our study suggests that the suppression of JA pathway may be one of reasons why elevated CO2 decreased both resistance and tolerance of tomato when damaged by H. armigera.

Conclusion

This study has generated a number of significant findings. First, the results support the view that the JA signaling pathway is important in both resistance and tolerance to chewing insects. Second, the results are consistent with the GRM model, which predicts that tolerance will be greater in resource-limited than in resource-unlimited environments. Third, a trade-off between resistance and tolerance as predicted by classical theory was not evident in our study [52]. Finally and perhaps most importantly, the results suggest that plants may suffer greater damage from herbivorous insects if levels of atmospheric CO2 continue to increase (Fig. 8).

thumbnail
Figure 8. The major results and conclusion of this study were summarized.

Elevated CO2 decreased resistance and tolerance of WT to H. armigera. In contrast, the resistance and tolerance of spr2 were not changed by elevated CO2. Elevated CO2 reduces the resistance and tolerance of WT plants by suppressing the JA signaling pathway.

https://doi.org/10.1371/journal.pone.0041426.g008

Supporting Information

Table S1.

F and P values from MANOVAs for the effect of CO2 level, tomato genotype and H. armigera infestation on growth traits, photosynthesis, and foliar chemical components of two tomato genotypes.

https://doi.org/10.1371/journal.pone.0041426.s001

(DOC)

Table S2.

F and P values from two-way ANOVAs for the effect of CO2 level and tomato genotype on MRGR and midgut proteolytic enzymes of H. armigera .

https://doi.org/10.1371/journal.pone.0041426.s002

(DOC)

Acknowledgments

We thank Dr. Haifeng Cao and Dr. Hongying Cui for help with the field OTCs experiment, and Prof. Bruce Jaffee (University of California at Davis) for reviewing a draft of the manuscript.

Author Contributions

Conceived and designed the experiments: HG FG LK CW KZ. Performed the experiments: HG QR. Analyzed the data: HG YS. Contributed reagents/materials/analysis tools: HG CL. Wrote the paper: HG YS KZ.

References

  1. 1. Intergovernmental Panel on Climate Change (2007) Climate Change 2007; the physical science basis. Summary for policy makers. Report of Working Group I of the Intergovernmental Panel on Climate Change. [WWW document]. Available: http://www.ipcc.ch/pub/spm18-02.pdf.
  2. 2. Bezemer TM, Jones TH, Newington JE (2000) Effects of carbon dioxide and nitrogen fertilization on phenolic content in Poa annua L. Biochem Syst Ecol 28: 839–846.
  3. 3. Sun YC, Cao HF, Yin J, Kang L, Ge F (2010) Elevated CO2 changes the interactions between nematode and tomato genotypes differing in the JA pathway. Plant Cell Environ 33: 729–739.
  4. 4. Couture JJ, Servi JS, Lindroth RL (2010) Increased nitrogen availability influences predator-prey interactions by altering host-plant quality. Chemoecology 20: 277–284.
  5. 5. Strauss SY, Agrawal AA (1999) The ecology and evolution of plant tolerance to herbivory. Trends Ecol Evol 14: 179–185.
  6. 6. Fornoni J, Valverde PL, Nunez-Farfan J (2003) Quantitative genetics of plant tolerance and resistance against natural enemies of two natural populations of Datura stramonium. Evol Ecol Res 5: 1049–1065.
  7. 7. Halitschke R, Baldwin IT (2004) Jasmonates and related compounds in plant-insect interactions. J Plant Growth Regul 23: 238–245.
  8. 8. Baldwin IT, Schwachtje J (2008) Why does herbivore attack reconfigure primary metabolism? Plant Physiol 146: 845–851.
  9. 9. Schwachtje J, Minchin PE, Jahnke S, van Dongen JT, Schittko U, et al. (2006) SNF1-related kinases allow plants to tolerate herbivory by allocating carbon to roots. Proc Natl Acad Sci USA 103: 12935–12940.
  10. 10. Belsky AJ, Carson WP, Jensen CL, Fox GA (1993) Overcompensation by plants - herbivore optimization or red herring. Evol Ecol 7: 109–121.
  11. 11. Babst BA, Ferrieri RA, Thorpe MR, Orians CM (2008) Lymantria dispar herbivory induces rapid changes in carbon transport and partitioning in Populus nigra. Entomol Exp Appl 128: 117–125.
  12. 12. Katjiua ML, Ward D (2006) Resistance and tolerance of Terminalia sericea trees to simulated herbivore damage under different soil nutrient and moisture conditions. J Chem Ecol 32: 1431–1443.
  13. 13. Leimu R, Koricheva J (2006) A meta-analysis of tradeoffs between plant tolerance and resistance to herbivores: combining the evidence from ecological and agricultural studies. Oikos 112: 1–9.
  14. 14. Bidart-Bouzat MG, Imeh-Nathaniel A (2008) Global change effects on plant chemical defenses against insect herbivores. J Integr Plant Biol 50: 1339–1354.
  15. 15. Yin J, Sun YC, Wu G, Ge F (2010) Effects of elevated CO2 associated with maize on multiple generations of the cotton bollworm, Helicoverpa armigera. Entomol Exp Appl 136: 12–20.
  16. 16. Karowe DN, Grubb C (2011) Elevated CO2 increases constitutive phenolics and trichomes, but decreases inducibility of phenolics in Brassica rapa (Brassicaceae). J Chem Ecol 37: 1332–1340.
  17. 17. DeLucia EH, Zavala JA, Casteel CL, Nabity PD, Berenbaum MR (2009) Role of cysteine proteinase inhibitors in preference of Japanese beetles (Popillia japonica) for soybean (Glycine max) leaves of different ages and grown under elevated CO2. Oecologia 161: 35–41.
  18. 18. Zavala JA, Casteel CL, DeLucia EH, Berenbaum MR (2008) Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects. Proc Natl Acad Sci USA 105: 10631–10631.
  19. 19. Hawkes CV, Sullivan JJ (2001) The impact of herbivory on plants in different resource conditions: a meta-analysis. Ecology 82: 2045–2058.
  20. 20. Wise MJ, Abrahamson WG (2007) Effects of resource availability on tolerance of herbivory: a review and assessment of three opposing models. Am Nat 169: 443–454.
  21. 21. Bidart-Bouzat MG, Portnoy S, DeLucia EH, Paige KN (2004) Elevated CO2 and herbivory influence trait integration in Arabidopsis thaliana. Ecol Lett 7: 837–847.
  22. 22. Bidart-Bouzat MG (2004) Herbivory modifies the lifetime fitness response of Arabidopsis thaliana to elevated CO2. Ecology 85: 297–303.
  23. 23. Marshall CB, Avila-Sakar G, Reekie EG (2008) Effects of nutrient and CO2 availability on tolerance to herbivory in Brassica rapa. Plant Ecol 196: 1–13.
  24. 24. Wu G, Chen FJ, Ge F (2007) Impacts of early-season square abscission on the growth and yield of transgenic Bt cotton under elevated CO2. Field Crops Res 102: 239–243.
  25. 25. Wu G, Chen FJ, Sun YC, Ge F (2007) Response of cotton to early-season square abscission under elevated CO2. Agron J 99: 791–796.
  26. 26. Chen FJ, Wu G, Ge F (2004) Impacts of elevated CO2 on the population abundance and reproductive activity of aphid Sitobion avenae Fabricius feeding on spring wheat. J Appl Entomol 128: 723–730.
  27. 27. Smith JL, De Moraes CM, Mescher MC (2009) Jasmonate- and salicylate-mediated plant defense responses to insect herbivores, pathogens and parasitic plants. Pest Manag Sci 65: 497–503.
  28. 28. Ge F, Liu XH, Ding YQ, Wang XZ, Zhao YF (2003) Life-table of Helicoverpa armigera in Northern China and characters of population development in Southern and Northern China. Chin J Appl Ecol 14(2): 241–245.
  29. 29. Chen FJ, Ge F (2004) An experimental instrument to study the effects of changes in CO2 concentrations on the interactions between plants and insects—CDCC-1chamber. Acta Entomol Sin 41(3): 37–40.
  30. 30. Li CY, Liu GH, Xu CC, Lee GI, Bauer P, et al. (2003) The tomato Suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. Plant Cell 15: 1646–1661.
  31. 31. Ren Q, Zhang QW, Cao LZ, Su JW, Xie MH, et al. (2010) Volatile emissions from the invasive weed Eupatorium adenophorum induced by Aphis gossypii feeding and methyl jasmonate Treatment. Weed Sci 58: 252–257.
  32. 32. Thaler JS, Stout MJ, Karban R, Duffey SS (1996) Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. J Chem Ecol 22: 1767–1781.
  33. 33. Mahanta PK, Boruah SK, Boruah HK, Kalita JN (1993) Changes of Polyphenol Oxidase and Peroxidase-Activities and Pigment Composition of Some Manufactured Black Teas (Camellia-Sinensis L). JAgri Food Chem 41: 272–276.
  34. 34. Tegelberg R, Julkunen-Tiitto R, Vartiainen M, Paunonen R, Rousi M, et al. (2008) Exposures to elevated CO2, elevated temperature and enhanced UV-B radiation modify activities of polyphenol oxidase and guaiacol peroxidase and concentrations of chlorophylls, polyamines and soluble proteins in the leaves of Betula pendula seedlings. Environ Exp Bot 62: 308–315.
  35. 35. Erlanger BF, Cohen W, Kokowsky N (1961) Preparation and properties of 2 new chromogenic substrates of trypsin. Arch Biochem Biophys 95: 271–278.
  36. 36. Blackwood CE, Erlanger BF, Mandl I (1965) A new test for quantitative determination of chymotrypsin chymotrypsin-like enzymes and their inhibitors. Anal Biochem 12: 128–136.
  37. 37. Chen FJ, Wu G, Parajulee MN, Ge F (2007) Long-term impacts of elevated carbon dioxide and transgenic Bt cotton on performance and feeding of three generations of cotton bollworm. Entomol Exp Appl 124: 27–35.
  38. 38. Bailey JK, Schweitzer JA (2010) The role of plant resistance and tolerance to herbivory in mediating the effects of introduced herbivores. Biol Invasions 12: 337–351.
  39. 39. Muola A, Mutikainen P, Laukkanen L, Lilley M, Leimu R (2010) Genetic variation in herbivore resistance and tolerance: the role of plant life-history stage and type of damage. J Evol Biol 23: 2185–2196.
  40. 40. Valladares F, Gianoli E, Gomez JM (2007) Ecological limits to plant phenotypic plasticity. New Phytol 176: 749–763.
  41. 41. Hartley SE, Jones CG, Couper GC, Jones TH (2000) Biosynthesis of plant phenolic compounds in elevated atmospheric CO2. Global Change Biol 6: 497–506.
  42. 42. Matros A, Amme S, Kettig B, Buck-Sorlin GH, Sonnewald U, et al. (2006) Growth at elevated CO2 concentrations leads to modified profiles of secondary metabolites in tobacco cv. SamsunNN and to increased resistance against infection with potato virus Y Plant Cell Environ 29: 126–137.
  43. 43. Vigue LM, Lindroth RL (2010) Effects of genotype, elevated CO2 and elevated O-3 on aspen phytochemistry and aspen leaf beetle Chrysomela crotchi performance. Agr Forest Entomol 12: 267–276.
  44. 44. Stout MJ, Workman J, Duffey SS (1994) Differential Induction of Tomato Foliar Proteins by Arthropod Herbivores. J Chem Ecol 20: 2575–2594.
  45. 45. Govind G, Mittapalli O, Griebel T, Allmann S, Bocker S, et al. (2010) Unbiased transcriptional comparisons of generalist and specialist herbivores feeding on progressively defenseless Nicotiana attenuata plants. PLoS One 5: e8735.
  46. 46. Yin J, Sun YC, Wu G, Parajulee MN, Ge F (2009) No effects of elevated CO2 on the population relationship between cotton bollworm, Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), and its parasitoid, Microplitis mediator Haliday (Hymenoptera: Braconidae). Agr Ecosys Environ 132: 267–275.
  47. 47. Ainsworth EA, Rogers A, Nelson R, Long SP (2004) Testing the “source-sink” hypothesis of down-regulation of photosynthesis in elevated CO2 in the field with single gene substitutions in Glycine max. Agr Forest Meteorol 122: 85–94.
  48. 48. Li CR, Gan LJ, Xia K, Zhou X, Hew CS (2002) Responses of carboxylating enzymes, sucrose metabolizing enzymes and plant hormones in a tropical epiphytic CAM orchid to CO2 enrichment. Plant cell environ 25: 369–377.
  49. 49. Baldwin IT, Zavala JA (2006) Jasmonic acid signalling and herbivore resistance traits constrain regrowth after herbivore attack in Nicotiana attenuata. Plant Cell Environ 29: 1751–1760.
  50. 50. Babst BA, Ferrieri RA, Gray DW, Lerdau M, Schlyer DJ, et al. (2005) Jasmonic acid induces rapid changes in carbon transport and partitioning in Populus. New Phytol 167: 63–72.
  51. 51. Cipollini D, Enright S, Traw MB, Bergelson J (2004) Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua. Mol Ecol 13: 1643–1653.
  52. 52. Fineblum WL, Rausher MD (1995) Trade off between resistance and tolerance to herbivore damage in a morning glory. Nature 377: 517–518.