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- Materials and Methods
- Supporting Information
Global atmospheric CO2 concentrations have been increasing at an accelerating rate from 280 ppm before industrialization to 396 ppm in Feburary 2013 (Mauna Loa Observatory: NOAA-ESRL), and are anticipated to reach at least 550 ppm by the year 2050 (IPCC, 2007). Elevated CO2 is expected to enhance crop yields by increasing photosynthetic rates and water-use efficiencies, particularly in C3 crops. Observed increases in yield, however, have not always matched theoretical expectations in CO2-enrichment experiments (Ainsworth & Long, 2005; Long et al., 2005), perhaps because the theory does not include interactions between plants and herbivorous insects.
The performance of aphids and other herbivorous insects is affected by bottom-up effects of host plants in terms of nutritional status and chemical and physical defenses (Awmack & Leather, 2002). With respect to nutritional status, aphids feed exclusively on phloem (Douglas, 2003), which provides a protein : carbohydrate ratio (mainly amino acids : sugars) as low as 1 : 10 (w/w) (Nowak & Komo, 2010). Although aphids have evolved to adapt to this nutrient-poor substrate, they are still able to discriminate among host plants with low and high nitrogen (N) concentrations and tend to prefer plants with higher N concentrations (Nowak & Komo, 2010). Moreover, N-fertilized plants enhance aphid population growth because of the increased concentration of amino acids in the phloem (Honek, 1991; Petitt et al., 1994; Ponder et al., 2000). Thus, it seems that the N nutritional status of the host plant is an important determinant of aphid development and fecundity.
With respect to plant defenses, once aphid stylets penetrate the epidermis, the plant triggers a common defensive response based on reactive oxygen species (ROS) by activating superoxide dismutase (SOD) and peroxidase (POD) (Moloi & van der Westhuizen, 2006). A further line of defense involves the rapid synthesis and polymerization of phenolic compounds in the cell wall (Matern & Kneusel, 1988). During this process, polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) are key secondary metabolism enzymes that mediate plant resistance against aphids (He et al., 2011). Effects of elevated CO2 on crop yields should consider how elevated CO2 alters host nutrition and host defenses relative to aphids and other herbivores.
Elevated CO2 reduces the nutritional quality of some nonleguminous C3 plants by decreasing the N concentration (Ainsworth & Long, 2005; Ainsworth & Rogers, 2007), which may consequently increase the developmental time and reduce the fecundity and fitness of leaf-chewing insects (Coll & Hughes, 2008). However, N concentrations in legumes were rarely affected by elevated CO2 because of the enhancement of biological N fixation (BNF), which counteracts the adverse effect of elevated CO2 on leaf-chewing insects (Karowe, 2007; Taub & Wang, 2008; Karowe & Migliaccio, 2011). For a sap-sucking insect like the pea aphid (Acyrthosiphon pisum), the increased BNF in legumes under elevated CO2 increases available N and thereby increases aphid numbers (Guo et al., 2013). When BNF is suppressed by artificial mutation, however, BNF cannot satisfy the increased demand for N that occurs under elevated CO2, and aphid numbers do not increase (Guo et al., 2013). Thus, it appears that enhanced BNF is necessary for the positive response of the pea aphid to elevated CO2.
In legumes, BNF is regulated by several hormone signaling pathways, including the ethylene signaling pathway. The involvement of the phytohormone ethylene in nodulation was initially proposed based on studies showing that the application of exogenous ethylene or its biosynthetic precursor 1-amino-cyclopropane-carboxylic acid (ACC) suppresses nodulation, and, conversely, that application of chemical inhibitors of ethylene perception (i.e. Ag+) or biosynthesis (i.e. the amino ethoxyvinyl glycine, AVG) increases nodule numbers (Ma et al., 2002; Penmetsa et al., 2003, 2008). Once the key gene Mtskl in the ethylene-perception pathway was mutated in Medicago truncatula, the resulting ethylene-insensitive mutant, sickle, produced more nodules than the wild-type, and its nitrogenase activity was increased about two times (Penmetsa & Cook, 1997).
In addition to having a key role in the regulation of BNF, ethylene is the most important hormone involved in plant resistance against pathogens and pests. The expression of genes involved in ethylene production and ethylene signaling (ACC oxidase and ethylene-responsive elements) are up-regulated in response to aphid infestation (Moran et al., 2002; Divol et al., 2005). Ethylene is also responsible for the regulation of ROS and downstream defensive enzymes against aphids (Jung et al., 2009). The sickle mutant showed increased sensitivity to Rhizoctonia solani and other pathogens on legumes and cereals (Penmetsa et al., 2008). Thus, it is reasonable to speculate that the ethylene-insensitive mutant sickle, which produces more nodules and has enhanced BNF as well as reduced resistance to aphids relative to the wildtype, will supply more N nutrition to aphids and be less resistant to aphids.
The results of recent studies indicate that elevated CO2 fine-tunes phytohormone signaling pathways when plants encounter biotic stress, as indicated by enhanced induced defenses derived from the salicylic acid signaling pathway and reduced jasmonic acid-dependent defense (Zavala et al., 2008; Sun et al., 2011; Guo et al., 2012). Furthermore, elevated CO2 tends to increase ethylene production in healthy plants but down-regulates the expression of downstream genes in the ethylene signaling pathway when plants are attacked by Japanese beetles (Seneweera et al., 2003; Casteel et al., 2008). Although it is well established that the ethylene signaling pathway mediates plant resistance against pathogens and aphids, it is unclear whether elevated CO2, by modulating the ethylene signaling pathway, simultaneously alters ethylene-dependent defense as well plant nutrition for aphids and other herbivores.
Here, we hypothesized that elevated CO2 would decrease the responses of the ethylene signaling pathway, which would directly reduce ethylene-dependent plant defenses while indirectly enhancing N availability for aphids via an increase in nodulation, such that pea aphid abundance would be greater under elevated CO2 than under ambient CO2. To test this hypothesis, we used sickle (a supernodulating mutant of M. truncatula that is insensitive to ethylene) and the wild-type A17 to determine how elevated CO2 affects the interaction between M. truncatula and the pea aphid via the ethylene signaling pathway. The specific goals were to determine whether the supernodulating mutant sickle grows better and has higher N metabolism than the wild-type under elevated CO2; whether elevated CO2 affects the resistance of the two genotypes against the pea aphid and consequently affects pea aphid feeding behavior and abundance; and whether the ethylene signaling pathway is involved in the regulation of the plant–aphid interaction under elevated CO2.
- Top of page
- Materials and Methods
- Supporting Information
Elevated CO2 affects herbivorous insects mainly by altering host plant nutritional quality and resistance (Awmack & Leather, 2002). Under ambient CO2, the ethylene-insensitive mutant sickle, which produces more nodules and exhibits a stronger BNF than the wild-type, grew better and was less resistant than the wild-type A17 and consequently supported higher numbers of pea aphids than A17. Although the increased growth under elevated CO2 could increase plant N demand (Daepp et al., 2000), the increased BNF in both genotypes under elevated CO2 provided sufficient N so that the plants could produce greater biomass and more pods than under ambient CO2. Our results indicate that elevated CO2 tends to suppress the ethylene signaling pathway in wild-type A17 plants, so that increased nodulation and BNF satisfy the increased N requirement for growth under elevated CO2. By decreasing the ethylene signaling pathway, however, elevated CO2 reduced plant resistance against the pea aphid. In summary, impairment of the ethylene signaling pathway by elevated CO2 has two important effects in M. truncatula: it up-regulates amino acid metabolism but reduces aphid resistance ability; and the increased plant growth and reduced resistance result in increased numbers of pea aphids per plant.
The response to elevated CO2 differs among insect feeding guilds (Robinson et al., 2012). Typically, elevated CO2 tends to prolong the development of chewing insects because it decreases the N content and increases secondary metabolites in host tissues (Coll & Hughes, 2008). By contrast, elevated CO2 has species-specific effects on phloem-sucking insects such as aphids, which obtain food from phloem sieve elements (Sun & Ge, 2011). Some aphid species exhibit increased fecundity, abundance, and survival under elevated CO2 (Pritchard et al., 2007). Although Newman proposed that aphid populations tend to be larger under elevated CO2 if host plants have higher N supplementation (Newman et al., 2003), evidence concerning how elevated CO2 affects both bottom effects on aphids (via host nutrition and resistance) has been lacking until the current study. Our previous study revealed that increases in pea aphid numbers under elevated CO2 depend on an increase in BNF and thus an increase in host plant amino acid metabolism (Guo et al., 2013). When BNF was suppressed by mutation, host plant amino acid metabolism was not increased by aphid infestation under elevated CO2. Because BNF is important in supporting the N nutrition of pea aphids, we speculated that pea aphids would be able to obtain more N from the supernodulating genotype sickle than from the wild-type A17.
As important indices of BNF, nodules were more abundant and expression of ENOD, nifH, and nodF was higher in sickle than in A17 plants. The increases in N metabolism leads to increased investment in Rubisco and other C assimilation-related enzymes (Gleadow et al., 1998), which in turn results in greater Chl content, biomass, and pod number in sickle than in A17 plants. Although elevated CO2 increased nodule number, gene expression involved in nodulation and BNF, Chl content, biomass and pod number for both genotypes, it did not change the growth advantages of sickle relative to A17. The improved growth traits of sickle had positive bottom-up effects on the aphid, as demonstrated by greater aphid abundance and feeding efficiency on sickle than on A17, regardless of CO2 concentrations.
Aphids have stylet-like mouthparts and feed mainly on phloem sap (Douglas, 2003). Most previous studies have measured aphid response to elevated CO2 as a function of whole-leaf composition rather than phloem sap composition (Robinson et al., 2012). The current study indicated that M. truncatula exhibited three patterns of individual amino acid concentrations in phloem sap in response to elevated CO2 and aphid infestation. When infested by aphids, the concentrations of amino acids loading on RF1 (seven essential amino acids and four nonessential amino acids) were higher in sickle than in A17 regardless of CO2 concentrations. However, elevated CO2 has a contrasting effect on the concentrations of amino acids loading on RF2 (mainly nonessential amino acids) in which sickle was higher than A17 plant under ambient CO2 but lower under elevated CO2. These results confirmed that sickle plants provide better N nutrition for aphids than A17 plants, and elevated CO2 tends to decrease this nutritional advantage of sickle when compared with A17. Although elevated CO2 increased the activities of N assimilation and transamination-related enzymes, and consequently increased amino acid concentration of infested plants of both genotypes, the pattern for this enhancement of amino acids differed between the two genotypes. Elevated CO2 increased individual amino acid concentrations loading onto RF1, RF2, and RF3 in A17 plants but only increased the concentration of amino acids loading onto RF1 in sickle plants. The relative improvement of N nutrition in response to elevated CO2 was less in sickle than in A17 plants because the basal N metabolism under ambient CO2 was much higher in sickle plants.
The regulation of nodule formation by ethylene signaling greatly affects the plant's ability to adapt to elevated CO2, in that enhanced BNF can satisfy the increased demand for N under elevated CO2 (Penmetsa & Cook, 1997). The current study showed that elevated CO2 down-regulated the expression of the ethylene signaling pathway genes ACC, SKL and ERF in A17 plants. Similarly, Casteel et al. (2012) found that elevated CO2 decreased the ethylene signaling pathway when attacked by Japanese beetles (Popillia japonica).This result suggests that, under elevated CO2, M. truncatula suppresses the ethylene signaling pathway so as to increase nodulation and BNF and thereby satisfy the increased demand for N. The ethylene signaling pathway in M. truncatula, however, has also been found to provide resistance against the pea aphid (Gao et al., 2008).
To access the amino acids in phloem sap, aphids must overcome a number of plant defense responses. One of the early plant responses to aphids is the release of ROS (Moloi & van der Westhuizen, 2006). Two other important defense enzymes are PPO and PAL, which are involved in the synthesis of phenolic compounds that may be absorbed by the salivary sheath of the aphid stylet. The further polymerization of phenolic compounds causes browning of cells in contact with the saliva, which is disadvantageous to aphid feeding (Jiang & Miles, 1993). In our study, aphid infestation increased the activities of SOD and POD (which are involved in ROS synthesis) and of PPO and PAL in A17 plants. In sickle plants, however, aphid infestation did not induce SOD or POD, which is consistent with a previous finding that the inhibition of ethylene synthesis or perception blocks the ROS response (de Jong et al., 2002). These results indicate that because its ethylene signaling pathway was mutated, sickle could not trigger the downstream ethylene-dependent defense in response to aphid infestation. This is consistent with our EPG finding that, relative to aphids feeding on A17, aphids feeding on sickle spend less time on salivation and more time on ingestion in phloem. Furthermore, elevated CO2 decreased the activities of SOD, POD, and PPO in infested A17 plants. As noted earlier, the decreased ethylene signaling pathway of M. truncatula under elevated CO2 affects the pea aphid in two ways, that is, by maintaining host N metablism and reducing host resistance.
Most plants are well adapted to process ‘extra’ carbon under elevated CO2, and this allows them to grow faster and larger. To satisfy the increased N demand under elevated CO2, legume plants evidently decrease their ethylene sensitivity so as to increase the formation of nodules and enhance BNF. Furthermore, considering the vital role of the ethylene signaling pathway in regulating plant resistance against aphid infestation, our results suggested that the down-regulation of the ethylene signaling pathway is accompanied by decreased plant resistance against the pea aphid. In other words, in decreasing the ethylene signaling pathway under elevated CO2 to match their N budgets and growth, plants sacrifice their resistance against aphids. Furthermore, given that the ethylene pathway was mutated in sickle plants, it is reasonable to expect that the mutated plants would lack one of the phytohormone signaling pathways that increases N metabolism in response to elevated CO2. Unexpectedly, although elevated CO2 only increased amino acids in RF1 for sickle plants infested with aphids, but increased amino acids in RF1, RF2, and RF3 for A17 plants infested with aphids, nodule numbers, expression of genes related to N fixation, growth traits, and N nutrition for pea aphids were all increased in sickle by elevated CO2. This suggests that the ethylene signaling pathway may not be the only phytohormone pathway regulating plant response to elevated CO2. The interaction between plant and aphid is coordinated by other interacting signaling phytohormones (such as jasmonic acid and salicylic acid pathways), except for ethylene (Felton & Korth, 2000). These signaling pathways are cross-talked in a complex network, which supports plants rapidly adapting to biotic and abiotic stresses by triggering an enormous regulatory mechanism. Among the three signaling pathways, jasmonic acid and ethylene signaling pathway has an antagonistic interaction with salicylic acid signaling pathway. The emerging data suggest that elevated CO2 tends to modulate these phytohormone signaling pathways, such as the jasmonic acid and salicylic acid pathways, that affect responses to insect herbivores (DeLucia et al., 2012; Sun et al., 2013; Zavala et al., 2013). Thus, additional research is needed to determine how multiple phytohormone signaling pathways are coordinately regulated by elevated CO2.
In summary, elevated CO2 increased pea aphid abundance on M. truncatula by affecting both host plant nutritional quality and resistance. Elevated CO2 decreased the ethylene-dependent resistance of wild-type M. truncatula against the pea aphid. On the other hand, the decrease in the ethylene signaling pathway increased the nodulation and BNF and thereby increased the phloem amino acids supporting aphid reproduction. The two effects of the ethylene signaling pathway would synergistically increase the fitness of pea aphids under elevated CO2. Because the supernodulating genotype sickle has higher amino acid metabolism and lower resistance, it is more suitable for the pea aphid than the A17 plant under ambient CO2, and the greater suitability of sickle in comparison to A17 is not changed by elevated CO2.