Discussion
In the current study, constitutive antibiosis was higher in the susceptible than in the resistant inbred, but data collected on field resistance (always evaluated under artificial infestation) of these inbreds in previous studies have always shown better performance of the resistant inbred suggesting that induced responses could have an important role in resistance (Butron et al. , 1999, Ordas, Malvar, Santiago & Butron, 2010). According to that, stems of the resistant inbred pre-conditioned by continuous damage of MCB larvae reduced larval weight compared to the control; meanwhile larval effectors or a general decrease of the plant metabolism provoked by insect damage seems to disrupt constitutive defenses of the susceptible inbred because stems from susceptible plants pre-conditioned for a long period increased larval weights compared to the susceptible control plants. Dafoe et al (Dafoe et al. , 2011, Dafoe et al. , 2013) already showed that 24-48h feeding by Ostrinia nubilalis , the European corn borer, could increase stem susceptibility of a single genotype. In opposition, induction by MCB attack in the resistant inbred had a detrimental effect on the weight of larvae agreeing with the increased antibiosis of maize leaves infested with Ostrinia furnacalis , the Asian corn borer, recently reported (Guo, Guo, He, Bai, Zhang, Zhao & Wang, 2017, Guo, Qi, He, Wu, Bai, Zhang, Zhao & Wang, 2019). Therefore, in the current study, alteration of plant performance under subsequent conspecific attack due to previous insect damage was genotype-dependent as it has been previously reported in other plant-insect interactions (Su et al. , 2018).
Long-term attack to the resistant genotype caused stronger effect on the weight of larvae than short-term attack suggesting that defenses induced by MCB could rather be progressively accumulated, since the initiation of damage, than punctually increased. We hypothesize that, after a prolonged period of insect damage, progressive accumulation of induced defensive metabolites would allow the resistant inbred to perform better than the susceptible inbred against insect attack as it has been reported in previous studies (Butron et al. , 1999, Cao et al. , 2019). However, after nine days of insect feeding, although continuous insect damage has broken down constitutive defenses of the susceptible inbred, accumulated defenses in the resistant would not still be enough to outperform antibiosis on larval growth of the susceptible inbred. Even so, long term induced responses made the mortality of larvae fed on the resistant plants significantly higher than that of larvae fed on the susceptible ones. Therefore, better field performance of the resistant inbred against MCB attack would greatly depend on the accumulation of induced defenses by MCB feeding, accumulation being higher as exposure to insect damage is prolonged; while higher constitutive resistance does not guarantee good performance under MCB infestation because continuous insect damage could disrupt constitutive defenses. These results suggest that the level of field resistance of the studied inbred line rather depends on induced changes by MCB attack than on constitutive defenses and those changes are determined by the duration of insect feeding. Continuous damage by insect feeding seems to contribute to increased susceptibility or resistance depending on the genotype and those genotype-dependent changes could be as the result of reconfiguration of metabolism in attacked plants (Schuman & Baldwin, 2016).
Under the hypothesis that the level of field resistance rather depends on induced changes by MCB attack than on constitutive defenses and those changes are determined by the duration of insect feeding, ions implicated in the long term response could be important determinants of genotype induced resistance. Agreeing with other studies on induced metabolic plant changes by insect attack, the primary metabolism was downregulated in both maize genotypes by MCB 9-day feeding, including sugar, amino acid fatty acid and vitamin metabolism, as well as the tricarboxylic acid (TCA) or Krebs cycle which implies a disruption of energy production (Kang et al. , 2019, Liu et al. , 2010, Sabino, Tavares, Riffel, Li, Oliveira, Feres, Henrique, Oliveira, Correia, Nascimento, Hawkes, Santana, Holmes & Bento, 2019, Wanget al. , 2016). According to a general downregulation of energy production and metabolism, intermediates or end-products of the shikimate pathway were repressed by MCB infestation in the susceptible inbred since decreases of tryptophan, phenylalanine and beta-tyrosine levels and those of several phenolic and indole-related compounds derived from them were registered in that inbred after 9-day feeding. However, some compounds synthesized through the shikimate pathway, such as tryptophan, or derived from the end products of the pathway, such as the indole-derived DIMBOA glucoside (antibiotic against S. nonagrioides larvae) and indol-acrylate (plant hormone) or the phenylpropanoids methyl-4-methoxy-3-nitrobenzoate (probable insecticide), 4-hydroxy-6-methylcoumarin (biocide), sinapaldehyde (intermediate in lignin formation) or o-hydroxyhippurate [also known as salicylurate] (insect antifeedant), were up-regulated in the resistant inbred by MCB feeding (Cutler, Cutler, Wright & Dawson, 2002, Feng, Chen & Zhang, 2018, Ortego, Ruiz & Castanera, 1998, Stuart, Brooks, Prescott & Blackwell, 2000). In general, the levels of these compounds are higher in untreated stems of the susceptible compared to the resistant inbred and remain higher after 9-days of larvae feeding. In addition, a decrease of several nitrogen-containing compounds was observed in the susceptible inbred, while those compounds increased in the resistant inbred after 9-day feeding, but, again, levels were still higher in the susceptible. Therefore, the inbred susceptible at field conditions seemed to constitutively possess a resistance metabolic array that is broken down by continuous insect feeding, meanwhile the field resistant inbred, appeared to acquire induced resistance by channeling metabolism toward biosynthesis of defensive metabolites like the benzoxazinoid DIMBOA glucoside and methyl-4-methoxy-3-nitrobenzoate (Schuman & Baldwin, 2016). As metabolomics analyses were made using undamaged stem sections, herbivore challenged plants of the resistant inbred would be able of mounting systemically active defense responses meanwhile susceptible plants could not (Howe & Jander, 2008). Other authors have demonstrated that maize herbivory by Mythimna separata conferred resistance to the subsequently infested caterpillars through systemic changes of benzoxazinoids and probably other defensive metabolites (Malook, Qi, Hettenhausen, Xu, Zhang, Zhang, Lu, Li, Wang & Wu, 2019). Findings agreed with expectations because plant response to insect attack has been proved to be genotype-dependent, resulting in increased levels of phenylpropanoids, hydroxamic acids or/and nitrogen-containing secondary metabolites in resistant genotypes; meanwhile increased susceptibility after insect attack in other genotypes was associated to reduced levels of phenylpropanoids (Biyiklioglu, Alptekin, Akpinar, Varella, Hofland, Weaver, Bothner & Budak, 2018, Kang et al. , 2019, Liu et al. , 2010, Suet al. , 2018).
Enzymatic or chemical oxygenation of free or membrane-esterified polyunsaturated fatty acids produces oxylipins and, among them, jasmonic acid has been highlighted as a key phytohormone in mediating maize resistance response to herbivory by insects (Borrego & Kolomiets, 2016, Christensen, Huffaker, Kaplan, Sims, Ziemann, Doehlemann, Ji, Schmitz, Kolomiets, Alborn, Mori, Jander, Ni, Sartor, Byers, Abdo & Schmelz, 2015). Jasmonic acid is a 13-LOX α-linolenic acid-derived plant oxylipin, 13-HPOTrE[R], being the first intermediate in the synthesis of jasmonic acid. This phytohormone was upregulated and downregulated by 48h feeding in resistant and susceptible inbreds, respectively. In parallel, 13-hydroxylinolenic acid (13-HOTrE), that resulted from reducing HPOTrE[R] and is not a jasmonic acid intermediate, was downregulated by 48h feeding in the resistant inbred contributing to the precursor pool leading to cyclization and eventual synthesis of jasmonic acid (Farmer, Caldelari, Pearce, Walkersimmons & Ryan, 1994). Epoxy – and hydroxy – derivatives of linoleic acid resulting from the peroxygenase pathway have been described as fungitoxic oxylipins and the metabolite 13-HPODE that belong to that oxylipin group was reduced by 48h feeding in the resistant inbred and by 9-day feeding in both, meanwhile vernoleate diminished after 9-day feeding in both (Blee, 2002, Kato, Yamaguchi, Uyehara, Yokoyama, Namai & Yamanaka, 1983, Tsitsigiannis, Kunze, Willis, Feussner & Keller, 2005). Therefore, we speculate that maintenance of the upregulation of jasmonic acid precursors after 48h feeding in the resistant inbred could play an important role in inducing systemic resistance meanwhile resistance was not induced in the susceptible inbred because precursors were downregulated. Production of jasmonic acid is required for a systemic response to herbivory (Bosch, Wright, Gershenzon, Wasternack, Hause, Schaller & Stintzi, 2014). Systemic response to parasites involves transduction processes in which transient shifts of intracellular and apoplastic pH are essential: rapid alkalinization of the apoplast is combined with intracellular acidification, loss of K+, and influx of Ca2+ and followed by an oxidative burst and up-regulation of several pathways involved in defense (Viehweger, Dordschbal & Roos, 2002). In that context, authors proposed lysophosphatidylcholines as good candidates for transducing the elicitor-triggered signal; lysophosphatidylcholines would be intracytoplasmic messengers that would connect the activation of a stress-responding enzyme of the plasma membrane (phospholipase A2) with the production of vacuolar proton fluxes. According with that idea, levels of lysophosphatidylcholines in the stems of the resistant inbred, augmented after feeding by MCB larvae; meanwhile levels of lysophosphatidylcholines in the stems of the susceptible inbred, that showed increased susceptibility upon feeding, diminished after feeding.
In the stems of the resistant inbred, organic acids malate and, especially, malonate (only detected in stems of PB130 pre-conditioned by 9-day feeding) increased after 9-day feeding. This can be consequence of a high redox level in the stem cells of the resistant inbred because under those conditions, the TCA cycle in mitochondria is transformed to a “non-cyclic” partial TCA cycle supplying citrate for the synthesis of 2-oxoglutarate, glutamate, and malonate (citrate valve), while malate is stored and participates in the redox balance (via malate valve) (Igamberdiev & Eprintsev, 2016). In that scenario, the 3-Hydroxy-3-Methylglutarate that is an ”off-product” intermediate in the leucine degradation process and accumulated at high levels in the stem of the resistant inbred after continuous feeding by MCB larvae could be proposed as an important agent in causing and maintaining oxidative burst since this metabolite cause acute disruption of redox homeostasis in animal tissues (da Rosa, Seminotti, Amaral, Fernandes, Gasparotto, Moreira, Gelain, Wajner & Leipnitz, 2013). Agreeing with the hypothesis of high oxidative stress in the resistant inbred after 9-day feeding by MCB larvae, this inbred line presents a high ROS scavenging level that increased after long-term feeding by MCB larvae because glutathione is higher in the resistant than in the susceptible inbred at control conditions and glutathione levels were doubled in both inbreds after 9-day damage by MCB larvae; in parallel, oxoproline that is a reservoir of glutamate and participates in glutathione homeostasis decreased in the susceptible but augmented in the resistant inbred (Ohkama-Ohtsu, Oikawa, Zhao, Xiang, Saito & Oliver, 2008).
Longer maintenance of high jasmonic acid precursor levels and up-regulation of lysophosphatidylcholines could act as signal molecules of induced systemic resistance (ISR) to stem tunneling by stem borers. ISR would imply an oxidative burst (counterbalanced by increased ROS scavenging metabolites) and up-regulation of metabolites involved in defense. Therefore, the different resistance of the two inbreds to stem tunneling by MCB larvae could depend on their ability to stablish that systemic response. Similarly, the observed differential defense responses of two different switchgrass cultivars to fall armyworm herbivory indicate that the resistant cultivar mounted a more robust response with potential activation of pathways that could lead to the production of antifeedants as compared to the susceptible (Palmer, Basu, Heng-Moss, Bradshaw, Sarath & Louis, 2019).
We hypothesize that the level of field resistance rather depends on induced changes by MCB attack than on constitutive defenses and those changes are determined by the duration of insect feeding. Therefore, differential defense responses to continuous MCB feeding would result in resistance differences and ions that were differentially induced in both inbreds by long-term feeding could play an important role in resistance. A limit number of differentially induced features could be assigned to known metabolites but point to the ability of the inbred to establish a systemic response, involving oxidative burst and up-regulation of defense compounds, as determinant for limiting damage by MCB larvae.