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.