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Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro-economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling is required for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.
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In response to insect attack, plant tissues undergo significant reprogramming of genetic and metabolic processes, resulting in direct and indirect defense responses (Maffei et al., 2006). As direct countermeasures, toxins and defensive proteins are produced to repel pests and impede digestibility. As a mode of indirect defenses, herbivore-induced plant volatile (HIPV) emissions attract insect predators and parasitoids (D'Alessandro and Turlings, 2006; Heil, 2008; Dicke, 2009). While our knowledge of the detailed molecular and biochemical regulation of these responses is still incomplete, it is widely known that much of the essential defense signals are produced in the fatty acid oxidation pathways, including metabolites derived from lipoxygenases (LOXs; Matsui, 2006; Howe and Jander, 2008; Mosblech et al., 2009).
Initiation of the LOX pathway begins when polyunsaturated fatty acids [linoleic (18:2) and α-linolenic (18:3) acids] are cleaved from cell membranes by diverse lipases and dioxygenated by either 9- or 13-LOXs to form 9- and 13-hydroperoxides, respectively. These hydroperoxides act as a substrate for seven downstream branches of the LOX pathway, namely peroxygenases, divinyl ether synthases, reductases, epoxy alcohol synthases, hydroperoxide lyases (HPLs), allene oxide synthases (AOSs) and additional LOX reactions, which collectively produce numerous oxylipins (Feussner and Wasternack, 2002; Mosblech et al., 2009). Although there is still much to unveil in terms of the physiological roles of specific oxylipins in response to stresses, the HPL and AOS pathways responsible for green leaf volatile (GLV) and jasmonic acid (JA) production, respectively, are the predominant and better understood pathways activated in the wound and herbivore response.
The synthesis of GLVs begins with the cleavage of 13-hydroperoxy octadecatrienoic acid (13-HPOTE) by HPL to form (3Z)-hexenal, which is enzymatically converted to other C6 compounds, including (3Z)-hexenol and (3Z)-hexenyl acetate (Blée, 2002; D'Auria et al., 2002; Matsui, 2006). GLVs act as signals that induce the expression of defensive genes (Bate and Rothstein, 1998), regulate plant–plant communication after insect elicitation (Arimura et al., 2000; Farag and Pare, 2002; Engelberth et al., 2004) and attract parasitoid wasps (Whitman and Eller, 1990). The exposure of plants to exogenous GLVs induces JA and HIPV production, and, more importantly, enhances the JA response to herbivore attack (Farag and Pare, 2002; Engelberth et al., 2004). GLVs also possess antimicrobial properties, although in some plant pathogen systems they may serve as signals to facilitate pathogenesis processes (Prost et al., 2005; Christensen and Kolomiets, 2011). These herbivore- and microbial-related examples show the large number of organisms that respond to GLVs, and demonstrate the varied effects GLVs can have, based on the co-evolution of the plant and interacting species involved.
Similar to GLVs, the formation of JA begins with LOX-derived 13-HPOTE, which is catalyzed into allene oxide by AOS and subsequently transformed into a cyclopentenone by allene oxide cyclase. The resulting (+)-12-oxo-phytodienoic acid (OPDA) is further reduced by 12-oxo-phytodienoate reductase isoform 3 (OPR3), and then truncated by three beta-oxidation steps to form (+)-7-iso-JA (Wasternack, 2007). JA and/or its derivatives play important roles in growth and development (Creelman and Mullet, 1997; Acosta et al., 2009; Yan et al., 2012a,b), and in microbial- and herbivore-induced defense responses (Browse, 2009; Koo and Howe, 2009). The rapid accumulation of JA in response to wounding or insect attack is essential for direct defenses at the site of attack and systemic defense signaling (Howe and Jander, 2008). A major ecological significance of JA is found in its regulation of HIPVs (Dicke et al., 1999; Koch et al., 1999; Bruinsma et al., 2009), which play an important role in indirect defense. The majority of HIPVs are synthesized via the isoprenoid pathway, where terpene synthases enzymatically convert their pyrophosphate substrates into diverse homo-, mono-, sesqui- and diterpenes, which are sensed as foraging cues for natural enemies, i.e. predators (Kessler and Baldwin, 2001; Turlings et al., 2004). An interesting feature of HIPVs is their diurnal emissions during herbivory. Although this emissions pattern has long been established (Turlings et al., 1995; Martin et al., 2003), the mechanism behind it is not fully understood.
The significance of lipid derivatives in diverse biological and ecological functions has been demonstrated by studies of LOX mutants in several plant species. For example, analysis of transgenic lines in tomato TomLOXC, potato LOX-H1, tobacco NaLOX2 and rice OsLOX1 show that these genes specialize in GLV biosynthesis (Leon et al., 2002; Chen et al., 2004; Wang et al., 2008 and Allmann et al., 2010). Furthermore, the measurement of wound- and/or herbivore-induced JA accumulation has shown that Arabidopsis LOX2, rice OsHI-LOX and OsLOX1, and tobacco NaLOX3 all contribute to JA biosynthesis (Bell et al., 1995; Halitschke and Baldwin, 2003; Wang et al., 2008; Zhou et al., 2009). Beyond functions in GLVs and JA biosynthesis, LOXs also regulate downstream defense responses and biological resistance. Antisense plants downregulated for Arabidopsis LOX2 are more susceptible to Pieris rapae (Zheng et al., 2011) than their corresponding wild type (WT). Silenced Nicotiana attenuata plants at the NaLOX3 locus are impaired in nicotine, trypsin protease inhibitors (TPIs) as well as terpene volatile emissions (Halitschke and Baldwin, 2003). OsHI-LOX and OsLOX1 were also shown to be involved in resistance through assays with stem borers or brown plant hoppers (Wang et al., 2008; Zhou et al., 2009). Although the collective characterization of these and other JA- and GLV-producing LOXs has contributed to our understanding of plant insect defense in various plant species, there is a dearth of genetic evidence for the functions of defense genes in the economically important crop Zea mays (maize). Moreover, despite the diverse functions of LOXs in regulating different aspects of the GLV, JA or HIPV pathways, evidence for a single LOX isoform that modulates all three remains unseen.
Although GLVs and JA are commonly synthesized by the LOX pathway, the mode by which the final products are produced is diverse and appears to be species dependent. For instance, the chloroplast-localized rice LOX, OsLOX1, has been shown to supply both the AOS and HPL branches of the LOX pathway (Wang et al., 2008). In other reported cases, separate LOX isoforms provide pathway-specific 13-hydroperoxide substrates for either the JA or the GLV cascade (Halitschke and Baldwin, 2003; Halitschke et al., 2004; Chehab et al., 2008; Allmann et al., 2010). Despite the variation that exists in the number of LOXs required to feed substrate to the GLV and JA pathways, the HPL and AOS branches are commonly co-localized to the same organelle: the chloroplast (Froehlich et al., 2001; Farmaki et al., 2007). To the best of our knowledge, the only reported crosstalk between these co-localized pathways has been at the linolenic fatty acid or 13-hydroperoxide substrate levels (Halitschke et al., 2004; Chehab et al., 2008; Wang et al., 2008). Surprisingly, the intermediates and final products of these branches are known for their function as potent signaling molecules, yet there is no evidence suggesting signaling interaction between these pathways. For example, GLVs have been shown to be strong inducers of JA when pharmacologically applied (Engelberth et al., 2004, 2007), but the endogenous presence or absence of these metabolites because of anti sense suppression or overexpression (Halitschke et al., 2004; Chehab et al., 2008; Allmann et al., 2010) does not seem to affect the production of JA, even though both pathways reside in the same organelle.
Here we provide evidence that, in maize, GLV and JA biosynthesis occurs in separate and distinct organelles, and show that wound-induced JA is dependent on signaling from GLVs and/or oxylipins derived from the GLV-producing LOX. Specifically, we show that LOX10 provides substrate to the GLV biosynthesis pathway, and is localized to organelles distinct from chloroplasts. Furthermore, we demonstrate that the chloroplast-localized LOX8 (also known as tasselseed 1, LOX-ts1; Acosta et al., 2009) is responsible for wound-induced JA, yet LOX8-mediated JA production is dependent on signaling from LOX10-derived oxylipins. The elimination of LOX10 by Mu transposon insertional mutagenesis compromises resistance to insects under both laboratory and field conditions, and leads to the impairment of ecologically important HIPV emissions, making plants less appealing to the parasitoid wasp Cotesia marginiventris. Collectively, our results show that ZmLOX10 is a key modulator of insect defense in maize by regulating JA, GLV and HIPV production in response to herbivory.
Generation of transposon-insertional lox10 mutants
Nemchenko et al. (2006) reported on the two segmentally duplicated LOX paralogs, ZmLOX10 and ZmLOX11, which share 90% identity at the amino acid level. To identify putative mutant alleles, we screened the Mutator-transposon insertional genetics resource at Pioneer Hi-Bred International, Inc. (http://www.pioneer.com) for insertions in these two genes. Unfortunately, there were no Mu insertions identified in the ZmLOX11 gene; however, three independent alleles were identified at the ZmLOX10 locus (lox10-1, lox10-2 and lox10-3). Sequencing of the regions flanking insertion sites showed that the lox10-1 allele harbored a Mu element in the first intron, whereas lox10-2 and lox10-3 had Mu insertions in exon III (Figure 1a). The original mutants were backcrossed into the B73 (lox10-2 and lox10-3) and W438 (lox10-2) genetic backgrounds, and then genetically advanced to create near-isogenic mutant and WT lines (BC3F4–BC5F4; as designated below) suitable for functional analysis. To test whether lox10-2 and lox10-3 are null alleles, transcript accumulation was measured 8 h after mechanical wounding, the time point at which LOX10 is expressed at the greatest levels (Nemchenko et al., 2006). The WT plants showed a clear induction of ZmLOX10 transcripts, whereas no detectable hybridization signal was observed for both lox10-2 and lox10-3 alleles (Figure 1b). These data demonstrate that lox10-2 and lox10-3 mutants are null alleles.
The characterization and subcellular localization of LOX10
The clustering of LOX10 with other GLV-producing isoforms (Figure S1; Nemchenko et al., 2006) prompted the hypothesis that LOX10 feeds substrate to the HPL pathway. As GLVs are emitted rapidly upon mechanical damage, we cut leaf tissue and collected head-space volatiles from WT, lox10-2 and lox10-3 mutant seedlings for 10 min. Gas chromatography mass spectrometry (GC/MS) analysis showed no detectable levels of (3Z)-hexenal, (3Z)-hexenol and (3Z)-hexenyl acetate in wounded lox10-2 and lox10-3 mutant leaves, whereas WT plants emitted normal levels of all three volatiles (Figure 2a). GLV emission was further measured in response to Spodoptera exigua feeding on seedlings 2–4 h after infestation. Normal levels of total GLVs [(3Z)-hexenal, (3Z)-hexenol, (3Z)-hexenyl acetate and (2E)-hexenal] were observed in infested WT plants (156 ng h−1 – B73 background; 230 ng h−1 – W438 background), whereas lox10-2 and lox10-3 mutant emissions were comparable with background levels (Figure 2b). These results show that LOX10 is the sole 13-LOX isoform that provides substrate for the HPL pathway for GLV production in maize leaves in response to wounding or herbivory by S. exigua.
Because LOX10 was not observed in chloroplasts (Mohanty et al., 2009), the expected site for GLV and JA biosynthesis, we further characterized subcellular localization using a ZmLOX10:YFP fluorescent protein-tagged gene, driven by the endogenous ZmLOX10 promoter. Our results showed that fluorescence from the YFP-tagged protein is, indeed, emitted from organelles lacking chlorophyll autofluorescence (Figure 2c, panel E and F), indicating that LOX10 is localized to non-chloroplast organelles.
ZmLOX8 is responsible for wound-induced JA in maize
With the functional role of LOX10 in GLV production confirmed, our next step was to determine which LOX provides 13-hydroperoxy substrate to the JA biosynthesis pathway. To test this, we wounded WT seedlings and measured transcript accumulation in candidate maize 13-LOXs (ZmLOX7, ZmLOX8 and ZmLOX9). Of the maize genes that clustered with JA-producing LOXs from other plant species (Figure S1), ZmLOX8 was strongly induced by wounding at early time points (Figure 4c). These data, coupled with the knowledge that ZmLOX8 is required for JA-mediated tassel development (LOX8 was designated as ts1 in Acosta et al., 2009), led us to hypothesize that LOX8 provides substrate for wound-induced JA biosynthesis. Using the publicly available ts1-ref allele, a knock-out mutant of the LOX8 locus (Acosta et al., 2009), we measured wound-induced JA levels in homozygous WT and mutant seedlings from the F2 segregating family. Figure 3 shows that lox8/ts1-ref mutants (hereafter denoted as lox8) have 66% less JA than WT in response to wounding (P < 0.05). The JA levels between wounded and unwounded lox8 did not significantly differ, indicating that LOX8 provides substrate to the wound-induced JA biosynthesis pathway, but is not responsible for basal JA in unwounded leaves (Figure 3). Unfortunately, Mu insertion mutants in ZmLOX9 are not available, but analysis of mutants in the ZmLOX7 gene showed that LOX7 is not responsible for either basal or wound-induced JA in leaves.
LOX10-derived signaling is required for wound-induced JA via regulation of the JA biosynthesis genes ZmLOX8 and ZmOPR7/8
Because GLVs have been shown to play a strong signaling role for the induction of JA in maize (Engelberth et al., 2004, 2007), we hypothesized that lox10 mutants would have decreased wound-induced JA levels. To test this hypothesis, OPDA and JA levels were measured in the mechanically wounded leaves of the WT, and in lox10-2 and lox10-3 mutants. The accumulation of OPDA, the natural JA precursor, did not differ between mutants and WT at the 0 h time point (P ≥ 0.20), but was significantly higher in the WT compared with the mutants 2 h after wounding (P ≤ 0.01; Figure 4a). Twelve hours after wounding, the OPDA levels in WT plants were still significantly higher than lox10-2 and lox10-3 mutants (P ≤ 0.01). Similar to OPDA, there was no significant difference in the levels of JA for non-wounded (0 h time point) WT and lox10-2 and lox10-3 mutants (P ≥ 0.25; Figure 4b); however, 2 h after wounding, WT levels of JA were approximately 79% higher than lox10-2 and lox10-3 mutants (P ≤ 0.001). By 12 h post-treatment, WT JA levels were still 50% higher than levels in lox10-2 and lox10-3 mutants (P ≤ 0.05). These data strongly suggest that ZmLOX10 is involved in the wound-induced regulation of OPDA and JA biosynthesis, but not in the production of basal levels of these octadecanoids.
To elucidate the potential mechanism of LOX10 involvement in the regulation of wound-induced JA biosynthesis, the transcript accumulation of known JA-producing genes was measured in wounded leaves over a time course spanning 8 h. In addition to ZmLOX8, there are two other previously characterized JA-producing genes in the maize genome: ZmOPR7 and ZmOPR8 (Zhang et al., 2005; Yan et al., 2012a,b). These two genes are highly homologous (>95% nucleotide sequence homology), and are the only maize OPRs that phylogenetically cluster with the Arabidopsis JA-producing OPR3 (Zhang et al., 2005; Schaller et al., 2000; Yan et al., 2012a,b; hereafter denoted as ZmOPR7/8). Northern blot analysis showed that WT seedling transcript levels of ZmLOX8 increased strongly as early as 0.5 h after wounding, and then returned to basal levels by 4 h post-treatment (Figure 4c). Although similar in kinetics, there was a notable decrease in the expression levels of ZmLOX8 in lox10-2 and lox10-3 mutant plants, as compared with the WT. Similar differences between WT and lox10-2 and lox10-3 were seen in ZmOPR7/8 gene expression at 2 h after wounding, the time of maximal transcript accumulation for these genes in WT plants. Combined, these results suggest that LOX10-mediated signaling is required for the normal wound-induced expression of the JA-producing genes ZmLOX8 and ZmOPR7/8, and normal levels of JA biosynthesis in response to mechanical damage.
To determine if LOX10-derived GLVs regulate wound-induced JA biosynthesis, we attempted to complement JA levels in lox10 mutants with GLVs. Because of the rapid volatilization of GLVs and to better mimic the direct release of these compounds from damaged tissue, a substantial quantity of GLVs was applied directly to the wound site immediately after wounding. At 2 h post-treatment, we observed the partial restoration (approximately 50% recovery) of JA in lox10-2 mutant plants (Figure 4d). These data suggest that GLVs may play a role in wound-induced JA biosynthesis, but that signaling from additional LOX10 derivatives may be required.
LOX10 mediates HIPV emissions
To test whether the disruption of LOX10 alters HIPV emissions, WT, lox10-2 and lox10-3 seedlings in the B73 background were infested with third-instar S. exigua larvae, and GLVs, monoterpenes, homoterpenes and sesquiterpenes were collected over a 2-h period. As expected, total GLVs [(3Z)-hexenal, (2E)-hexenal, (3Z)-hexen-1-ol and (3Z)-hexenyl acetate] were essentially absent in lox10-2 and lox10-3 mutants, as compared with the WT plants (P < 0.001; Figure 2b). Emissions of monoterpenes (α-phellandrene, 3-carene, α-terpinene, limonene, ocimene, geranyl acetate, β-pinene and β-myrcene) and sesquiterpenes (copaene, cedrene, caryophyllene, bergamotene, E-β-farnesene and bicyclosesquiphellandrene) in lox10-2 and lox10-3 mutants were about 60% lower than emissions from WT plants (P ≤ 0.03 and P = 0.066, respectively; Figure 5a). Moreover, levels of homoterpenes (4,8-dimethyl-1,3,7-nonatriene and 4,8,12-trimethyl-1,3,7,11-tridecatetraene) were approximately 73% lower in lox10 mutants (P ≤ 0.02). In addition to B73, we ran a 7.5-h time course on WT and mutant plants from the W438 genetic background, which produced greater levels of GLVs during S. exigua feeding (Figure 2b). Here, even greater differences in HIPVs were observed between WT and lox10-2 mutant plants (Figure 5b). As expected, GLVs were essentially absent in lox10-2 mutants at all time points. Monoterpenes and homoterpenes both showed strong induction 2–4 h after infestation, demonstrating significant differences between the WT and mutants (P ≤ 0.0076 and P ≤ 0.001, respectively). Moreover, WT levels for all three classes of volatile terpenes were significantly higher than the mutant at 4–6 h (monoterpenes, P ≤ 0.001; homoterpenes, P ≤ 0.001; sesquiterpenes, P ≤ 0.001; Figure 5b) and 6–7.5 h (monoterpenes, P ≤ 0.001; homoterpenes, P ≤ 0.001; sesquiterpenes, P ≤ 0.001) post-infestation. The results from these two experiments in two genetic backgrounds strongly advocate that ZmLOX10 has an important function in the mediation of HIPV production in response to insect herbivory.
The circadian rhythm regulated ZmLOX10 and HIPV diurnal emissions
While running the 7.5-h time course in the W438 genetic background, we observed an increase in HIPV emissions during midday and then a decrease in emissions late in the afternoon and early evening, which was reminiscent of the daily volatile patterns observed in diurnal emission studies (Turlings et al., 1995; Martin et al., 2003). In our previous work, we showed that expression of ZmLOX10 is regulated by circadian rhythm, with transcript levels peaking in the afternoon at 14:00 h, and decreasing to their lowest levels in the early morning at 02:00 h (Nemchenko et al., 2006; Figure 6). With maize plants dependent on LOX10 for normal HIPV production, we questioned whether the significant reduction of HIPV emissions in lox10 mutants would be sustainable over an extended period of time. To test whether mutant plants could recover and produce diurnal emissions despite the loss of LOX10, we infested WT and lox10-2 mutants with Spodoptera littoralis and monitored HIPV emissions over a time course of 2.5 days. Other than modest increases in WT GLVs, HIPV emissions remained low during the first night. During the second day, levels of HIPV in WT infested plants were increased to high levels by the afternoon (12:30–15:30 h; GLVs, >15 ng h−1; monoterpenes, >80 ng h−1; homoterpenes, >80 ng h−1; sesquiterpenes, >170 ng h−1), and then tapered off to low levels again during the evening (Figure 6). On the third day, levels of GLVs, monoterpenes and homoterpenes again increased to high levels in WT plants during the day; however, only a minor increase was observed in sesquiterpenes. The levels of all four classes of volatiles were reduced to low levels by the evening on the third day. For the lox10-2 mutant infested plants, levels of homoterpenes remained low throughout the time course. Increases in monoterpenes and sesquiterpenes were observed during the second day (<40 and <150 ng h−1, respectively), but remained low throughout the remainder of the time course. There were no GLVs observed throughout the 2.5-day period for lox10-2 infested plants, and both WT and lox10-2 controls remained relatively low for all classes of volatiles measured throughout the time course. Collectively, these results suggest that mutation in ZmLOX10 is sufficient to maintain the disruption of herbivore-induced diurnal emissions over several days.
ZmLOX10 mediates parasitoid attraction to HIPVs
To determine the behavioral significance of LOX10 in parasitoid attraction, we subjected naive C. marginiventris females to volatiles emitted by W438 WT and lox10-2 mutants infested with S. littoralis in a six-arm olfactometer system. Odors emitted from WT plants were significantly more attractive to C. marginiventris compared with those produced by mutants (Figure 7a). Non-infested WT and mutant genotypes were comparable with mutant infested plants in appeal, but more attractive than empty controls. Although plant-emitted volatiles are known to be attractive to parasitoids, the specific maize-derived signal(s) that wasps use to home in on their prey remains unknown (D'Alessandro et al., 2009). To genetically test for the impact that maize GLVs have on wasp attraction, we used only mutant plants in the six-arm olfactometer, allowing wasps to choose between odors emitted from infested and non-infested lox10 mutants. In the absence of WT plants, the infested mutants were significantly more attractive than non-infested mutants and empty controls (Figure 7b). These results suggest that GLVs alone are not responsible for parasitoid attraction. Volatile collections during the olfactometer assay revealed several other HIPVs that may be significant attractants for wasps (Figure 7c). HIPVs that had significantly higher emissions in the infested mutants compared with non-infested mutants include linalool (P = 0.036), 2-phenethyl acetate (P = 0.036), indole (P = 0.036) and β-farnesene (P = 0.016). Collectively the data indicate that the volatile suite responsible for C. marginiventris attraction is made up of a complex blend, in which GLVs play an important role. The results also implicate that infested lox10 mutants are not repellant by nature, but are less attractive because of the absence of GLVs and reduced levels of other volatile attractants.
lox10 mutants are more susceptible to chewing insects under laboratory and field conditions
The orchestrating effect of LOX10 on herbivore-induced defense-related compounds prompted us to measure its biological relevance to insect resistance. We infested near-isogenic WT in the B73 background and lox10-2 and lox10-3 mutant plants with second instar S. exigua and monitored caterpillar performance by measuring weight gain 6 days after feeding. The S. exigua specimens that fed on WT plants gained significantly less weight than those fed on both lox10-2 (P = 0.0265) and lox10-3 (P = 0.0407) mutants (Figure 8a). To measure herbivore damage on the infested plants, we compared the fresh weight of the control non-infested WT and lox10-2 and lox10-3 mutant plants with their counterpart infested plants 6 days after feeding. Although the weight of the WT infested plants was not significantly different from that of the control non-infested plants after S. exigua feeding (Figure 8b; P = 0.5456), both infested lox10-2 and lox10-3 mutant plants weighed significantly less than their non-infested equivalents (P = 0.03 and P = 0.05, respectively), indicating that the S. exigua consumed more plant tissue when feeding on mutant plants than on the WT. The feeding preference of S. exigua on WT and lox10 mutants was measured by placing larvae in pots with WT and lox10-2 plants, or with WT and lox10-3 plants. Six days post-infestation, weights of WT and mutant infested plants were compared with non-infested plants to estimate the weight loss incurred by larval feeding. Figure 8c shows that both lox10-2 and lox10-3 mutant plants decreased in weight over the 6-day feeding period, whereas WT plants showed increased weight gain. To see how lox10 mutants would perform under field conditions, we planted WT and lox10-2 and lox10-3 mutant plants in the B73 background in the field at College Station, TX, USA, in 2010, and WT and lox10-2 mutants in the W438 background in 2011. In 2010, larvae consumed 36 and 39% more leaf tissue on lox10-2 and lox10-3 mutant plants, respectively, than on the WT (Figure 8d). In 2011, the insects consumed 76% more leaf tissue from lox10-2 mutants than from WT plants in the W438 background. These field results implicate a role for LOX10 in both direct and possibly indirect defense responses. Collectively, our laboratory and field results show that ZmLOX10 plays a significant biological role in resistance to herbivory.
Plants have evolved the ability to resist insect attack by inducible direct and indirect defense responses. The characterization of these responses in maize is fragmentary, yet emerging evidence suggests that the major strategy of maize involves diverse lipid oxidation products that serve as defense signals, including those derived from the HPL and AOS branches of the LOX pathway (Farag and Pare, 2002; Engelberth et al., 2004; Ton et al., 2007; Engelberth, 2011). Several studies have described inducible genes associated with these defense signals in an effort to exploit the information to enhance agronomic prosperity. Although these advances in scientific research have shed a much needed light on the potential biological and ecological roles of these genes, genetic studies elucidating the specific genes that generate and regulate these defense signals in the agronomically important crop maize remain unknown. In this study, we provide genetic and biochemical evidence for the individual LOX isoforms that provide substrate for the GLV and JA biosynthesis pathways, and elucidate the significant modulating role that ZmLOX10 plays in direct and indirect defense responses, both under laboratory and field conditions.
One of the principle findings of this study is that LOX10 is the sole isoform, among the 13 individual LOX genes identified in the maize genome, responsible for generating 13S-HPOTE for the GLV biosynthesis pathway. Evidence for this was generated in both wounding- and herbivore-related treatments where lox10-2 and lox10-3 mutants were GLV-deficient. Surprisingly, this occurs in the presence of five other functional 13-LOXs, including ZmLOX11, which is a segmentally duplicated gene that shares 90% identity at the deduced amino acid level with LOX10 (Nemchenko et al., 2006). Nemchenko et al. (2006) showed that ZmLOX10 and ZmLOX11 are differentially expressed under diverse treatments and in an organ-specific manner, which suggests that the biochemical and perhaps physiological functions of these paralogs diverged since their duplication event, possibly as a result of minimal selection pressure on ZmLOX11 in the presence of a functional ZmLOX10. The fact that ZmLOX10 is uniquely responsible for GLV biosynthesis in leaves in the presence of ZmLOX11 and the four other 13-LOXs underscores the functional significance of the differential transcriptional regulation, subcellular localization and tissue-specific spatial organization that exists among different LOX isoforms in planta (Bannenberg et al., 2009).
Differential LOX function arising from distinct localizations is further exemplified by the diverse roles of LOX8 and LOX10. In dicots, the JA and HPL pathways have been shown to compete for the same substrate: 13-hydroperoxide of linolenic acid. Evidence of this can be seen when one pathway is shut down and the other produces more product because of diminished substrate competition (Halitschke and Baldwin, 2003; Halitschke et al., 2004; Chehab et al., 2008). This negative correlation most likely results from the co-localization of JA- and HPL-associated enzymes in dicots, with both being present in the chloroplast (Froehlich et al., 2001; Farmaki et al., 2007). The GLV and JA pathways in maize, however, are physically separated from each other, as evidenced by the localization of JA-producing LOX8 to chloroplasts (Acosta et al., 2009) and GLV-producing LOX10 to organelles distinct from chloroplasts (Figure 2c). The physical separation of these two LOXs in distinct organelles prevents competition for the JA and GLV pathway substrate, suggesting that any interaction between these two LOX pathway branches would be the result of signaling crosstalk. Decreased wound-induced transcript levels of the JA-producing genes ZmLOX8 and ZmOPR7/8 (Figure 4c) in lox10 mutants, plus the partial restoration of wound-induced JA in lox10 mutants with GLVs (Figure 4d), indicates that wound-induced JA production is mediated by oxylipin signals derived from LOX10. To date, the intermediates and final products of the JA and GLV LOX branch pathways have never been reported to engage in signaling crosstalk. This is rather surprising given the well-known potency of these signals, their induction by the same stresses and their proximity in cell. This study presents evidence that in maize, the products of LOX10 have a dramatic impact on wound-induced JA biosynthesis.
Another important discovery of this study was the finding that lox10 mutants are reduced in HIPV production. Mono-, homo- and sesquiterpene levels were reduced in lox10 mutants during herbivore feeding assays run in both the B73 and W438 genetic backgrounds. During an extended time course we also tested for a possible function of LOX10 in diurnal emissions. Our monitoring of HIPV emissions from infested WT and lox10 mutant plants over a 2.5-day period showed that clock-regulated expression of ZmLOX10 (Nemchenko et al., 2006) may be a mechanistic link for circadian GLV emissions, and explains, at least in part, the well-characterized HIPV diurnal emissions. Our study genetically shows that GLVs may contribute to the induction of HIPVs, supporting previous results obtained using exogenous GLVs (Farag and Pare, 2002; Engelberth et al., 2004, 2007). Other reports have shown the significance of JA in mediating HIPV emissions (Dicke et al., 1999; Koch et al., 1999; Bruinsma et al., 2009), and a recent study showed that JA production follows a diurnal pattern during herbivory (Goodspeed et al., 2012). Whether or not GLVs have a direct impact on HIPV production in maize or if HIPVs are solely JA-regulated needs further investigation, using appropriate mutants and/or double mutants in the respective pathways.
As LOX10 is required for GLV, JA and HIPV production, it is not surprising that lox10 mutants were more susceptible to insect feeding. Under laboratory conditions, the increase in mutant plant consumption paralleled the increase in larval weight gain, and the mutants were significantly preferred over the WT by S. exigua, indicating a role for LOX10 in direct defense. This preference for, and lack of resistance in, lox10 mutants was also observed under field conditions, which may have been a result of both direct and indirect defense responses. In parasitoid attraction assays, WT plants had a significantly greater appeal to C. marginiventris than lox10-2 mutants, genetically confirming the importance of HIPVs for wasp attraction in WT maize plants. Further tests using GLV-deficient lox10 mutants (Figure 9) showed that HIPVs other than GLVs are important for parasitoid attraction in maize. Indeed, attraction appears to be the result of a complex volatile blend, in which GLVs play an important role. Collectively, our parasitoid data suggest that LOX10 has a strong ecological function in parasitic wasp behavior.
Based on our finding that LOX10 regulates, at least in part, JA- and HIPV-mediated defense responses for resistance to insect attack, we propose a model that describes this interaction (Figure 9). It is proposed that α-linolenic acid is catalyzed by LOX10 to produce 13S-HPOTE, which is used in the HPL branch of the LOX pathway for GLV production. Upon wounding or herbivory LOX10 derivatives are produced in damaged tissue, inducing the transcription of ZmLOX8 and ZmOPR7/8 for increased levels of JA production. Because both exogenous GLVs and JA have been proposed to induce HIPV emissions (Farag and Pare, 2002), it is further suggested that GLVs may, along with JA, contribute to the release of HIPVs for indirect defense. The collective induction of these important defensive compounds results in heightened wasp attraction and increased resistance to herbivory.
In summary, although maize is an important agro-economical crop with broad applications in food, chemical, livestock and biofuel industries, little is understood in terms of defensive gene function because of the difficulty in generating knock-out mutants and the resulting lack of genetic studies. In this report, we show that LOX10 provides substrate to the GLV biosynthesis pathway, and is localized to organelles distinct from chloroplasts. Furthermore, we demonstrate that chloroplast-localized LOX8 is responsible for wound-induced JA, yet LOX8-mediated JA production is dependent on signaling from LOX10-derived oxylipins. The functional role that LOX10 plays in direct defense responses is evident from the susceptibility levels of lox10 mutants to insect feeding during the described biological assays in this study. Additionally, LOX10 plays a key role in indirect defense, as evidenced by the reduced levels of HIPV in lox10 mutants and the resulting diminished attraction of C. marginiventris. These findings denote ZmLOX10 as an important herbivore defense-related gene that plays a central role in herbivore-induced defense mechanisms in maize. Knowledge gained from the characterization of ZmLOX10 in plant defense against herbivory will contribute to improving our knowledge about the evolution of plant–insect interactions, and may help to develop marker-assisted selection strategies in breeding for insect resistance.
Plant materials and subcellular localization
The reverse genetics resource (Trait Utility System for Corn, TUSC) at Pioneer Hi-bred Inc. (http://www.pioneer.com) was used to generate mutant alleles as described by Gao et al. (2007), using a Mutator (Mu)-specific primer and ZmLOX10 gene-specific primers (see Appendix S1). Three Mu insertional alleles were detected for the ZmLOX10 locus (lox10-1, lox10-2 and lox10-3), and the flanking regions of each insertion site were cloned with a pCR2.1 TOPO vector (Invitrogen, http://www.invitrogen.com) and sequenced to determine the precise location of the insertion sites. The Mu insertion site for lox10-1 was positioned 342 bp inside the first intron, and the lox10-2 and lox10-3 insertion sites were positioned 253 and 278 bp inside the third exon, respectively. Original mutants were backcrossed into the B73 and W438 genetic backgrounds, and were genetically advanced to eliminate unwanted Mu insertions throughout the genome and to create near-isogenic mutant and WT lines suitable for functional analysis (BC3F4–BC5F4, as designated below). The lox8 mutant was acquired from the Maize Genetics Cooperation Stock Center at The University of Illinois at Urbana-Champaign (Maize COOP, http://maizecoop.cropsci.uiuc.edu) as a segregating 1:1 heterozygous:mutant population. Heterozygous individuals were selfed and a subsequent 1:2:1 segregating population was genotyped with gene-specific primers (see Appendix S1). For the ZmLOX10pro:ZmLOX10-YFP transgenic line, the construct was generated using the MultiSite Gateway® Three-Fragment Vector Construction Kit (Invitrogen). The complete coding sequence of ZmLox10 plus 2.609 kb of upstream and 1.589 kb of downstream genomic sequence were recombined into the maize pTF101.1 binary vector, tagging ZmLox10 with YFP citrine at the C terminus. Agrobacterium-mediated transformation of maize HiII was performed by the Iowa State University Plant Transformation Facility. T0s were crossed to the inbred line B73 and T1s were imaged live using a Zeiss 510 confocal laser-scanning microscope (Zeiss, http://corporate.zeiss.com). Construct sequences and image metadata are available at http://maize.jcvi.org/cellgenomics/index.php.
A maximum-likelihood tree was built by aligning protein sequence from maize 13-LOXs (ZmLOX7, ZmLOX8, ZmLOX9, ZmLOX10, ZmLOX11 and ZmLOX13) and other GLV- or JA-relevant 13-LOXs in Solanum lycopersicum (tomato; TomLOXC and TomLOXD), Solanum tuberosum (potato; StLOXH-1 and StLOXH-3) and Nicotiana tabacum (tobacco; NaLOX3) using ClustalW (Guindon and Gascuel, 2003). Phyologeny was reconstructed using the LG substitution model implemented in phylm 3.0.
Quantification of GLVs and HIPV
Two grams of leaf cuttings from V2-stage WT and lox10-2 and lox10-3 maize plants were placed into glass cylinders connected to a Super Q filter trap (Alltech Associates, http://www.alltech.com/), and methods followed those described by Engelberth et al. (2004). For HIPV emission collections, S. exigua were reared on a pinto-based artificial diet (Southland Products Inc., http://www.tecinfo.com/~southland/) at 25°C to the third instar developmental stage. The night before volatile collection, S. exigua were fed in individual diet cups on corn leaf cuttings. Experimental treatments (consisting of infested and non-infested WT and lox10 mutant plants) and sample extractions were carried out in the J. Tumlinson Laboratory at Penn State University, as previously described by Cardoza and Tumlinson (2006).
Oxylipin profiling of wounded and GLV-treated plants
Both WT (BC5F2) and mutant allele lox10-2 and lox10-3 (BC4F2 and BC5F2, respectively) maize seedlings were grown at 25–28°C in commercial soil (Metro-Mix 366; Scotts-Sierra Horticultural Products, http://www.everris.com) under a 12-h photoperiod (120 μmol m−2 sec−1; Quantum Meter; Apogee Instruments, http://www.apogeeinstruments.com). V2 seedlings were placed in the dark for a period consisting of two consecutive nights to circumvent the circadian rhythm-regulated gene expression of ZmLOX10 (Nemchenko et al., 2006). Under green light, seedlings were wounded seven times in the mid portion of the second leaf (with three wound sites on one side of the midvein and four on the other side), using a hemostat (wound sites approximately 1–2 cm apart). The second leaves from WT and lox10-2 and lox10-3 seedlings were harvested in liquid N2 at 0, 2 and 12 h, and subsequently analyzed for free oxylipins (JA and OPDA) as outlined in the methods described on the University of Göttingen oxylipin database website (http://www.oxylipins.uni-goettingen.de), with minor modifications. For GLV complementation and ZmLOX8 analysis, plants were treated and, JA was measured by LC/MS in negative phase mode using the methods outlined by Pan et al. (2008), with minor modifications (Appendix S2).
Northern blot analysis
For northern blots, total RNA was extracted using the standard TRI reagent protocol (Molecular Research Center Inc., http://www.mrcgene.com). Following extraction, RNA (12 μg) was separated by a 1.5% formaldehyde/1X 3-(N-morpholino)propanesulfonic acid (MOPS) gel and transferred to a MagnaGraph nylon membrane (Micron Separations Inc., Westboro, MA, USA) in 10X SSC (1500 mm sodium chloride, 150 mm sodium citrate) overnight. Blots were pre-hybridized with UltraHyb hybridization solution (Ambion, now Invitrogen, http://www.invitrogen.com) and probed overnight with a 32P-labeled ZmLOX10, ZmLOX8 or ZmOPR7/8 gene-specific probe, as indicated below. Blots were washed twice for 5 min (or twice for 15 min for ZmLOX8) with 2 × SSC and 0.1% sodium dodecyl sulfate (SDS) solution (an additional 2 × 5-min or 3 × 15-min wash with 0.1× SSC and 0.1% SDS was performed for the blots hybridized to ZmLOX8 gene-specific and ZmOPR7/8 probes, respectively) and exposed to BioMax X-ray film (Kodak, http://www.kodak.com) at −80°C for 1–6 days prior to developing the films. rRNA loading controls were visualized with ethidium bromide staining and UV transillumination. For gene expression of ZmLOX8 and ZmOPR7/8 in both WT (BC4F2) and lox10-2 and lox10-3 (BC4F2 and BC3F7, respectively) mutant backgrounds, seedlings were grown at 25–29°C in commercial soil (SB300 Universal Mix; SunGro Horticulture, http://www.sungro.com) under a 12-h photoperiod. V2-stage plants were wounded using the treatment described above and immediately harvested in liquid N2 at 0, 0.5, 1, 2, 4 and 8 h for northern blot analysis.
Biological assays and field trials
Preference and performance feeding assays were carried out with WT and lox10-2 and lox10-3 seedlings in the B73 genetic background. Five replicates of seedlings planted in 5-inch pots, were randomly arranged to have treated and untreated lines consisting of individual WT, lox10-2 or lox10-3 seedlings (performance test), or all three genotypes together (preference test), in each replicate. When seedlings reached the V3 stage, the three plants were loosely put together to form a trio. A cage sleeve was then carefully placed over the pot/plants and an individual second instar S. exigua larva was weighed and placed in the middle of the trio before the cage was fastened at the top. At 6 days post-infestation S. exigua larvae were removed and weighed to determine total weight gain, and seedlings were cut at the soil level and the fresh weight was taken. For field studies, between two and four WT and mutant rows (7.62 m per row) were planted (with 25 seeds per row) in test plots and exposed to natural infestation. Leaf-chewing herbivores identified in the College Station field include: fall armyworm, sugarcane corn borer, European corn borer and south-western corn borer. The percentage damage was estimated by a scoring system ranging from 0 to 6: 0, no damage; 1, approximately 7%; 2, approximately 14%; 3, approximately 21%; 4, approximately 28%; 5, approximately 35%; 6, approximately 42% damaged area.
Wasp attraction and circadian rhythm assays
The rearing of the caterpillar S. littoralis and the endoparasitoid C. marginiventris was carried out as described by Turlings et al. (2004). Plants for wasp attraction and circadian rhythm assays were grown according to the methods described by Erb et al. (2010), and olfactometer bioassays and odor collections were performed as previously described by Turlings et al. (2004). For circadian rhythm, volatiles were collected for 3-h periods over a time course of 2.5 days at 19:30, 01:30, 07:30 and 13:30 h under continuous feeding with 10 sec instar capterpillars of S. littoralis or no treatment (control) (n = 6).
We would like to thank Dr Eric Schmelz for his helpful suggestions for this article. We would also like to thank Pia Meyer and Sabine Freitag for technical assistance with oxylipin profiling. Dr Charles Greenwald, Quinten Cazares, Sarah Kieth, Travis Gordon and Gretta Sharp are acknowledged for their help with molecular and phylogenetic analyses. This work was supported by the Texas Agricultural Experiment Station Cropping Systems Program and NSF grants IOB-0544428, IOS-0951272, IOS-0925561 and USDA National Institute of Food and Agriculture to Dr Michael Kolomiets, and by NSF grant IOS 0925615 to Dr Jurgen Engelberth.