JAZ Transcript Levels were Temporally Induced in Maize by FAW Infestation. To explore the potential role of ZmJAZ in the JA-regulated defense response, we first investigated the expression profile of JAZ genes in response to FAW feeding. To establish the timing of the feeding response, gene expression was monitored up to 12 hr after FAW infestation in both Mp708 and Tx601. The early time points (10 and 30 min) were selected because as a primary defense regulator, JAZ genes are known for their rapid induction following mechanical wounding and herbivore feeding in Arabidopsis (Chung et al. 2008; Koo et al. 2009). Figure 1a shows that all JAZ transcripts accumulated in leaves in response to FAW feeding, but their individual expression patterns were different. Overall, ZmJAZ1 transcripts accumulated to a much greater level than ZmJAZ2 or ZmJAZ3 (see y-axis scales in Fig. 1a). In Mp708, ZmJAZ1 transcripts started to accumulate as early as 30 min after infestation. After a slight dip at 1 hr, ZmJAZ1 transcript levels increased again at 6 hr and remained high at 12 hr. Although ZmJAZ1 expression was upregulated in Tx601 upon feeding, there was no peak expression at 30 min and there was no dramatic change throughout the remainder of the time course. Expression of ZmJAZ1 was significantly higher in Mp708 than Tx601 at 30 min and 6 and 12 hr. Unlike ZmJAZ1, ZmJAZ2 transcripts did not have the early 30 min peak, but gradually increased to peak at 1 hr in Mp708 and 6 hr in Tx601. However, there were no significant differences in ZmJAZ2 expression between the two genotypes and its overall expression levels were the lowest of the three JAZ genes tested. ZmJAZ3 transcripts had similar temporal profiling as ZmJAZ2 and peak induction was seen at 1 hr for both Mp708 and Tx601, except the expression was significantly higher at 30 min in Mp708. We also tested the JAZ gene expression in another caterpillar susceptible maize inbred line B73 in response to FAW feeding (Supplemental Fig. 1a-c). Similar results were observed as in Tx601, except the expression levels continued to increase at 12 hr. Taken together, these results confirmed that FAW feeding could induce the expression of ZmJAZ1-3, and expression of ZmJAZ1 was regulated differently in Mp708 than in Tx601.
Faw Feeding can Increase Endogenous JA Levels in Maize Leaves. To test the effect of feeding on JA accumulation, time courses of JA production from leaves of Mp708 and Tx601 in response to FAW infestation were measured, and the amounts of active (cis-JA), inactive (trans-JA), and total JA were determined (Fig. 2). In both inbreds, cis-JA (Fig. 2a) levels rapidly accumulated within 10 min of FAW feeding, and Mp708 had a significantly greater induction level than Tx601; cis-JA levels increased to approximately 648 and 374 ng/g fresh weight in Mp708 and Tx601, respectively. Cis-JA levels kept increasing until a peak was reached at 1 hr, and they dropped significantly at 6 hr. Following herbivory, although cis-JA levels were higher in Mp708 than Tx601, they were only statistically different after 10 min of FAW infestation. Trans-JA levels were significantly higher in Mp708 at the resting stage but tended to be higher in Tx601 after feeding (Fig. 2b). Interestingly, the ratio of cis/trans-JA varied in maize inbreds as well. In undamaged control plant, ~50% of the JA from Mp708 leaves was in trans-conformation, but within 10 min after insect damage, ~80% of the JA was found in the cis-conformation, compared with ~40% and ~60% from Tx601 leaves, respectively. When combined, no dramatic differences in total JA content were seen between the two genotypes, except constitutively (0 min) and 10 min after feeding, where total JA levels were significantly higher in Mp708 (Fig. 2c). In the un-fed control, the constitutive levels of cis- and trans-JA in undamaged Mp708 leaves (controls) were approximately 132 and 124 ng/g fresh weight, respectively. As expected, these values were 2 to 4-fold higher than those in Tx601, which were approximately 54 and 31 ng/g fresh weight, respectively. Our results supported the finding of Shivaji et al. (2010) reporting significantly higher concentrations of both cis- and trans-JA in undamaged Mp708 leaves. In combination with the qRT-PCR data, the results show ZmJAZ expression levels correlate with JA accumulation at the feeding sites. In addition, the ability of Mp708 to rapidly accumulate cis-JA as soon as 10 min after infestation may help to account for its increased resistance because it can modulate defenses more quickly.
JA Biosynthesis Genes were Upregulated in Maize by FAW Infestation. As stated previously, elevated JA levels were observed between 10 and 30 min near the caterpillar feeding sites, and remained high at least for an hour. Next, we compared changes in the transcript levels of key genes potentially involved in the JA biosynthesis pathway. ZmLOX1 (AF271894), ZmAOS (AY488135), and ZmOPR2 (AY921639) were selected because they are the closest homologs in maize compared with the JA biosynthesis pathway in rice and Arabidopsis (Ankala et al. 2013; Zhang et al. 2005). As shown in Figure 3a, ZmLOX1, ZmAOS, and ZmOPR2 were up-regulated by herbivore feeding. For ZmLOX1, known for its up-regulation by wounding and MeJA (Kim et al. 2003), there was a gradual increase in transcript levels up to 12 hr and there were no significant differences in expression between Mp708 and Tx601. ZmAOS is considered a rate-limiting step in JA biosynthesis (Wasternack 2007), and its transcripts increased throughout the feeding periods, and only were significantly higher in Mp708 than Tx601 at 30 min (Fig. 3a). Previous groups have reported that constitutively higher levels of OPDA and JA were observed in Mp708 than Tx601 (Shivaji et al. 2010; Varsani et al. 2019), surprisingly, ZmOPR2 expression tended to be higher in Tx601 than in Mp708 at all times tested (Fig. 3 and 5). Although ZmOPR2 expression responded to feeding, it might not be a major contributor to JA biosynthesis upon FAW feeding in Mp708 (Borrego and Kolomiets 2016; Pingault et al. 2021; Zhang et al. 2005) and it was not responsive to MeJA treatment (Fig. 3b). Again, gene expression was also measured in B73 (Supplemental Fig. 1g-i) and the trends were similar to those in Tx601. Our results agreed with previous reports showing that genes in JA-biosynthesis pathways were under positive feedback regulation upon herbivore feeding (Wasternack and Song 2016).
Identification of COI1 and MYC2 Homologs in the Maize Genome. COI1, an F-box protein, serves as a receptor for JA signaling by binding directly to bioactive JA-Ile and is critical for all JA-mediated responses (Yan et al. 2009). In Arabidopsis, expression of primary responsive genes (i.e. JAZs) is induced in a COI1-dependent manner by wounding or feeding (Chung et al. 2008). There is only one AtCOI1 gene in the Arabidopsis genome (Devoto et al. 2002; Xie et al. 1998), but three closely related homologs are present and expressed in the rice genome (Hu et al. 2006; Lee et al. 2013; Ye et al. 2012). Two out of three COI1 genes-OsCOI1a and OsCOI1b-could be the result of a duplication event, due to high sequence identity. Arabidopsis point mutation coi1-1 does not respond to JA-Ile, while either OsCOI1a or OsCOI1b can complement this mutation and restore JA signal transduction (Lee et al. 2013; Lee et al. 2015). After a BLAST search using homologous rice sequences, a total of five putative ZmCOI genes were identified in the maize genome, and these homologs were named based on synteny information (Supplemental Table 1). However, in this study one sequence (GRMZM2G035314) was manually deleted due to lack of critical F-box sequence. ZmCOI1b-1 and ZmCOI1b-2 were possibly the products of a recent duplication event in maize because they have located in the maize syntenic region and shared a 93% amino acid sequence identity.
Phylogenetic analysis was further performed using the deduced amino acid sequences from maize, rice, and Arabidopsis (Supplemental Fig. 2b). Based on the sequence alignment, these plant COI1 homologs shared high sequence conservation at the amino acid level and the characteristic features of F-Box motif and leucine-rich repeats (LRR) were observed in all COI1s (Xie et al. 1998). The essential amino acid residues required for COI1-interaction were conserved across all COI1s (Lee et al. 2013), as marked in Supplemental Figure 2a. It showed that the COI1 sequences from rice were more closely related to maize rather than Arabidopsis (Supplemental Fig. 2b). For example, for each syntenic group (COI1 and 2), the ZmCOI1 shared an approximate 84% amino acid sequence identity with OsCOI1, but only approximately 56% identity with AtCOI1. Results from an in silico study showed that, among four ZmCOI1 genes, only ZmCOI1a had a predicated nuclear localization. For these reasons, ZmCOI1a was chosen for expression analyses.
MYC2 is from the family of the basic helix-loop-helix (bHLH) transcription factors (TF), which is a direct target for JAZ proteins during JA-induced gene expression (Cheng et al. 2011). In Arabidopsis, AtMYC2 is well characterized as the master regulator in JA repression and point of crosstalk with other signaling pathways (Kazan and Manners 2013; Wang et al. 2015). In addition to the active nuclear localization signal, MYC2 TF contains three important domains: an N-terminal transcriptional activation domain (TAD) and adjacent JAZ interaction domain (JID) which required for JAZ protein binding, and a C-terminal conserved plant bHLH domain (Cheng et al. 2011; Fernandez-Calvo et al. 2011). Earlier research shows that one maize putative TF from the bHLH family named MYC7E shares high sequence similarity with Arabidopsis MYC2 (Abe et al. 2003; de Pater et al. 1997; Loulergue et al. 1998), and it was induced upon wounding and JA-Ile treatment in maize leave (Engelberth et al. 2012; Fu et al. 2020). A rice homologous sequence (bHLH137) from the bHLH family was also detected and tested (Toda et al. 2013; Zhu et al. 2005). These MYC2 homologs all show high sequence similarity and have the same gene structure as AtMYC2 with only one exon, unlike other rice homologs (bHLH148) that have three exons (Li et al. 2006; Seo et al. 2011). This finding was confirmed in another study by Wang et al. (2015). In this study, we found an additional homologous sequence (GRMZM2G001930) using the previously identified MYC7E sequence, and paralogous information was obtained from the maize genome database (http://www.maizesequence.org) (Monaco et al. 2014). Supplemental Table 2 listed the candidate maize MYC2 homologous, and both genes (ZmMYC2a and 2b) had predicated nuclear localization. The two ZmMYC2 homologs shared over 90% sequence identity at amino acid level, and over 80% and 50% identity to OsMYC2 and AtMYC2, respectively. The phylogenetic data (Supplemental Fig. 3) showed high conservation for ZmCOI1 and ZmMYC2 genes at the nucleotide level and suggested the potential role of ZmMYC2 genes in JA-related plant responses and therefore, they were selected for expression analyses.
Gene Expression of ZmCOI1 and ZmMYC2 was Responsive to FAW Infestation in Maize Leaves. Since there is limited expression data about ZmCOI1 and ZmMYC2, and no report of feeding data in maize to date, to better understand the regulation of these signaling pathway genes, we measured their expression levels in response to caterpillar feeding. Transcript levels of ZmCOI1a (GRMZM2G125411), ZmMYC2a (GRMZM2G001930), and ZmMYC2b (GRMZM2G049229) were tested. These genes were chosen since they were the most closely related maize homologs to those in rice and Arabidopsis (Lee et al. 2013; Loulergue et al. 1998) and had a predicted nuclear localizationResults from feeding-induced expression (Fig. 4a) revealed that ZmCOI1a transcripts were up-regulated as soon as 10 min (2.9-fold change) after FAW infestation in Mp708 compared to the undamaged control. After this transient peak, the transcript levels decreased dramatically and then increased slightly in Mp708 at 6 hr and 12 hr. But other than 10 mins, there were no significant differences in ZmCOI1a transcript levels between Mp708 and Tx601 during the remainder of the time course. ZmMYC2 expression also was induced by FAW feeding and similarly, transcripts of ZmMYC2a and ZmMYC2b both accumulated within 10 min of feeding, and gradually increased over time until they began to diminish at 12 hr. Similar trends but with weaker induction levels were observed in Tx601, and there were no significant differences in the expression of these two genes between Tx601 and Mp708. The expression levels of ZmCOI1a and ZmMYC2 were analyzed in B73 as well (Supplemental Fig. 1d-f) and were generally similar to those of Tx601. In summary, the data show that ZmCOI1a and ZmMYC2a/b exhibited an inducible expression pattern in response to herbivore feeding at very early stage (10 min), which indicated their potential involvement in JA-related defense responses in maize.
Effects of JA, Wounding, and Ethylene (ET) Treatments on JA-Related Gene Expression in Maize. To further investigate if exogenous hormone application could induce JA-related defense gene expression, plants were first sprayed with MeJA, and leaf samples were collected from both inbreds at the same time intervals used in the feeding experiments. MeJA was used since it can penetrate the cell membranes easily, and also can be transmitted by airborne diffusion due to its volatility (Farmer and Ryan 1990). After MeJA treatments, qRT-PCR data shown an inducible expression pattern of all selected genes tested in this study. These results are shown in panel B of Figures 1, 3, and 4. In general, gene expression responded more slowly in MeJA-treated Mp708 and Tx601 compared with FAW-fed plants. For example, JAZ transcript level increased dramatically at 6 hr after MeJA treatment and remained relatively constant at 12 hr (Fig. 1b). This is in contrast to FAW feeding where JAZ transcript accumulation occurred as early as 30 min. The only time point showing significant differences between Mp708 and Tx601 was 12 hr after MeJA treatment when the accumulation of all three ZmJAZ remained higher in Mp708 than Tx601. Similar results were seen for JA biosynthesis gene (ZmLOX1) and JA signaling (ZmCOI1a and ZmMYC2a/b) genes; transcript levels of these marker genes were induced by MeJA, but induction time and intensity differed when compared with FAW feeding (Fig. 3b and Fig. 4b). However, in Tx601 MeJA-induced ZmAOS expression was similar to feeding-induced levels, whereas in Mp708 ZmAOS transcript levels appeared to have a typical MeJA-induced pattern: a delayed peak of induction but greater fold-change at later time points. Transcript levels of ZmOPR2 were also differently regulated as previously mentioned (Louis et al. 2015; Zhang et al. 2005) and it was not responsive to MeJA treatment in Mp708, while there was only a weak expression in Tx601 (see y-axis scales in Fig. 3a and 3b).
Since MeJA treatment alone could not effectively mimic the same induction response as FAW infestation, we then examined the effect of mechanical wounding combined with MeJA application. The expression level of JA-related genes was examined first in mechanically wounded Mp708 and Tx601 plants without applying exogenous MeJA. Analysis 30 min after wounding showed no differences in expression relative to the controls (data not shown), so two additional time points (1 hr and 6 hr) were then tested. Since only subtle changes were seen at 1 hr post wounding (data not shown), qRT-PCR results for the two maize genotypes at 6 hr post wounding were presented in Figure 5. The results indicated that selected gene expression levels for JA signaling and biosynthesis pathways increased at 6 hr after wounding similar to feeding for 6 hr, except for ZmJAZ1 and ZmMYC2a/b. For these genes, wounding alone would not effectively induce transcript accumulation to the same level as feeding, and our results differ from the previous founding in Arabidopsis (Fu et al. 2020). When the treatments of wounding and JA application were combined, the transcript levels of all tested genes dramatically increased at 6 hr compared to wounding only (Fig. 5). However, the induction of ZmCOI1a transcripts was largely due to wounding instead of MeJA, since there was no difference at expression level between wounding with MeJA and wounding with buffer treatments (Fig. 5d).
In addition to JA, ethylene (ET) is also associated with defense against pests and pathogens (Glazebrook 2005; Trujillo and Shirasu 2010). JA and ET have been reported to have either synergistic (Ankala et al. 2009; Harfouche et al. 2006) or antagonizing (Rojo et al. 1999; Tian et al. 2014) roles in plant response regulations, and ET-related genes were also regulated by MYC2 in Arabidopsis. Louis group (2015) found Mp708 plants use ET instead of JA to regulate mir1 expression in response to corn leaf aphid. However, the regulatory mechanisms for crosstalk between JA and ET in herbivore resistance remain inconclusive (Onkokesung et al. 2010). Since EAR (ethylene-response factor amphiphilic repression) motif was found in maize JAZ genes (Han and Luthe 2021), considering the possible involvement of JAZ proteins in hormone crosstalk, we also examined the effect of exogenous ET on gene expression. Mechanically wounded maize leaves were treated with either Ethephon, an ethylene generating compound (Ankala et al. 2009), or a combination of MeJA and Ethephon, and samples were harvested and analyzed at 6 hr (Fig. 5) after treatments. In the ET and wounding combined treatments, expression levels of tested genes were either equivalent to (ZmJAZ2, ZmMYC2, ZmLOX1, ZmAOS, and ZmOPR2) or less than (ZmJAZ1) the undamaged controls, and only two genes-ZmJAZ3 and ZmCOI1a-were positively induced. When JA, ET, and wounding treatments were combined the results differed, JA not only rescued the transcription activation that was reduced by ET, in some cases (e.g. ZmCOl1, ZmLOX1, and ZmAOS), ET appeared to enhance the JA response. The data suggested that ET alone was not as effective as JA in enhancing defense gene expression in maize, but it did enhance the JA regulated defense responses, which is unlike the antagonistic role of ET in Arabidopsis (Wasternack and Song 2016) and tomato (Tian et al. 2014). The effects of mechanical wounding and hormone treatment were different between Mp708 and Tx601. In general, gene expression levels were significantly higher for wounding alone or wounding with hormone treatments in Mp708 than Tx601, except for ZmOPR2 (Fig. 5i). Collectively, the results indicate that JA is critical for the up-regulation of maize defense responses, and ET could facilitate the induction of the JA-related gene expression.