Defense of the Aristolochia contorta against its specific herbivore

In general, polyphenol oxidase (PPO) and proteinase inhibitors (PIs) are known as the herbivore resistance components (Constabel et al., 2000; Gatehouse, 2002). Homologs of polyphenol oxidase AcPPO (c64496_g1_i4) was upregulated under W+OS treatment rather than the W+DW treatment (Fig. 4-11f). Induced expression level of polyphenol oxidase differs among the herbivore species (Bosch et al., 2014). In tomato, specialist herbivore could suppress the expression of PPO (Bosch et al., 2014). Expression levels of some proteinase inhibitors were also upregulated under W+OS treatment rather than the W+DW treatment (Table 4-8). In addition, other genes which are involved in general response such as chitinase, MYB transcription factors, and jasmonate O-methyltransferase were up-regulated under W+OS treatments.

Tyrosine-derived alkaloids are commonly known to be synthesized in limited clades (Khan et al., 2013). The biosynthetic pathway and the intermediate compounds have been assessed for pharmacological use, while the enzymes which involves in the biosynthetic pathway are not clear (Attaluri et al., 2014). On the other hand, highly induced gene expression of AcNCS (S-norcoclaurine synthase) could be followed by isoquinoline alkaloids biosynthesis such as magnoflorine (Fig. 4-8;

Canedo-Téxon et al., 2019). Therefore, some tyrosine-derived isoquinoline alkaloids

seemed to be induced under herbivory. In case of A. thaliana, some glucosinolate biosynthesis-related gene expressions were induced by specific herbivory of P. rapae, which are classified as family Brassicaceae specific-secondary metabolite group (Reymond et al., 2004).

However, most of the specific defense mechanisms are known to role as

“constitutive” defense mechanism (Gatehouse, 2002). Regardless of its toxicity, which secondary metabolites are “inducible” or “constitutive” seemed to be different from each final product. Aristolochic acid, which is known as the specific compound in the family Aristolochiaceae, is classified as the member of isoquinoline alkaloids or aporphine alkaloids (Shamma and Guinaudeau, 1985). Specific secondary metabolite aristolochic acid in Asarum species is cumulated constitutively both in roots and leaves (Wang et al., 2018). In similar, aristolochic acids are cumulated at diverse tissues in Aristolochia species (Heinrich et al., 2009). Previous study revealed that the expression level of tyrosine decarboxylases (TyrDCs) is correlated to the aristolochic acid contents, whereas the other genes which are involved in the aristolochic acid biosynthesis are unclear (Wang et al., 2018). From the expression levels of TyrDCs under simulated herbivory, biosynthesis of the aristolochic acids seemed to be “constitutive” process rather than “induced” response in the present study (Fig. 4-11). In the other study, quantification of some tyrosine-derived alkaloids revealed that some specific secondary metabolites including aristolochic acids were not significantly induced by wounding with specific herbivore elicitor in A. controta (Fig. 4-12; Park, 2020). This results are similar to the previous study on the Nicotiana species wounded with the caterpillar regurgitants, which showed that the herbivore elicitor did not induce the biosynthesis of specific alkaloid nicotine (McCloud and Baldwin, 1997). Although aristolochic acids are fatal to the generalist

herbivores, some Aristolochia-eating specialist herbivore has detoxification enzyme activity (Nishida, 1994). Thus, the role of aristolochic acids might be “constitutive”

defense against the generalist herbivore rather than “induced” defense response.

Fig. 4-12. Secondary metabolite content in control (undamaged) and induced (wounded and rubbed with the oral secretion of S. montela and sampled after 48 hours) in greenhouse experiment. All metabolites did not show the statistical significance in t-test (p ≥ 0.05) (Park, 2021).

In summary, defense response of A. contorta to specialist herbivore could be inferred from the transcriptome under artificial herbivory treatment. Major responses are similar to the other eudicot species. For example, signaling pathway by ROS, MeJA, and ethylene seemed to be induced. Also, further gene expression was expected to be regulated by the induction of transcription factor MYBs and WRKYs. There were various genes which are involved in the affect the insect metabolism such as chitinase, PIs, and some xenobiotic compound detoxifying genes.

Lignin biosynthesis also seemed to be induced from the expression of some phenylpropanoid biosynthesis-related genes.

Biosynthesis of some distinctive secondary metabolites seemed to be induced by herbivory. While family Aristolochiaceae-specific secondary metabolites such as aristolochic acid seemed not to be induced by herbivory, some alkaloid compounds such as reticuline (from AcCYP80B2 and AcNCSs) seemed to be synthesized after herbivory, which could be the precursor of morphine or magnoflorine (Canedo-Téxon et al., 2019). Biosynthesis of these inducible secondary metabolites could induce the prevention of generalist herbivore, rather than specialist herbivore. Simulated herbivory seemed to be able to induce the biosynthesis of phenolic compounds such as the bitter-taste by flavonoid biosynthetic pathway and the other phenylpropanoids metabolism. Also, volatile sesqeuiterpene compounds such as geraniol-derived (e.g.

loganin; Battersby et al., 1970) and nerolidol-derived (from AcNES; Schnee et al., 2002) compounds could be involved in the induction the tri-trophic interaction. For assess the defensive effect of induced secondary metabolites, validation by the secondary metabolite biosynthesis which are predicted to be induced by transcriptome analysis should be conducted.

4.5. Conclusion

Under the simulate herbivory of specialist herbivore S. montela, elicitor seemed to induce gene expression of defense response and secondary metabolite biosynthesis of A. contorta leaves rather than the simple wounding (Fig. 4-13). This results suggest the defense mechanism against specialist herbivore of basal angiosperms was mostly similar to the previous studied eudicots. Systemic response against herbivory also occurred partially compared to the local damaged leaf.

Biosynthesis of some volatile compounds and alkaloids were predicted to be induced under herbivory, which could prevent the generalist herbivores. However, specific secondary metabolite biosynthesis of Aristolochia such as aristolochic acids seemed to did not induced by both simple wounding and specific herbivory, which implies that specific secondary metabolite in A. contorta might be involved in the constitutive defense. For more understanding the relationship between A. contorta and S. montela, multi-layer approach should be further conducted such as deep sequencing and quantification of the secondary metabolite content.

Fig. 4-13. Summary of the transcriptomic change of A. contorta under artificial herbivory of S. montela.