Murakami, S.; Nakata, R.; Aboshi, T.; Yoshinaga, N.; Teraishi, M.; Okumoto, Y.; Ishihara, A.; Morisaka, H.; Huffaker, A.; Schmelz, E.A.; Mori, N. Insect-Induced Daidzein, Formononetin and Their Conjugates in Soybean Leaves. Metabolites 2014, 4, 532-546.
Abstract
In response to attack by bacterial pathogens, soybean (Gylcine max) leaves accumulate isoflavone aglucones, isoflavone glucosides, and glyceollins. In contrast to pathogens, the dynamics of related insect-inducible metabolites in soybean leaves remain poorly understood. In this study, we analyzed the biochemical responses of soybean leaves to Spodoptera litura (Lepidoptera: Noctuidae) herbivory and also S. litura gut contents, which contain oral secretion elicitors. Following S. litura herbivory, soybean leaves displayed an induced accumulation of the flavone and isoflavone aglycones 4’,7-dihyroxyflavone, daidzein, and formononetin, and also the isoflavone glucoside daidzin. Interestingly, foliar application of S. litura oral secretions also elicited the accumulation of isoflavone aglycones (daidzein and formononetin), isoflavone 7-O-glucosides (daidzin, ononin), and isoflavone 7-O-(6’-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin). Consistent with the up-regulation of the isoflavonoid biosynthetic pathway, folair phenylalanine levels also increased following oral secretion treatment. To establish that these metabolitic changes were the result of de novo biosynthesis, we demonstrated that labeled (13C9) phenylalanine was incorporated into the isoflavone aglucones. These results are consistent with the presence of soybean defense elicitors in S. litura oral secretions. We demonstrate that isoflavone aglycones and isoflavone conjugates are induced in soybean leaves, not only by pathogens as previously demonstrated, but also by foliar insect herbivory.
Discussion
We identified daidzin (1), 4’,7-dihyroflavone (2), daidzein (3), and formononetin (4) as S. litura inducible metabolites following herbivory in soybean leaves. Additionally malonyldaidzin (5), ononin (6), and malonylononin (7) were also accumulated in soybean leaves treated with S. litura gut content extracts. Given that many analytes were present at 3–10 fold greater levels in leaves treated with gut contents compared to S. litura herbivory, we suspect that the additional detection of malonyldaidzin (5), ononin (6), and malonylononin (7) occurred due to a greater overall level of rapid elicitation. These conjugates are considered to be latent forms of isoflavonoids and upon cleavage are ultimately converted to the aglycones, daidzein (3) and formononetin (4) [25]. Another possibile difference between S. litura herbivory and application of gut contents is that soybean isoflavone conjugate-hydrolyzing β-glucosidases may exhibit greater activation in soybean leaves damaged by continuous herbivory compared to a single damage time point. In general the induced defense responses of soybean leaves to S. litura herbivory were largely reproduced by application of S. litura gut content extracts.
When damaged by S. exigua, corn plants release volatile compounds which can serve as host location cues for natural enemies of the herbivores. Synthesis and release of plant volatiles is elicited by specific substances present in herbivore oral secretions and gut contents. The best known of these insect-produced elicitors are the fatty acid amino acid conjugates (FACs), first identified from S. exgua larvae [20]. The FACs are also found in several other lepidopteran species [26,27,28,29,30,31,32,33]. FACs also induce soybean to release volatile compounds [34] and tobacco plants to produce trypsin proteinase inhibitors [23]. We add to this body of knowledge by demonstrating that elicitors present in S. litura gut contents induce the production of flavonoid biosynthesis in soybean leaves. The future identification of S. litura elicitors responsible for this response will further contribute to our understanding of soybean defense regulation.
Furthermore, it is important to investigate whether these induced compounds are accumulated locally or systemically in response to the insect feeding and elicitor treatment. For example, cotton plants Gossypium hirsutum L. damaged by herbivorous insects release volatiles, which are released, not only at the damaged site, but from the entire cotton plant [35]. All systemically released compounds are known to be induced by caterpillar feeding. Larval oral secretions of Manduca sexta or synthetic FACs significantly increase trypsin proteinase inhibitor elicitation in both treated (local) and systemic untreated leaves in Nicotiana attenuata [23]. Further studies are necessary to evaluate whether these induced flavone and isoflavones are produced locally or systemically following the treatments in the near future.
Nicotine, an inducible defense compound in Nicotiana species, is produced in the roots after insect herbivory and mechanical damage, and then transported to aerial parts of the plant [36]. In soybean roots, significant levels of daidzin (1), daidzein (3), and malonyldaizin (5), are also reported [7]. To investigate whether or not flavonoid synthesis occurs at the site of elicitation, we applied 13C-labeled phenylalanine to damaged leaves in the presence of gut content elicitors and searched for isotopic incorporated into daidzein (3) and formononetin (4). As shown in Figure 7, labeled phenylalanine was incorporated into 4’,7-dihydroxyflavone (2), daizdzein (3), and formononetin (4) 48 hours after the elicitor treatment.
When subjected to bacterial or fungal challenge, soybean leaves also accumulate glyceollins [1,4,5,37,38,39,40]. No increase in the aglycones, the glucoside conjugates, the malonylglucoside conjugates, or glyceollins occurs after leaf tissues are injured by physical stress [2]. Given that the accumulation of glyceollins are believed to play a significant role in the resistance of soybean cultivars to Phytophthora megasperma [4,5] and that daidzein (3) is a biosynthetic precursor of glyceollins [38], regulation of the daidzein is important early step in disease resistance. Similar to established pathogen induced responses, S. litura attack on soybean leaves results in the accumulation of daidzein (3) and formononetin (4). In contrast to pathogen elicitation, glyceollins have not been identified in leaves damaged by S. litura or those treated with gut contents, while glyceollins are reported to accumulate in soybean (var. Tamahomare) cotyledons [41] treated with one of polysaccharides, such as elicitors derived from fungal cell wall [42]. These results support the accumulation of daidzein (3) and formononetin (4), but not glyceollins, following S. litura herbivory. This selective plant response is likely to be important defensively as Zhou et al. demonstrated that daidzein can significantly inhibit the larval growth of S. litura [43]. Although no direct functions of the inhibitory activity of formononetin (4) against S. litura larvae has also been reported thus the accumulation of daidzein (3), without transference to glyceollins, likely represents an adaptive anti-herbivore defensive strategy of soybean.
In nature, plants are constantly challenged by a multitude of biotic stresses, including herbivorous insects and phytopathogens. However, most studies focus on responses to a single attacker and without seeking to understand synergies and trade-offs between insect and pathogen resistance in plants. In the present study we demonstrate that, in additional to established responses to pathogens, soybean leaves also synthesize and accumulate a subset of flavonoids following herbivore attack. Future investigations will include the analysis of gene expression and biosynthetic enzymes to better understand the role of flavonoid defenses against diverse numerous biotic attackers that operate in terrestrial ecosystems both above and belowground.
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Daidzein belongs to the group of isoflavones.Daidzein can be converted to its end metabolite S-equol in some humans based on the presence of certain intestinal bacteria. Based on several decades of research, S-equol has potential for significant health benefits. Daidzein
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