Dynamic regulation of phenylpropanoid pathway metabolites in modulating sorghum defense against fall armyworm

Abstract

Plants undergo dynamic metabolic changes at the cellular level upon insect infestation to better defend themselves. Phenylpropanoids, a hub of secondary plant metabolites, encompass a wide range of compounds that can contribute to insect resistance. Here, the role of sorghum (Sorghum bicolor) phenylpropanoids in providing defense against the chewing herbivore, fall armyworm (FAW), Spodoptera frugiperda, was explored. We screened a panel of nested association mapping (NAM) founder lines against FAW and identified SC1345 and Ajabsido as most resistant and susceptible lines to FAW, respectively, compared to reference parent, RTx430. Gene expression and metabolomic studies suggested that FAW feeding suppressed the expression level of genes involved in monolignol biosynthetic pathway and their associated phenolic intermediates at 10 days post infestation. Further, SC1345 genotype displayed elevated levels of flavonoid compounds after FAW feeding for 10 days, suggesting a diversion of precursors from lignin biosynthesis to the flavonoid pathway. Additionally, bioassays with sorghum lines having altered levels of flavonoids provided genetic evidence that flavonoids are crucial in providing resistance against FAW. Finally, the application of FAW regurgitant elevated the expression of genes associated with the flavonoid pathway in the FAW-resistant SC1345 genotype. Overall, our study indicates that a dynamic regulation of the phenylpropanoid pathway in sorghum plants imparts resistance against FAW.

Keywords: fall armyworm; flavonoids; plant defense; regurgitant; sorghum.

Figure 1

SC1345 provided enhanced resistance to fall armyworm (FAW). (A) Mean larval weight of FAW collected from sorghum lines, 10 days after the introduction of newly hatched larvae on sorghum (three-leaf stage) plants under greenhouse conditions (n = 14). (B) Mean larval weight of FAW collected from sorghum lines, 12-14 days after the FAW egg masses infestation on sorghum (nine-leaf stage) plants under field conditions. Data from 2018 and 2019 field experiments were pooled to represent mean data (n = 45-49). (C) Mean head capsule width of FAW caterpillars collected from sorghum lines, 10 days after the introduction of newly hatched larvae on sorghum (three-leaf stage) plants under greenhouse conditions (n = 15-18). (D) Mean head capsule width of FAW caterpillars collected from sorghum lines, 12-14 days after infestation on sorghum (nine-leaf stage) plants under field conditions in 2019 (n = 21-22). Error bars represent ± SE. Different letters indicate significant difference relative to each other (P < 0.05).

Figure 2

Schematic representation of phenylpropanoid pathway in sorghum (modified from Scully et al., 2016). Enzyme abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; HCT, p-hydroxycinnamoyltransferase; C3H, 4-coumarate hydroxylase; CSE, caffeoyl shikimate esterase; CCoAOMT, caffeoyl-CoA-O-methyltransferase; CCR, cinnamoyl-CoA reductase; F5H, ferulate 5-hydroxylase; COMT, caffeic acid O-methyl transferase; CAD, cinnamyl alcohol dehydrogenase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthasae; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase.

Figure 3

Fall armyworm (FAW) feeding suppressed the accumulation of phenolic intermediates of the monolignol biosynthetic pathway in sorghum. (A) Principal component analysis (PCA) of soluble and cell-wall bound phenolic compounds quantified by GC-MS after 10 days of FAW infestation on RTx430, SC1345 and Ajabsido plants. FAW-uninfested plants were used as controls. (B–F) Relative abundances of intermediates of the monolignol biosynthetic pathway were determined by GC/MS analysis of soluble phenolics extracted from sorghum leaves. Peak area was normalized to the internal standards using 4-methyl cinnamic acid for soluble phenolics. The relative abundances of soluble (B) syringic acid, (C) p-coumaric acid, (D) ferulic acid, (E) caffeic acid, and (F) sinapic acid are presented (n = 4). Error bars represent ± SE. Different letters indicate significant difference relative to each other (P < 0.05).

Figure 4

Principal component analysis (PCA) of flavonoid compounds quantified after 10 days of fall armyworm (FAW) infestation on RTx430, SC1345 and Ajabsido plants. FAW-uninfested plants were used as controls.

Figure 5

Fall armyworm (FAW) infestation enhanced the levels of several flavonoid compounds in SC1345 plants. Absolute abundances of flavonoid compounds, (A) Apigenin, (B) Epicatechin, (C) Genistein, (D) Luteolin, (E) Naringenin, (F) Phloretin, (G) Quercetin-3-glucoside, (H) Quercetin-3-galactoside, (I) Quercetin, and (J) Rutin, in RTx430, SC1345, and Ajabsido plants before and after FAW infestation for 10 days (n = 3-4). FW, fresh weight. Error bars represent ± SE. Different letters indicate significant difference relative to each other (P < 0.05).

Figure 6

Correlation plot of phenolics and flavonoid compounds differentially changed after 10 days of fall armyworm (FAW) infestation on sorghum plants. A Pearson pairwise correlation was calculated for differentially induced phenolic and flavonoid compounds in JMP Pro 14. Asterisks are used to show significant correlations between compounds (P < 0.05).

Figure 7

Sorghum flavonoids affected the fall armyworm (FAW) growth adversely. Mean larval weight of FAW collected from (A) sorghum tan and purple lines (n = 11-15 for each sibling line, total n = 63-79), and (B) RTx430 (wild-type) and HCT (p-hydroxycinnamoyltransferase)-overexpression (OE) events (n = 19-21), 10 days after the introduction of newly hatched larvae on sorghum (three-leaf stage) plants under greenhouse conditions. (C) Total flavonoids estimated using spectrophotometer on two-week-old RTx430 and HCT OE plants (n = 3-4). Error bars represent ± SE. Different letters indicate significant difference relative to each other (P < 0.05).

Figure 8

Application of fall armyworm (FAW) regurgitant on sorghum wounded plants enhanced the expression levels of flavonoid pathway genes. RT-qPCR analysis of flavonoid pathway genes (A) FNSII, flavone synthase II; (B) FNR, flavanone 4-reductase; (C) CHS, chalcone synthase; and (D) DFR3, Dihydroflavonol-4-reductase. (E) Total flavonoids estimated using spectrophotometer in leaves of sorghum SC1345 plants after four applications of FAW regurgitant every 24 hours. Undamaged and wounded plants were used as controls (n = 3-4). Error bars represent ± SE. Different alphabets indicate significant difference relative to each other (P < 0.05). W + W, treatments in which water was applied to wounds; W + R, treatments in which FAW regurgitant was applied to wounds.