Phytochemical Shift from Condensed Tannins to Flavonoids in Transgenic Betula pendula Decreases Consumption and Growth but Improves Growth Efficiency of Epirrita autumnata Larvae

Abstract

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni-and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.

Genetic modification of the flavonoid pathway alters growth and reveals flexible responses to enhanced UVB – Role of foliar condensed tannins

“…Accumulation of trans-flavan-3-ols in ANSi leaves and stems (Table 1) supports the interpretation that partial silencing of BpANS caused an accumulation of its substrates, which the leucoanthocyanidin reductase (LAR) converted into catechin and gallocatechin (Figure 1d). The decreased growth of flavonoid-accumulating DFRi and ANRi plants (Figures 2a and 3a) is consistent with earlier experiments using the same plant material (Kosonen et al, 2015;Thitz et al, 2020).…”

“…The effects of RNAi on LMW phenolics in leaves were similar to the earlier results obtained from the same plant lines (Thitz et al, 2020), and stem flavonoids followed a corresponding pattern:…”

“…F-values related to main and interactive effects of RNAi and UVB are indicated, as well as significant differences from the ambient UVB treatment at p < .001 (***), 0.001 < p < .01 (**), and 0.01 < p < .05 (*) (Barnes et al, 1996;Tattini et al, 2000). The increased density of glandular trichomes under enhanced UVB supports the hypothesis that they may have UVB-protective functions in addition to their antiherbivore properties in B. pendula (Martemyanov et al, 2015;Thitz et al, 2020).…”

“…RNAi constructs based on the coding sequences of BpDFR1, BpANS and BpANR (Methods S1) were transferred into BPM5 (hereafter the "control line") by Agrobacterium-mediated gene transfer as described in Kosonen et al (2015) and Thitz et al (2020).…”

“…We used RT-qPCR to confirm the decreased expression of the silenced genes in leaves and stems of RNAi-modified plant lines (Table S1), using the Birch 18S ribosomal RNA gene (EMBL accession number AJ279693) as the internal reference gene. The primers used for RT-qPCR have been published in Thitz et al (2020) and Kosonen et al (2015). Three to four lines per RNAi construct (for the selected lines, see label in Figure S1) with decreased condensed tannin levels in preliminary acid butanol assays (see Analytical methods) in leaves and stems of micropropagated plantlets were selected for this study.…”