Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.

Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals

Upstream and downstream transcription regulation of PAP1-activated genes.

<p>A, Endogenous phytohormone levels were determined in leaves of GUS control and <i>PAP1</i> lines (PAP-8) not infested or infested with <i>Spodoptera litura</i> for 2 days. ABA, abscisic acid; JA, jasmonic acid; JA-Ile, jasmonoyl-L-isoleucine; SA, salicylic acid. B, The effect of treatment with methyl jasmonate (MeJA) on gene expression was determined in leaves of GUS and PAP-8 lines treated with 0.1 mM MeJA (in 0.1% ethanol solution) for 24 h. Plants treated with 0.1% ethanol solution for 24 h served as controls. Transcript levels of genes were normalized by comparing them to those of <i>NtEF1α</i>. Data are shown as the mean+standard error (<i>n</i> = 4–5). Means followed by different small letters are significantly different (<i>P</i><0.05). F3H, flavanone 3-hydroxylase; PI2, proteinase inhibitor 2.</p

Temporal patterns of <i>F3H</i> and <i>PI2</i> gene regulation in leaves of <i>PAP1</i> lines (PAP-8) plants during exposure to <i>Spodoptera litura</i> for 48 h and for up to another 48 h after removal of the larvae (recovery period).

<p>Transcript levels of genes were normalized by comparing them to those of <i>NtEF1α</i>. Data are shown as the means ± standard errors (<i>n</i> = 4–5).</p

Endogenous accumulation of flavonoids/phenylpropanoids.

<p>Values for the major anthocyanin compound (cyanidin-3-<i>O</i>-rutinoside), other flavonoid (kaempferol 3-<i>O</i>-rutinoside and rutin) and phenylpropanoid (chlorogenic acid) compounds were determined in leaves of wild-type (WT), GUS control and <i>PAP1</i> lines not infested or infested with <i>Spodoptera litura</i> for 2 days. Data are shown as the mean+standard error (<i>n</i> = 5). Means followed by different small letters are significantly different (<i>P</i><0.05).</p

Schematic presentation of effect of <i>Spodoptera litura</i> feeding on the PAP1-regulated phenylpropanoid biosynthetic genes in a manner independent of jasmonate signaling.

<p>Arrows and bars indicate positive and negative interactions, respectively. TFs, transcription factors.</p

The net body weight that <i>Spodoptera litura</i> larvae gained during 4 days on wild-type (WT), GUS control and <i>PAP1</i> lines.

<p>Data are shown as the mean+standard errors (<i>n</i> = 26–32). Means followed by different small letters are significantly different (<i>P</i><0.05).</p

Expression of genes for transcription factors PAP1 and MYBJS1, An1a, An1b and structural genes involved in the flavonoid/phenylpropanoid pathway.

<p>Relative transcript levels of genes were determined in leaves of GUS control and <i>PAP1</i> lines (PAP-8) not infested or infested with <i>Spodoptera litura</i> for 2 days. Transcript levels of genes were normalized by those of <i>NtEF1α</i>. Data are shown as the mean+standard errors (<i>n</i> = 4–5). Means followed by different small letters are significantly different (<i>P</i><0.05). Expression of genes in GUS and PAP-8 leaves after 4 days of damage are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108849#pone.0108849.s004" target="_blank">Figure S4</a>. ANS, anthocyanidin synthase; C3H, cinnamate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; 4CL, 4-coumarate coenzyme A ligase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; PAL, phenylalanine ammonia-lyase.</p