Variation in antiherbivore defense responses in synthetic Nicotiana allopolyploids correlates with changes in uniparental patterns of gene expression.

We examined the expression of Nicotiana attenuata (Na) and Nicotiana obtusifolia (No) herbivore-induced genes in synthetic autopolyploids (NaT and NoT) and five independent allopolyploid Nicotiana x obtusiata (Nxo) lines to understand how the expression of genes regulating complex polygenetic defense traits is altered in the early stages of allopolyploid hybridization. In Na, applying Manduca sexta oral secretions (OS) to wounds rapidly increased the transcript accumulation of wound-induced protein kinase (WIPK), lipoxygenase 3 (LOX3), nonexpressor of pathogenesis-related 1 (NPR1), and jasmonate-resistant 4 (JAR4) genes; these were correlated with increases in accumulation of jasmonic acid (JA), jasmonate-isoleucine, and trypsin protease inhibitors (TPIs). In No, OS elicitation reduced NPR1 transcripts and increased the level of salicylic acid (SA) that appeared to antagonize JA and JA-mediated defenses. OS elicited Nxo lines, accumulated high levels of the uniparental transcript of WIPK, LOX3, JAR4, and TPI, but low levels of both parental NPR1 transcripts that in turn were correlated with an increase in SA and a decrease in JA levels, suggesting SA/JA antagonism in the allopolyploid crosses. Methyl jasmonate treatment of Nxo lines elicited transcripts of both parental LOX3, JAR4, and TPIs, demonstrating that the uniparental pattern observed after OS elicitation was not due to gene inactivation. TPIs were induced at different levels among Nxo lines; some lines expressed high levels comparable to Na, others low levels similar to No, suggesting that synthetic neoallopolyploids rapidly readjust the expression of their parental defensive genes to generate diverse antiherbivore responses. Changes in the expression of key genes and posttranscriptional events likely facilitate adaptive radiations during allopolyploid speciation events.

Silencing NaTPI expression increases nectar germin, nectarins, and hydrogen peroxide levels and inhibits nectar removal from plants in nature.

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellent, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.