Silencing NaTPI Expression Increases Nectar Germin, Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

Abstract

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 repellant, 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 (H 2 O 2 ) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H 2 O 2 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 H 2 O 2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H 2 O 2 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 H 2 O 2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

Protein and alkaloid patterns of the floral nectar in some solanaceous species

“…[13,63] as in our study. Evaluating various Nicotiana taxa, N. rustica had higher nectar protein concentrations, compared to either the other two species involved in our study (Table 1), or to Nicotiana species investigated previously [13,23,63]. The 5 major proteins in the floral nectar of the ornamental tobacco N. langsdorffii × N. sanderae var.…”

“…In the nectar of N. attenuata the concentration of nicotine was 3 ± 0.35 µg/mL [13]. The mean concentrations of nicotine measured by us in the nectar of N. alata and N. rustica were within the range of 0.79-2.5 µg/mL which was found to be attractive and even addictive for honeybees [55], whereas nicotine concentrations in N. tabacum nectar slightly exceeded 5 µg/mL, which can already have aversive effect.…”

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

“…There is evidence that plant PIs protect specific tissues, act as storage proteins, regulate the activity of proteases, and direct their release (Ryan, 1973;Mosolov and Valueva, 2005). Plants altered in the expression of endogenous PIs display changes in growth rate (Zavala et al, 2004a;Xie et al, 2007), flower morphology and seed development (Sin et al, 2006), phloem structure (Xie et al, 2007), and nectar protein composition (Bezzi et al, 2010). These alterations in fitness-relevant traits showed that members of the potato inhibitor type II family (PI-II), formerly associated with defense, can have other physiological functions.…”

Pithy Protection: Nicotiana attenuata’s Jasmonic Acid-Mediated Defenses Are Required to Resist Stem-Boring Weevil Larvae

“…Why T. mucorea larvae prefer ir-pi plants in the pith choice tests remains unclear. One explanation could be an altered total protein content as it has been shown for the nectar of ir-pi plants (Bezzi et al, 2010).…”

“…attenuata uses a combination of toxins and digestibility reducing defenses, both of which are elicited by herbivore attack. Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes (van Dam et al, 2000;Glawe et al, 2003;Zavala and Baldwin, 2004;Horn et al, 2005;Zavala et al, 2008;Bezzi et al, 2010). Nicotine, a neurotoxin, generally reduces consumption by a variety of herbivores (Steppuhn et al, 2004).…”