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

Bezzi, Siham, Kessler, Danny, Diezel, Celia, Muck, Alexander, Anssour, Samir, Baldwin, Ian T.
Plant physiology 2010 v.152 no.4 pp. 2232-2242
proteins, nectar, sugars, Nicotiana, anthers, nicotine, hydrogen peroxide, pollinators, trypsin inhibitors, field experimentation, indigenous species, emissions, proteomics, attractants, insects, glucose oxidase, benzaldehyde
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 (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.