|SCHMITT, ANTHONY - University Of Minnesota|
|SATHOFF, ANDREW - University Of Minnesota|
|HOLL, CATHERINE - University Of Minnesota|
|BAUER, BRITTANY - University Of Minnesota|
|Samac, Deborah - Debby|
|CARTER, CLAY - University Of Minnesota|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2018
Publication Date: 11/27/2018
Citation: Schmitt, A., Sathoff, A., Holl, C., Bauer, B., Samac, D.A., Carter, C.J. 2018. The major nectar protein of Brassica rapa is a non-specific lipid transfer protein with strong antifungal activity. Journal of Experimental Botany. 69(22):5587-5597. https://doi.org/10.1093/jxb/ery319.
Interpretive Summary: Bee-pollinated flowers secrete sugar-containing nectars that often contain other compounds, including a small number of proteins, most with unknown functions. The nectar from flowers of rapid cycling Brassica, closely related to turnip and napa cabbage, was collected and the major protein isolated and characterized. The protein is extremely heat stable and binds saturated free fatty acids. It has broad antimicrobial activity that inhibits growth of fungal plant pathogens at low concentrations and also inhibits growth of some bacterial plant pathogens to a lesser degree. These results suggest that the protein functions to prevent microbial growth in nectars or possibly may be involved in the movement of free fatty acids into nectar. This research extends knowledge on the makeup of nectar and role of compounds found in nectar that are important in pollination of crops by honeybees and other pollinating insects.
Technical Abstract: Nectar is one of the key rewards mediating plant-mutualist interactions. In addition to sugars, nectars often contain many other compounds, including proteins. SDS PAGE analysis of raw Brassica rapa nectar revealed the presence of ~10 proteins, with a major band at ~10 kDa. This major band was found to contain a non-specific lipid transfer protein (nsLTP) by LC MS/MS analysis, with multiple peptides mapping to the B. rapa locus Bra028980, previously termed BrLTP2.1. Sequence analysis of BrLTP2.1 predicted the presence of a signal peptide required for secretion from the cell, eight cysteines, and a mature molecular weight of 7.3 kDa. Constitutively expressed BrLTP2.1-GFP in Arabidopsis displayed accumulation patterns consistent with secretion from cells. BrLTP2.1 was also found to have relatively high sequence similarity to nsLTPs with known functions in plant defense, including Arabidopsis DIR1. Using the crystal structure of AtDIR1 as a template, mature BrLTP2.1 was predicted to have four alpha-helices, four disulfide bonds, and a hydrophobic lipid-binding pocket. Heterologously expressed and purified BrLTP2.1 was extremely heat stable and strongly bound to both saturated free fatty acids, but not methyl jasmonate. Recombinant BrLTP2.1 also had direct antimicrobial activity against multiple plant pathogens, being particularly effective against necrotrophic fungi. Cumulatively, these results suggest that BrLTP2.1 may function to prevent microbial growth in nectars or possibly be involved in the movement of free fatty acids into the nectar.