The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity

Abstract

Nectar is one of the key rewards mediating plant-mutualist interactions. In addition to sugars, nectars often contain many other compounds with important biological functions, including proteins. This study was undertaken to assess the proteinaceous content of Brassica rapa nectar. SDS-PAGE analysis of raw B. 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 encoded by B. rapa locus Bra028980 and subsequently 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 mass of 7.3 kDa. Constitutively expressed BrLTP2.1-GFP in Arabidopsis displayed accumulation patterns consistent with secretion from nectary cells. BrLTP2.1 was also found to have relatively high sequence similarity to non-specific lipid-transfer proteins with known functions in plant defense, including Arabidopsis DIR1. Heterologously expressed and purified BrLTP2.1 was extremely heat stable and bound strongly to saturated free fatty acids, but not methyl jasmonate. Recombinant BrLTP2.1 also had direct antimicrobial activity against an extensive range of plant pathogens, being particularly effective against necrotrophic fungi. Taken together, these results suggest that BrLTP2.1 may function to prevent microbial growth in nectars.

Fig. 1.

A lipid transfer protein (LTP) is the major protein in Brassica rapa nectar. (A) Whole B. rapa flower (left) beside one from its close relative, Arabidopsis. (B) Example of a nectar droplet collected from B. rapa flowers for protein identification. LN, lateral nectary; Ov, ovary; Pe, petal. A short stamen was removed from the flower to visualize the nectar droplet. (C) Protein profile of raw B. rapa nectar after separation by 12% SDS-PAGE and silver staining. The major protein band (arrowhead) was excised from the gel and processed for protein identification. (D) Peptides identified from the major protein band [arrowhead in (C)] by LC-MS/MS. (E) BLAST searches identified the major protein band as Bra028980, a putative lipid-transfer protein. Peptides identified by MS/MS are shaded in gray, cysteines are highlighted, and a putative signal peptide required for secretion from the cell is underlined.

Fig. 2.

Constitutively expressed BrLTP2.1–GFP is secreted from Arabidopsis cells. (A, B) Full-length BrLTP2.1–GFP driven under control of the 35S-CaMV promoter leads to secretion from rosette leaf pavement cells. Arrowheads in (A) and (B) point to stomata. (C–E) BrLTP2.1–GFP preferentially accumulates in the stoma formed by guard cells in a lateral nectary (LN). Note that a similar accumulation of BrLTP2.1–GFP is not observed in the stomatal pores of rosette leaves (arrowheads).

Fig. 3.

Phylogenetic analysis of Bra028980 (BrLTP2.1). The protein sequence of Bra02890 (BrLTP2.1) was subjected to CLUSTAL Omega multiple sequence alignment and tree analysis with all members of LTP family encoded by B. rapa genome, as well as select LTPs from other species with known or implicated biological function (in bold, see Supplementary Table S1 for detailed list). One of the nearest Arabidopsis LTPs with implicated function, AtAZI7, was also previously found to have enriched expression in nectaries by microarray analyses, suggesting conservation of BrLTP2.1 function, at least within the Brassicaceae.

Fig. 4.

Structural prediction of BrLTP2.1. (A) AtDIR1, a close homolog to BrLTP2.1 involved in plant defense responses, was used as a template to model BrLTP2.1 structure. This analysis predicted the presence of four α-helices (labeled H1–H4 from N- to C-terminus) and four disulfide bonds. (B) Model of BrLTP2.1 with the lipid LP3 (1-stearoyl-sn-glycero-3-phosphocholine) bound. The specific sidechains predicted to coordinate lipid binding are Leu37, Gln38, Cys40, Ser15, Leu65, Leu68, Cys69, Ile82, Ser95, and Leu97. The models shown in both (A) and (B) were predicted by iTASSER using Arabidopsis DIR1 (2rknA) as a threading template. The sequence of the predicted mature BrLTP2.1 without signal peptide was used as the input, but the amino acid numbering includes the predicted 26 amino acid signal peptide.

Fig. 5.

Heterologously expressed BrLTP2.1 has lipid binding activity. (A) Heterologously expressed and purified BrLTP2.1 from E. coli. Lane 1: pre-induction E. coli lysate; lane 2: 4 h post-induction lysate; lane 3: cell media; lane 4: flow-through from Co²⁺ affinity column; lanes 5–7: column washes; lanes 8–10: elutions with 300 mM imidazole; lane 11: pure protein after concentration and desalting. (B) TNS, a lipophilic fluorophore binds to BrLTP2.1. TNS concentration ranged from 0 to 10 μM, while BrLTP2.1 concentration was held constant at 2.5 μM, with excitation at 320 nm and emission recorded at 437 nm. Inset: TNS-dependent fluorescence in wild-type and two mutant versions of BrLTP2.1 (protein and TNS both at 2.5 μM). (C) Lipids present in Brassica nectars competitively displaced TNS from BrLTP2.1. TNS and BrLTP2.1 concentration were each held constant at 2.5 μM, while myristic acid (C14), palmitic acid (C16), stearic acid (C18), and methyl jasmonate (MeJA) ranged from 0 to 10 μM. Inset: Dot blot analysis of BrLTP2.1 binding to myristic (C14), pentadecanoic (C15), heptadecanoic (C17), and stearic (C18) acid, as well as phosphatidylcholine (PC). A 2:1:0.8 solution of methanol:chloroform:water, the solvent for all lipids, was used as a negative control (neg). BrLTP2.1 binding was detected with anti-His antibodies.

Fig. 6.

BrLTP2.1 is extremely heat stable. (A) One milliliter of E. coli cell culture was harvested 4 h after induction of BrLTP2.1 expression with IPTG and directly boiled for the indicated times. Boiled samples were incubated on ice for 15 min and centrifuged at 17000 g for 10 min to precipitate denatured protein and cell debris. Remaining proteins were evaluated by SDS-PAGE. (B) SDS-PAGE analysis of the clarified supernatant of cell cultures boiled for 15 min (left lane) and further purified BrLTP2.1 via Co²⁺ affinity chromatography (right lane). (C) Western blot analysis of boiled and purified BrLTP2.1 from (B) as detected by anti-His-tag antibodies. Arrowheads indicate the multiple bands corresponding to BrLTP2.1 post-boil and affinity purification. (D) Boiled BrLTP2.1 retains lipid-binding activity after purification [from (B)], as determined by displacement of TNS with palmitic acid.

Fig. 7.

Heterologously expressed BrLTP2.1 has direct antimicrobial activity. Spores harvested from a battery of fungal plant pathogens were incubated with BrLTP2.1 from 0 to 300 μg ml⁻¹ and monitored for growth over 48 h. In the examples shown, Alternaria solani (A, B) and Bipolaris oryzae (C, D) were either mock treated (A, C) or incubated with 50 μg ml⁻¹ BrLTP2.1 (~5 μM; B, D). Summarized data are provided in Table 1.