Modulation of the biological activity of a tobacco LTP1 by lipid complexation

Abstract

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.

Figure 1.

Biochemical properties of the recombinant tobacco LTP1. (A) Circular dichroism spectrum. (B) Effect of fatty acids on the fluorescence level of the LTP1-TNS complex. FAs or JA (16 μM) and TNS (3 μM) were incubated together for 1 min and then LTP1 (250 nM) was added. Results are expressed as the percentage of the fluorescence of the LTP1-TNS (control, no FA or JA added, fluorescence level; 100 ± 6.01%). Experiments were performed in triplicate and results are expressed as the mean values ± SD.

Figure 2.

Dixon's plot of the binding competition between JA and TNS to LTP1. Various amount of JA (0–30 μM) and TNS (1–10 μM) were incubated together for 1 min and then LTP1 (250 nM) was added. From top to bottom, linear regressions lead to the following cr² values: 0.989, 0.972, 0.993, 0.981, and 0.915 for TNS concentrations of 1, 2, 3, 5, or 10 μM, respectively. Inset: replot of the slopes of the Dixon plots and linear regression (cr² = 0.993). Experiments were performed in triplicate and results are expressed as the mean values ± SD.

Figure 3.

Specific interactions between tobacco LTP1 and high-affinity sites located on tobacco plasma membranes. Plasma membrane preparations were incubated with various concentrations of ¹²⁵I-labelled LTP1. Specific binding was determined by subtracting nonspecific binding from total binding. Experiments were repeated at least five times, and results are expressed as the mean values ± SD (fmol bound LTP1/mg plasmalemma proteins). •, LTP1; ▴, LTP1-LA; and ▵, LTP1-JA. Inset: displacement of ¹²⁵I-labelled LTP1 specifically bound to plasma membrane by lysozyme (A), unlabeled LTP1 (B), or unlabeled cryptogein (C), 30 min after the addition of the unlabeled protein. The experiments were repeated three times, and results are the mean values ± SD.

Figure 4.

Level of tobacco resistance to P. parasitica induced by cryptogein and tobacco LTP1. Ethanolic elicitor solutions were applied on the stem section of freshly decapitated plants (cryptogein 0.1 nmol/plant, LA or JA 1 nmol/plant, LTP1 or LTP1-LA or LTP1-JA 0.5 nmol/plant). Ethanol (1%) was added on control plants. Twenty-four hours later, a mycelial plug was dropped on the cut petiole of the third fully expanded leaf from the apex. Seven days after inoculation, stems were split in two, and the internal necroses, corresponding to the brown or desiccated zones, measured. Results are expressed as the means of percentage of protection ± SD from three replicate experiments and five plants for each treatment. Small letters represent the result of a Student-Newman-Keuls test at 0.05.