Comparison of the ability of partially N-acetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves

Abstract

Chitin, a linear polysaccharide composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1-->4)-linked GlcNAc and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1-->4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1-->4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1-->4)-linked GlcNAc with a degree of polymerization >/= 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Figure 1

Influence of the DP on the activity of purified GlcNAc oligomers (1 mg mL⁻¹; 24 h after injection) as elicitors of PAL and POD in wheat leaves. Data are means ± sd of triplicate samples from one representative of two independent experiments. The dotted lines indicate enzyme activities of water-treated control plants. prot., Protein.

Figure 2

Dose-response curves of PAL and POD elicitation by the GlcNAc octamer (▪) and the GlcN heptamer (•) 24 h after injection into the intercellular spaces of primary wheat leaves. Data are means ± sd of triplicate samples from one representative of two independent experiments. The dotted lines indicate enzyme activities of water-treated control plants. prot., Protein.

Figure 3

Influence of the DA on the activity of chitosan polymers (_n = 540–1100; 100 μg mL⁻¹; 24 h after injection) as elicitors of PAL and POD activities in wheat leaves. Data are means ± sd of triplicate samples from one representative of two independent experiments. The dotted lines indicate enzyme activities of water-treated control plants. prot., Protein.

Figure 4

Dose-response curves of PAL and POD elicitation by polymeric chitosans with DAs of 1%, 35%, and 60% 24 h after injection into the intercellular spaces of primary wheat leaves. Data are means ± sd of triplicate samples from one representative of two independent experiments. The dotted lines indicate enzyme activities of water-treated control plants. prot., Protein.

Figure 5

Time-response curves of PAL and POD elicitation by polymeric chitosans with DAs of 1% (circles), 35% (squares), and 60% (triangles) at 100 μg mL⁻¹ upon injection into the intercellular spaces of primary wheat leaves. Data are means ± sd of triplicate samples from one representative of two independent experiments. The dotted lines indicate enzyme activities of water-treated control plants. prot., Protein.

Figure 6

Macroscopically visible symptoms induced in wheat leaves by polymeric chitosans with DAs of 1%, 15%, 35%, 49%, and 60%, at 1, 10, and 100 μg mL⁻¹, 24 h after injection into the intercellular spaces of primary wheat leaves.

Figure 7

Correlation between the extent of foliar necroses and the induction of PAL and POD activities by chitosans with DAs of 1% (a–c), 35% (d–f), and 60% (g–i), at 1 (a, d, and g), 10 (b, e, and h), and 100 (c, f, and i) μg mL⁻¹, 24 h after injection into the intercellular spaces of primary wheat leaves. Necroses were arbitrarily quantified by macroscopic and UV-fluorescence microscopic examination on a necrosis index scale from 0 to 6. Macroscopic effects: 0, no symptoms visible; 2, central part of the injected area chlorotic; 4, central part of injected area necrotic, single chlorotic spots toward the periphery; 6, all of the injected area necrotic. Microscopic effects: 0, only cells directly at the injection site showed yellow autofluorescence under UV light; 2, many collapsed and yellow autofluorescing cells in the central part of the injected area, sparse clusters of such cells toward the periphery; 4, all of the cells in the central part of the injected area and many cells in the periphery collapsed and exhibited yellow autofluorescence; 6, all of the cells in the injected area collapsed and exhibited yellow autofluorescence.