Cucumber hypocotyls respond to cutin monomers via both an inducible and a constitutive H(2)O(2)-generating system

Abstract

Hypocotyls from etiolated cucumber (Cucumis sativa L.) seedlings were gently abraded at their surface to allow permeation of elicitors. Segments from freshly abraded hypocotyls were only barely competent for H(2)O(2) elicitation with fungal elicitor or hydroxy fatty acids (classical cutin monomers). However, elicitation competence developed subsequent to abrasion, reaching an optimum after about 4 h. This process was potentiated in seedlings displaying acquired resistance to Colletotrichum lagenarium due to root pretreatment with 2,6-dichloroisonicotinic acid or a benzothiadiazole. Induction of competence depended on protein synthesis and could be effected not only by surface abrasion, but also by fungal spore germination on the epidermal surface or by rotating the seedlings in buffer. Inhibitor studies indicated that the inducible mechanism for H(2)O(2) production involves protein phosphorylation, Ca(2+) influx, and NAD(P)H oxidase. In contrast, a novel cucumber cutin monomer, dodecan-1-ol, also elicited H(2)O(2) in freshly abraded hypocotyls without previous competence induction. This finding suggests the presence of an additional H(2)O(2)-generating system that is constitutive. It is insensitive to inhibitors and has, in addition, a different specificity for alkanols. Thus, dodecan-1-ol might initiate defense before the inducible H(2)O(2)-generating system becomes effective.

Figure 1

Surface abrasion and a subsequent conditioning period are required for H₂O₂ elicitation by FE. Cucumber seedlings were grown either on water (control; white symbols) or in the presence of INA or BTH (SAR; black symbols). Their hypocotyl surface was gently abraded and segments were either cut immediately (0 time) or after rotating the whole abraded seedlings in buffer for the indicated time periods (conditioning). The segments were adapted for 45 min in the H₂O₂ elicitation assay before the addition of 20 μg mL⁻¹ FE. The H₂O₂ concentration at the burst maximum (45 min after elicitation) was corrected against values determined in a parallel batch of segments without elicitor. The curves (○ and •) refer to one representative experiment in which SAR was induced by root pretreatment with INA. For the 4-h time point, means ± sd are given from 15 experiments with controls (□) and 13 experiments with seedlings rendered systemic resistant by root pretreatment with BTH (▪). For further notes on the variability in the time course of elicitation competence induction, see text.

Figure 2

Influence of a conditioning period on the time course of H₂O₂ elicitation by the hydroxy fatty acids DHSA and HPA. Seedlings were either grown on water (controls; □ and ▵) or on INA (SAR; ▪, ▴, and ▾). Segments were cut from hypocotyls of freshly abraded seedlings (▾) or from seedlings conditioned for 4 h subsequent to abrasion (▪ and ▴). DHSA (□ and ▪) or HPA (▵ and ▴) were used as elicitors at 50 μm. In freshly abraded segments only the values from SAR seedlings elicited with DHSA are shown (▾), but those from control segments elicited with any of the two elicitors were similarly low. One representative experiment is given. In additional experiments, the absolute level of the H₂O₂ burst maximum (120 min) differed for susceptible (control) segments between 1 and 4 μm, and for systemic resistant segments between 3 and 10 μm. An increase in the elicited H₂O₂ burst due to SAR induction was seen in any individual experiment performed. For instance, upon elicitation with DHSA in four independent experiments with INA this increase was 3.2 ± 1.2-fold, and in seven experiments with BTH it was 2.4 ± 0.8-fold. For further notes on the variability between experiments, see text.

Figure 3

Time course for H₂O₂ elicitation by the novel cucumber cutin monomer DDO. Hypocotyl segments were from either freshly abraded susceptible seedlings grown on water (•) or from seedlings grown on INA and conditioned for 4 h subsequent to abrasion (○). One experiment is given as an example. The variability between experiments is given in Table II.

Figure 4

H₂O₂ elicitation with DHSA, DDO, and FE is differently inhibited in freshly abraded and conditioned hypocotyls. Segments from either freshly abraded control hypocotyls or from systemic-resistant (INA) abraded seedlings that had been conditioned for 4 h were used. DPI (10 μm), K-252a (1 μm), or La³⁺ (0.8 mm) were applied 15 min prior to elicitation in assays performed as for Figures 1–3. With freshly cut segments, H₂O₂ elicitation was determined 20 min after DDO addition. In conditioned seedlings, H₂O₂ elicitation was determined 2.5, 2, or 1 h after addition of DHSA, DDO, or FE, respectively. The number (n) of individual experiments performed is given in the top. Negative values indicate that in addition to a full inhibition of the elicited H₂O₂ production, the slight non-elicited H₂O₂ production was also affected by the respective inhibitor.

Figure 5

Germinating spores of a melanin-deficient C. lagenarium mutant and rotating the seedlings in buffer can partially replace surface abrasion with regard to the stimulus needed for induction of elicitor competence. SAR was induced in the seedlings by root pretreatment with either 100 μm INA (A) or 40 μm BTH (B). As in Figure 1, the hypocotyls of one batch of seedlings were abraded and the seedlings conditioned for 4 h. The other part of the seedlings was rotated in a suspension of spores that adhere to the surface and germinate during the 4-h period. A third part of seedlings (controls) was rotated for 4 h in buffer only. Both the samples with spores and the controls were abraded after rotating for 4 h. Segments from all three batches were then cut and elicited with either FE (20 μg mL⁻¹) or DHSA (100 μm). Means ± sd from three (A) or five (B) independent experiments are given. Note that in contrast to the freshly abraded segments used in Figures 1 and 2, the controls also exhibited considerable elicitor competence. In four independent experiments similar to B, we confirmed 6 months later that the stress presumably caused by rotating the nonabraded seedlings in buffer can indeed induce some elicitor competence. In these experiments, freshly abraded hypocotyls directly from the growth box exhibited 1.0% ± 0.7%, whereas the buffer-treated hypocotyls had 8.7% ± 3.2% competence for elicitation with FE compared with abraded and conditioned seedlings (100%).