Figure 1.
Laboratory pre-treatment procedures to produce conditioned and control algal sporophytes.
“Source" sporophytes were either elicited by application of GG in filtered seawater, either handled in the same way without elicitation for control procedure. Control and conditioned “target" sporophytes were co-incubated with non-elicited or elicited source algae, respectively. After 24 hours, the defense responses of each target sporophyte were tested by a subsequent oligoguluronate-elicitation.
Figure 2.
Elicitor-response patterns in laboratory-grown and wild sporophytes of Laminaria digitata.
A. Laboratory-grown (right scale) and harvested wild (left scale) L. digitata sporophytes were elicited with oligoguluronates in filtered seawater (FSW) and the concentration of H2O2 was recorded. Sample size was n = 2–3 thalli and values represent means +/− Standard Errors of Means (SE) on two different scales. B. Kinetics of defense-related gene expression in laboratory-grown and harvested wild L. digitata sporophytes. Fold variations of transcript levels quantified by RT-qPCR were calculated from different individual thalli (n = 3) between control and elicited sporophytes. For each of the defense related genes, differences between the six conditions (algal origin*time interaction) were tested using Tukey-Kramer test for multiple comparisons of means presented in Table 1 (Letters above the error bars indicate groups that are not significantly different, p<0.05). C. Values of the maximum of H2O2 concentrations reached during the oxidative burst by wild L. digitata sporophytes elicited either immediately after harvest from their natural habitats or after laboratory incubation in FSW. Values are mean ± SE (n = 3).
Table 1.
Effects of algal origin, time and their interactions on the intensity of gene induction after elicitation by GG.
Figure 3.
Effects of transplantation to a kelp field of laboratory-grown L digitata sporophytes on elicitor-response patterns.
Laboratory-grown sporophytes were kept in filtered seawater (FSW) in the laboratory (white) or transferred to a kelp population, either in a hermetically sealed plastic bag filled with FSW (light grey) or in a net allowing direct contact with natural seawater (NSW) (dark grey). Wild sporophytes were harvested from the same kelp bed (black). Sporophytes were taken back to the laboratory and subsequently elicited with GGs. A. Values of the maximum amount of H2O2 detected in FSW after elicitation in laboratory-grown sporophytes, previously transferred (or not) in the kelp bed for 90 min, and wild-type L. digitata sporophytes. Values are means ± SE (n = 3). Letters above the error bars indicate groups that are not significantly different (Tukey-Kramer test for multiple comparisons of means, p<0.05). B. Expression of defense-related genes in laboratory-grown L. digitata sporophytes transplanted either in a net or a sealed bag in the kelp bed for 24 hours and subsequently elicited with GGs for 3 h in laboratory. Fold variations of transcript levels quantified by RT-qPCR were calculated between control and elicited sporophytes. Values are means ± SE (n = 3). For each defense related genes, differences of fold variations were tested using a t-test between algae previously kept in a sealed bag or maintained in a net allowing direct contact with natural seawater (the results of the tests are indicated above the error bars, ns: non-significant, p>0.05; *: p<0.05; **: p<0.01).
Figure 4.
GG-induced oxidative burst in conditioned and unconditioned L. digitata sporophytes.
Sporophytes were elicited with GGs in seawater and the concentration of H2O2 was recorded. Experiments were replicated three times and a typical result is shown. Inset: Means and standard errors (n = 3) of the time required to reach the maximum of H2O2 concentrations in the medium after elicitation. The two means were significantly different for conditioned (black bar) and unconditioned (white bar) L. digitata sporophytes (t test: *, P≤0.05).
Figure 5.
Change in transcript levels of defense-related genes in conditioned and unconditioned L. digitata sporophytes after elicitation with GGs.
Transcript levels were quantified by RT-qPCR before elicitation (t = 0) and after 1.5 h, 3 h and 6 h. Values represent the fold changes in transcript levels at one time point compared to t = 0 (t/t0, means ± SE, n = 3). For each defense related genes, differences between the six conditions (treatment *time interaction) were tested using Tukey-Kramer test for multiple comparisons of means presented in Table 2 (letters above the error bars indicate groups that are not significantly different, p<0.05). For 6pgd2, statistical analyses were based on two time kinetics (1.5 and 6 h).
Table 2.
Effects of conditioning treatment, time and their interactions on the intensity of gene induction after elicitation by GG.
Figure 6.
Release of VOCs by conditioned and unconditioned L. digitata sporophytes after elicitation with GGs.
VOCs were quantified in the medium surrounding L. digitata 1 h after challenge or not with GGs. For each compound, the non elicited unconditioned control level was set to a relative unit of 1 to express the fold-variation in the other conditions (absolute concentration values are provided in Tables S1 and S2). 4-HHE, 4-hydroxy-(E)-2-hexenal; C7:2, (E,E)-2,4-heptadienal; C8:2, 2,4-octadienal; C8:3, 2.4.7-octatrienal; 4-HNE, 4-hydroxy-(E)-2-nonenal; C10:3, 2.4.7-decatrienal; 4-HDDE, 4-hydroxydodecadienal; CH3CH2I, iodoethane; CH2I2, diiodomethane; CHBr3, bromoform; CHBr2Cl, dibromochloromethane; CHBrCl2, bromodichloromethane; CH2Br2, dibromomethane. Values are means of three independent replicates.