2.2. Response to IE and MW Treatment after Priming with Z-3-HAC
We monitored the growth of treated leaves (in-leaf responses) over 3 days after exposure to Z-3-HAC (as well as in controls), with the MW and IE treatment performed on day 2 (Figure 2
). We analyzed the growth response in controls and after Z-3-HAC priming of actively growing leaves of 7-, 8-, 12- and 13-day-old plants by treating the 2nd, 3th, 4th and 5th leaf in the respective maize seedling. The results show that the growth responses to Z-3-HAC priming and subsequent treatment with MW and IE were much more diverse when compared to their corresponding control plants. For in-leaf responses in control maize seedlings we found that treatment with MW and IE reduced the growth rate significantly in 7-, 8- and 12-day-old maize seedlings while 13-day-old maize seedlings did not experience significantly reduced growth rates after MW treatment (Figure 2
) but responded to IE treatment with a growth reduction.
However, when maize seedlings were first treated with Z-3-HAC overnight and then with MW or IE, we found a significant reduction in growth rates only in 7- and 13-day-old seedlings while 8- and 12-day-old plants did not display any significant alterations in their growth rates.
The similar growth responses of MW- and IE-treated plants in controls as well as after Z-3-HAC priming came as a surprise considering the differential defense responses of maize seedlings to these treatments. IE treatment is very similar in its outcome to actual insect herbivory and induces much stronger defense responses than MW alone [37
]. Furthermore, in maize leaves, these responses occur both around the damage site and in distal parts of the treated leaf while at the same time no significant activation of defense responses in basal parts of the leaf was detected [17
]. In contrast, treatment with MW only induced responses in the immediate vicinity of the damage site, with no distal or basal effects. Thus, the investments in defense are significantly higher in IE-treated maize seedlings when compared to MW alone, which would suggest a much stronger reduction in growth. However, with the exception of 13-day-old control plants treated with IE, no significant differences between these treatments were found. The results in Figure 2
also clearly demonstrate that at least some signal or metabolic component during these responses affects the basal parts of the treated leaf, which is an area of active growth and results in a significant reduction of growth. However, the efficiency of this signal also seems to depend on the age/developmental stage of the plant. Furthermore, it appears that the mechanical wounding part of both, the MW- and the IE treatment, is mainly responsible for the observed growth reduction and that IE does not further reduce growth in the basal areas of the treated leaves.
The previous experiment confirmed that both MW and IE treatments produced a basal signal in at least some ages/developmental stages, with a previous exposure to Z-3-HAC further modulating these responses. Thus, we also analyzed the effects of these treatments on systemic leaves. As described above for the in-leaf treatment group, we found that control plants at all ages/developmental stages showed a significantly reduced growth rate after treatment with MW and IE in the systemic leaf (Figure 3
). Furthermore, both IE and MW caused the same reduction in growth rate. However, in Z-3-HAC-treated maize seedlings, the effects of MW and IE on the systemic response again differed greatly. Although IE treatment caused a significant reduction in the growth rate of 8-day-old seedlings compared to its control, MW-treated plants showed a growth rate that was similar to control plants. For 10-day-old maize seedlings, we found that both MW- and IE-treated plants had significantly reduced growth rates. In contrast, 13-day-old seedlings showed no difference in growth rate between MW, IE and control plants. However, the growth rates of MW- and IE-treated 14-day-old maize seedlings were again significantly lower than those of the controls and even stayed lower for two days after this treatment. Although these results again demonstrate an apparent response from a site located basally from the MW- and IE treatment site that resulted in systemic growth alteration, they also demonstrate a modulating effect of priming with Z-3-HAC on growth in systemic leaves.
Although exposure to Z-3-HAC caused a decrease in growth rate, subsequent treatment with MW or IE surprisingly did not further reduce growth in maize seedlings. When comparing the growth rates on day 2 after direct MW and IE treatments, Z-3-HAC pre-treated maize seedlings showed similar or even increased growth rates after MW and IE treatment when compared to their respective non-Z-3-HAC controls (Figure 4
). This distinct growth rate behavior is surprising when considering that Z-3-HAC-primed maize seedlings subsequently challenged with IE produce nearly twice as much JA and HIPV when compared to only IE-treated controls [29
]. The increased investment in defense caused by priming is not reflected in the observed growth rates in either the treated leaf or systemic leaf. Furthermore, growth responses to treatments with MW alone were also positively affected by a previous exposure to Z-3-HAC in several treatment groups. In fact, it is surprising that IE treatment generally does not cause a stronger reduction in growth than MW does with only the 13-day-old plants with the treated leaf deviating from this trend. This suggests that basal signaling from the damage site after MW and IE treatments does not depend on the abundance of elicitors but instead is mainly caused by the damage alone. Only minor attenuations in growth rates were observed after IE treatment.
One factor may be the sink–source relationship in the growing maize seedling. Older leaves generally support younger leaves with assimilates. Therefore, one might argue that by damaging the vasculature system, this transport may have become interrupted and thus may lead to reduced growth in the expanding part of the treated leaf or in the systemic leaves. However, considering the responses of 12- to 14-day-old seedlings after previous exposure to Z-3-HAC, it appears as if those leaves, although similarly damaged, respond with normal growth rates to MW and IE treatments. Furthermore, Z-3-HAC-treated control plants often show an increased growth rate when compared to their controls. For example, 7- and 12-day-old in-leaf treated maize seedlings as well as 10- and 14-day-old systemic leaves showed a significantly increased growth rate after Z-3-HAC treatment. Therefore, other yet to be identified alterations in metabolism might explain these rather normal or sometimes even increased growth rates in maize seedlings after exposure to GLVs.
However, by analyzing the overall growth affected by these treatments over a period of three days, we found very few effects of either treatment on growth (Figure 5
). Significant differences were only found between 7-day-old in-leaf treated Z-3-HAC Control and Control IE plants (Tukey’s HSD test, q = 4.337; p = 0.0452) and between 14 day-old Z-3-HAC and Z-3-HAC MW and Control IE plants (q = 4.502, p = 0.03 and q = 6.396, p = 0.001, respectively). Overall, induced differences in plant growth rates appear to level off after an extended period, suggesting that neither a single stimulus nor a combination of Z-3-HAC and subsequent MW or IE treatment instigates a long-lasting effect on the growth of maize seedlings. This is surprising since all three stimuli by themselves caused a significant reduction in growth in the immediate aftermath of their application. One can only conclude that mechanisms must be in place that help to alleviate those consequences. However, these mechanisms are yet to be discovered.
To summarize, we found that treatment of maize seedlings with Z-3-HAC as our model GLV caused a significant growth reduction over the first 16 hours of treatment. However, Z-3-HAC-treated maize seedlings increased their growth rate on day 2, which resulted in similar or even slightly enhanced overall growth compared to control plants. MW and IE treatments also reduced growth in treated leaves as well as systemically. Priming with Z-3-HAC was found to modulate growth responses depending on the developmental stage and/or age of the plant. However, over a period of three days, the overall growth was found to be very similar between all of these treatments, suggesting that maize seedlings have developed physiological mechanisms to compensate for losses caused by single treatments as described herein. We are currently investigating potential mechanisms that may allow maize seedlings as well as possibly other plants to alleviate the costs of metabolic investments in defenses, including those incurred by priming.