Ethylene has been studied as a volatile phytohormone in experiments using exogenous applications (Lill et al. 1979; Locke et al. 2000; reviewed by Pierik et al. 2006); here with experiments using WT and transgenic N. attenuata seedlings that lack the ability to produce ethylene, we establish that the natural release of ethylene influences the root growth of neighbors. This is the first report demonstrating that the natural release of a volatile chemical inhibits the growth of neighboring siblings, a process called allelopathy. N. attenuata is a fire-chaser that germinates in the first growing season after a fire from long-lived seedbanks and therefore frequently initiates growth in dense seedling populations where intense intra-specific competition is the main determinant of Darwinian fitness (Preston and Baldwin 2000). Because of the close proximity of competitors during seedling growth, the demographic importance of seedlings and their sensitivity to naturally released phytohormones, seedling growth is a commonly used parameter for evaluating growth-suppressing activities of phytohormones (Locke et al. 2000). The positive correlation observed between root growth inhibition of WT receivers, the number of WT emitter seedlings that shared the common headspace, and the ethylene levels in the common headspace (Fig. 1) is consistent with the hypothesis that the release of ethylene from WT emitters inhibits the root growth of WT receivers. Ethylene accumulation in Petri dishes by N. attenuata seedlings is reported to reduce root growth of its seedlings (Hummel et al. 2008). It has also been observed that ethylene, at low, physiologically relevant concentrations, tends to stimulate growth (reviewed in Pierik et al. 2006). However, some studies have shown that ethylene inhibits elongation, but stimulates radial swelling, of roots thereby improving their mechanical strength in resistant soil (Clarke et al. 2003).
Ir-aco emitters produce significantly less ethylene than do WT plants (Fig. 3, inset; von Dahl et al. 2007). We found that WT emitters, which released significantly more ethylene into the headspace, had a much stronger effect on ir-aco receiver seedlings than ir-aco emitters (Fig. 3). The pronounced growth response of ir-aco receivers is consistent with the hypothesized greater ethylene sensitivity of ethylene-silenced ir-aco plants (von Dahl et al. 2007). However, in the absence of any emitter seedlings we did not observe any significant (t = 1.783, P
2-tailed = 0.081) differences between root length of ir-aco and WT seedlings (Fig. 3), which is likely due to the fact that ethylene emitted by five WT seedlings (11.62 ± 2.4 nl/cuvette) was not significantly (t = 1.172, P
2-tailed = 0.280) different from background emission (7.87 ± 1.73 nl/cuvette), thus did not have significant (P < 0.001) impact on root growth of WT receivers (see Fig. 1). Furthermore, it is likely that ir-aco plants adapt to their endogenous ethylene concentrations and are able to grow normally in the absence of exogenous ethylene. Relative growth rates of ethylene-insensitive genotypes of Arabidopsis thaliana, Petunia hybrida and Nicotiana tabacum were similar to those of WT growth rates under near optimal growth conditions (Tholen et al. 2004), suggesting that these ethylene-insensitive plants adjusted their growth “normally” in a stress-free environment. Although root lengths of WT receiver was not significantly different when the emitter was WT or ir-aco, the root lengths of WT receivers grown with WT emitters were significantly reduced when compared to no-emitter treatment (Fig. 3). This observation in conjunction with the reduced root lengths of ir-aco receivers grown with WT emitters discussed above establishes that WT emitters influence root growth of receivers (WT or ir-aco) by emitting higher amounts of ethylene compared to ir-aco emitters (Fig. 3, inset). The relative weak silencing of ethylene emission in ir-aco plants, however, may not always have been sufficient to significantly to significantly influence the growth of WT receivers.
The exogenous manipulation of the ethylene concentrations by ACC supplementation and by ethylene scrubbing using KMnO4 only partially supported our hypothesis of ethylene-mediated growth inhibition of WT receivers. Although ACC supplementation of the growth media of WT emitters resulted in a large ethylene emission (993.8 ± 217.2 nl/cuvette; Supplementary Figure 2a), only negligible effects on the root growth of WT receiver seedlings was observed (Fig. 2). However, hardly any ethylene was detected from the media (38.2 ± 19.1 nl/cuvette) or WT seedlings grown in the presence KMnO4 (21.4 ± 9.2 nl/cuvette; Supplementary Figure 2a), and here scrubbing of ethylene by KMnO4 restored the root growth of WT receivers regardless of the presence of WT emitter seedlings (Fig. 2). Surprisingly the effects on ir-aco receivers by these exogenous applications were the opposite and supplementing the growth media of WT emitter seedlings with ACC inhibited the root growth of ir-aco receivers (Fig. 4). These observations reflect the challenges of meaningfully interpreting the effects of high concentration ethylene treatments.
Though various mechanisms for ethylene-mediated root growth suppression are possible, its diffusion through the air from emitters and subsequently through the receiver plant to its root system appears most likely. Root systems are known to be sensitive to volatile and non-volatile chemicals (Inderjit et al. 2005), and ethylene could as well travel through the medium or soil system. A striking example of this sensitivity occurred in the second half of the 20th century when many trees in or near the major cities of Germany died apparently as a result of ethylene leaking from subterranean pipelines (Grümmer 1955).
A series of experiments with cultivated tobacco analyzed the role of ethylene in competition for light and nutrients using transgenic plants over expressing a mutant ethylene receptor (etr1-1) from Arabidopsis thaliana that rendered the plants ethylene insensitive (Pierik et al. 2003, 2004, 2006). These studies elegantly demonstrate that ethylene signaling was required for cultivated tobacco to compete with its neighbors (Pierik et al. 2003), specifically due to its function in the shade-avoidance response of plants (Pierik et al. 2004). However, the responses of the Tetr-1 plants and the WT plants were compared and therefore the experiments could not differentiate between the effects of endogenous ethylene and the potential effects of exogenous ethylene from neighboring plants. In these studies ethylene levels in the air of densely grown cultivated tobacco stands were sufficient to induce growth responses in neighbors. Our study specifically aimed to exclude other factors such as resource competition and shade-avoidance effects and concentrated on the effects of exogenous ethylene. Procedures commonly used in allelopathy research do not isolate the effects of chemicals from other processes that could inhibit the growth of neighbors (Inderjit and Weiner 2001; Lau et al. 2008) and this problem could be resolved by the use of plants that lack the ability to produce a particular chemical.
Transgenics that do not produce a putative allelochemical are excellent tools to determine whether an allelopathic transgenic crop could suppress unwanted neighbors by the release of allelochemicals. For example, sorgoleone and m-tyrosine have been suggested as promising candidates for the construction of transgenic allelopathic crops (Bearson et al. 2008; Duke 2007) but conclusive evidence for the allelopathic potential of these compounds in natural environments is lacking. It is important to establish their allelopathic potential before commercializing allelopathic transgenic crops, and we believe that the approach presented in this study can be used to achieve this end.