Ethylene and auxin interaction in adventitious rooting in Arabidopsis thaliana

Adventitious roots (ARs) are roots that develop from non-root tissues, mostly from aerial plant parts (Verstraeten et al, 2014). ARs are controlled by multiple endogenous and environmental factors, with auxin exhibiting a central role in the process (Pacurar et al, 2014). The auxin indole-3-acetic acid (IAA) is a potent growth regulator, but recent studies have demonstrated the importance of the natural auxin-precursor IBA (indole-3-butyric acid) because IBA-derived IAA is a significant part of the auxin necessary for seedling development (Strader and Bartel, 2011). Ethylene could be another hormone involved in AR-formation, because it affects a variety of processes during the plant lifetime, and influences many features of auxin-dependent plant growth by altering auxin signaling, synthesis and/or transport (Muday et al, 2012). However, in A.thaliana, presently there is limited information, and contrasting data are available for other species, highlighting many unresolved questions concerning the possible role(s) of ethylene in AR-formation (Li et al, 2009). Instead, there is information about lateral roots (LRs), i.e., the other post-embryonic roots sharing developmental stages with ARs (Della Rovere et al, 2013). In A.thaliana, studies in planta show an inhibitory effect of ACC (1-aminocyclopropane-1-carboxylic acid), the direct ethylene precursor, on LR-formation (Negi et al, 2008). However, this effect seems related to the concentration (about 10-8M), because lower concentrations stimulate the process (Ivanchenko et al, 2008). The present research investigated ethylene effects on AR-formation in seedlings of A. thaliana, and the possible relationship of ethylene with auxins (IBA and IAA). For this reason, AR-density in the hypocotyl was evaluated with/without ACC (0.1µM), and with/without IBA (10 µM), by the use of wild type (wt) seedlings, mutants and transgenic GUS lines. Wt seedlings showed a significant reduction in AR-density in the presence of ACC, whereas in the presence of IBA, the same ACC concentration caused a synergistic effect in AR proliferation compared to IBA alone, suggesting a possible opposite effect of ACC on endogenous IAA and exogenous IBA. Investigation on DR5::GUS seedlings supported the hypothesis, because a weaker signal was shown in the presence of ACC compared to the hormone-free condition, and a stronger signal in the presence of IBA plus ACC compared with IBA alone. To better investigate the hypothesis of a contrasting effect of ethylene with the two auxins, AR-production was evaluated in knockout mutants under the same treatments, i.e. the ethylene insensitive mutants ein2-1 and ein3eil1, the IAA-biosynthetic double mutant wei2wei7, the IAA partially insensitive double mutant tir1afb2, and the double mutant blocked in IAA-influx lax3aux1. The AR-response of these mutants showed that ethylene acts on the AR-process by modulating IAA biosynthesis and reception. The hormone does not affect auxin influx, as shown by lax3aux1 response and confirmed by LAX3::GUS and AUX1::GUS lines showing a similar signal expression with/without ACC. All together, results suggest that ethylene perception affects AR-formation in seedlings, possibly through a regulation of IAA biosynthesis and perception, but does not act on auxin influx. Moreover, ethylene seems to interact positively with exogenous IBA possibly favouring its conversion into IAA, thus promoting AR-rooting.