Structure-activity relationship of 2,4-D correlates auxin activity with the induction of somatic embryogenesis in Arabidopsis thaliana

Abstract

2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic analogue of the plant hormone auxin that is commonly used in many in vitro plant regeneration systems such as somatic embryogenesis (SE) and organogenesis. The effectiveness of 2,4-D in inducing SE, compared to the natural auxin indole-3- acetic acid (IAA), has been attributed to the stress triggered by this compound rather than its auxin activity. However, this hypothesis has never been thoroughly tested. Here we used a library of 40 2,4- D analogues to test the structure-activity relationship with respect to the capacity to induce SE and auxin activity in Arabidopsis thaliana. Four analogues induced SE as effectively as 2,4-D and 13 analogues induced SE but were less effective. Based on root growth inhibition and auxin response reporter expression, the 2,4-D analogues were classified into different groups, ranging from very active auxins to not active. A halogen at the 4-position of the aromatic ring is important for auxin activity, whereas a halogen at the 3-position is accepted, but results in reduced activity. Moreover, a small substitution at the carboxylate chain, a methyl or ethyl group, is tolerated, as is extending the carboxylate chain with two but not with one carbon. Molecular dynamics simulations showed that the auxin activity of the 2,4-D analogues correlated well with their TIR1-Aux/IAA coreceptor binding characteristics. In the process, we identified two 2,4-D analogues as efficient inducers of adventitious root formation and several possible anti-auxins. Moreover, we observed a strong correlation between SE induction efficiency and auxin activity, indicating that the stress-related effects that have been reported to be triggered by 2,4-D and are considered important for SE induction are down-stream of auxin signaling.

Transient efflux inhibition improves plant regeneration by natural auxins

“…These results are in line with our recent observation that SE induction by 2,4‐D primarily requires a strong auxin response that is dependent on the canonical auxin signaling pathway (Karami et al., 2023a). Analysis of the auxin‐responsive reporters pDR5:GUS and pDR5:GFP (Benková et al., 2003; Ulmasov et al., 1997) showed that incubation of IZEs for 7 days on 2,4‐D medium induces a strong auxin response (Figure 1F; Figure S2), especially at the adaxial side of cotyledons on positions where embryonic cell clusters form (Karami et al., 2023b).…”

“…Many SE and de novo shoot regeneration protocols make use of the synthetic auxin 2,4‐D to induce SE because of its effectiveness in inducing plant regeneration. Our previous results and the results presented here show that 2,4‐D, as with natural auxins, acts through the canonical auxin response pathway (Karami et al., 2023a) and that the ‘secret’ of 2,4‐D's efficiency lies in it being an inefficient substrate for the auxin efflux machinery (Delbarre eta al., 1996; this work). Due to these properties, 2,4‐D is able to accumulate in the target cells to much higher levels than natural auxins and thus trigger a stronger (optimal) auxin response.…”

“…We used the efficient SE system of 2,4‐D‐treated Arabidopsis immature zygotic embryos (IZEs) (Gaj, 2001; Karami et al., 2023a,b) to investigate why 2,4‐D is a much more effective inducer of SE than natural auxins or other synthetic auxins. In this system, somatic embryos are derived from epidermal cells at the adaxial side of IZE cotyledons.…”

“…Although 2,4‐D is used in many SE protocols because of its efficient somatic embryo induction, these protocols produce many embryos that do not develop into normal plants. In our Arabidopsis SE protocol, IZE explants are cultured for 2 weeks on hormone‐containing medium to induce proembryonic masses (PEMS) and subsequently transferred to hormone‐free medium for 1 week to allow proper patterning and development of somatic embryos (Karami et al., 2023a,b). When we left the explants for an additional 2 weeks on hormone‐free medium, shoot development was observed, which lagged behind for 2,4‐D treated explants compared to those treated with natural auxins + NPA (Figure S4).…”

“…Previously we have shown that efficient SE induction on immature zygotic embryos of Arabidopsis thaliana (Arabidopsis) by 2,4‐D initially relies on the activation of the canonical auxin signaling pathway by 2,4‐D, but later requires the induction of endogenous auxin biosynthesis to maintain embryogenic cell identity (Karami et al., 2023a,b). Here we used the same experimental system to investigate the reason for the higher effectiveness of 2,4‐D as an SE inducer compared to natural auxins or other synthetic analogs.…”