Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

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

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). Its effectiveness 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 auxinic activity. However, this hypothesis has never been thoroughly tested. Here we used a library of forty 2,4-D analogues to test the structure-activity relationship with respect to the capacity to induce SE and auxinic 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 to not active auxin analogues. A halogen at the 4-position of the aromatic ring was important for auxinic activity, whereas a halogen at the 3-position resulted in reduced activity. Moreover, a small substitution at the carboxylate chain was tolerated, as was extending the carboxylate chain with an even number of carbons. The auxinic activity of most 2,4-D analogues was consistent with their simulated TIR1-Aux/IAA coreceptor binding characteristics. A strong correlation was observed between SE induction efficiency and auxinic activity, which is in line with our observation that 2,4-D-induced SE and stress both require TIR1/AFB auxin co-receptor function. Our data indicate that the stress-related effects triggered by 2,4-D and considered important for SE induction are downstream of auxin signalling.

Regulatory networks of hormone-involved transcription factors and their downstream pathways during somatic embryogenesis of Arabidopsis thaliana.

Somatic embryogenesis (SE) depends on a variety of developmental pathways that are influenced by several environmental factors. Therefore, it is important to understand the relationship between environmental and genetic factors by identifying the gene networks involved in SE through gene set enrichment analysis (GSEA). For determination of SE effective transcription factors, upstream sequences of core-enriched genes were analyzed. The results indicated that response to hormones is one of the biological pathways activated by the enriched TFs at all stages of somatic embryogenesis and about half of the hormonal pathways were enriched. On the fifth day after 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment, the activity of hormone-affecting genes reached its maximum. At this time, more transcription factors regulated the enriched genes compared to the other stages of somatic embryogenesis. MYBs, AT-HOOKs, and HSFs are the main families of transcription factors which affect core-enriched genes during SE. CCA1, PRR7, and TOC1 and their related genes at the center of protein-protein interaction of SE-key transcription factors, involved in the regulation of the circadian clock. Gene expression analysis of CCA1, PRR7, and TOC1 revealed that the genes involved in circadian clock reached their maximum activity when embryonic cells formed. Also, auxin response elements were identified at the upstream of SE-circadian clock transcription factors, indicating that they might mediate between auxin signaling and SE-related hormonal pathways as well as SE marker genes such as AGL15, BBM, and LECs. Based on these results, it is possible that the cellular circadian rhythm activates various developmental pathways under the influence of auxin signal transduction and their interactions determine the induction of somatic embryogenesis. According to the results of this study, modifying pathways affected by SE-related transcription factors such as circadian rhythm may result in cell reprogramming and increase somatic embryogenesis efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03546-7.