A 2018 mass-spectrometric atlas followed more than 30 endogenous phytohormones — including the non-indole auxin phenylacetic acid (PAA), indole-3-acetic acid (IAA) and its conjugates, cytokinins, abscisic acid, jasmonates, salicylic acid (SA), and benzoic acid (BzA) — through proliferation, maturation, desiccation, and germination of Norway spruce (Picea abies) somatic embryos, but could only correlate hormone levels with developmental events. The source explicitly leaves open why PAA, the most abundant auxin, shows a temporal profile distinct from indole auxins (peaking at maturation week 3 and rising sharply during germination while IAA stays low) and what developmental roles it fulfils; which molecular mechanisms mediate the auxin–cytokinin interactions governing meristem formation and organ differentiation; what biological function the high SA levels during proliferation and high BzA levels during maturation serve; and which transporters, receptors, and downstream targets interpret the measured hormonal changes. Later literature (2019–2026) has advanced PAA metabolism in model plants and produced transcriptomic resources for spruce embryogenesis, but no work assigns causal developmental roles to these endogenous hormone dynamics in conifer somatic embryogenesis. The problem is to convert the correlative hormone atlas into a mechanistic, gene-anchored account of how the profiled hormones drive, or permit, each stage of conifer somatic embryogenesis.

Background

Somatic embryogenesis (SE) is an in vitro process in which somatic plant cells are reprogrammed into embryos that develop into plants; in conifers it proceeds from a proliferating embryogenic suspensor mass (ESM, a culture of early embryos with meristematic heads and suspensor cells) through maturation, desiccation, and germination, and it is the main route for scaling up elite Norway spruce (Picea abies) planting stock. Auxins are plant hormones that control cell division and differentiation; the best-known auxin is indole-3-acetic acid (IAA), which is actively moved through tissues by polar auxin transport (directed carrier-mediated transport that forms concentration gradients) and is perceived through the TIR1/AFB receptor family (Transport Inhibitor Response 1/Auxin-signaling F-Box proteins, the F-box receptors whose binding of auxin activates auxin-responsive gene expression). Phenylacetic acid (PAA) is a phenylalanine-derived, non-indole natural auxin: it regulates an overlapping set of genes via the same TIR1/AFB pathway, is often more abundant than IAA endogenously, but is much weaker in activity and, unlike IAA, does not undergo polar transport (Sugawara et al. 2015). Cytokinins (CKs) are cell-division-promoting hormones; the auxin/CK ratio is decisive for embryo meristem and organ formation (Su et al. 2011), yet the molecular mechanisms of this interaction remain poorly characterized. Abscisic acid (ABA) dominates maturation; salicylic acid (SA) and benzoic acid (BzA) are phenolic compounds with roles in defense signaling and, for SA, established immunity functions. Using HPLC-ESI-MS/MS (high-performance liquid chromatography coupled to tandem mass spectrometry), Vondráková et al. (2018) provided the most comprehensive hormone atlas of conifer SE to date. Striking unexplained observations included: PAA was the most abundant auxin (up to ~12,650 pmol/g dry weight) with maxima at week 3 of maturation (when embryos polarize) and again during germination, while indole auxins stayed low; the auxin/CK ratio was high during maturation and very low during desiccation; SA was high during proliferation and BzA peaked at maturation week 3, correlating with auxins; and massive exogenous 2,4-D (a synthetic auxin used in proliferation medium) accumulated in the cultures. The authors stated that the role of SA and BzA during proliferation 'remains unclear and has yet to be elucidated', that 'the molecular mechanisms underlying auxin–CK interactions remain largely unknown', and that the distinct biosynthetic, transport and degradation pathways of PAA versus indole auxins 'may underlie their contrasting concentration changes during Norway spruce embryo germination' — a suggestion, not a demonstrated mechanism. Newer model-plant work clarified pieces of PAA biology: PAA biosynthesis proceeds through phenylalanine-derived routes distinct from IAA biosynthesis, PAA and IAA share core conjugation machinery (GH3 acyl-amido synthetases — GRETCHEN HAGEN3 enzymes that conjugate auxins to amino acids — and newly found PAA-amino acid and PAA-glucose conjugates; Hladík et al. 2025), and a 2023 review concludes that PAA homeostasis and especially its physiological roles remain poorly understood (Perez et al. 2023). None of this work engages conifer SE. In spruce itself, follow-up studies have re-profiled hormones during desiccation (Eliášová et al. 2022), shown that different exogenous auxins generate different SE outcomes in two Picea species (Hazubska-Przybył et al. 2020), and built transcriptome and single-nucleus RNA-seq atlases of spruce embryogenesis (Stojkovič et al. 2024; Zhu et al. 2026) that expose expression of hormone-related genes such as PIN-FORMED (PIN) auxin efflux transporters — but no study has causally connected the endogenous hormone dynamics to developmental outcomes.

Problem Statement

The question, as posed by Vondráková et al. (2018) and still unanswered, is to convert their correlative phytohormone atlas of Norway spruce somatic embryogenesis into a causal, mechanistic account connecting endogenous hormone dynamics to developmental outcomes. Its linked components are: (1) establish the functional distinction between the non-indole auxin phenylacetic acid (PAA) and indole auxins during conifer somatic embryogenesis — determine which specific developmental roles PAA fulfils, whether its sharp maxima at maturation week 3 and during germination (while indole auxins remain low) are required for embryo polarization, germination, and growth of emblings (plantlets regenerated from somatic embryos), and which biosynthetic, transport, and degradation mechanisms produce its temporal profile; (2) identify the molecular mechanisms mediating the auxin–cytokinin interactions, and in particular the high auxin/CK ratio at maturation, that govern meristem formation and organ differentiation in conifer embryos; (3) determine the biological function of the high endogenous salicylic acid concentrations during proliferation and benzoic acid during maturation, including their possible impact on embryo immunity, stress tolerance, or developmental signaling; and (4) identify the transporters, receptors, and downstream target genes that interpret the measured hormonal changes, so that the connection between the hormone profiles and developmental outcomes across proliferation, maturation, desiccation, and germination is mechanistically closed. A complete answer establishes, by genetic, pharmacological, or biochemical causal evidence in the conifer SE system (or an equivalent conifer embryogenesis system), which hormone species and which molecular components are necessary and/or sufficient for the staged developmental transitions that the 2018 profiles track. Each of the four components may be investigated independently, but all four are jointly required for a complete answer; resolving a subset of them constitutes a partial result rather than a complete resolution.

References

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