Somatic embryogenesis (SE) — the development of embryos from cultured somatic tissues — proceeds through genetically separable stages: acquisition of embryo identity, formation of multicellular proembryos, and patterning of the somatic embryo. Each stage has distinct auxin transport requirements, yet the molecular identity of the auxin influx and efflux carriers operating at each stage is only partially defined. In the Arabidopsis immature zygotic embryo (IZE) SE systems induced by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of the AHL15 (AT-HOOK MOTIF NUCLEAR LOCALIZED 15) transcription factor, only PIN1 (efflux) and AUX1 (influx) have been directly detected among tested reporters, with LAX2/LAX3 involvement inferred only genetically; work from 2024 identified PIN1, PIN2, ABCB1 and ABCB19 as the efflux carriers whose depletion of natural auxins limits SE induction, explaining why 2,4-D (a poor efflux substrate that accumulates intracellularly) is a stronger inducer than indole-3-acetic acid (IAA), the natural auxin. What remains undefined is the complete, stage-resolved transporter complement — including PIN3/PIN6, LAX2/LAX3, endoplasmic-reticulum (ER)-localized PIN-LIKES (PILS) carriers, and notably the bidirectional, auxin-concentration-dependent transporter ABCB21, whose role in SE has never been examined in Arabidopsis — together with a mechanistic account of why sustained 2,4-D arrests development at the proembryo stage while endogenous IAA maintains embryonic identity and supports patterning. The problem is to define this machinery stage by stage without presupposing any particular carrier family.

Background

Somatic embryogenesis (SE) is the process by which differentiated somatic cells cultured in vitro reacquire embryonic identity — an expression of plant cell totipotency — and develop into embryos without fertilization. In the model system built on Arabidopsis immature zygotic embryos (IZEs), SE can be induced either by culturing explants on medium containing the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of the transcription factor AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Auxin (principally the natural hormone indole-3-acetic acid, IAA) accumulates asymmetrically in tissues through polar auxin transport, which is mediated by plasma-membrane carriers: PIN-FORMED (PIN) proteins are asymmetrically localized efflux carriers that give auxin transport its direction; AUXIN1/LIKE-AUX1 (AUX1/LAX) proteins are influx permeases that import auxin into cells; ATP-binding cassette subfamily B (ABCB) transporters are a second class of efflux (and, for some members, conditionally bidirectional) carriers; and PIN-LIKES (PILS) proteins are endoplasmic-reticulum (ER)-localized putative carriers that buffer intracellular auxin availability. Classic pharmacology uses N-1-naphthylphthalamic acid (NPA) to block PIN/ABCB-mediated efflux and 1-naphthoxyacetic acid (1-NOA) to block AUX1/LAX-mediated influx. Karami et al. (2023a) used the embryonic-cell-identity reporter pWOX2:NLS-YFP (WOX2, a WUSCHEL-related homeobox gene marker of embryonic cells, fused to nuclear yellow fluorescent protein) and the auxin-response reporter pDR5:GFP to distinguish three SE stages — (i) acquisition of embryo identity, (ii) formation of proembryos, and (iii) somatic embryo patterning — and showed that endogenous IAA biosynthesis and auxin influx are dispensable for stage (i) but required to maintain embryonic identity and form proembryos, while both influx and PIN1-mediated efflux are required for stage (iii) patterning. Among the reporter lines they examined (PIN1, PIN2, PIN4, PIN7, AUX1, LAX1), only PIN1 and AUX1 were detected during SE; genetic tests with aux1 lax double mutants suggested AUX1 acts redundantly with LAX2 and LAX3, whose expression was never directly examined. The same paper explicitly asked whether PIN1 is the only efflux carrier operating during SE, noting that PIN3 and ABC-family efflux transporters had not been tested, and posed two mechanistic questions: how 2,4-D can trigger embryo-identity acquisition if endogenous auxin is not needed for it, and why 2,4-D itself cannot maintain embryonic identity the way endogenous IAA does. A parallel line of work in Lilium pumilum motivates the same gap from the transporter side: Song et al. (2020a) showed by transcriptomics that ABCB, PIN/PILS and AUX/LAX family genes are dynamically regulated during Lilium SE, and Song et al. (2020b) provided preliminary gain- and loss-of-function evidence that LpABCB21 (a homolog of the Arabidopsis bidirectional transporter ABCB21) regulates the timing of early somatic embryo formation while LpPILS7 affects induction efficiency. ABCB21 is of particular interest because, as characterized by Kamimoto et al. (2012) and Jenness et al. (2019), it is a facultative auxin importer/exporter whose transport direction switches with cytoplasmic auxin concentration and which regulates auxin levels in cotyledons, root pericycle and leaves — properties directly relevant to a developmental process that hinges on precise intracellular auxin thresholds, yet its role in SE has never been examined in Arabidopsis. Subsequent work by the same Leiden group partially resolved the initiation-stage picture: a structure–activity analysis of forty 2,4-D analogues showed that SE induction correlates with auxinic activity mediated by the TIR1/AFB (TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX) family of intracellular auxin co-receptors rather than with stress effects (Karami et al. 2023b), and an efflux-inhibition study (Karami et al. 2024) demonstrated that natural auxins become efficient SE inducers when efflux is transiently blocked by NPA, that pin2 and abcb1 abcb19 mutants show enhanced SE under IAA treatment, and that 2,4-D's effectiveness rests on its being a poor substrate for efflux (PIN1, PIN2, ABCB1, ABCB19), causing intracellular accumulation. These advances define the induction-stage efflux logic but leave the complete stage-resolved transporter complement — influx carriers, later-stage efflux carriers, ABCB21, PILS, and direct transport-activity measurements in embryogenic tissue — undefined.

Problem Statement

Define the stage-specific auxin transport machinery of somatic embryogenesis, without presupposing any particular carrier family. In the Arabidopsis immature zygotic embryo (IZE) systems where SE is induced by 2,4-D or by AHL15 (AT-HOOK MOTIF NUCLEAR LOCALIZED 15) overexpression — and, as a comparative extension, in other species' SE systems such as Lilium pumilum — determine, for each of the three recognized stages (acquisition of embryo identity; formation of multicellular proembryos; patterning and differentiation of the somatic embryo), the complete complement of auxin influx and efflux carriers that operate in the embryogenic tissue. This includes establishing, by direct expression/protein data and functional tests rather than inference, which PIN proteins beyond PIN1 (e.g., PIN2, PIN3, PIN6), which AUX1/LAX influx carriers (LAX1, LAX2, LAX3), which ABCB transporters (ABCB1, ABCB4, ABCB14, ABCB19, and in particular the facultative importer/exporter ABCB21, whose bidirectional, cytoplasmic-auxin-concentration-dependent transport makes it a specifically plausible but unexamined component), and which ER-localized PILS carriers are present, where they localize, and what they transport at each stage. The definition should establish whether and how the stage-resolved transporter complement accounts for the observed behavioral asymmetry between exogenous 2,4-D and endogenous IAA — in particular why sustained 2,4-D application induces embryo-identity acquisition but arrests development at the proembryo stage until 2,4-D removal, while endogenous IAA, acting with AUX1-mediated influx, maintains embryonic identity and supports patterning and differentiation. The induction-stage portion of this question has been partially resolved (PIN1, PIN2, ABCB1 and ABCB19 deplete natural auxins during induction; 2,4-D accumulates because it is a poor efflux substrate); the surviving task is the complete stage-resolved machinery and its mechanistic consequences.

References

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