During somatic embryogenesis (SE) induction from cultured Arabidopsis immature zygotic embryo (IZE) explants, local expression of YUCCA (YUC) flavin-monooxygenase genes — and thus local de novo auxin biosynthesis — is required, and it is enabled by a concurrent down-regulation of ethylene biosynthesis and ethylene responses. However, the classical ethylene-insensitive mutants etr1-3 and ein2-1 and the ACC (1-aminocyclopropane-1-carboxylic acid, the ethylene precursor) synthase double mutant acs2-1 acs6-1 did not significantly alter SE frequency, leaving unknown which ethylene perception and signaling components actually mediate the repression of YUC transcription; the molecular mechanism of that repression (direct action of ethylene-responsive transcription factors such as EIN3/EIL1 on YUC promoters versus indirect hormonal crosstalk) is undetermined; the functional relevance of the accompanying down-regulation of cytokinin and abscisic acid metabolism/signaling genes is untested; and whether this ethylene-repression module operates in other species and other SE culture regimes — where ethylene can instead promote SE — remains unresolved. The problem is to identify the ethylene-signaling pathway that gates YUC-dependent auxin biosynthesis during SE initiation and to establish the molecular mechanism of that repression.

Background

Somatic embryogenesis (SE) is the process by which somatic (vegetative) plant cells cultured in vitro are reprogrammed into embryo-like structures that can develop into whole plants, and it is a principal experimental model of plant cell totipotency as well as the basis of clonal propagation and transformation protocols. A standard Arabidopsis SE system uses immature zygotic embryos (IZEs) excised from developing seeds as explants: a first period on medium containing the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) produces embryogenic tissue, and transfer to auxin-free medium then triggers somatic embryo formation. Key to this second phase is local biosynthesis of the natural auxin indole-3-acetic acid (IAA) by YUCCA (YUC) genes, which encode flavin monooxygenases of the indole-3-pyruvic acid (IPA) auxin biosynthesis pathway; in this system YUC1, YUC4, and YUC10 are activated at the sites where somatic embryos form. Bai et al. (2013) showed that YUC function is required for SE induction and that ethylene acts as a negative regulator of this step: ethylene levels fall progressively during SE initiation, whereas application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene overproducer mutation eto1, or the constitutive ethylene-response mutation ctr1 disrupts SE and inhibits YUC expression. Here eto1 affects an ACC synthase regulator (ethylene overproduction 1), and CTR1 (constitutive triple response 1) is a kinase that, in the canonical ethylene signal transduction pathway, represses downstream signaling when ethylene is absent; the canonical pathway proceeds from five receptor proteins (of which ETR1 is one) through CTR1 and the membrane protein EIN2 (ethylene insensitive 2) to the EIN3 (ethylene insensitive 3)/EIL1 (EIN3-like 1) family of transcription factors, whose stabilization by ethylene activates ethylene responses such as the seedling triple response (the ethylene-induced seedling morphology of a short, thickened hypocotyl and root). The puzzle left by the study is that single ethylene-signaling mutants (etr1-3, ein2-1) and the ACC-synthase double mutant acs2-1 acs6-1 did not significantly affect SE regeneration frequency, so the components that actually transduce the ethylene signal to repress YUC transcription during SE initiation were not identified, and no direct molecular link between ethylene signaling and the YUC promoters was demonstrated. The same microarray study found cytokinin and abscisic acid (ABA) metabolism/signaling genes down-regulated at SE initiation, but their functional role was not tested. Later work makes the question sharper rather than moot: in the same IZE system, nitric-oxide/phytoglobin (phytoglobins are plant hemoglobins that scavenge nitric oxide) manipulations that alter ethylene levels change both SE output and YUC4/IAA accumulation, and the ethylene-insensitive mutants ein2-1 and ein3-1 were reported by another laboratory to increase SE output — an unreconciled discrepancy with the null results of Bai et al.; conversely, in an Arabidopsis seed-derived SE regime under continuous 2,4-D and in species such as Coffea arabica, ethylene signaling promotes SE, with EIN3/EIL1 directly activating the embryonic-identity genes LEAFY COTYLEDON1/2 (LEC1/LEC2) rather than repressing YUC. Understanding this node matters because the ethylene–auxin crosstalk gating local auxin biosynthesis is one of the decisive switches of SE induction and of plant regeneration capacity in vitro.

Problem Statement

The problem is to determine the regulatory mechanism by which ethylene gates local YUCCA-mediated auxin biosynthesis during somatic embryogenesis (SE) induction in Arabidopsis — the open mechanistic gap left by the findings of Bai et al. (2013), whose published results demonstrate that YUC-dependent local auxin biosynthesis is required for SE induction, that ethylene acts as a negative regulator of this step, and that ethylene inhibits YUC expression, yet do not identify the components or the molecular mechanism of that repression: (1) identify the ethylene perception and signaling components — among the five ethylene receptors, downstream effectors such as EIN2/EIN3/EIL1, and ACC-synthase isoforms — that actually mediate the negative regulation of YUC1/YUC4/YUC10 transcription during SE initiation, and explain why the single mutants etr1-3 and ein2-1 and the ACC-synthase double mutant acs2-1 acs6-1 did not significantly change SE regeneration frequency while ACC treatment, eto1, and ctr1 suppress SE, including reconciling or refuting the conflicting reports for ein2-1/ein3-1 in the same IZE system; and (2) establish whether ethylene-dependent repression of YUC transcription is direct (binding of an ethylene-responsive transcription factor, e.g. EIN3/EIL1 or an ERF — ethylene response factor, a member of the AP2/ERF family of plant transcription factors, many of which act downstream of ethylene signaling — to YUC promoters in the SE-initiating tissue) or indirect via crosstalk with other hormonal pathways. An answer should specify the mediating components — accounting for the null versus suppressive mutant and chemical phenotypes, including reconciliation or refutation of the conflicting ein2-1/ein3-1 reports — together with the directness and molecular targets of the repression. Both parts (1) and (2) are required: a response that resolves only one of them does not resolve the problem. The functional relevance of the cytokinin and ABA metabolism/signaling changes and the operation of this repression module in other species or other SE culture regimes — where ethylene can instead promote SE — are background context for the problem and are not required for a complete answer.

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