In Arabidopsis somatic embryogenesis (SE), the auxin response (DR5 reporters) marks the future shoot pole where WUSCHEL (WUS) is induced, while a cytokinin response (ARR7/ARR15 reporters, AHK-receptor dependent) becomes restricted to the opposite, basal region where WOX5 and PLETHORA establish the root apical meristem; disturbed cytokinin signaling disperses WOX5 expression and blocks embryo formation. Su et al. (2015) established these complementary hormone-response domains as correlates of early axis formation but left their causal mechanism open, closing with the statement that the mechanisms of hormonal regulation of SE initiation 'remain a major challenge for the future'. A decade of follow-up work has dissected auxin's embryo-induction function (auxin biosynthesis, chromatin rewiring, and the transcription factors LEAFY COTYLEDON 2 (LEC2), WRINKLED 1 (WRI1), and MONOPTEROS/AUXIN RESPONSE FACTOR 5 (MP/ARF5) isoforms) and staging of endogenous auxin requirements, but no study has identified the molecular mechanism that partitions auxin and cytokinin responses into opposite, stage-specific domains along the somatic embryo axis. The open problem is to determine that mechanism: what localizes and induces the cytokinin response in future root-meristem cells, what auxin–cytokinin crosstalk positions the two response domains at opposite poles and spatially restricts WOX5, and whether auxin's embryo-induction function is mechanistically separable from its later axis-patterning function.

Background

Somatic embryogenesis (SE) is the process by which differentiated plant cells cultured in vitro reprogram into totipotent cells that develop into embryos without fertilization; it is both a leading model for plant cell totipotency and the basis of clonal propagation and transformation systems. In a well-characterized Arabidopsis system (Su et al. 2009), immature zygotic embryos first form primary somatic embryos and then embryonic callus on medium containing the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D); transferring the callus to hormone-free embryo-inducing medium (SEIM) triggers secondary somatic embryos. Auxin and cytokinin are the two central plant growth regulators: auxin acts through its TIR1/AFB (transport inhibitor response 1/auxin signaling F-box) receptors and AUXIN RESPONSE FACTOR (ARF) transcription factors, and its local activity is visualized by the DR5 reporter, a synthetic auxin-responsive promoter construct; cytokinin acts through histidine-kinase receptors (AHK2, AHK3, AHK4) and a phosphorelay that induces type-A ARABIDOPSIS RESPONSE REGULATOR genes (ARR7, ARR15), which feed back negatively on the pathway, so ARR7/ARR15 promoter activity serves as a cytokinin-response reporter. The embryonic shoot apical meristem (SAM) is specified by WUS expression (WUSCHEL, a homeobox transcription factor maintaining stem cells) and the root apical meristem (RAM) by WOX5 (a WUS-related homeobox gene active in the quiescent center, the mitotically inactive organizing center of the root meristem) together with PLETHORA (PLT) and SCARECROW (SCR) transcription factors; together they define the apical–basal (shoot–root) axis of the embryo. In zygotic embryogenesis, auxin signaling positions the root stem-cell niche by directly inducing ARR7/ARR15 to repress cytokinin response in the basal lineage (Müller and Sheen 2008), and in de novo shoot organogenesis ARF3/ETTIN shapes complementary auxin and cytokinin response domains by repressing cytokinin biosynthesis (Cheng et al. 2013). For SE, Su et al. (2009) had shown that auxin-induced WUS expression and PIN-mediated auxin gradients (PIN proteins are plasma-membrane auxin efflux carriers that polarize auxin flow) are required to trigger SE, and Su et al. (2015) then found that WUS and WOX5 are activated nearly simultaneously in overlapping callus-edge cells within ~24 h of transfer to SEIM, that the auxin response occupies the apical (WUS/future-SAM) region and is absent from the early basal region (later reappearing basally overlapping PLT2), and that the cytokinin response instead becomes restricted to the basal region overlapping WOX5, at the pole opposite WUS. Overexpressing ARR7 or ARR15 (attenuating cytokinin signaling) or mutating the AHK receptors disperses WOX5 expression and severely impairs RAM initiation and SE formation, establishing necessity but not mechanism: which upstream inputs create and position the two hormone-response domains, and how the two hormones interact to do so, was left unresolved.

Problem Statement

Determine the molecular mechanism by which auxin and cytokinin responses become partitioned into complementary stage- and domain-specific patterns during somatic embryogenesis, so that the embryonic shoot–root axis is established. Concretely, in the Arabidopsis SE system where the question was defined (embryonic callus transferred from 2,4-D-containing medium to hormone-free embryo-inducing medium) or an equivalent SE system, the answer should establish: (i) what upstream inputs — cytokinin biosynthesis, transport, perception, or signal transduction, and their regulation upon withdrawal of exogenous auxin — create and restrict the cytokinin response (ARR7/ARR15-reported, AHK-dependent) to the basal callus cells that will form the root apical meristem, where it confines WOX5 expression; (ii) what crosstalk between auxin and cytokinin signaling positions the auxin response (DR5-reported) at the apical, WUS-expressing future shoot pole early in induction and later redistributes it to the basal PLT-expressing domain, i.e. how the two hormone-response domains come to occupy opposite poles of the nascent embryo; and (iii) whether auxin's embryo-induction function (triggering WUS expression and SE initiation) is mechanistically separable from its later axis-patterning function. A complete answer identifies the molecular components and causal regulatory interactions, and accounts for the established observations that WUS and WOX5 are first co-induced in nearly overlapping cells, that the auxin response is initially excluded from the early basal region, and that attenuated cytokinin signaling (ARR7/ARR15 overexpression, ahk receptor double mutants) disperses WOX5 and blocks RAM initiation and SE formation.

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