Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus

The clone EMB-2 of the interspecific hybrid Helianthus annuus H. tuberosus providesan interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, inaddition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leavesbearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression ofH. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishmentof embryogenic competence.† Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. Theendogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gaschromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach.† Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EPleaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves wereinduced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather thanin absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal wasdetected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied theearly stages of embryo development. Changes in auxin level and distribution were observed in NEP leavesinduced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcriptlevels in spite of an enhancement of the regeneration frequency.†Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although theectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated thatlocalized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somaticembryogenesis displayed by the epiphyllous EMB-2 clone.