title: Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus.
abstract: OBJECTIVE
  The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence.
  
  
  METHODS
  Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach.
  
  
  RESULTS
  Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels 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 the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.
authors.affiliation: Università della Calabria, Dipartimento di Ecologia, Via P. Bucci, I-87036 Arcavacata di Rende (CS), Italy.
authors.forename: A
authors.initials: A
authors.lastname: Chiappetta
authors.forename: M
authors.initials: M
authors.lastname: Fambrini
authors.forename: M
authors.initials: M
authors.lastname: Petrarulo
authors.forename: F
authors.initials: F
authors.lastname: Rapparini
authors.forename: V
authors.initials: V
authors.lastname: Michelotti
authors.forename: L
authors.initials: L
authors.lastname: Bruno
authors.forename: M
authors.initials: M
authors.lastname: Greco
authors.forename: R
authors.initials: R
authors.lastname: Baraldi
authors.forename: M
authors.initials: M
authors.lastname: Salvini
authors.forename: C
authors.initials: C
authors.lastname: Pugliesi
authors.forename: M B
authors.initials: MB
authors.lastname: Bitonti
mesh_terms: D003433:Crosses, Genetic; D047108:Embryonic Development / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant; D006368:Helianthus / Q000187:drug effects / Q000196:embryology* / Q000235:genetics*; D006824:Hybridization, Genetic / Q000187:drug effects; D007210:Indoleacetic Acids / Q000378:metabolism* / Q000494:pharmacology; D019441:Plant Epidermis / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D018515:Plant Leaves / Q000166:cytology / Q000187:drug effects / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012038:Regeneration / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D007210:Indoleacetic Acids; D010940:Plant Proteins; D012333:RNA, Messenger