Auxin-mediated expression of a GH3 gene in relation to ontogenic state in Chestnut

The CsGH3 - 1 gene was isolated from chestnut microshoots. Expression analysis during induction of adventitious roots indicates an alteration in shoot auxin homeostasis during maturation that negatively affects adventitious rooting. A new auxin inducible gene isolated from chestnut microshoots was found to encode a protein belonging to group II of the Gretchen Hagen 3 (GH3) family. The gene was, therefore, named CsGH3-1. Predicted protein sequence analysis revealed the presence of conserved domains involved in the conjugation of amino acids to indole-3-acetic acid (IAA). Modelling of the protein and molecular docking of IAA, indole-3-butyric-acid (IBA), 1-naphthaleneacetic acid (NAA), and benzothiazole-2-oxyacetic acid (BTOA) into the active site of CsGH3-1 indicated a high and similar binding affinity for the four substrates. Expression analysis by qPCR indicated that CsGH3-1 is regulated by wounding, darkness, and auxin in chestnut microshoots, in an ontogenetic-dependent manner. Under IBA treatment, upregulation of CsGH3-1 was higher in mature than in juvenile shoots and was negatively correlated with the ability of microshoots to form roots. High levels of auxin-induced expression of CsGH3-1 were detected in mature shoots 24 h after the IBA treatment, whereas transcript levels decreased in rooting-competent shoots with cell-type-specific expression. CsGH3-1 transcripts were specifically localized in cells involved in the initiation of adventitious roots only in rooting-competent shoots at the time when cells switch their fate to root initial cells. Thus, these data show a correlation between the specific localization of transcripts and rooting competence, and they also suggest a role for CsGH3-1 in regulating auxin homeostasis during the onset of adventitious root formation.