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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.

Plant GH3 proteins are a group of enzymes whose activity is tightly linked with hormone homeostasis and signaling pathways, and therefore their complete characterization is of major relevance for a better understanding of the influence of hormones in plant responses and development programs. According to sequence similarities and substrate specificities, the family is divided in three major subfamilies. In the present work, we have identified a new GH3 Subfamily (IV) with specific sequence characteristics that suggests activity over non-yet described substrates. Our results show that this Subfamily is absent in relevant species like Arabidopsis or maize. The gene promoter analysis of members of this Subfamily in several woody species implies that the activity of these proteins is mainly related to light responses, biotic and abiotic stresses, among other processes. Characterization of the substrates of these enzymes is necessary for a complete understanding of the activity of the GH3 family.

The present study investigated how auxin concentration and the method of application affected the formation of adventitious roots in microshoots of chestnut (Castanea sativa) and oak (Quercus robur). The activity of two urea derivatives (2, 3-MDPU and 3, 4-MDPU) was also evaluated. Microshoots were derived from basal sprouts of two mature chestnut trees (P1 and P2) and one adult oak genotype (Sainza). In chestnut, rooting percentage was positively affected by auxin in a dose -dependent manner, particularly in shoots treated with the hormone for 24 h. The effect of auxin on rooting also differed depending on the application method. In shoots treated for 24 h, the highest concentration of auxin produced the healthiest rooted plantlets, in terms of the root system and shoot quality. By contrast, in shoots treated by the basal quick-dip method, the shoot quality was best at the lowest auxin concentration. The effect of urea derivatives on the root system depended on the species as well as on the auxin concentration and application period. Use of the MDPUs improved the root system architecture of auxintreated shoots by promoting lateral root development and triggering the synchronous initiation of root primordia at the base of the shoot. Shoot quality was also improved by MDPUs, which promoted resumption of growth and reduced shoot-tip necrosis.

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

Trabajo presentado en el 11Congresso Ibérico do Castanheira = II Iberian Congress on Chestnut, celebrado en Vila Real (Portugal), del 20 al 22 de junio de 2007, We have isolated a full-length cDNA (CsCPE) from chestnut somatic embryos and analyzed its expression in zygotic and somatic embryos at different developmental stages. The biological features of the CsCPE-encoded protein are very similar to the putative protein encoded by the oak QrCPE cDNA. The highest accumulation of CsCPE mRNA was detected at the globular stage of somatic embryos and also at early developmental stages of zygotic embryos, with the lowest levels being detected at the early cotyledonary stage. Abundant levels of mRNA were also detected in roots in comparison to seedlings shoots. These results indicate that the CsCPE gene is involved in somatic embryogenesis and seems to be developmentally regulated., This work has been supportcd by grants of Xunta de Galicia (PGIDIT06PXIB400003PR) and Spanish Ministry of Education and Science (AGL 2006O 1387/FOR)

This paper reports for the first time the identification of SCARECROW-LIKE genes from two distantly-related forest species, Pinus radiata and Castanea sativa, which are auxin-induced in rooting-competent cuttings before the activation of cell division that gives rise to adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family, and show a strong similarity to the SCARECROW-LIKE proteins, which indicates conserved functions of these proteins. Quantitative RT-PCR analysis shows that these genes are expressed at relatively high levels in roots. In addition, an induction of the mRNA levels in rooting-competent cuttings from P. radiata and C. sativa in response to exogenous auxin is observed within the first 24 h of the root induction process, a time when cell reorganization takes place, prior to the resumption of cell division and to the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes may play a role at very early stages of adventitious root formation., Ministry of Education and Science (Spain) projects: (AGL2002-04225-CO2-01 AGR/FOR to C.S. and AGL2002-04225-CO2-02 AGR/FOR to C.D.-S.) Xunta de Galicia (Spain) project:(PGIDT03PXIC40002P)

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT-ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT-ROOT (SHR) proteins. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings., Spanish Ministry of Education and Science (AGL2002-04225-CO2-01 AGR/FOR and AGL2005-07228-CO2-02 AGR/FOR to C.S. and AGL2002-04225-CO2-02 AGR/FOR and AGL2005-07228-CO2-01 AGR/FOR to C.D.-S.) Partially supported by Xunta de Galicia (PGIDIT06PXIB400003PR to C.S.) University of Alcala (UAHCG2006-028 to C.D.-S.).