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13 páginas, 5 figuras, Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids., This work was supported by a research contract from Phytoplant Research (Ref. 20190548), and by the Xunta de Galicia (Spain) through the projects IN607A 2021 and “Contrato Programa” 2021 (AGI/CSIC I+D+I 2021, Ref- ACAM 20210200033).

20 páginas, 6 figuras, Maturation imposes several changes in plants, which are particularly drastic in the case of trees. In recalcitrant woody species, such as chestnut (Castanea sativa Mill.), one of the major maturation-related shifts is the loss of the ability to form adventitious roots in response to auxin treatment as the plant ages. To analyze the molecular mechanisms underlying this phenomenon, an in vitro model system of two different lines of microshoots derived from the same field-grown tree was established. While juvenile-like shoots root readily when treated with exogenous auxin, microshoots established from the crown of the tree rarely form roots. In the present study, a transcriptomic analysis was developed to compare the gene expression patterns in both types of shoots 24 h after hormone and wounding treatment, matching the induction phase of the process. Our results support the hypothesis that the inability of adult chestnut tissues to respond to the inductive treatment relies in a deep change of gene expression imposed by maturation that results in a significant transcriptome modification. Differences in phytohormone signaling seem to be the main cause for the recalcitrant behavior of mature shoots, with abscisic acid and ethylene negatively influencing the rooting ability of the chestnut plants. We have identified a set of related MADS-box genes whose expression is modified but not suppressed by the inductive treatment in mature shoots, suggesting a putative link of their activity with the rooting-recalcitrant behavior of this material. Overall, distinct maturation-derived auxin sensibility and homeostasis, and the related modifications in the balance with other phytohormones, seem to govern the outcome of the process in each type of shoots., This work was funded by Xunta de Galicia (Spain) through the projects IN607A and “Contrato Programa” 2021 (AGI/CSIC I + D + I 2021, Ref-ACAM 20210200033), and also by the CSIC program I-COOP + 2020 (RefCOOPB20584).

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, β-A and β-H, all supplemented with 2-μM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in β-A medium supplemented with 2-μM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species., This work was supported by a research contract from Phytoplant Research (Ref. 20190548) and by the Xunta de Galicia (Spain) through the projects IN607A 2021/06 and Contrato Programa 2021 (AGI/CSIC I+D+I 2021, Ref- ACAM 20210200033).

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.

In eukaryotes, trithorax group proteins play critical roles in the regulation of transcription, cell proliferation, differentiation and development. In this work we report the molecular cloning and characterization of SEPR11, a cDNA from the conifer Monterrey pine (Pinus radiata) encoding a polypeptide homologue of a trithorax group member described in animals and yeast. A full-length clone was isolated from RNA prepared from somatic embryos and contained a 1,239 bp ORF encoding 412 amino acids. Characterization of the isolated sequence revealed that it contains a SPRY domain in the C-terminal region. A comparison of the pine sequence with homologous proteins from plants, animals and yeast revealed that SEPR11 is phylogenetically related to the trithorax group members and not a SPRY-domain containing protein. RT-PCR analyses of transcript abundance in pine tissues demonstrated that SEPR11 is particularly abundant in embryos, suggesting that this gene could be involved during embryo development. The spatial localization of SEPR11 transcripts revealed that gene expression was restricted to the vascular bundle and apical and radicular meristems, suggesting a possible function of this gene in meristem control and vascular bundle development. This work is the first report of the presence of a trithorax group homologue gene in gymnosperm.

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.