Phase change refers to the process of maturation and transition from the juvenile to the adult stage. In response to this shift, certain species like chestnut lose the ability to form adventitious roots, thereby hindering the successful micropropagation of adult plants. While auxin is the main hormone involved in adventitious root formation, other hormones, such as ethylene, are also thought to play a role in its induction and development. In this study, experiments were carried out to determine the effects of ethylene on the induction and growth of adventitious roots. The analysis was performed in two types of chestnut microshoots derived from the same tree, a juvenile-like line with a high rooting ability derived from basal shoots (P2BS) and a line derived from crown branches (P2CR) with low rooting responses. By means of the application of compounds to modify ethylene content or inhibit its signalling, the potential involvement of this hormone in the induction of adventitious roots was analysed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterise the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. The findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modulation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour.

Wounding and exogenous auxin are needed to induce adventitious roots in chestnut microshoots. However, the specific inductive role of wounding has not been characterized in this species. In the present work, two main goals were established: First, we prompted to optimize exogenous auxin treatments to improve the overall health status of the shoots at the end of the rooting cycle. Second, we developed a time-series transcriptomic analysis to compare gene expression in response to wounding alone and wounding plus auxin, focusing on the early events within the first days after treatments. Results suggest that the expression of many genes involved in the rooting process is under direct or indirect control of both stimuli. However, specific levels of expression of relevant genes are only attained when both treatments are applied simultaneously, leading to the successful development of roots. In this sense, we have identified four transcription factors upregulated by auxin (CsLBD16, CsERF113, Cs22D and CsIAA6), with some of them also being induced by wounding. The highest expression levels of these genes occurred when wounding and auxin treatments were applied simultaneously, correlating with the rooting response of the shoots. The results of this work clarify the genetic nature of the wounding response in chestnut, its relation to adventitious rooting, and might be helpful in the development of more specific protocols for the vegetative propagation of this species.

Chestnut is a highly valuable species at both the ecological and economic levels, but vegetative propagation protocols have not been optimized for mature material due to its recalcitrant behaviour, thus limiting potential biotechnological applications. In this study, we focused on the formation of adventitious roots in mature chestnut microshoots, which show low rooting responses. Silver thiosulfate, an ethylene-signalling inhibitor, was applied as a pretreatment before auxin induction to study the role of ethylene in this developmental process. Rooting rate was significantly improved in response to the pretreatment, suggesting that ethylene negatively influences the induction of adventitious roots in mature shoots. Transcriptomics and real-time PCR analyses suggest that the improvement in the rooting response was mainly due to the activity of different auxin transport genes, whose expression seems to be repressed by ethylene. However, other hormones might also be negatively influencing rooting in mature shoots, although their specific role remains to be characterized.

Wounding and exogenous auxin are needed to induce adventitious roots in Chestnut microshoots. However, the specific inductive role of wounding has not been characterized in this species. In the present work two main goals were established: First, we prompted to optimize exogenous auxin treatments to improve overall health status of the shoots at the end of the rooting cycle. Second, we developed a time-series transcriptomic analysis to compare gene expression in response to wounding alone and wounding plus auxin, focusing in the early events within the first days after treatments. Results suggest that the expression of many genes involved in the rooting process is under direct or indirect control of both stimuli. However, specific levels of expression of relevant genes are only attained when both treatments are applied simultaneously, leading to the successful development of roots. In this sense, we have identified four transcription factors upregulated by auxin (CsLBD16, CsERF113, Cs22D, CsIAA6), with some of them being also induced by wounding. The highest expression levels of these genes occurred when wounding and auxin treatments were applied simultaneously, correlating with the rooting response of the shoots. Results in this work clarify the genetic nature of the wounding response in chestnut, its relation to adventitious rooting and might be helpful in the development of more specific protocols for the vegetative propagation of this species.

Vegetative propagation of recalcitrant woody species is a challenging task that limits biotechnological exploitation of economically relevant trees. In the case of chestnut, a severe decline in its ability to form adventitious roots is imposed during maturation. In previous reports, both juvenile-like and mature microshoots were subject to different hormonal treatments to gain deeper insights into the molecular processes driving the formation of adventitious roots while allowing us to increase our understanding of failed treatments. In the present work, RNAseq libraries from those treatments were used to develop a Weighted Gene Co-expression Network Analysis in order to identify gene modules correlated with the formation of adventitious roots. Hub genes within relevant modules were then used for promoter region analysis to identify transcription factor families that may play a role in the process by controlling the expression of the genes within those modules. bZIP and bHLH families in juvenile-like microshoots and WRKY and SBP families in mature microshoots were found to be particularly relevant for this developmental process. Specific transcription factors, like CsbHLH30 and CsbZIP44, seem to play key roles in the rooting process, while the activity of CsWRKY75 is putatively related to recalcitrant responses in mature shoots.