WUSCHEL-RELATED HOMEOBOX (WOX) genes are key players controlling stem cells in plants and can be divided into three clades according to the time of their appearance during plant evolution. Our knowledge of stem cell function in vascular plants other than angiosperms is limited, they separated from gymnosperms ca 300 million years ago and their patterning during embryogenesis differs significantly. For this reason, we have used the model gymnosperm Pinus pinaster to identify WOX genes and perform a thorough analysis of their gene expression patterns. Using transcriptomic data from a comprehensive range of tissues and stages of development we have shown three major outcomes: that the P. pinaster genome encodes at least fourteen members of the WOX family spanning all the major clades, that the genome of gymnosperms contains a WOX gene with no homologues in angiosperms representing a transitional stage between intermediate- and WUS-clade proteins, and that we can detect discrete WUS and WOX5 transcripts for the first time in a gymnosperm.

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Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster.

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Key message Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Abstract Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2–6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.