Cytokinin signaling contributes to the regulation of multiple fundamental processes active in plant development. These include cell division, meristem maintenance, shoot initiation and growth, vascular patterning, flower and seed development, nutrient uptake, chloroplast differentiation and light perception
[1–3]. Additionally, this hormone plays a role in regulating several developmental programs defining the life of perennial woody plants, including the activity of vascular cambium, branching pattern of the shoot, and the onset of leaf senescence. The long life span and extensive radial growth contribute to the large size and massive amount of wood present in a tree, creating a stark contrast to the much smaller herbaceous annuals. However, only few studies have thus far been published about the role of cytokinin in the regulation of woody plant development. To facilitate this research, we are now presenting a comprehensive description of cytokinin signaling and homeostasis gene families in two hardwood tree species: Populus trichocarpa and Prunus persica. Gene identification in tree genomes was based on homology with Arabidopsis genes, as cytokinin homeostasis and signal transduction pathways have been extensively studied and well-characterized in this species
Structurally, cytokinins are adenine derivatives; based on side chain identity they can be classified into four groups representing isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin, and aromatic cytokinins. iP and tZ are the bioactive forms of this hormone, to which plants respond through a multistep two-component histidine-aspartate (His-Asp-His-Asp) phosphorelay system
[4–6]. The phosphorelay is initiated when a cytokinin ligand binds to a histidine kinase receptor, which triggers autophosphorylation of a His residue. After an intramolecular transfer of the phosphoryl to an Asp residue, it will be transferred to a His in a cytosolic histidine phosphotransfer (HPt) protein. The HPts provide a mobile connection between the cytosol and nucleus; they continuously cycle between these two compartments. In the nucleus, the HPt transfers the phosphoryl onto an Asp in a phospho-accepting response regulator (RRs). RRs can be classified into several different types according to their structure and function. Type-B RRs, which belong to the Myb-transcription factors, activate the transcription of cytokinin primary response genes. Among them are the type-A RRs, which are involved in a negative feedback mechanism that helps to fine-tune the function of cytokinin signaling pathway. Type-A RRs repress activity of type-B RRs
[4, 7] and are stabilized by HPt mediated phosphorylation (To et al.
). Adding further flexibility to the signaling pathway, many of its components are capable of forming both homo- and heterodimers
[9–13]. Different combinations of the two-component elements presumably add diversity into the process and outcome of the phosphorelay.
Cytokinin signaling represents an ancient hormonal pathway. All of its components are already present in the genome of moss Physcomitrella patens[14, 15], indicating that the cytokinin phosphorelay was already functional prior to the development of a well-defined plant vasculature. As compared to the moss, the cytokinin signaling pathway has, however, become more diverse during the evolution of land plants. The number of members in most cytokinin signaling gene families is much higher in the genomes of vascular plants than in Physcomitrella[14, 15]. In general, the dynamic nature of plant genomes has influenced the evolution of all gene families in vascular plants. All angiosperm lineages have undergone reoccurring genome duplications, indicating that polyploidization confers a fitness advantage for plant species. Each advent of a whole genome duplication is subsequently followed by a gradual gene loss; this rediploidization ultimately promotes a new duplication, allowing the process to repeat in a cyclical manner
To study the structure of cytokinin signaling and homeostasis genes families in woody plants, we sought to characterize and compare them between two hardwood tree species. For the first species in our phylogenetic study, we chose the most common model tree for molecular biology: Populus trichocarpa, black cottonwood. Populus is a fast growing a dioecious tree, which can reach reproductive maturity in four to six years. Populus trees provide a wood source for the pulp and paper industry and have the potential to be developed into a biofuel feedstock
. P. trichocarpa has a relatively small diploid (2n = 38) genome with the haploid size of 485 Mbp. The first version of genome assembly was published in 2006 by Tuskan et al.
. Due to the challenges of genome assembly in a highly heterozygous tree species, only the current, third genome assembly of P. trichocarpa, has been able to resolve a large number of reads that were previously published as unassembled scaffolds. Specific loci identities have only recently been assigned to all predicted genes. Thanks to these improvements, we have now for the first time been able to reliably recover a complete set of cytokinin signaling and homeostasis genes from a tree species. Accordingly, we will discuss how our analysis differs from previously published reports of P. trichocarpa cytokinin signaling genes
[14, 19, 20].
The second hardwood tree species used in this study is the economically important fruit tree peach, Prunus persica. In terms of cultivated surface area, P. persica is the third most important temperate fruit crop. Additionally, it is a member of the economically important Rosaceae family, which includes important crops such as peaches, apples, pears, cherries, plums, apricots, strawberries, almonds, and roses. An international effort has led to the genome sequencing and development of Prunus persica as a genomic model for the Rosaceae family
[21–23]. This hardwood tree is a self-pollinating diploid (2n = 16), with a short juvenile period (2–3 years) and a genome size of 265 Mbp
Currently only a little is known about the role of cytokinin signaling in the regulation of tree or fruit development in Rosaceae. The available data indicates that cytokinins are important for fruit development: high hormone levels have been measured in growing peach fruits
. It has also been demonstrated that exogenous application of cytokinin on sweet cherry fruits significantly increases fruit size and weight
. Additionally, cytokinin treated fruits showed increased fruit firmness, increased fruit soluble solid concentrations and a delay in exocarp coloration
. Similar results have also been seen in apples and pears that have been treated exogenously with cytokinin
[26–29]. Taking together, these observations indicate that the cytokinin signaling and homeostasis pathways can provide candidate genes for the breeding of fast growing and high quality Rosaceae fruits.
The third species chosen for our study is the most common herbaceous model plant, Arabidopsis thaliana. Arabidopsis provides an excellent reference genome, as its cytokinin homeostasis and signal transduction pathways have been characterized in detail
[1–3]. In addition to the contrast between the woody perennial versus herbaceous annual life cycles, the selected three model species differ in their reproductive strategies. Both Prunus and Arabidopsis have hermaphroditic flowers, whereas Populus is a dioecious tree whose genomic sequence was derived from a female plant
All three model species belong to the rosid clade of angiosperm plants. Populus (Malpighiales) and Prunus (Rosales) belong to the eurosids I subclade (Fabidae), whereas Arabidopsis (Brassicales) belongs to the eurosids II (Malvidae)
. They display diverse genome duplication histories: since their last common ancestor, Populus lineage has undergone one whole genome duplication, Arabidopis two, and Prunus none
[18, 31, 32]. Based on the genome duplication history and number of synonymous nucleotide substitutions, the molecular-clock rate has been calculated to be faster in Arabidopsis than in Populus. Due to the genome duplication history and gene evolution rate, the Populus genome has on average 1.5 orthologs for each Arabidopsis gene
, and Prunus 0.85
 (http://www.rosaceae.org/projects/peach_genome/v1.0/homology). The differences in the cytokinin signaling and homeostasis related gene family sizes are consistent with the general genomic trends. We identified a total of 85 genes from the Populus trichocarpa genome and 45 genes from Prunus persica, as compared to the 60 Arabidopsis genes. The gene family structures between the two tree species and Arabidopsis were compared through phylogenetic analyses.