The Solanum chacoense Fertilization-Related Kinase 3 (ScFRK3) is involved in male and female gametophyte development.

BACKGROUND: The Fertilization-related kinases (FRK) form a class that belongs to the MEKK subfamily of plant MAPKKKs. It was recently shown that FRK class kinases expanded during angiosperm evolution, reaching their maximum numbers in the lineage leading to solanaceous species and culminating in the Solanum genus where they account for more than 40% of the total MEKKs. The first members studied, ScFRK1 and ScFRK2 were shown to play a pivotal role in gametophyte development in the wild potato species Solanum chacoense. RESULTS: ScFRK3 is also involved in gametophyte development. ScFRK3 is expressed in developing pollen and young ovules, reaching its highest level immediately after meiosis and during the mitosis steps in both gametophytes. Hence, three independent lines of ScFRK3 RNAi mutant plants showed decreased number of seeds per fruit. We also observed an important number of degenerated embryo sac in mature ovary. Analysis of ovule development showed that most embryo sac did not enter mitosis I in ScFRK3 RNAi mutant plants. Severe lethality was also observed during male gametophyte development, pollen being arrested before mitosis I, as observed in the female gametophyte. Obvious defects in vegetative organs were not observed, emphasizing the reproductive roles of the FRK class kinases. To isolate MAP kinases acting downstream of ScFRK3, a de novo S. chacoense transcriptome from male and female reproductive organs was assembled. Of the five ScMKKs and 16 ScMPKs retrieved, only the ScMKK3 interacted with ScFRK3, while only the ScMPK13 interacted with ScMKK3, leading to an apparent single three-tiered canonical MAP kinase cascade combination involving ScFRK3-ScMKK3-ScMPK13. CONCLUSIONS: The ScFRK3 MAPKKK is involved in a signaling cascade that regulates both male and female gamete development, and most probably act upstream of ScMKK3 and ScMPK13.

Genome-wide analysis of MAPKKKs shows expansion and evolution of a new MEKK class involved in solanaceous species sexual reproduction.

BACKGROUND: Members of the plant MAP Kinases superfamily have been mostly studied in Arabidopsis thaliana and little is known in most other species. In Solanum chacoense, a wild species close to the common potato, it had been reported that members of a specific group in the MEKK subfamily, namely ScFRK1 and ScFRK2, are involved in male and female reproductive development. Apart from these two kinases, almost nothing is known about the roles of this peculiar family. METHODS: MEKKs were identified using BLAST and hidden Markov model (HMM) to build profiles using the 21 MEKKs from A. thaliana. Following protein sequence alignments, the neighbor-joining method was used to reconstruct phylogenetic trees of the MEKK subfamily. Kinase subdomains sequence logos were generated with WebLogo in order to pinpoint FRK distinct motifs. Codon alignments of the FRKs kinase subdomains and maximum-likelihood phylogenetic trees were used in the codon substitution models of the codeml program in the PAML package to detect selective pressure between FRK groups. RESULTS: With the recent progress in Next-Generation Sequencing technologies, the genomes and transcriptomes of numerous plant species have been recently sequenced, giving access to a vast amount of data. With the aim of finding all members of the MEKK subfamily members in plants, we screened the genomes of 15 species from different clades of the plant kingdom. Interestingly, the whole MEKK subfamily has significantly expanded throughout evolution, especially in solanaceous species. This holds true for members of the FRK class, which have also strongly expanded and diverged. CONCLUSIONS: Expansion and rapid evolution of the FRK class members in solanaceous species support the hypothesis that they have acquired new roles, mainly in male and female reproductive development.

The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.