Signalling events through small peptides are essential in multiple aspects of plant reproduction. The ScRALF3 Solanum chacoense Rapid Alkalinization Factor (RALF) peptide was previously shown to regulate multiple aspects of cell–cell communication between the surrounding sporophytic tissue and the female gametophyte during ovule development. We analysed the global expression pattern of ScRALF3 with GUS reporter gene under control of the ScRALF3 promoter and validated it with in situ hybridisation. To better understand the role of ScRALF3 we used three different RNA interference (RNAi) lines that reduced the expression of ScRALF3 during pollen development. Both expression methods showed the presence of ScRALF3 in different tissues, including stigma, style, vascular tissues and during stamen development. Down‐regulation of ScRALF3 expression through RNAi showed drastic defects in early stages of pollen development, mainly on the first mitosis. These results suggest that the ScRALF3 secreted peptide regulates the transition from sporogenesis to gametogenesis in both male and female gametophytes.

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 Sc FRK3 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. Electronic supplementary material The online version of this article (10.1186/s12870-019-1804-0) contains supplementary material, which is available to authorized users.

Fertilization-related kinase (FRK) is a group of the mitogen-activated protein kinase kinase kinase (MAP3K or MEKK) that has proliferated in Solanaceae species. Studies on the wild potato Solanum chacoense have shown that three ScFRKs are directly involved in female gametophyte development. Decreasing the expression of ScFRK1 and ScFRK3 by RNA interference lead to embryonic sac development arrest at the functional megaspore (FM) stage. As for ScFRK2, the first FRK studied, antisense and co-suppression lines showed no abnormality, while overexpression lines lead to a drastic decrease in seed numbers, presumably caused by a conversion of the ovule into a carpellike structure. Here we show that in ScFRK2 overexpression lines, carpel-like structures from the ovule cannot explain the drastic decrease in seeds considering the low percentage of these carpellike structures but occurs in early ovule development as observed in Scfrk1 and Scfrk3 knockdown mutants were most ovules are arrested at the FM stage. The highly similar phenotype from knockdown mutants (Scfrk1 and Scfrk3) and ScFRK2 overexpression lines suggests that these MAP kinases could operate antagonistically through a balance between ScFRK1 and 3 on one side and ScFRK2 on the other. This study strongly suggests the importance of the FRK family expression levels during early stages of ovule development in Solanum chacoense embryo sac.