Investigating the role of StFKF1 in tuberisation of potato (Solanum tuberosum)

Potato (Solanum tuberosum) is one of the most significant global food crops. Given their origin in cool Andean regions, potato is susceptible to higher temperatures. In light of the rising temperatures resulting from climate change, it is crucial to gain insight into the molecular processes governing tuber formation. The objective of this study was to elucidate the role of the circadian clock output and blue-light dependent Flavin-binding, Kelch-repeat, F-box protein 1 (StFKF1) in tuberisation and flowering of potato. StFKF1 targets Cycling-Dof Factor 1 (StCDF1) for degradation via the proteasome, which is bound to the Constans-like 1 (StCOL1) promoter, repressing its expression. StCOL1 negatively regulates the activation of the Self-Pruning 6A (StSP6A) through the activation of Self-Pruning 5G (StSP5G). StSP6A is activating tuber formation and growth as shown in various studies. First, the localisation and expression pattern of StFKF1 in potato plants (cv Solara) was examined. The gene was predominantly expressed in green tissue, particularly in source leaves, and its promoter exhibited high activity in mesophyll cells of leaves, as well as in the vasculature of petioles and stems. Furthermore, the promoter exhibited greater sensitivity to heat than light. In addition, the protein was found to localise in the nucleus. The highest mRNA was detected in the afternoon, with a relatively constant level of expression throughout the development of the plant. F-box proteins, like StFKF1, act primarily at the protein level. Therefore, potential interaction partners were investigated by yeast-two-hybrid (Y2H) and mass-spectometry (MS). The results corroborated the previously documented interaction with StCDF1 and underscored its involvement in an E3 ubiquitin ligase complex through its interaction with a StSKP1-like protein, which targets other proteins for degradation. Furthermore, a potential link to the tricarboxylic acid (TCA) cycle was identified, as the GFP-StFKF1 protein was found to interact with a succinyl-semialdehyde reductase and a glutamate decarboxylase. Next, transgenic potato plants with altered StFKF1 expression were analysed. Overexpression of StFKF1 showed no phenotypic changes when compared with WT plants. The overexpression of a GFP-StFKF1 variant in plants resulted in significant phenotypic alterations, including a dwarf-like morphology, premature tuberisation onset, and the absence of floral development. In contrast, the downregulation of StFKF1 by RNAi in potato plants resulted in early flowering. However, no other major significant alterations in phenotype or leaf transcriptome were found compared to WT plants. In contrast, overexpression of GFP-StFKF1 resulted in significant alterations in the transcriptome profile with regard to hormonal responses, including those associated with the hormones cytokinin, jasmonate and abscisic acid. Genes coding for enzymes of the TCA cycle exhibited differential gene expression in GFP-StFKF1 when compared to WT and RNAi-StFKF1. In addition to modifications in the hormone response, alterations were observed in the transcriptome involved in photosynthesis, which may have caused an increase in the chlorophyll content in GFP-StFKF1 plants. Further analysis revealed that protein metabolism was also altered in GFP-StFKF1, with regard to its function as an E3 ubiquitin ligase. To corroborate its functionality, the GFP-StFKF1 protein was introduced in Arabidopsis thaliana WT Col-0 and fkf1 mutants, and the flowering time was observed, as the fkf1 mutants exhibit a late flowering phenotype. Thus far, a definitive conclusion regarding the functionality of the GFP-StFKF1 protein has not been drawn, as the flowering experiment did not yield an unambiguous outcome. As a final approach, single-cell RNA sequencing (scRNA-Seq) of potato leaf protoplasts was initiated to determine the exact tissue type where StFKF1 is present. Previously, promoter activity of StCDF1 and StSP6A was detected in the vascular system of potato leaves, whereas in this study, promoter activity of StFKF1 was mainly located in the mesophyll cells of leaves. Protoplasts were isolated and sequenced to verify these results and to determine how these genes might interact to regulate tuber formation. Unfortunately, during the library construction process, cells were burst and mainly chloroplasts were sequenced. Most of the remaining genes were associated with stress and photosynthesis, which may be related to the protoplast isolation process and high chloroplast contamination. Nevertheless, scRNA-Seq can be a very powerful tool, but for future experiments, the protocol needs to be improved to ensure stabilised potato protoplasts or nuclei. Overall there is collective evidence, that indicates an involvement of StFKF1 in a range of regulatory processes within potato plants, including the regulation of flowering time, the onset of tuberisation, and the potential regulation of the TCA cycle.