WP1. Study cell cycle in the developing tuber
Using flow cytometry, we determined that cells in the growing tuber of a diploid potato variety gradually increased their ploidy level from 2C to 32C, suggesting that the tuber development is linked to endoreduplication. A set of tissue-specific RNA-seq analysis was performed and several genes were identified that are highly expressed in the tuber meristem including core cell cycle, hormonal regulatory- and homeobox genes that typically play role in basic developmental transitions. Cell cycle phase-specific expression of many of these genes was identified in an in-vitro experimental system in sprout tips.
WP2. Study the role of TOR signaling in tuberization
We generated transgenic potato plants with silenced TOR expression. A high-throughput phenotyping was carried out to monitor physiological properties of theses TOR-silencing lines by comparing them to the parental line. We found that lowering the TOR expression primarily affected three parameters: biomass, flowering time and tuber number in soil grown potato plants. Another important experimental system was set up in which we could in-vitro monitor tuber initiation (“microtuber” production) to investigate how the onset of tuberization regulated by the TOR pathway. We found that either applying TOR-kinase specific inhibitors or the silencing of TOR gene expression could enhance the production of microtubers, suggesting that the TOR signaling pathway is a negative regulator of tuber initiation. Additionally, our research revealed that microtubers cultivated under TOR-inhibited conditions were increased in size. Analysis of cell measurements indicated that while these cells were smaller individually, their number was higher, highlighting the role of TOR signaling in tuber size regulation. Finally, we performed transcript profiling on laser-dissected tissues of early tubers to identify the transcription of regulatory genes that are specific to certain cell layers at the onset of tuber formation. With this experiment, we identified regulators that are possibly central to determine the new meristem identity e.g. a Wuschel-related and many of the core cell cycle genes were among the very highly expressed transcript in the cortex layer of the stolon after tuber initiation.
WP3. Identifying allelic variations of TOR signaling
The primary phosphorylation targets of the TOR kinase are the S6K proteins. It is also known that endoreduplication and cell cycle gene expression is regulated by the TOR signaling through RBR/E2F protein complexes. Therefore, we analyzed available sequence information of potato haplotypes to identify allelic variations of conserved TOR signaling components including these genes: TOR, S6Ks, RBR, E2Fs. The extensive sequence data set consisted of the known reference- and de novo/re-sequenced genomes of cultivated landraces. We identified a number of single nucleotide polymorphisms (SNPs) that can affect transcriptional regulation of these key regulatory genes or alter the conformation and function of the gene products. We are correlating this information with phenotypic variations found within breeding companies and publicly accessible databases. Another approach that we followed was to identify SNPs using quantitative genetic tools such as QTL mapping. In certain environmental conditions, some potato varieties form tubers above ground on the axils of the stem. When overexpressed or silenced, many of the genes known to regulate tuberization induce the formation of such aerial tubers. A diploid backcross population was generated and used for QTL mapping of tuber properties. Among these plants, we found that a QTL for aerial tuber formation is located at the end of chromosome 1, where the TOR gene is localized. Further examination (fine mapping) needs to be designed and carried out in order to confirm that indeed an allelic variation of TOR is responsible for this trait.
These research outcomes were presented in dutch and international outreach events: ADAPT meeting, 2021, Wageningen, Netherlands; SOLANACEAE2022 conference, 2022, Thessaloniki, Greece; Plantum symposium, 2023, Lunteren, Netherlands; Annual Meeting Experimental Plant Sciences, 2023, Lunteren, Netherlands. Furtermore, there is a manuscript under revision about the role of the TOR pathway in the regulation of potato tuber development.
