Periodic Reporting for period 1 - TorCrop (Linking the Target of Rapamycin signalling to cell cycle progression during tuber initiation and its impact on potato yield parameters)
Reporting period: 2021-09-01 to 2023-08-31
Potato is one of the most important food crops, therefore producing high yields of high-quality potatoes are in great demand worldwide. The consumable potato tubers are underground organs that were evolved to store nutrients and energy for the plant. A better understanding in the regulation of tuber formation is crucial to advance our biological understanding for improving potato yields and developing more resilient potato cultivars. In the TORCrop project we focused on the molecular mechanisms involved in the process of tuber initiation. Tubers are normally developing on underground stems called stolons. The systemic signals that promote tuber formation are produced in the leaves and transported to the stolon tips. We know that during this process a rapid activation of cell cycle occurs in the subapical region of the stolon that will create the “tuber meristem”. Several genes have been identified as playing a role in the initiation and regulation of tuber development in potato, however, the complex interplay between these genes and environmental factors remains an ongoing area of research. In plants, the evolutionary conserved Target of Rapamycin (TOR) pathway emerged as a central regulator of cell cycle and meristem activity. TOR research in various crops is an emerging field due to its conserved role in regulating organ growth, metabolism, and stress responses, this prompted us to investigate the possible involvement of TOR signaling in tuber initiation. The workflow of the project was organized in 3 work packages that correspond to project objectives.
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.
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.
We identified the TOR signaling pathway as a negative regulator of tuber initiation. We hypothesized that for the formation of the tuber meristem, the shoot apical meristem of the stolon needs to be deactivated. Since the TOR signaling controls the shoot apical meristem, reducing TOR signaling presumably can serve as the switch that initiate the formation of the tuber meristem. We also found that TOR regulates tuber size, possibly through the control of endoreduplication. Recent findings highlight that TOR signaling is implicated in starch biosynthesis and amyloplast development. These processes, together with endoreduplication, are likely to form the basis for tuber bulking, suggesting that the TOR pathway is also involved in the regulation of this stage of tuber development. Studying allelic variants of TOR signaling components provided valuable information for phenotypic variations. This acquired knowledge will enable us to choose and target specific genes or genetic loci that potentially determine desirable traits in potato. Such genetic alleles as molecular markers can aid breeders in selecting genotypes with enhanced tuber traits, consequently we discussed our findings and the possibilities for future collaborations with potato breeding companies such as PepsiCo and Solynta.