Biology 101: Target of Rapamycin (TOR)
Target of Rapamycin (TOR) is a conserved serine/threonine kinase found in all eukaryotic organisms, that has been known to be a part of the TOR pathway. The TOR pathway is a ubiquitous signaling pathway that has been known to regulate nutrient, energy, and stress signaling networks as well gene expression by the coordination of transcriptional and translational control. (Bakshi et al. 2021). Moreover, TOR regulates areas within the framework of pathogen defense, cell development, and cell wall remodeling in plants. . (Florian et. al, 2011). TOR was first identified in Saccharomyces cerevisiae , a type of yeast, through genetic screens for resistance against rapamycin. (Xiong & Sheen, 2014).
Rapamycin, an immunosuppressive and antiproliferative drug, has been known to target the TOR kinase. (Florian et. al, 2011). Rapamycin is known to be produced by the bacteria Streptomyces hygroscopicus and its inactivation is mediated by the formation of of a ternary complex, in which rapamycin forms noncovalent links between the peptidyl-prolyl isomerase 12-kDa FK506-binding protein (FKBP12) and the FKBP rapamycin-binding domain (FRB) of TOR. (Menand et. al, 2002). This ternary complex is known to inactivate the activity of the TOR kinase in a strict manner, since rapamycin has no other known cellular targets. (Ennar et. al, 2006). In the model organism Arabidopsis thaliana, it was seen that a single TOR encoding gene and its inactivation led to a cease in embryo development at early stages of development.
In addition, TOR, having a Ser/Thr kinase domain, is preceded by several HEAT repeats, which interact with the numerous TOR proteins. (Ennar et. al, 2006). In the N-terminal region, TOR consists of 20 HEAT repeats, followed by the FAT domain, the FRB domain, the kinase domain, and the FATC domain. As seen in Figure 1, the TOR protein consists of a catalytic domain at the N-terminus by the FAT and FRB domain and latter being the binding site of rapamycin. (John et. al, 2011).
Fig 1. Structure of the TOR protein with the HEAT repeats, FAT domain, FRB domain, FATC domain, and region of the rapamycin binding-site.
Specifically, rapamycin binds to FKBP12, which in turn, interacts with the FRB domain of TOR, inactivating the TOR pathway. ( Ren et. al, 2012). Studies with Arabidopsis have made it evident that treatment of rapamycin resulted in decrease of root, shoot, and leaf growth, which causes poor nutrient uptake and light energy utilization. Furthermore, it was seen that in one of the signaling pathways TOR is known to regulate is the ABA signaling system, which regulates several protective plant responses including stomatal closure, which are openings in the guard cells of plants that limit the quantity of water that leaves the plant via transpiration. This makes the TOR gene important to study as it plays a significant role in plant responses to stress. Although the effects of the TOR pathway have been well understood in seed plants, its characterization and role in seedless plants is not fully understood. Understanding the role of the TOR pathway in seedless plants can assist in the characterization of the gene, and can also assist in yielding crops, since it is known that it influences root, shoot, and leaf growth.
To characterize the role of the TOR pathway in seedless plants, the fern Ceratopteris richardii was analyzed. Ceratopteris is a seedless plant that contains alterations through generations and cycles between two free living stages, which is a trait unique to plants. The first stage is the haploid gametophyte stage, which is similar to pollen and ovule in seed plants. In the haploid gametophyte stage, Ceratopteris grows as a flat, two-dimensional layer of cells that are plainly apparent or highly visible. This allows the monitoring and collecting of quantitative data on the effects of rapamycin on gametophytes much easier, without the need for tissue manipulation. The diploid sporophyte stage, in which spores are formed, is the second stage. This is the standard three-dimensional stage that seed plants experience. The gametophytes are also easily and cheaply grown and require low maintenance, making them an ideal organism to experiment on. The observations seen in the gametophytes can be used to compare to the pollen and ovule in seed plants, which in turn, will result in information that might be valuable regarding vegetation and agricultural output.
The effects of rapamycin in Ceratopteris richardii were very different from those seen in seed plants. In several eukaryotes, inhibiting the TOR signaling system through nutritional deprivation, rapamycin therapy, or genetic mutation causes fast downregulation of protein synthesis and ribosome biogenesis. (Li et. al, 2006) Subcellular localization of TOR was discovered to be found in the nucleus as well as the cytoplasm and cytosol. (Ren et. al, 2011). It was observed in the model organism Arabidopsis thaliana that the TOR gene was repressed when using an inhibitor. The metabolism, biosynthesis of amino acids, and other metabolic processes as well as genes associated with plant hormone signal transduction were affected, demonstrating that TOR plays an important role in regulating phytohormones in Arabidopsis (Zhu et al, 2020). It has also been known that the inhibition of the TOR gene in Arabidopsis influenced that embryonic development as well. (Menand, et al, 2001). Arabidopsis has been found to be insensitive to rapamycin at concentration up to 10 uM. (Menand, et al, 2001). In addition, disruption of the TOR gene in Arabidopsis led to embryonic arrest, similarly in Drosophila larvae where disruption of the gene led to growth arrest and defects in protein synthesis. (Menand, et al, 2001).
The TOR gene being impacted at embryonic and early stages of development in Arabidopsis is a stark contrast between the TOR gene in Ceratopteris richardii. It has been shown that during seed germination under light, applying TOR inhibitors or suppressing TOR expression of TOR, inhibits its functions in promoting cotyledon greening, chloroplast development and seedling growth (Dong et al, 2015). Despite this, it was seen that the inhibition of the TOR kinase had no significant results regarding germination and was not influenced by the dose of rapamycin given. This signifies that while in Arabidopsis, TOR was influenced by rapamycin in early phases, TOR does not play a significant role during early stages of ferns and seedless plants. Both TOR proteins, in seed and seedless plants, do have the commonality of playing a vital role in stem cell fate, differentiation, and proliferation (Shi et. al, 2018). Inhbition of the gene resulted in retarded root elongation, and blocked meristem growth, cell proliferation, and root hair expansion and similarly in Ceratopteris, it was observed that high concentrations of rapamycin led to decreased cell growth, which decreased the overall gametophyte area, showing that TOR played a role in proliferation in both seed and seedless plants.
The expression of TOR in gametophytes is not consistent at all stages of development, according to RT-PCR analysis. The expression of TOR is consistent across all periods of development in Arabidopsis, according to a microarray analysis of the TOR gene as seen in Figure 8, and TOR has been documented to have a function in both embryonic and post embryonic development in Arabidopsis. (Shi et al., 2018) Because the expression of the gene differs at that moment in development, this shows that TOR plays a larger role in early embryonic phases in seed plants than in seedless plants.
Fig 9. Microarray Analysis of TOR in Arabidopsis thaliana
Another noteworthy distinction between Ceratopteris and Arabidopsis is that Arabidopsis is rapamycin-insensitive. FKP12 appears to be unable to form a complex with rapamycin and TOR, according to research. It was postulated that structural modifications in Arabidopsis FKP12 developed to avoid the formation of the inhibitory complex with TOR and rapamycin. (Xiong & Sheen, 2011). In Ceratopteris, the TOR pathway was blocked in the gametophytes after the plants were treated to rapamycin, but not in the gametophytes. Despite the fact that Arabidopsis is resistant to rapamycin, alternative inhibitors, such as AZD8055, an ATP-competitive TOR kinase inhibitor, have been found to be effective. (Zhang et. al, 2016).
In conclusion, our hypothesis was supported. It was seen that rapamycin affected Ceratopteris richardii in a dose dependent manner. While there were no significant effects seen in terms of germination, high concentrations of 10uM and 20uM significantly reduced the cell number and overall area of the gametophytes. It was also seen that rapamycin induces gametophyte death at specific stages of development, correlating with the expression of CrTOR. Because there was an increased presence of CrTOR in the gametophyte, this could explain why later stages of the gametophytes were susceptible to rapamycin.
Future study of this project could include identifying the full length sequence of TOR in Ceratopteris richardii as well as characterizing how various abiotic and biotic affect its expression and also to determine the role of TOR in the cellular senescence of gametophytes. Additionally, another future study would be to identify and characterize mutants that are insensitive to the effects of rapamycin. This is important because screening for mutants' tolerance to high doses of rapamycin as well as understanding how the inhibition of growth helps gametophytes survive can help develop crops that are resistant to the inhibition of TOR. Future studies would also include identifying different transcription factors and the amount of introns and exons present in the TOR gene in Ceratopteris richardii.
BY: Tauba Ashrafi