About the Project
Summary: A strong shift from animal farming to nutritious plant crops is essential to sustain the growing human population on Earth. Global warming and increased CO2 levels are thought to lower the overall protein yield of the major staple crops (Myers, S. S. et al. (2017) Annu Rev Public Health 38, 259-277.). This research project aims at providing the key to unlocking the enormous potential for improving crops for protein and polymer production using synthetic biology to induce the formation of designer protein bodies (PB) and protein storage vacuoles (PSV) in high yielding starch crops.
Scientific foundation: The project is ground-breaking because it builds on a better understanding of the basis for the two key organelles in plants which are responsible for the synthesis and storage of the most nutritionally relevant proteins on Earth, the endoplasmic reticulum (ER) and the plant protein storage vacuole (PSV).
The plant ER has been shown to accommodate 100-fold increased levels of ER resident proteins and extremely dilated ER compartments without any damage to growth and development (Crofts, A. J. et al. (1999) Plant Cell 11, 2233-2247). Dilated ER regions behaved like independent ER subdomains without interfering with the rest of the ER function, making them an ideal target for engineered protein storage in vegetative tissues such as potatoes. Recent work in the our laboratory established a strong gain-of-function effect of ectopic ERD2 expression in vivo (Lima-Alvim et al., (2018) Plant Cell, submitted) that boosts the ER retention capacity of plants beyond those limits previous established.
Research at Leeds has also established a ground breaking advance in our understanding of the protein transport route to the plant vacuole. We discovered the late prevacuolar compartment (LPVC) as an intermediate between the prevacuolar compartment (PVC) and the lytic vacuole (Foresti, O. et al. (2010) Plant Cell 22, 3992-4008; Gershlick, D. C. et al. (2014) Plant Cell 26, 1308-1329). We can induce LPVC formation synthetically in vegetative tissues with engineered rab7 GTPases (Bottanelli, F. et al. (2012) Traffic 13, 338-354). This approach appears to mimic the natural process in seeds that develop storage vacuoles, because recently we established that LPVCs have properties identical to those reported for PBs and PSVs on sucrose gradients (Gershlick et al., (2018), Plant Cell, submitted).
Proposed project: By exploring the protein storage capacity of the ER and PSVs via synthetic biology, the project aims at developing a food crop with drastically enhanced nutritional value. The model crop potato has been chosen due to 1) its suitability for large scale cultivation in temperate climate zones representing the vast majority of arable land on Earth, 2) the enormous yields exceeding 50 tonnes per hectar in 5 months, 3) the fact that protein content has not been specifically targeted by breeders and can be drastically increased and 4) the ease with which the crop can be harvested and stored locally for up to 8 months underground without further energy input.
The research work will start with the generation of transgenic potato with enhanced ER retention capacity and tuber-specific expression of nutritionally balanced storage proteins and rab7 variants to induce PSV formation. Training will involve recombinant DNA technology, genetic and protein engineering, molecular cell biology, transient expression technology, plant tissue culture, regeneration, and stable transformation, protein detection and quantitative analysis of tuber properties to assess yield, composition and storage properties.
Expected outcomes: The project is of both strategic and fundamental nature. It will test several working hypotheses about the nature of storage vacuoles in plants which will lead to publications in peer reviewed journals. It also potentially offers the design of a new crop plant which could offer a step-change in protein-productivity and have an impact well beyond the current incremental efforts to improve crop protection.
References
Recent Publications
1) Foresti O, Denecke J. (2008). Intermediate organelles of the plant secretory pathway: identity and function. Traffic 9: 1599–1612
2) Rojo E, Denecke J. (2008). What is moving in the secretory pathway of plants? Plant Physiol. 147(4):1493-503.
3) Snowden CJ, Leborgne-Castel N, Wootton LJ, Hadlington JL, Denecke J.(2007). In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain. Plant J. 52(6):987-1000.
4) Foresti, O., daSilva, L.L.P. and Denecke, J. (2006). Overexpression of the Arabidopsis Syntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo. The Plant Cell, 18, 2275–2293.
5) DaSilva, L.L.P., Foresti, O., and Denecke, J. (2006). Targeting of the plant vacuolar sorting receptor BP80 is dependent on multiple sorting signals in the cytosolic tail. The Plant Cell, 18, 1477–1497
6) Pimpl, P., Taylor, J.P., Snowden, C.J., Hillmer, S., Robinson, D.G. Denecke, J. (2006). Golgi-Mediated Vacuolar Sorting of the Endoplasmic Reticulum Chaperone BiP May Play an Active Role in Quality Control within the Secretory Pathway. The Plant Cell, 18, 198–211.
7) DaSilva, L.L., Taylor, J.P., Hadlington, J.L., Hanton, S.L., Snowden, C.J., Fox, S.J., Foresti, O., Brandizzi, F., and Denecke, J. (2005). Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. The Plant Cell 17, 132-148.
8) Luis L.P. daSilva, Erik L. Snapp, Jürgen Denecke, Jennifer Lippincott-Schwartz, Chris Hawes, and Federica Brandizzi (2004) Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells. The Plant Cell 16, 1735-1771.
9) Pimpl, P., Hanton, S. L., Taylor, J.P., Pinto-daSilva, L.L., and Denecke, J. (2003). The GTPase ARF1p Controls the Sequence-Specific Vacuolar Sorting Route to the Lytic Vacuole. The Plant Cell 15, 1242-1256.
10) Hadlington, J. L., Santoro, A., Nuttall, J., Denecke, J., Ma, J. K.Vitale, A., Frigerio, L. (2003). The C-terminal extension of a hybrid immunoglobulin A/G heavy chain is responsible for its Golgi-mediated sorting to the vacuole. Mol Biol Cell 14, 2592-2602.
11) Brandizzi, F., Hanton, S., DaSilva, L. L., Boevink, P., Evans, D., Oparka, K., Denecke, J., Hawes, C. (2003). ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants. Plant Journal 34, 269-281.
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