F-actin associated proteins implicate new mechanisms involved in SI-PCD

Self-incompatibility (SI) is an important mechanism used by flowering plants to prevent self-fertilization, which would otherwise result in undesirable inbreeding and loss of plant fitness. For this reason, SI has made a significant contribution to the evolutionary success of flowering plants. After pollination, SI utilizes cell-cell recognition to prevent self-fertilization by inhibition of pollen tube growth, which is crucial for the delivery of sperm cells to the egg cell inside the pistil. This involves a highly specific interaction between a pistil-expressed protein and a cognate pollen protein that results in recognition and inhibition of genetically identical or self- (incompatible) pollen, but not cross (compatible) pollen. In Papaver rhoeas (field poppy), the stigma of the pistil secretes a small protein (PrsS) which acts as a signalling "ligand". Upon pollination, PrsS interacts specifically with "self" pollen expressing the SI receptor (PrpS), allowing pollen to distinguish between "self" and "non-self" female partners. This interaction is the critical step in cell-cell recognition and determining acceptance or rejection which triggers a complex network of signalling in the incompatible pollen and results in pollen being inhibited and "told" to commit suicide: "Programmed Cell Death" (PCD).

PCD is essential for a range of processes in all higher organisms. It is vital for normal plant development, playing a decisive role in the life cycle of plants, including fertilisation, embryo development, and rejection of self-pollen. They all depend on tightly controlled and executed PCD. The scientists involved have played a pioneering role in our understanding of plant PCD. Major breakthroughs have come from establishing that key core components of animal PCD machinery are similar to those in plants. However, our understanding of the detailed molecular regulation and downstream processes of plant PCD are still largely unknown and lag behind that of PCD in animal cells.

We have made several recent breakthroughs in our PCD studies in Papaver SI that form the basis of this project. SI triggers dramatic changes of the actin cytoskeleton, an internal protein structure that helps a cell with shape, support, and movement. We recently discovered that SI leads to dramatic acidification of the cell content (cytosol). Other recent findings suggest the involvement of a special type of endocytosis, a process by which cells absorb molecules. This project will carry out the first live-cell imaging studies to discover exactly what happens to the actin cytoskeleton during SI. Other studies, using genetics, microscopy and biochemistry will investigate exactly how these different processes mechanistically control SI-induced PCD.

These fundamental studies are likely to generate excitement in the scientific community as they will provide important mechanistic insights into the role of actin in SI-PCD and the role of [pH]cyt in mediating this. Identifying links between some of these processes will be completely novel for plant cells. Analyzing key molecular mechanisms involved in regulating SI-PCD will be important for our general understanding of evolutionary conservation of PCD.

On a practical note, understanding the mechanisms involved in SI-PCD can lead to applications useful to plant breeding. Fertility and seed set are critical for crop yield and thus Food Security. The transfer of SI-PCD traits into food crops could potentially help plant breeders develop F1 hybrid seeds, which produce bigger and more productive F1 hybrid plants, more efficiently and economically. Currently, hand-emasculation is used to produce F1 hybrid seeds, which is time-consuming and expensive. Introducing SI-PCD into a crop species allows it to be crossed without any emasculation, as no self-pollen can fertilize these plants. Thus, utilization of knowledge on SI-PCD provides a potential alternative means to breed F1 hybrid crops.

We will investigate how actin and actin-associated proteins are mechanistically involved in mediating SI-PCD and how cytosolic acidification regulates this. We will investigate involvement of Clathrin-Mediated Endocytosis (CME) and Elongation Factor-1 alpha (EF1A) in SI-PCD, and investigate how these components link to SI-induced actin rearrangement. A major approach will be using Arabidopsis "SI" lines co-expressing genetically encoded markers, crossed to T-DNA mutant lines, or gene-silencing/overexpression lines.

(A) Live-cell imaging of "SI" lines expressing the actin marker Lifeact-mRuby will, for the first time, characterize these changes in real time. Biochemical studies will establish how pH affects actin polymer status and how this regulates entry into PCD.

We will study the subcellular localization of CAP and ADF, through fusions with pH-stable fluorescent proteins expressed in "SI"/Lifeact-mRuby lines.

F-actin co-sedimentation assays for CAP at various pHs will test if its actin-binding activity is altered by pH.

(B) We will cross our Arabidopsis "SI" lines with EF1A T-DNA mutants and see if knockdowns are defective in PCD, showing functional involvement of EF1A in SI-PCD.

We will artificially alter [pH]i using propionic acid and see if pH affects EF1A localization in pollen co-expressing EF1A-pHst-FP and Lifeact-mRuby.

Phalloidin pulldowns of pollen at different pHs will show if EF1A binds F-actin in a pH-dependent manner.

(C) We will characterize and dissect endocytosis during SI using FM4-64 labelling and live-cell imaging. Using Arabidopsis "SI" lines expressing pHst-FP CME markers at various pHs will reveal the impact of SI-PCD and pH on the localization and dynamics of CME.

Monitoring DEVDase activity in the presence of endocytosis/CME inhibitors will establish if CME is involved in PCD. Monitoring seed set in these lines will provide a clear measure of SI functionality and evidence for the involvement of CME in SI and PCD.

Economic & Social Impact:
Longer term, knowledge about self-incompatibility triggered PCD (SI-PCD) may provide solutions to currently expensive F1 breeding systems. F1 hybrids are generally better and more productive than their parents, offering significant benefits to growers in terms of yield improvement, agronomic performance and consistency of end-use quality. Not surprisingly, hybrid seed comprises ~40% of global seed sales (worth billions p.a.). In the UK, for example, sugar beet, forage maize and many vegetable crops are all grown from F1 hybrid seed. Hybrid varieties also account for an increasing share of the rapeseed and winter barley market, and new hybrid wheat varieties have recently been introduced. Currently plant breeders have to hand-emasculate flowers to produce F1 hybrids. This is time-consuming and expensive. SI-PCD provides a potential route to produce F1 hybrids more easily and more economically. If a crop has an SI-PCD system it does not need to be emasculated, as all crosses will result in hybrid seed. The successful transfer of the Papaver SI system to Arabidopsis raises the possibility that a similar functional transfer is possible to other dicot crops, or even to the more distantly related grasses. This would mark a highly significant biotechnological break-through that could lead to a change in public-good and commercial breeding practises around the world. The ability to more effectively capture hybrid vigour in food crops would have profound food-security implications.

Another area through which knowledge about SI-PCD can potentially contribute to Economic and Social Impact is the development of new herbicides. Efficient herbicide systems for weed control are essential to safeguard crop yield. However, weeds are increasingly resistant to currently used herbicides. PCD constitutes a source of unexplored molecular targets for new herbicidal modes of action. Such new targets would avoid the use of toxic chemicals, benefiting the farmers, the agro-chemical industry, the environment and the wider public.

Fulfilling BBSRC strategic aims:
Use of SI could impact on the BBSRC research priority of Food Security. Providing enough food for the world is a major challenge. More economic ways to make F1 hybrid crops with better yields could provide an important contribution. Likewise, increasing our knowledge of PCD in plants can lead to the development of an innovative weed resistance management strategy. Thus, this project contributes to one of BBSRC's principal strategic aims; "underpinning practical solutions to major challenges such as food security" and by "securing national research capability in a strategically important area", namely, food security. Another BBSRC strategic aim is "maintaining world-class UK bioscience by supporting the best people and best ideas and by securing national research capability in strategically important areas".

The use of Arabidopsis as a model system for studies on SI-PCD is based on a Current Biology paper (2012) and a recent Science paper (2015). The proposed collaborative project is novel, cutting edge, internationally competitive science and builds on well-established, high impact research, which underpins possible solutions to food security. Importantly, funding of this project is essential for the continuation of this high impact, and mostly BBSRC funded, research on plant cell signalling and PCD for the future.

Public/Education:
With Food Security being an important global issue, and a key strategic priority for the BBSRC, the general public needs to be better informed about the importance of plant reproduction for our food production. NFT and MB have a good track record of being involved in public engagement activities and will be actively involved in several public engagement activities aimed at making plant reproductive biology and Food Security better appreciated and understood by the general public (see Pathways to Impact).