Elucidating the role of ROS in mediating self-incompatibility induced 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 signalling network in incompatible pollen and results in pollen tubes being inhibited and "told" to commit suicide: "Programmed Cell Death" (PCD).

Reactive oxygen species (ROS) are unstable molecules that easily react with other molecules in the cell. If a cell contains too many of these ROS molecules (often hydrogen peroxide) they can cause damage to proteins and may even cause cell death. Low levels of tip-localized ROS are important for regulating normal tip growth of pollen. However, we have shown that SI triggers a rapid increase of the ROS levels in another part of the pollen tube and that these high levels of ROS trigger changes of the actin cytoskeleton (crucial for a cell's shape and movement) and that this type of ROS increase activates SI-induced PCD.

We recently discovered that these high levels of SI-induced ROS cause changes/damage to a range of different pollen proteins that fulfil important functions in pollen tube growth. The effect of high levels of ROS molecules on protein function and cellular processes has been extensively studied in animals, often in relation to diseases, in particular cancers. However, we know very little about the damaging effect that high levels of ROS can have on the function of plant proteins and their associated cellular processes. The Papaver SI system, that we can mimic in the laboratory by growing pollen tubes in dishes with growth medium and adding the PrsS proteins to trigger the SI response, provides a great opportunity to study these aspects in full detail.

Using biochemistry, genetics, and microscopy this project will investigate how high levels of ROS, triggered by SI, affect the function of a range of selected proteins and cellular processes. These fundamental studies are likely to generate excitement in the scientific community as they will not only provide important mechanistic insights into the role of ROS in SI-PCD but also more broadly for our understanding of the consequences of ROS induced protein damage in plant cells.

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, with high levels of ROS as an essential component, provides a potential alternative means to breed F1 hybrid crops.

(A) Live-cell imaging of Arabidopsis SI lines expressing genetically encoded markers, including roGFP-Orp1 H2O2 sensors, in combination with markers/probes for various cellular organelles, will establish the intracellular sources of SI-induced ROS.

We will use the cell permeable sulfenic acid probe BCN-E-BCN, which traps sulfenic acids and, through linkage with Alexafluor dyes, allows for the fluorescence detection of sulfenylation in situ, to track the subcellular location of protein oxidation in pollen during SI.

(B) We will conduct 14C-glucose labelling assays to determine the SI-ROS induced changes in glycolytic metabolism in pollen tubes.

Using fluorometric and luminescence-based assays, we will determine if the activity of GAPDH and enolase, both subject to oxPTMs, are affected by SI-induced ROS. We will measure glycolytic/TCA cycle intermediates using LC-MS and GC-MS.

We will measure SI-induced changes in pollen energy charge using an ATP luminescence assay with luciferin. In addition, we will use a FRET-based biosensor for ATP, ATeam1.03-nD/nA, to monitor dynamics of ATP production in Arabidopsis pollen tubes and determine how this changes after SI.

We will use pharmacological inhibitors for ATP synthase, hexokinase and GAPDH, in combination with caspase-activity assays, to establish a link between reduced energy metabolism and PCD.

PM H+-ATPase (AHA7/8) T-DNA mutants in the Arabidopsis SI background will be used to evaluate their functional involvement in SI-PCD. The ratiometric pH sensor (phGFP) will be used to investigate a mechanistic link with SI-induced acidification.

We will determine SI-induced changes in the localization of GAPC in Arabidopsis "SI" plants by expressing an FP version.

(C) A series of biochemical assays with recombinant Papaver actin and ABPs, both containing oxidation resistant substitutions, will determine if oxidative modifications affect actin assembly/disassembly, and interactions with ABPs in vitro.

Economic & Social Impact:
Longer term, knowledge about self-incompatibility (SI), including an understanding of how SI-induced ROS are mechanistically involved in SI-PCD, may provide solutions to currently expensive F1 breeding systems. F1 hybrids offer significant benefits to growers in terms of yield improvement, agronomic performance and end-use quality. Not surprisingly, hybrid seed comprises ~40% of global seed sales. In the UK, e.g. 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 hybrid wheat varieties have been introduced. Currently, plant breeders have to hand-emasculate flowers to produce F1 hybrids. This is time-consuming and expensive. 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 be a significant biotechnological break-through that could lead to a change in public-good and commercial breeding practices. 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. 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:
This proposal sits firmly within the "Understanding the rules of life - promoting creative, curiosity-driven frontier bioscience to address fundamental questions in biology" remit of BBSRC described in the Forward Look for UK Bioscience document (see earlier). While the science we propose is fundamental, there are potential applications for SI in the future (see above). Use of SI could impact on the BBSRC research priorities of "Sustainably enhancing agricultural production" and "Agriculture and 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. In addition, elements of the proposed research associate with "Systems approaches to the biosciences" and "Technology development for the biosciences". The proposal benefits from an "International partnership" with Shanjin Huang, Tsinghua University, Beijing.

The proposed research also aligns with the three themes highlighted in BBSRC's forward look for the UK bioscience: "Advancing the frontiers of bioscience discovery", "Tackling strategic challenges" (in particular "Bioscience for sustainable agriculture and food"), and "Building strong foundations".

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 BBSRC priority, the general public needs to be better informed about the importance of plant reproduction for our food production. The PIs have an excellent 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.