Elucidating the role of ROS in mediating self-incompatibility induced PCD

(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.

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.