A novel approach for studying programmed cell death in living plant tissues

Programmed cell death (PCD) is a highly regulated process in which cells are killed as part of developmental programmes or as defence mechanisms against pathogens, but the process is less well understood in plant cells compared to animal cells. Reactive oxygen species (ROS) are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCDhas not yet been fully elucidated due to the involvement of complex signalling networks. Elucidation of these mechanisms and signalling pathways will allow manipulation of cell death in plants, which could help to improve yield and quality of crops and thus contribute to solving the increasing food demands of the planet. Examples of this could be the development of cultivars with enhanced and/or faster response to pathogen attacks, or cultivars with increased grain filling and hence increased starch content through delayed cell death of the endosperm. The barley aleurone layer is a generally accepted model system for studying phytohormone signalling, enzyme secretion, and PCD during seed germination. However, two main issues affect PCD-related research in the barley aleurone layer: Firstly, the current knowledge about ROS signaling and the cellular redox environment in aleurone layers undergoing PCD is mainly based on analyses of cell extracts, which do not evaluate the overall cellular redox environment in intact plant cells. Secondly, analyses based on cell extracts are end-point measurements, which are limited by the fact that each analysis is performed on a different pool of samples, as each tissue sample or population of cells can only be analysed at a single time point. This is of great importance for studies of time-dependent processes such as PCD, as time course experiments can be affected by biological and experimental diversity between independent samples, which could distort the interpretation of the results. Time course experiments on the same tissue or population of cells can be enabled by the use of assays that do not destroy cellular integrity, which could also facilitate the combination of multiple assays. However, time course studies with analysis of multiple parameters using different detection techniques remain challenging, as the different assays under consideration may be incompatible, and some assays may affect the plant tissue and therefore influence the outcome of simultaneous or subsequent analyses.

A previously described optical method was used to monitor PCD, while a previously described method for electrochemical detection of intracellular redox activity was tested and optimised for use with the aleurone layers. The electrochemical method had not previously been used inplant biology, and provided new insight by determining both the intra- and extracellular reducing capacity in living cells rather than using cell extracts. The reducing capacity of aleurone cells was shown to increase over time in parallel with the increase in cell death. Use of the flavoenzyme inhibitor diphenyleneiodonium chloride (DPI) provided evidence that the gibberellic acidinduced increase in reducing capacity is dependent on the plasma membrane-bound NADPH oxidase. A preliminary proteomics investigation also showed indirect effects of DPI on the abundance of glyceraldehyde-3-phosphate dehydrogenase 2 but not on the very similar paralogueiv glyceraldehyde-3-phosphate dehydrogenase 1. Further investigations are needed to clarify these effects and to determine which other enzymes are affected by DPI. A 24-well multiplate incubation system for immobilised plant tissues was developed to allow time course studies on the same tissues and to enable parallel use of multiple non-destructive assays. Immobilisation of the tissues in the lid of a 24-well plate facilitated easy combination ofmultiple assays by movement of the plate lid, lessened the workload by decreasing the amount of aleurone layers to be dissected 25-fold, and enabled a higher throughput. The system wasused for parallel time course studies of cell viability, intracellular reducing capacity and transient expression profiles in immobilised tissue under multiple incubation conditions. Immobilisation resulted in decreased rates of cell death due to the lower exposure of immobilised tissues to the incubation buffer, but tendencies for both cell viability and reducing capacity remained the same for both non-immobilised and immobilised tissues. The parallel studies of cell viability and reducing capacity also revealed that PCD is induced by different mechanisms for tunicamycin, an inducer of protein unfolding in the endoplasmic reticulum, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide.

Using optical and electrochemical detection techniques, this project has obtained new knowledge of increases in intra- and extracellular reducing capacity taking place in parallel with PCD, and proposed this increased reducing capacity as a mechanism for holding the ’oxidative window’ for germination open. The involvement of the NADPH oxidase or other flavoenzymes in determining the level of gibberellic acid-induced reducing capacity was also shown using the inhibitor DPI. The new incubation system for immobilised aleurone layers enabled simple, user friendly handling of plant tissue incubations and facilitated transient expression studies in plant tissues by particle bombardment as well as time course studies on the same population of cells combining multiple non-destructive assays. The immobilised approach allowed single transformed cells to be followed over time and provided an insight into the cell-to-cell variability of the actual transformation event, yielding a more detailed picture of transient expression profiles compared to traditional approaches. Future applications of this type of setup could be used for other types of plant tissues such as leaves or germinating embryos for studying the effects of e.g. biotic and abiotic stresses or for screening of compounds for biological effects. Due to the ease of use and many possibilities of assay combinations, the setup has great potential in the area of plant science.