Arabinogalactan-proteins and programmed cell death

Arabinogalactan-proteins (AGPs) are complex glycoproteins localized at the cell surface and in the cell wall of all plants. AGPs have recently been implicated in various biological processes such as pattern formation, cell expansion, salt tolerance, disease response, plant-microbe interactions and programmed cell death (PCD). However, the modes of action by which AGPs fulfil these diverse roles in planta are still elusive. PCD in both plants and animals is controlled by reactive oxygen species (ROS), Ca2+-flux, mitochondrial protein release and proteolytic cascades, but the upstream-controls of these regulators of PCD are largely unknown. The hypothesis supported by my recent observations predicts that AGPs are involved in the control of PCD in a ROS dependent fashion. Furthermore I hypothesize that the genetic link between AGPs and PCD might be controlled by an as yet unknown cell wall integrity control system which is also involved in the control of biotic and abiotic stress resistance and growth, and that AGPs might be an integral component of this important control system. To establish a molecular- physiological link between AGPs and PCD, I propose to study the relationship between AGPs, Ca2+-flux and ROS in roots, and to clone a genetic modifier of AGP-damage-triggered PCD in Arabidopsis thaliana. Because of the general importance of cell wall integrity control for plant cells, the physiological characterization and molecular identification of novel loci involved in AGP-dependent PCD will provide new insights into both the control of cell growth and stress resistance, and will potentially lead to the development of new approaches to plant disease and stress management.

Every plant cell is surrounded by a network of fibrous polymers called the cell wall. Cell walls are required for mechanical stability, for growth, for resistance to pathogens and to establish tolerance to environmental stress. The cell wall consists of the carbohydrates cellulose, hemicellulose and pectin and contains a large number of complex glycoproteins. One of the largest and most complex families of cell wall glycoproteins are the arabinogalactan- proteins (AGPs) that are needed for correct cell division and expansion, for cell mechanics and communication and ultimately participate in the decision on cell survival or cellular suicide also known as programmed cell death (PCD). Still, regardless little is known on how AGPs interact with other components and processes in the cell. In this project we asked the question how PCD is induced by alterations in AGPs. Apparently, both positive and negative genetic regulators have to act in a highly balanced way. To find regulators for AGP-related PCD and we employed the tools of genetic and pharmacological analysis. Mutant Arabidopsis plants that produce abnormal AGPs because of a general defect in glycosylation (the uge4 mutant) or that contain a single abnormal AGP (the fla4 mutant) develop necrotic lesions in their roots resulting in abnormal root growth. We mutagenized these mutant plants and screened for new mutations that would either further enhance this growth abnormality or that would suppress it. In another approach, generally known as pharmacogenetics, we used a compound that binds to AGPs, interferes with their function and thereby induces cell death, and we tested the response of more than 150 different mutants in genes that are known or expected to act in signal perception, signal transduction and gene expression. Reciprocally, we analysed the growth of fla4 plants in the presence of different compounds that are known to stimulate or suppress specific signalling pathways in the cell. Moreover, we expressed modified versions of receptors that might be important for sensing the state of AGPs and the cell wall, in plants, in order to interfere with their normal sensing function. Pharmacogenetics on the one hand identified novel roles for brassinosteroid signalling and abscsicic acid, two well-known plant growth regulators in the pathway of cell wall integrity signalling. On the other hand pharmacogenetics and transgenic analyses identified new roles for several genes in the plants response to cell wall variation. Some of the genes previously had not been associated with any function at all, while for others, we revealed a novel role. Most significantly, we identified several genetic loci that are essential for PCD and also control abnormal growth in the fla4 mutant. Three of these loci were mapped to chromosomal regions and characterized using genome sequencing and for the first locus two possible candidate genes, a receptor protein and a regulator for gene expression were identified. The knowledge of regulatory genes that monitor and balance cell wall structure, cell growth and survival eventually could help to better understand how plants grow and survive and under difficult conditions.