Apoplastic proteins, enzymes and radicals
The soluble and readily extractable part of the plant extracellular matrix has been termed, the apoplast and contains a wide range of components such as, complex carbohydrates, structural proteins, enzymes and radicals that are known to be responsive to stress and developmental pressures. This thesis describes the development of a technique for the selective enrichment of apoplastic components for a range of subsequent analyses. Using this technique a number of apoplastic proteins were N-terminally sequenced and revealed 2 cell wall related enzymes, an antifungal protein and 3 auxin-binding/germin-like proteins. This technique also provided a novel approach to the further study of auxin-binding proteins via the use of affinity chromatography at their putative site of action, the apoplast. Three potential auxin-binding protiens were identified. Many attempts were made to subject the material extracted from the apoplast to the highly resolving technique of 2-dimensional electrophoresis, and during the process two unusual 2D systems were developed. These systems could be run in a small format that permitted very rapid analysis and/or using an in-gel loading strategy to subject up to 500µg of protein to 2D separation therefore permitting N-terminal sequencing from single 2D gels. Unfortunately 2D separation of apoplastic proteins was never fully achieved within the time frame of this study due to the vast degree of heterogenity present in the sample material. It did however demonstrate the very complex nature of apoplastic components. A series of experiments revealed that the tobacco leaf apoplast contained compartment specific antioxidant enzymes, some of which share physical characteristics with similar enzymes from other species. The activity of these enzymes altered in response to stress and according to the developmental age of the tissue. The reduced activity of these enzymes directly correlated to the degree of oxidative modification of apoplastic proteins illustrating that these enzymes are important in the detoxification of apoplastic radicals. Follow on experiments following the apoplastic generation of the superoxide anion and nitric oxide from impact stressed potato tuber tissue showed that radicals play important roles in the responses of plant tissue to stress, and show the first involvement of nitric oxide in plants in response to abiotic stress.