Characterization of carrot arabinogalactan proteins

Arabinogalactan proteins (AGPs) are highly glycosylated proteins. Besides galactose and arabinose the carbohydrate part of AGPs contains other neutral sugars and uronic acids. AGPs are widely distributed in the plant kingdom, probably occurring in all tissues of every plant. Yariv phenylglycoside is a synthetic molecule and can form a complex with AGPs and this property of Yariv is used to isolate AGPs. Exposure of cell cultures or seedlings to Yariv phenylglycoside indirectly showed that AGPs have a biological function. For the study of embryogenesis cell cultures have been used.The cells of a cell culture can differentiate, form embryos and finally develop into plants. The growth conditions can be changed and the effect on the development of the embryos can be investigated. By adding AGPs to cell cultures the amount of embryos formed can be manipulated.This research showed that especially AGPs that were derived from carrot seeds were able to increase the number of embryos formed. Pre-treatment of the seed AGPs with chitinase increased the number of embryos formed when compared to the untreated AGPs. Chitinase is an enzyme able to hydrolyse the glycosidic bounds between acetylglucosamine and glucosamine. For the determination of an AGP that showed sensitivity to chitinase and the formed products, AGPs have been isolated from carrot cell culture medium and carrot seeds. In the AGPs that were isolated from the cell culture no glucosamine could be detected. The seed AGP extract contained a very low concentration of glucosamine. After fractionation of the seed AGP extract it was not possible to detect glucosamine in the obtained fractions. It could be possible that the glucosamine containing compound present in crude AGP extracts was coprecipitated with Yariv phenylglycoside during the isolation of AGPs.AGPs that were isolated from carrot cell cultures and the cell walls of different carrot tissues show in general different molecular weight fractions. Linkage analysis of the carbohydrates of two main fractions and additional protein analysis showed that bothfractionsposses the chemical characteristics of AGPs. Analysis of AGPs that were isolated with Yariv shows a large variation in sugar composition, depending on the purification procedure used. When AGPs from carrot cell culture medium and cell wall fractions were further purified with copper ions, galacturonic acid rich fractions were identified. Homogalacturonan hydrolysing enzymes were used to test whether the galacturonic acid was organised as homogalacturonan or present in the side chains of the AGPs. Homogalacturonan is a characteristic structural element of pectin. Oligogalacturonans were found after incubation of the galacturonic acid rich AGP fractions with polygalacturonase and pectin methylesterase. This result indicates that the galacturonic acid present in AGPs is organised as homogalacturonan. AGPs that were isolated from carrot tap root cell walls also showed a galacturonic acid rich fraction that was sensitive to homogalacturonan hydrolysing enzymes.The chemical analysis of carrot cell wall AGPs from two different tissues showed that a small fraction of AGPs is present that contains galacturonic acid in the form of homogalacturonan. The interaction of AGPs and pectin has been suggested earlier and could be due to non-covalent interactions or a covalent linkage. The galacturonic acid rich AGP fractions were isolated from the cell walls with EDTA buffer and it is very unlikely that the interaction between AGPs and pectin found in this study is accomplished by ionic interactions. This research has shown that AGP-pectin complexes exist in carrot tissues and this finding could be a starting point for a more precise determination of the linkage