Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 24, 1998
Publication Date: N/A
Interpretive Summary: A plant's ability to survive hostile environmental conditions depends on its capability to store food when excess is available and to retrieve this stored material when food supplies are limited. Most plants store excess food as large carbohydrates. Retrieval is dependent upon the breakdown of these stored carbohydrates to small sugars that cells can easily use for renergy. The objective of this work was to determine how barley retrieves the carbohydrates it stores in its stems. Barley stores fructan, long polymers of fructose, that contain two different chemical bonds. This work successfully identified the protein responsible for degrading one of these two bonds. As this protein is the only one in barley that can degrade this type of bond and because fructan degradation is correlated with survival of episodes of drought or low temperature, it is likely to be an important component of the plant's biochemical defense against a hostile environment. Hence, the protein identified and characterized is considered a good candidate for manipulation with classical genetics or biotechnical procedures.
Technical Abstract: Barley (Hordeum vulgare cv. Morex) fructan exohydrolase was purified by precipitation with ammonium sulfate and chromatography on anion exchange and lectin affinity columns. The final enzyme preparation was homogenous as determined by the presence of a single band on silver stained SDS-PAGE and IEF gels. The purified protein had a mol mass of 33 kD and a pI of 7.8. Analyses of relative hydrolytic rates of various fructans was determined by measuring released fructose by pulsed electrochemical detection after separation of reactions by HPLC. The purified enzyme hydrolyzed beta-2,1-linkages in 6g,1-kestotetraose, 1 & 6g-kestotetraose, 1,1-kestotetraose, and 1-kestotriose with relative rates of 100:96:85:88. This enzyme slowly hydrolyzed the beta-2,6-linkages in 6g-kestotriose and in 6g,6-kestotetraose and sucrose with relative rates of 5:4:3 compared to 6g,1-kestotetraose hydrolysis rates arbitrarily set at 100. The substrate attack pattern, determined by identifying products from hydrolysis of purified fructan tetrasaccharides, was of the multichain type. Sucrose was a mixed-type inhibitor of hydrolysis of inulin.