|Michiels, An - UNIVERSITY OF MARYLAND|
|Van Laere, Andre - BELGIUM|
|Van Den Ende, Wim - BELGIUM|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 29, 2003
Publication Date: June 29, 2003
Citation: Michiels, A., Van Laere, A., Van Den Ende, W., Tucker, M.L. 2003. Expression analysis of a chicory fructan 1-exohydrolase gene reveals complex regulation by cold. Journal of Experimental Botany. 55:1325-1333. Interpretive Summary: The most prominent storage carbohydrate in the plant kingdom is starch; nevertheless, fructan (a fructose polymer) is used as a storage compound in approximately 15% of flowering plants. Inulins, a specific type of fructan, are widely used in food as healthy alternatives for low-calorie sweeteners, dietary fiber and fat substitutes. It has been suggested that the human intestinal flora is improved by a daily intake of low amounts of inulin that stimulate the growth of Bifidobacteria and Lactobacilli in the colon. During the past decade, industrial fructan production has increased from 1 kiloton to 100 kilotons annually. However, further scaling up of inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH)that degrades inulin and limits its yield. We have identified and sequenced the gene that encodes the enzyme that degrades inulin in chicory and begun identification and characterization of sequence elements that control its expression in the plant. A better understanding of the molecular regulation of 1-FEH expression will provide important information and tools necessary for scientists and industrial partners to develop the methods to increase inulin yield.
Technical Abstract: We isolated and cloned the gene for a recently identified cDNA, 1-FEH IIa, encoding a fructan 1-exohydrolase from Cichorium intybus and characterized a 1093 bp promoter fragment. An analysis of the genomic 1-FEH IIa sequence indicated that the gene consists of 6 introns and 7 exons similar to plant invertase genes. Like invertase genes, 1-FEH IIa also contains the 9 nt mini-exon encoding the tripeptide DPN. A database search for cis-acting response elements within its promoter identified multiple elements that appear to have relevance to cold induced expression of the gene in field grown roots. Promoter analysis by transient expression assay demonstrated that the 1-FEH IIa gene promoter is highly expressed in etiolated Cichorium leaves and cold stored roots, which correlated well with the high level expression detected by RNA blot analysis. Cold also enhanced 1-FEH IIa reporter gene expression in green leaves, however the reporter gene activity was much lower compared to similar induction experiments in etiolated leaves. Promoter deletion analysis demonstrated the presence of potential cold-responsive ABRE and/or CRT/DRE elements in the '22 to '116 region, while regions '877 to '661 and '437 to '222 contain elements that can down regulate expression at the conditions used. Characterization of the 1-FEH IIa promoter may provide tools to study cold induced expression and to increase freezing tolerance in agricultural crops.