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Title: EXPRESSION OF UDP-GLUCOSE DEHYDROGENASE REDUCES CELL-WALL POLYSACCHARIDE CONCENTRATION AND INCREASES XYLOSE CONTENT IN ALFALFA STEMS

Author
item Samac, Deborah - Debby
item LITTERER, LYNN - UNIV OF MINNESOTA
item TEMPLE, GLENA - VITERBO COLLEGE
item Jung, Hans Joachim
item SOMERS, DAVID - UNIV OF MINNESOTA

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2003
Publication Date: 4/1/2004
Citation: Samac, D.A., Litterer, L., Temple, G., Jung, H.G., Somers, D.A. 2004. Expression of UDP-glucose dehydrogenase reduces cell-wall polysaccharide concentration and increases xylose content in alfalfa stems. Applied Biochemistry and Biotechnology. 116(1):1167-1182.

Interpretive Summary: Alfalfa is a very nutritious feed for dairy and beef cattle because of its high protein content. The proteins in alfalfa are rapidly broken down in the cow's first stomach, the rumen. Microorganisms in the rumen take up the degraded proteins and use them to grow and reproduce. The microorganisms are then digested in the cow's small intestine. Unfortunately, a large amount of the protein in alfalfa is often not captured by microbial growth because alfalfa does not contain enough rapidly digested carbohydrates that the microorganisms need to flourish. The lost protein is then excreted, creating a potential source of environmental pollution. One remedy, feeding starch along with alfalfa, can lead to serious health problems in cattle. Our goal is to increase the amount of pectin, an easily digested carbohydrate, in alfalfa stems to decrease protein wastage and improve health of dairy cattle. Alfalfa plants expressing a soybean gene for an enzyme that has a central role in pectin synthesis were generated and grown in a field test for 2 years. In initial greenhouse experiments, enzyme activity in some alfalfa plants was up to 7-fold greater than in control plants; however, field-grown plants had only a maximum of 1.9-fold more activity than the control. Expression of the soybean gene reduced yields during the first year but did not affect yield in the second year of the trial. The total amount of carbohydrates was lower in plants with the soybean gene and there was no change in the amount of pectin. Expression of the soybean gene did result in an increase in several sugars that are found in pectin resulting in a change in the composition of alfalfa pectin. The biochemical pathways leading to pectin production are very complex. Our results indicate that the activity of more than one enzyme will be required to change the amount of pectin in alfalfa stems. If successful, a modified alfalfa with increased pectin would increase the feed value of alfalfa, decrease costs for dairy producers, and reduce potential environmental pollution from animal wastes.

Technical Abstract: The primary cell-wall matrix of most higher plants is composed of large amounts of uronic acids, primarily D-galacturonic acid residues in the backbone of pectic polysaccharides. UDP-glucose dehydrogenase, catalyzing the irreversible conversion of UDP-glucose to UDP-glucuronic acid, is a key enzyme in the biosynthesis of uronic acids as UDP-glucuronic acid is a precursor for several of the major pectic monosaccharides. With the goal of increasing the amount of pectin in alfalfa (Medicago sativa L.) stems, we produced transgenic alfalfa plants expressing a soybean UDP-glucose dehydrogenase cDNA under the control of two promoters active in alfalfa vascular tissues. In initial greenhouse experiments, enzyme activity in transgenic lines was up to 7-fold greater than in non-transformed Regen-SY control plants; however, field-grown transgenic plants had only a maximum of 1.9-fold more activity than the control and only two transformant lines showed statistically increased activity in both years of the experiment. Alfalfa yield was lower for all transformed lines in 1999 than the control line; however, in 2000 yield did not differ among the lines. Cell-wall polysaccharide content was lower and Klason lignin content was higher in transgenics compared to the non-transformed control. No significant increase in uronic acids in the polysaccharide fraction was observed in any line. Xylose, which is downstream of uronic acid synthesis, increased 15% in most transgenic lines compared to the control and mannose concentration decreased slightly in all lines. Cellulose biosynthesis did not appear to be affected by the transgenes. Higher UDP-glucose dehydrogenase activity did not result in increased pectin accumulation in transgenic alfalfa. Because of the complexity of pectic polysaccharides and sugar biosynthesis, it may be necessary to manipulate multiple steps in carbohydrate metabolism to alter pectin content of alfalfa.