UDP-glucuronic acid decarboxylase (UXS) in alfalfa: A target to improve ruminal digestibility of stem cell walls

Authors: FANELLI, AMANDA - Orise Fellow; SMITH, REBECCA - University Of Wisconsin; Reinhardt, Laurie; WEIMER, PAUL - University Of Wisconsin; Sullivan, Michael

Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2025
Publication Date: 12/18/2025
Citation: Fanelli, A., Smith, R.A., Reinhardt, L.A., Weimer, P.J., Sullivan, M.L. 2025. UDP-glucuronic acid decarboxylase (UXS) in alfalfa: A target to improve ruminal digestibility of stem cell walls. Journal of Experimental Botany. https://doi.org/10.1093/jxb/eraf541.
DOI: https://doi.org/10.1093/jxb/eraf541

Interpretive Summary: Alfalfa is an important forage crop in the United States wtih 2024 production of alfalfa hay worth $ 8.2 billion. Known as “the queen of forages”, alfalfa is widely used to feed dairy cattle and other ruminants. However, although the leaves are rich in protein and vitamins, the stems have low digestibility, which represents a loss of the full potential of the forage. The cell walls of the stems are composed mainly of polysaccharides (cellulose, pectins, xylans) and lignin, a large polymer that provides no nutritional value. Cellulose and pectins are good energy sources for ruminants, whereas xylans and lignin contribute to poor digestibility. Therefore, decreasing xylan and increasing pectin content in cell walls of alfalfa could make them more digestible. In plants, an enzyme named UXS makes xylose, the major building block of xylans. We have used a transgenic approach to decrease UXS enzyme activity in alfalfa, which resulted in decreased levels of xylans and lignin and increased levels of the more digestible pectin. In experiments using dairy cattle rumen fluid, stems of the transgenic alfalfa showed increased digestibility. These results demonstrate the potential of reducing UXS activity in alfalfa, using either transgenic or conventional breeding approaches, to enhance the crop, providing more value to farmers and the dairy industry. The use of such crops to feed dairy cows could increase efficiency in milk production, decreasing the need to supplement the diet with other energy sources.

Technical Abstract: Alfalfa (Medicago sativa) has a high nutritional value, but poor digestibility of the stems limits its value as an energy source in ruminant diets. Xylan and lignin negatively impact cell wall digestibility, whereas pectins have high digestibility in the rumen. In plants, UDP-xylose synthase (UXS) catalyzes the decarboxylation of UDP-glucuronic acid to form UDP-xylose in an irreversible step that is key for xylan synthesis. We functionally characterized two UXS genes in alfalfa (MsaUXS2 and MsaUXS4) and investigated their impact on ruminal digestibility. Both genes are more highly expressed in stems than leaves, and the enzymes have UDP-glucuronic acid decarboxylase activity in vitro. Silencing MsaUXS2/MsaUXS4 via RNAi altered plant growth and resulted in a 40% decrease in xylose, a 115% increase in arabinose, and a 60% increase in galacturonic acid in the polysaccharide matrix as well as a 20% decrease in lignin in the cell wall. Together, our data show a major role for MsaUXS2 and MsaUXS4 in xylan synthesis and secondary cell wall deposition in alfalfa. Additionally, silenced lines had on average 30% increased gas production at 24 h in in vitro rumen digestibility assays, demonstrating the potential of targeting UXS genes to increase stem digestibility.