Altering uxs expression in alfalfa to potentially increase cell wall digestibility by decreasing xylan and increasing cellulose or pectin content

Authors: FANELLI, AMANDA - Oak Ridge Institute For Science And Education (ORISE); Sullivan, Michael

Submitted to: Gordon Research Conferences
Publication Type: Abstract Only
Publication Acceptance Date: 4/12/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Alfalfa (Medicago sativa) is a forage crop widely used for ruminant feed in temperate climates. The stem cell walls have great potential as an energy source, as they contain large amounts of carbohydrates, which make up cellulose, hemicellulose (mainly xylan) and pectic (rich in galacturonic acid) polysaccharides. Nonetheless, neither cellulose nor xylans are completely digested in the rumen. Among many factors, xylan interactions with cellulose and lignin contribute to the overall low digestibility of the cell wall. On the other hand, pectic materials are more efficiently digested by rumen microorganisms. One approach to increase digestibility of alfalfa stems is to reduce xylan levels while increasing cellulose and pectin levels. In plants, the building blocks that constitute the cell wall polysaccharides are synthesized via a nucleotide sugar interconversion pathway. Xylose, the main monosaccharide in the xylan backbone, is formed through the decarboxylation of UDP-D-glucuronic acid (UDP-D-GlcA) by UDP-D-GlcA decarboxylase (also referred to as UDP-D-xylose synthase [UXS]). In arabidopsis, mutants in the cytosolic versions of UXS (AtUXS3, AtUXS5 and AtUXS6) impact monosaccharide composition of the stem cell wall and digestibility. Therefore, uxs genes are an attractive target for downregulation in alfalfa, as this could decrease xylan levels, improving overall digestibility, while increasing cellulose or pectin levels due to restricted flow of sugar nucleotides through the pathway. To identify genes putatively encoding cytosolic versions of UXS in alfalfa, we performed BLASTn searches of its genome (CADL, available at legumeinfo.org) using five Medicago truncatula uxs genes as query. The output genes were annotated and translated into proteins. A maximum likelihood phylogeny with the identified amino acid sequences from M. sativa, as well as the UXS from M. truncatula and Arabidopsis thaliana, was inferred using PhyML. In this tree, four alfalfa sequences were in the clade containing the cytosolic UXS from arabidopsis. The identified genes correspond to two pairs of homologs, and each pair encodes a unique amino acid sequence. Analysis of published RNAseq data from alfalfa showed that the four genes are more highly expressed in stems than leaves. To assess the role of these genes in plant cell wall synthesis, a hairpin RNAi construct designed to silence the alfalfa uxs genes, and another two constructs for overexpression of a member of each homolog pair were transformed into the Regen-SY27 alfalfa genotype. We will analyze these transgenic plants to assess the impacts of altered uxs expression on cellulose, xylan, pectin and lignin levels, as well as in vitro digestibility.