|Storey, Kathleen - UNIVERSITY OF MINNESOTA|
|Jung, Hans Joachim|
|Somers, David - UNIVERSITY OF MINNESOTA|
Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 26, 2006
Publication Date: September 5, 2006
Citation: Schnurr, J.A., Storey, K.K., Jung, H.G., Somers, D.A., Gronwald, J.W. 2006. UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis. Planta. 224:520-532. Interpretive Summary: Alfalfa, the largest forage legume crop in the U.S., is important because of its high feed value for livestock and because it has considerable potential for development as a bioenergy crop. For both of these uses, a modification of the cell walls of alfalfa stems is desirable. Pectin is a matrix polysaccharide (MPS) found in the cell walls of plants. An increase in pectin in stem cell walls would improve alfalfa’s value as a livestock feed, while decreasing pectin would improve its value as a bioenergy crop. In order to develop new alfalfa varieties with modified stem cell walls, we need a better understanding of the metabolic pathways that regulate biosynthesis of pectin and other cell wall MPS. The myo-inositol oxidation (MIO) pathway plays a key role in the synthesis of MPS in plants and UDP-sugar pyrophosphorylase (USP) is an important protein in this pathway. In earlier research conducted with the model plant Arabidopsis, we identified the gene that makes the USP protein. To more clearly define the role of USP in cell wall formation and the synthesis of MPS, we examined Arabidopsis knockout lines where the USP gene has been turned off. In the knockout lines, we examined plant appearance, cell wall composition, and the amount of USP protein produced by the gene. Turning off the USP gene did not alter the appearance of Arabidopsis plants. In leaves and stems of the knockout lines, the amount of USP protein was reduced by about 50%, but this did not interfere with cell wall formation or the amount of MPS in cell walls. These results indicate that normal levels of USP protein in leaves and stems are more than adequate for producing the amount of MPS needed to make cell walls. In contrast, turning off the USP gene completely blocked the formation of cell walls in pollen. The lack of a cell wall resulted in collapsed, nonviable pollen grains. Our research provided the first report that the USP gene plays a critical role in cell wall synthesis in pollen. In contrast, the USP gene appears to be less important in MPS synthesis and normal cell wall development in stems. The results of this research have advanced understanding of metabolic pathways that regulate cell wall formation and composition in plants. This new knowledge will be used to plan future research to modify cell walls in alfalfa stems and increase the value of alfalfa as a livestock feed and bioenergy crop.
Technical Abstract: Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) is a broad substrate enzyme that synthesizes nucleotide sugars. The products of the USP reaction can act as precursors for the synthesis of cell wall components including pectin and hemicellulose. AtUSP has no close homologs in Arabidopsis and its biological function in vivo has not been clearly defined. We identified two T-DNA knockout lines for AtUSP, usp-1 and usp-2. Seedlings from selfed heterozygous individuals segregated in a 1:1 ratio under marker- assisted selection, and a homozygous line was not identified. Despite decreased levels of both AtUSP transcript and USP activity, heterozygous plants were indistinguishable from wild type at all stages of development. Reciprocal test crosses indicated the source of the segregation distortion was lack of transmission through the male gametophyte. Analysis of pollen tetrads from usp-1 in the quartet background revealed a 2:2 ratio of normal:collapsed pollen grains. These collapsed pollen grains were not viable as determined by pollen germination tests, Alexander’s viability, and DAPI staining. The pollen phenotype of usp-1 was complemented by transformation of usp-1 with the USP cDNA sequence. Surface and ultrastructural analysis of pollen from wild-type and usp mutants demonstrated that the mutation had no effect on the outer wall (exine) but prevented the synthesis of the pectocellulosic inner wall (intine). Evidence presented here shows that AtUSP has a critical role in pollen development.