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United States Department of Agriculture

Agricultural Research Service

Research Project: Removing Limitations to the Efficient Utilization of Alfalfa and Other Forages in Dairy Production, New Bio-Products, and Bioenergy to... Title: Identification and suppression of the p-coumaroyl-CoA: hydroxycinnamyl alcohol transferase in Zea mays L.

Authors
item MARITA, JANE
item HATFIELD, RONALD
item RANCOUR, DAVID
item Frost, Kenneth -

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 12, 2014
Publication Date: May 20, 2014
Citation: Marita, J.M., Hatfield, R.D., Rancour, D.M., Frost, K. 2014. Identification and suppression of the p-coumaroyl-CoA: hydroxycinnamyl alcohol transferase in Zea mays L. Plant Journal. doi:10.1111/tpj.12510.

Interpretive Summary: Grasses, including corn, are valuable sources of nutrition for ruminant animals worldwide. Corn silage is extensively utilized in the United States, especially in dairy operations, as a good source of nutritional energy. The fiber portion (cell walls) of the plant makes up a large portion (45 to 50%) of this potential energy source, but it is not completely utilized by dairy cows. Structural carbohydrates in the cell wall could be fully utilized by the animal if the inhibitory effects of lignin and related phenolic components could be eliminated or reduced. The complex nature of cell wall formation that makes up the fiber portion of the plant can be difficult to understand. Special enzymes are involved in the incorporation of phenolic materials into the cell wall. It has been proposed that the incorporation of one of these phenolic compounds, p-coumaric acid (pCA), into the cell wall may be important in lignin formation in grass cell walls. To investigate this hypothesis, work was undertaken to isolate the enzyme and the gene responsible for adding pCA to grass cell walls. This knowledge was then used to decrease the expression of this enzyme in corn, and the plants were then analyzed for cell wall characteristics. The enzyme did not appear to alter total lignin formation, but it did change the composition. Further work will be needed to determine to what degree such compositional changes alter secondary functions such as digestibility and utilization by ruminants.

Technical Abstract: Grasses are unique in the relatively high levels of p-coumarates (pCA) within their cell walls, though the role of pCA within the wall matrix remains unclear. Corn (Zea mays L.) contains large amounts of pCA, especially in stem tissues (15% of total isolated lignin). Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols (primarily sinapyl alcohol residues). To understand the role of pCA in corn development, work was undertaken to isolate and purify the p-coumaroyl:CoA hydroxycinnamyl alcohol transferase (pCAT). Proteins were isolated from corn stem rind tissues that contain the highest levels of transferase activity on a per gram fresh weight basis. The pCAT was purified to a single band on SDS-PAGE gel electrophoresis using a combination of DEAE, size exclusion, and affinity chromatography. Purified pCAT was subjected to partial Trypsin digestion, and peptides were sequenced to determine primary amino acid composition. A basic local alignment search tool (BLAST) of the acquired peptide sequences against published maize sequences revealed one unidentified sequence that contained all peptide sequences from the purified enzyme. A gene sequence was then identified for a putative p-coumaroyl:CoA hydroxycinnamyl alcohol transferase. The gene sequence for the putative transferase was used to construct a RNAi anti-sense gene for down-regulating pCAT in corn (Iowa State University, Plant Transformation Facility, [PTF]). Transformants with reduced levels of the putative transferase gene were cultured to produce seed. T1 corn plants generated from the seed obtained from PTF were analyzed. We report here on the first down-regulation of the p-coumaroyl:CoA hydroxycinnamyl alcohol transferase in a monocot. Primary screening of corn seedling leaves by HPLC following a modified quick alkaline hydrolysis was used to identify plants with decreased amounts of pCA. A follow-up screening on mature leaves from fully developed plants was used to confirm that the same plants contained reduced pCA. Detailed chemical analysis of isolated cell walls for structural components from mature transgenic stems and leaves revealed that lignin levels and neutral sugar composition did not change, but pCA levels decreased and the lignin composition was altered. In general, the transgenics with the lowest levels of pCA correspondingly had decreased levels of sinapyl alcohol. These results indicated that altering the levels of pCAT can lead to altered lignin composition but did not appear to alter the total amount of lignin present in the cell walls.

Last Modified: 8/19/2014
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