Impact of expressing p-coumaryl transferase in Medicago sativa L. on cell wall chemistry and digestibility

Authors: Marita, Jane; Rancour, David; Hatfield, Ronald; Weimer, Paul

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/27/2016
Publication Date: 12/30/2016
Publication URL: http://handle.nal.usda.gov/10113/5607533
Citation: Marita, J.M., Rancour, D.M., Hatfield, R.D., Weimer, P.J. 2016. Impact of expressing p-coumaryl transferase in Medicago sativa L. on cell wall chemistry and digestibility. American Journal of Plant Sciences. 7:2553-2569.

Interpretive Summary: The fiber component of forages is made up of the plant cell walls. Cell walls of alfalfa, an important feedstuff for dairy cows, is made up of structural carbohydrates (about 78%), protein (4%), and lignin (18%). Stem fraction of alfalfa is much less digestible than the leaf fraction. The structural carbohydrate fraction is an important energy source for the cow, but only about 50% in the stems can be digested in the rumen and used efficiently. The lignin fraction is not digested at all by the cow and prevents a large portion of the carbohydrate fraction from being digested in the rumen. A phenolic component, p-coumaric acid ([pCA], typically found attached to lignin in grass cell walls) is thought to alter lignin structure, allowing the cell wall to be more digested. Alfalfa stems contain only trace amounts of pCA in their cell walls. We inserted a gene into alfalfa that leads to the addition of pCA to lignin. The gene was highly expressed, and levels of pCA increased 20- to 200-fold in the cell wall. Total lignin concentration did not change, but composition was changed. Digestibility of the carbohydrate fraction did not improve over the unaltered plants. This would suggest that more drastic changes must occur in the lignin in order to improve digestibility. Changes in composition alone and a decrease in total lignin must occur to improve digestion of this forage in dairy cows.

Technical Abstract: The addition of p-coumaric acid (pCA) to lignin molecules is frequently found in members of the grass family. The role of this addition is not clearly understood, but is thought to potentially aid in the formation of syringyl type lignin. This is because the incorporation is as a conjugate of pCA ester linked to sinapyl alcohol, a major component of lignin. The forage legume, alfalfa (Medicago sativa L.), does not contain appreciable levels of pCA in its more heavily lignified stem tissues. The maize p-coumaryl transferase (pCAT) gene was used to transform alfalfa to determine its impact upon lignin composition and its potential to alter cell wall digestibility. A constitutive expression vector using the cassava vein mosaic virus (CsVMV) promoter was used to drive expression of maize pCAT in alfalfa. Expression of the pCAT transgene was detected in both leaves and stems. Although there was a range of pCA values in transformed alfalfa stems (0.2 – 1.79 micrograms mg-1 FW), this was a clear increase over bound pCA in control stems (0.15-0.2 mean=0.17 micrograms mg-1 FW). This did not lead to consistent responses concerning total lignin in the stem tissues. On the other hand, leaf tissues already had a relatively high level of pCA (0.85-1.2 mean= 0.99 micrograms mg-1 FW) and those expressing pCAT gene showed, on average, a small increase. However, there was a wide range of values among the transformants (0.38-1.55, mean= 1.064 micrograms mg-1 FW). Lignin in leaves did not appear to be significantly impacted. However, incorporation of pCA into the wall appeared to cause a shift in lignin composition. Testing the pCAT-expressing stem cell walls for digestibility using a rumen in vitro system showed there was no change in the digestibility of the stem compared to empty vectors and control alfalfa stems. Although expression of pCAT gene in alfalfa changes the amount of wall bound pCA, it does not appear to change lignin levels and impact digestibility.