Condensed tannins in white clover (Trifolium repens) foliar tissues expressing the transcription factor TaMYB14-1 bind to forage protein and reduce ammonia and methane emissions in vitro

Authors: ROLDAN, MARISSA - Agresearch; COUSINS, GREIG - Pyne Gould Guinness Limited And Agricom Limited (PGG) Wrightson Seeds; MUETZELA, STEFAN - Agresearch; Zeller, Wayne; FRASER, KARL - Agresearch; SALMINEN, JUHA-PEKKA - University Of Turku; BLANC, ALEXIA - Agresearch; KAUR, RUPINDER - Pyne Gould Guinness Limited And Agricom Limited (PGG) Wrightson Seeds; RICHARDSON, KIM - Agresearch; MAHER, DOROTHY - Agresearch; JAHUFER, ZULFI - Agresearch; WOODFIELD, DEREK - Pyne Gould Guinness Limited And Agricom Limited (PGG) Wrightson Seeds; CARADUS, JOHN - Grasslanz Technology; VOISEY, CHRISTINE - Agresearch

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2021
Publication Date: 1/6/2022
Citation: Roldan, M.B., Cousins, G., Muetzela, S., Zeller, W.E., Fraser, K., Salminen, J., Blanc, A., Kaur, R., Richardson, K., Maher, D., Jahufer, Z., Woodfield, D., Caradus, J.R., Voisey, C.R. 2022. Condensed tannins in white clover (Trifolium repens) foliar tissues expressing the transcription factor TaMYB14-1 bind to forage protein and reduce ammonia and methane emissions in vitro. Frontiers in Plant Science. 12. Article 777354. https://doi.org/10.3389/fpls.2021.777354.
DOI: https://doi.org/10.3389/fpls.2021.777354

Interpretive Summary: Condensed tannins (CTs) are polyphenolic compounds that occur abundantly in many vascular plants such as birdsfoot trefoil, sainfoin and sulla and have proven efficacy in increasing nitrogen use efficiency, reducing methane emissions and preventing pasture bloat when fed to ruminants. Despite these advantages, the utility of these forage species is marred by poor persistence in year-to-year harvests and under year-round grazing and suffer from low dry matter yields. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover and alfalfa, but can be found in flowers and seed coats. The discovery that expression of a transcription factor from rabbit’s foot clover in white clover, alfalfa and tobacco could induce condensed tannin accumulation in foliar tissues provided the opportunity to develop persistent forage legumes containing therapeutic levels of CTs. Transformation of white clover with the transcription factor from rabbit’s foot clover resulted in production of CTs in white clover leaves of up to 1.2% of dry matter (DM) in the primary transgenic plant and greater that 2% after successive selected breeding. This level of CT concentration in forages is considered sufficient to induce the beneficial effects on ruminants. The structure and composition of the CTs were determined using advanced nuclear magnetic resonance spectroscopy and mass spectrometry techniques. In vitro studies showed that foliar CTs, either purified or in crude extracts, bound to protein at pH 6.5, and were released at pH 2.0-2.5, suggesting that white clover CTs should protect and then release dietary protein in the rumen and abomasum of ruminants, respectively. Using cow rumen fluid in vitro assays, milled white clover leaves containing CTs of 1.6 – 2.4% of DM reduced methane production by 19% and ammonia production by 60% relative to non-transformed wild type controls after 6 h of incubation. These studies document significant steps accomplished toward the incorporation of CTs into leaves of important forage legumes such white clover and alfalfa.

Technical Abstract: Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75–93%) and procyanidins (PC, 17–25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6–2.4% of DM significantly reduced methane production by 19% (p<0.01) and ammonia production by 60% (p<0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.