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.

Marissa B Roldan,Juha-Pekka Salminen,Stefan Muetzel,Christine R Voisey,Alexia Blanc,Dorothy Maher,John R Caradus,Kim Richardson,Greig Cousins,Derek R Woodfield,Karl Fraser,Wayne E Zeller,Zulfi Jahufer,Rupinder Kaur

doi:10.3389/fpls.2021.777354

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.

Highlights

Anthropogenic greenhouse gas emissions pose a serious environmental challenge due to their effects on climate change, with methane 30 times more effective than carbon dioxide in trapping heat in the atmosphere (Stocker et al, 2013), and nitrous oxide (N2O) emissions being a key player in ozone depletion (Revell et al, 2015)
Transgenic plants were initially screened for TaMYB14-1 by PCR, and CTexpressing plants were identified by staining leaflets using the chromogenic reagent dimethylaminocinnamaldehyde (Li et al, 1996) (DMACA; Supplementary Methods section “White Clover Transformation”)
This study has demonstrated that the condensed tannins (CTs) trait in the leaves of transformed elite white clover cultivar is stable over at least two generations

Summary

Introduction

Anthropogenic greenhouse gas emissions pose a serious environmental challenge due to their effects on climate change, with methane 30 times more effective than carbon dioxide in trapping heat in the atmosphere (Stocker et al, 2013), and nitrous oxide (N2O) emissions being a key player in ozone depletion (Revell et al, 2015). Ruminant livestock fed on lush pastures rich in soluble proteins can suffer from bloat. This potentially fatal digestive disorder (Wang et al, 2012) is caused by gasses released during forage fermentation that can become trapped in a stable foam in the rumen, preventing eructation and affecting lung and heart function. CTs are formed through the covalent linkage of flavan-3-ol subunits in their trans (afzelechin, catechin, and gallocatechin) or cis (epiafzelechin, epicatechin, and epigallocatechin) configurations (Figure 1A). Afzelechin and epiafzelechin are referred to as propelargonidin subunits, catechin and epicatechin as procyanidin (PC) subunits, and gallocatechin and epigallocatechin as prodelphinidin (PD) subunits In this interflavan3-ol bond arrangement, covalent bonds are formed between C8 and the oxygen atom connected to C7 of the A-ring of one flavan-3-ol subunit to the C4 and C2 atoms of the C-ring, respectively, of the adjacent flavan-3-ol subunit (Figure 1B, right panel)

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Discussion

Conclusion