Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

Abstract

Summary The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.

The effects of PPO activity on the proteome of ingested red clover and implications for improving the nutrition of grazing cattle

“…The inefficient utilisation of dietary forage protein could potentially be improved by decreasing the extent of protein degradation that occurs within the rumen, and PPO containing forages could be a sustainable mechanism which contributes to achieving this. The subsequent increased flow of non-degraded proteins to the small intestine will improve the nutritional value of the forage feed [31] , [48] , contributing to overall improved animal productivity. Within the rumen, protein breakdown occurs rapidly during the first 6 h of ingestion [31] , with transition from primary to secondary microbial colonisation occurring at 4 h [35] .…”

“…Sulphur containing amino acids (methionine and cysteine) may act as PPO induced quinone binding sites to form protein bound phenol [16] . It is well established that increasing the content of sulphur-amino acids would increase delivery of what are essential dietary amino acids for ruminants [48] , [61] suggesting a possible further role for PPO in protecting these sulphur containing amino acids from metabolism within the rumen [62] thereby making them relatively more available for ruminant nutrition and less prone to wasteful deamination. However, it is unknown whether the acid conditions within the lower gut would be sufficient to reverse the quinone binding of peptides to prevent onset of anti-nutritional effects [63] .…”

Recent Progress of Transgenic Technology Development for Alfalfa

“…Being of a perennial nature that covers agriculture fields all seasons, alfalfa plants play important roles in soil conservation and soil ecology [3] [4]. In addition, due to its high biomass and non-human food nature, alfalfa is an attractive crop for producing recombinant proteins and other biomaterials from plants via molecular farming approaches [5] [6] [7] [8]. Like many other crops, genetic engineering is an important approach for alfalfa trait modification and improvement.…”