The Hunt for the “Holy Grail”: Condensed Tannins in Perennial Forage Legumes

A recent advance using molecular biology has identified a transcription factor or master switch that can ‘turn on’ the condensed tannin pathway present in white clover, and with the appropriate promoters allows biologically significant levels of condensed tannin expression in leaf tissue. In vitro tests have demonstrated that the condensed tannins produced in white clover leaves can bind protein at a pH 6.5, as found in the rumen, and then release them at pH 2.5, the pH in the abomasum, before entering the small intestine for amino acid absorption. Additional tests have demonstrated that these condensed tannins can reduce methane production by up to 25% in the first 6 hours of incubation. The journey to this point and the challenges ahead to deliver white clover cultivars with condensed tannin expression will be described

Grasses as Biofactories: Scoping out the Opportunities

Plant biopharming is set to dominate commercial recombinant protein expression for specific proteins. The choice of plant species depends on a multitude of factors and is determined on a case- by-case basis. As a leaf based expression system grasses would have to compete predominantly with tobacco and alfalfa. The grass-endophyte symbiosis offers a number of unique possibilities for biopharming

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

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

Seed Transmission of Epichloë Endophytes in Lolium perenne Is Heavily Influenced by Host Genetics

Vertical transmission of symbiotic Epichloë endophytes from host grasses into progeny seed is the primary mechanism by which the next generation of plants is colonized. This process is often imperfect, resulting in endophyte-free seedlings which may have poor ecological fitness if the endophyte confers protective benefits to its host. In this study, we investigated the influence of host genetics and environment on the vertical transmission of Epichloë festucae var. lolii strain AR37 in the temperate forage grass Lolium perenne. The efficiency of AR37 transmission into the seed of over 500 plant genotypes from five genetically diverse breeding populations was determined. In Populations I–III, which had undergone previous selection for high seed infection by AR37, mean transmission was 88, 93, and 92%, respectively. However, in Populations IV and V, which had not undergone previous selection, mean transmission was 69 and 70%, respectively. The transmission values, together with single-nucleotide polymorphism data obtained using genotyping-by-sequencing for each host, was used to develop a genomic prediction model for AR37 seed transmission. The predictive ability of the model was estimated at r = 0.54. While host genotype contributed greatly to differences in AR37 seed transmission, undefined environmental variables also contributed significantly to seed transmission across different years and geographic locations. There was evidence for a small host genotype-by-environment effect; however this was less pronounced than genotype or environment alone. Analysis of endophyte infection levels in parent plants within Populations I and IV revealed a loss of endophyte infection over time in Population IV only. This population also had lower average tiller infection frequencies than Population I, suggesting that AR37 failed to colonize all the daughter tillers and therefore seeds. However, we also observed that infection of seed by AR37 may fail during or after initiation of floral development from plants where all tillers remained endophyte-infected over time. While the effects of environment and host genotype on fungal endophyte transmission have been evaluated previously, this is the first study that quantifies the relative impacts of host genetics and environment on endophyte vertical transmission

Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses

White Clover (<em>Trifolium repens</em> L.) Benefits in Grazed Pastures and Potential Improvements

White clover has been, and continues to be, a valuable component of grazed pastures through improving feed quality and nutritive value, improving seasonal dry matter distribution, and providing biologically fixed nitrogen that benefits not only white clover itself but also the surrounding plants. The contribution of white clover to sustainability and environmental goals is a growing focus of breeding programs. The use of genome mapping and genotyping by sequencing to determine genetic variation and population structure in clover improvement programs needs to be expanded to improve breeding efficiencies. Seed yields also need to be improved while maintaining the selected agronomic performance traits to ensure that commercial cultivars remain cost-effective with other crops and land uses. Beneficial traits not available within the white clover genome may be provided through genetic modification and gene editing, particularly traits that contribute towards addressing challenges associated with animal nutrition and health, water quality and climate change. The inherent benefits of white clover as well as the potential for including additional beneficial traits will be described

Effects of ingestion of a Bacillus thuringiensis toxin and a trypsin inhibitor on honey bee flight activity and longevity

To assess potential impacts of transgenic pest-resistant plants, newly-emerged adult honey bees from ten colonies were tagged, placed in cages at 33 oC, and fed with 625 $\mu$g/g Cry1Ba Bacillus thuringiensis (Bt) toxin or 2.5 mg/g aprotinin proteinase inhibitor in pollen-food (equivalent to 0.25% or 1% of total soluble protein). Control bees were given similar food without additive. All foods were consumed at similar rates. After seven days, all bees were returned to their hives. Subsequent observations showed that Cry1Ba-fed bees did not differ significantly from control bees in the timing of their first flight, the period during which flights took place or in estimated longevity. However, aprotinin-fed bees began to fly and also died about three days sooner than Cry1Ba-fed or control bees. Their flight periods were similar to those of the other bees. The effects of transgenic aprotinin-plants on honey bees will thus depend on gene expression levels in pollen