SummaryAnthocyanin pigments accumulate to form spatially restricted patterns in plants, particularly in flowers, but also occur in vegetative tissues. Spatially restricted anthocyanin leaf markings are poorly characterised in plants, but are common in forage legumes.We hypothesised that the molecular basis for anthocyanin leaf markings in Trifolium spp. is due to the activity of a family of R2R3-MYB genes.R2R3-MYB genes were identified that are associated with the two classic pigmentation loci in T. repens. The R locus patterns 'red leaf', 'red midrib' and 'red fleck' are conditioned by a single MYB gene, RED LEAF. The 'diffuse red leaf' trait is regulated by the RED LEAF DIFFUSE MYB gene. The V locus was identified through mapping two V-linked traits, 'V-broken yellow' (Vby) and 'red leaflet' (Vrl ). Two highly similar R2R3-MYB genes, RED V-a and RED Vb, mapped to the V locus and co-segregated with the RED V pigmentation pattern. Functional characterisation of RED LEAF and RED V was performed, confirming their function as anthocyanin regulators and identifying a C-terminal region necessary for transactivation.The mechanisms responsible for generating anthocyanin leaf markings in T. repens provide a valuable system to compare with mechanisms that regulate complex floral pigmentation.
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.
Improving the genetic merit of temperate forage legumes helps ensure profitability and sustainability of our Australasian pastoral industries. Today’s plant breeders are supported by a range of underpinning research activities including genetic resources exploration and enhancement, plant physiology, plant health, feed quality, agronomy, quantitative genetics and plant biotechnology; and have collaborative interfaces with animal and farm systems science. Lifting the rate of gain by integration of molecular tools, innovative breeding strategies, and new genetic resources is the major objective of our white clover breeding network. This paper, presented at the Australasian Grassland Association’s recent Legume Symposium, focuses on the key research and development achievements in white clover breeding for Australasia, and on the success and future of an Australasian collaboration to breed improved cultivars for the region’s temperate environments. The paper reports on successful developments in the areas of improving white clover root systems for phosphate uptake, pest tolerance, development of novel inter-specific hybrids and marker-aided breeding. The successful trans-Tasman collaboration in white clover breeding and future work is also discussed.
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