Responses of Lotus corniculatus to environmental change 3: The sensitivity of phenolic accumulation to growth temperature and light intensity and effects on tissue digestibility.

MAIN CONCLUSION: Growth temperature and light intensity are major drivers of phenolic accumulation in Lotus corniculatus resulting in major changes in carbon partitioning which significantly affects tissue digestibility and forage quality. The response of plant growth, phenolic accumulation and tissue digestibility to light and temperature was determined in clonal plants of three genotypes of Lotus corniculatus (birdsfoot trefoil) cv Leo, with low, intermediate or high levels of proanthocyanidins (condensed tannins). Plants were grown from 10 °C to 30 °C, or at light intensities from 20 to 500 µm m-2 s-1. Plants grown at 25 °C had the highest growth rate and highest digestibility, whereas the maximum tannin concentration was found in plants grown at 15 °C. Approximately linear increases in leaf flavonol glycoside levels were found with increasing growth temperature in the low tannin genotype. Tannin hydroxylation increased with increasing growth temperature but decreased with increasing light intensity. The major leaf flavonols were kaempferol glycosides of which kaempferol-3-glucoside and kaempferol-3,7-dirhamnoside were the major components. Increases in both tannin and total flavonol concentrations in leaves were linearly related to light intensity and were preceded by a specific increase in the transcript level of a non-legume type chalcone isomerase. Changes in growth temperature and light intensity, therefore, result in major changes in the partitioning of carbon into phenolics, which significantly affects tissue digestibility and nutritional quality with a high correlation between tannin concentration and leaf digestibility.

Responses of Lotus corniculatus to environmental change. 4: Root carbohydrate levels at defoliation and regrowth climatic conditions are major drivers of phenolic content and forage quality.

MAIN CONCLUSION: Differential accumulation of root carbohydrates at defoliation have a higher impact than regrowth environmental conditions on the phenolic content and feed quality of the perennial forage legume Lotus corniculatus. The unpredictable nature of proanthocyanidin (condensed tannin) accumulation in regrowth vegetation of the perennial forage legume Lotus corniculatus represents a dilemma to the wider use of this species in agriculture, and a potential problem in the nutritional ecology of some terrestrial herbivores, as variable condensed tannin levels can result in either beneficial or detrimental effects on animal nutrition. However, the source of this variation has not been extensively explored. High levels of carbon allocation to roots during low-temperature preconditioning of clonal plants were found to significantly increase condensed tannin and flavonol levels in regrowth foliage, while low levels of carbon allocation to roots during periods of high-temperature preconditioning significantly decreased condensed tannin and flavonol levels. Phenolic accumulation and tissue digestibility were also differentially affected by regrowth of these defoliated plants at high CO2 concentrations and by drought. Lower rates of digestion generally paralleled increases in tannin levels in regrowth leaves under the different environmental conditions, with rates of digestion falling in high tannin plants, despite correspondingly higher levels of leaf carbohydrates. Differential accumulation of root carbohydrates between seasons and years may therefore explain some of the variability found in the nutritional quality of the forage of this species.

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification.

BACKGROUND: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. RESULTS: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. CONCLUSIONS: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.

Genome-wide association studies and prediction of 17 traits related to phenology, biomass and cell wall composition in the energy grass Miscanthus sinensis.

• Increasing demands for food and energy require a step change in the effectiveness, speed and flexibility of crop breeding. Therefore, the aim of this study was to assess the potential of genome-wide association studies (GWASs) and genomic selection (i.e. phenotype prediction from a genome-wide set of markers) to guide fundamental plant science and to accelerate breeding in the energy grass Miscanthus. • We generated over 100,000 single-nucleotide variants (SNVs) by sequencing restriction site-associated DNA (RAD) tags in 138 Micanthus sinensis genotypes, and related SNVs to phenotypic data for 17 traits measured in a field trial. • Confounding by population structure and relatedness was severe in naïve GWAS analyses, but mixed-linear models robustly controlled for these effects and allowed us to detect multiple associations that reached genome-wide significance. Genome-wide prediction accuracies tended to be moderate to high (average of 0.57), but varied dramatically across traits. As expected, predictive abilities increased linearly with the size of the mapping population, but reached a plateau when the number of markers used for prediction exceeded 10,000-20,000, and tended to decline, but remain significant, when cross-validations were performed across subpopulations. • Our results suggest that the immediate implementation of genomic selection in Miscanthus breeding programs may be feasible.

Genotype, development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus.

BACKGROUND AND AIMS: Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock. METHODS: Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol. KEY RESULTS: Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent. CONCLUSIONS: It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene-trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.

Identification of secondary metabolites containing a diketopiperazine core in extracts from myxobacterial strains with growth inhibition activity against a range of prey species.

Myxobacteria produce a variety of bioactive secondary metabolites, and with a wealth of under-researched species they hold vast potential for undiscovered compounds. With the ever-increasing need for new antibiotics, the development of novel therapeutics is vitally important. Therefore, this study aimed to extract and elucidate antimicrobial metabolites from the following myxobacteria: Myxococcus xanthus CA010 and AB022; Corallococcus exiguus DSM 14696T; Myxococcus stipitatus DSM 14675T; and Corallococcus aberystwythensis AB050AT. Metabolite mixtures were extracted in acetone from XAD-16 resin incubated in liquid cultures and analysed using GC-MS. Bioactivity was identified using a growth inhibition assay against a panel of clinically relevant prey species including Gram-positive and Gram-negative bacteria and a fungus. Growth of Klebsiella pneumoniae and Enterococcus faecalis was most affected by the metabolite mixtures and the mixtures from AB022 and AB050AT were effective against the most prey. GC-MS analysis revealed metabolites with roles in the synthesis and degradation of amino acids and fatty acids, but also identified compounds A and B with a diketopiperazine (DKP) core. With previously confirmed bioactivity of compound A, it is suggested that these DKP compounds are contributing to the antimicrobial activity observed. Furthermore, many compounds could not be identified and so these unknowns present further potential for novel bioactive compounds.

Variability of cell wall recalcitrance and composition in genotypes of Miscanthus from different genetic groups and geographical origin.

Miscanthus is a promising crop for bioenergy and biorefining in Europe. The improvement of Miscanthus as a crop relies on the creation of new varieties through the hybridization of germplasm collected in the wild with genetic variation and suitable characteristics in terms of resilience, yield and quality of the biomass. Local adaptation has likely shaped genetic variation for these characteristics and is therefore important to quantify. A key biomass quality parameter for biorefining is the ease of conversion of cell wall polysaccharides to monomeric sugars. Thus far, the variability of cell wall related traits in Miscanthus has mostly been explored in accessions from limited genetic backgrounds. Here we analysed the soil and climatic conditions of the original collection sites of 592 Miscanthus genotypes, which form eight distinct genetic groups based on discriminant analysis of principal components of 25,014 single-nucleotide polymorphisms. Our results show that species of the genus Miscanthus grow naturally across a range of soil and climate conditions. Based on a detailed analysis of 49 representative genotypes, we report generally minor differences in cell wall characteristics between different genetic groups and high levels of genetic variation within groups, with less investigated species like M. floridulus showing lower recalcitrance compared to the other genetic groups. The results emphasize that both inter- and intra- specific variation in cell wall characteristics and biomass recalcitrance can be used effectively in Miscanthus breeding programmes, while also reinforcing the importance of considering biomass yield when quantifying overall conversion efficiency. Thus, in addition to reflecting the complexity of the interactions between compositional and structural cell wall features and cell wall recalcitrance to sugar release, our results point to traits that could potentially require attention in breeding programmes targeted at improving the Miscanthus biomass crop.

Alterations in the metabolic fingerprint of Cladonia portentosa in response to atmospheric nitrogen deposition.

Nitrogen availability has profound ecological consequences in nutrient-limited systems. In terrestrial settings these would include the upland heaths, sand dunes and blanket bogs of temperate latitudes. Understanding the physiological consequences of nitrogen enrichment is a first critical step in predicting possible consequences. Results are presented from a metabolic fingerprinting study using Fourier transform-infrared spectroscopy (FTIR) to detect biochemical differences in the lichen Cladonia portentosa collected from 25 sites across mainland Britain varying in their nitrogen input. Partial least-squares regression analysis of the FTIR data demonstrated that changes in broad biochemical classes were consistently correlated with mean annual wet inorganic nitrogen deposition loads. These results demonstrated a direct coupling of a broad range of metabolic processes in C. portentosa to nitrogen deposition.