A large-scale forward genetic screen for maize mutants with altered lignocellulosic properties.

The development of efficient pipelines for the bioconversion of grass lignocellulosic feedstocks is challenging due to the limited understanding of the molecular mechanisms controlling the synthesis, deposition, and degradation of the varying polymers unique to grass cell walls. Here, we describe a large-scale forward genetic approach resulting in the identification of a collection of chemically mutagenized maize mutants with diverse alterations in their cell wall attributes such as crystalline cellulose content or hemicellulose composition. Saccharification yield, i.e. the amount of lignocellulosic glucose (Glc) released by means of enzymatic hydrolysis, is increased in two of the mutants and decreased in the remaining six. These mutants, termed candy-leaf (cal), show no obvious plant growth or developmental defects despite associated differences in their lignocellulosic composition. The identified cal mutants are a valuable tool not only to understand recalcitrance of grass lignocellulosics to enzymatic deconstruction but also to decipher grass-specific aspects of cell wall biology once the genetic basis, i.e. the location of the mutation, has been identified.

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities.

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan-xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the 'ancestral' XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link ß-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.

Dynamic co-culture metabolic models reveal the fermentation dynamics, metabolic capacities and interplays of cheese starter cultures.

In this study, we have investigated the cheese starter culture as a microbial community through a question: can the metabolic behaviour of a co-culture be explained by the characterized individual organism that constituted the co-culture? To address this question, the dairy-origin lactic acid bacteria Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Leuconostoc mesenteroides, commonly used in cheese starter cultures, were grown in pure and four different co-cultures. We used a dynamic metabolic modelling approach based on the integration of the genome-scale metabolic networks of the involved organisms to simulate the co-cultures. The strain-specific kinetic parameters of dynamic models were estimated using the pure culture experiments and they were subsequently applied to co-culture models. Biomass, carbon source, lactic acid and most of the amino acid concentration profiles simulated by the co-culture models fit closely to the experimental results and the co-culture models explained the mechanisms behind the dynamic microbial abundance. We then applied the co-culture models to estimate further information on the co-cultures that could not be obtained by the experimental method used. This includes estimation of the profile of various metabolites in the co-culture medium such as flavour compounds produced and the individual organism level metabolic exchange flux profiles, which revealed the potential metabolic interactions between organisms in the co-cultures.

DOF, MYB and TCP transcription factors: Their possible roles on barley germination and seedling establishment.

Seed germination is a multi-staged complex process during seed plant life cycle, and it is tightly regulated through a coordinated expression of diverse genes in diverse tissues. As regulatory molecules of gene expression, determination of transcription factors is crucial to understanding molecular basis and regulatory network of germination process and seedling establishment. However, limited data on the contributions of these transcription factors to the germination of crop barley (Hordeum vulgare L.) are available. Here, we investigated the expression profiles of selected transcription factors from different families (DOF, MYB and TCP) with qRT-PCR analysis in various tissues including coleoptiles, leaves and roots following the germination. Analysis of MYB and DOF gene expression profiles indicated that there were differing expressions in different aged tissues, HvMYB5 and HvDOF2 being the most outstanding one in the oldest tissue, 15-day-old root. On the other hand, investigated TCP genes were lowly expressed compared to selected MYB and DOF genes, except HvTCP3, where the highest expression was observed in 15-day-old root tissue. The obtained expression profiles illustrate the importance of potential regulatory roles of transcription factors in early developmental stages of barley germination and seedling establishment.