Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants.

Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce crops with cell walls that are more susceptible to hydrolysis to reduce preprocessing and enzyme inputs. To deepen our understanding of the molecular genetic basis of lignocellulose recalcitrance, we have screened a mutagenized population of the model grass Brachypodium distachyon for improved saccharification with an industrial polysaccharide-degrading enzyme mixture. From an initial screen of 2,400 M2 plants, we selected 12 lines that showed heritable improvements in saccharification, mostly with no significant reduction in plant size or stem strength. Characterization of these putative mutants revealed a variety of alterations in cell-wall components. We have mapped the underlying genetic lesions responsible for increased saccharification using a deep sequencing approach, and here we report the mapping of one of the causal mutations to a narrow region in chromosome 2. The most likely candidate gene in this region encodes a GT61 glycosyltransferase, which has been implicated in arabinoxylan substitution. Our work shows that forward genetic screening provides a powerful route to identify factors that impact on lignocellulose digestibility, with implications for improving feedstock for cellulosic biofuel production.

Unlocking the potential of lignocellulosic biomass through plant science.

The aim of producing sustainable liquid biofuels and chemicals from lignocellulosic biomass remains high on the sustainability agenda, but is challenged by the costs of producing fermentable sugars from these materials. Sugars from plant biomass can be fermented to alcohols or even alkanes, creating a liquid fuel in which carbon released on combustion is balanced by its photosynthetic capture. Large amounts of sugar are present in the woody, nonfood parts of crops and could be used for fuel production without compromising global food security. However, the sugar in woody biomass is locked up in the complex and recalcitrant lignocellulosic plant cell wall, making it difficult and expensive to extract. In this paper, we review what is known about the major polymeric components of woody plant biomass, with an emphasis on the molecular interactions that contribute to its recalcitrance to enzymatic digestion. In addition, we review the extensive research that has been carried out in order to understand and reduce lignocellulose recalcitrance and enable more cost-effective production of fuel from woody plant biomass.

Exploring PIF4 's contribution to early flowering in plants under daily variable temperature and its tissue-specific flowering gene network.

Molecular mechanisms of how constant temperatures affect flowering time have been largely characterized in the model plant Arabidopsis thaliana; however, the effect of natural daily variable temperature outside laboratories is only partly explored. Several flowering genes have been shown to play important roles in temperature responses, including PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC), the two genes encoding for the transcription factors (TFs) that act antagonistically to regulate flowering time by activating and repressing floral integrator FLOWERING LOCUS T (FT), respectively. In this study, we have taken a multidisciplinary approach to explore the contribution of PIF4 to the early flowering observed in the daily variable temperature (VAR) and to broaden its transcriptional network using publicly available transcriptomic data. We observed early flowering in the natural accessions Col-0, C24 and their late flowering hybrid C24xCol grown under VAR, as compared with a constant temperature (CON). The loss-of-function mutation of PIF4 exhibits later flowering in VAR in both the Col-0 parent and the C24xCol hybrid, suggesting that PIF4, at least in part, contributes to acceleration of flowering in the VAR condition. To investigate the interplay between PIF4 and its flowering regulator counterparts, FLC and FT, we performed transcriptional analyses and found that VAR increased PIF4 transcription at the end of the day when temperature peaked at 32°C, when FT transcription was also elevated. On the other hand, we observed a decrease in FLC transcription in the 4-week-old plants grown in VAR, as well as in the plants with PIF4 overexpression grown in CON. These results raise a possibility that PIF4 might also regulate FT indirectly through the repression of FLC, in addition to the well-characterized direct control of PIF4 over FT. To further expand our view on the PIF4-orientated flowering gene network in response to temperature changes, we have constructed a coexpression-transcriptional regulatory network by combining publicly available transcriptomic data and gene regulatory interactions of PIF4 and its closely related flowering genes, PIF5, FLC, and ELF3. The network model reveals conserved and tissue-specific regulatory functions, which are useful for confirming as well as predicting the functions and regulatory interactions between these key flowering genes.

An RNA thermoswitch regulates daytime growth in Arabidopsis.

Temperature is a major environmental cue affecting plant growth and development. Plants often experience higher temperatures in the context of a 24 h day-night cycle, with temperatures peaking in the middle of the day. Here, we find that the transcript encoding the bHLH transcription factor PIF7 undergoes a direct increase in translation in response to warmer temperature. Diurnal expression of PIF7 transcript gates this response, allowing PIF7 protein to quickly accumulate in response to warm daytime temperature. Enhanced PIF7 protein levels directly activate the thermomorphogenesis pathway by inducing the transcription of key genes such as the auxin biosynthetic gene YUCCA8, and are necessary for thermomorphogenesis to occur under warm cycling daytime temperatures. The temperature-dependent translational enhancement of PIF7 messenger RNA is mediated by the formation of an RNA hairpin within its 5' untranslated region, which adopts an alternative conformation at higher temperature, leading to increased protein synthesis. We identified similar hairpin sequences that control translation in additional transcripts including WRKY22 and the key heat shock regulator HSFA2, suggesting that this is a conserved mechanism enabling plants to respond and adapt rapidly to high temperatures.

Ptychography--a label free, high-contrast imaging technique for live cells using quantitative phase information.

Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis.