Consolidated Pretreatment and Hydrolysis of Plant Biomass Expressing Cell Wall Degrading Enzymes

Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.     

Composition and functional analysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat

Background

The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin.

Results

Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17.

Conclusion

We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.     

Major quantitative trait loci for seminal root morphology of wheat seedlings

Vigorous early root growth at seedling stage has been shown to be important for efficient acquisition of nutrients in wheat (Triticum aestivum L.). Identifying quantitative trait loci (QTL) for early root growth can facilitate the selection of wheat varieties with efficient nutrient use. A recombinant inbred line population derived from two Chinese wheat varieties, Xiaoyan 54 and Jing 411, was grown hydroponically at seedling stage. The maximum root length (MRL), primary root length (PRL), lateral root length (LRL), total root length (TRL), and root tip number (RN) of seminal roots were measured using the WinRHIZO Root Analyser. Numerous QTL for the investigated root traits were detected with QTL numbers varying from two to six, depending on the traits. Among them, two loci had major effects on primary (MRL and PRL) and lateral (LRL and RN) root parameters, respectively. The QTL (namely qTaLRO-B1) between Xgwm210 and Xbarc1138.2 on chromosome 2B explained 68.0 and 59.0% of phenotypic variations in MRL and PRL, respectively; the major QTL between Xgwm570 and Xgwm169.2 on chromosome 6A explained 30.5 and 24.5% of phenotypic variations in LRL and RN, respectively. These two major loci showed linkage with previous reported QTL for yield component and nutrient uptake. Detailed analysis of qTaLRO-B1 indicated that the positive allele of qTaLRO-B1 showed dominance over the negative allele, which showed impairment in primary root elongation. The existence of major QTL for root trait and their linkage with agronomic traits and nutrient uptake will facilitate the design of root morphology for better yield performance and efficient nutrient use.     

Characterization of the promoter of phosphate transporter TaPHT1.2 differentially expressed in wheat varieties

TaPHT1.2 is a functional, root predominantly expressed and low phosphate （Pi） inducible high-affinity Pi transporter in wheat, which is more abundant in the roots of P-efficient wheat genotypes （e.g., Xiaoyan 54） than in P-inefficient genotypes （e.g., Jing 411） under both Pi-deficient and Pi-sufficient conditions. To characterize TaPHT1.2 further, we genetically mapped a TaPHT1.2 transporter, TaPHT1.2-D1, on the long arm of chromosome 4D using a recombinant inbred line population derived from Xiaoyan 54 and Jing 411, and isolated a 1,302 bp fragment of the TaPHT1.2-D1 promoter （PrTaPHT1.2-D1） from Xiaoyan 54. TaPHT1.2-D1 shows collinearity with OsPHT1.2 that has previously been reported to mediate the translocation of Pi from roots to shoots. PrTaPHT1.2-D contains a number of Pi-starvation responsive elements, including P1BS, WRKY-binding W-box, and helix-loop-helix-binding elements. PrTaPHT1.2-D1 was then used to drive expression of 13-glucuronidase （GUS） reporter gene in Arabidopsis through Agrobacterium-mediated transformation. Histochemical analysis of transgenic Arabidopsis plants showed that the reporter gene was specifically induced by Pi-starvation and predominantly expressed in the roots. As there is only one SNP between the TaPHT1.2-D1 promoters of Xiaoyan 54 and Jing 411, and this SNP does not exist within the Pi-starvation responsive elements, the differential expression of TaPHT1.2 in Xiaoyan 54 and Jing 411 may not be caused by this SNP.     

pH-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors

The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.     

Neural crest regionalisation for enteric nervous system formation: Implications for Hirschsprung's disease and stem cell therapy

Midbrain, hindbrain and vagal neural crest (NC) produced abundant enteric nervous system (ENS) in co-grafted aneural hindgut and midgut, using chick-quail chorio-allantoic membrane grafts, forming complete myenteric and submucosal plexuses. This ability dropped suddenly in cervical and thoracic NC levels, furnishing an incomplete ENS in one or both plexuses. Typically, one plexus was favoured over the other. This deficiency was not caused by lower initial trunk NC number, yet overloading the initial number decreased the deficiency. No qualitative difference in neuronal and glial differentiation between cranial and trunk levels was observed. All levels formed HuC/D+ve, NOS+ve, ChAT+ve, and TH-ve enteric neurons with SoxE+ve, GFAP+ve, and BFABP+ve glial cells. We mathematically modelled a proliferative difference between NC populations, with a plexus preference hierarchy, in the context of intestinal growth. High proliferation achieved an outcome similar to cranial NC, while low proliferation described the trunk NC outcome of incomplete primary plexus and even more deficient secondary plexus. We conclude that cranial NC, relative to trunk NC, has a positionally-determined proliferation advantage favouring ENS formation. This has important implications for proposed NC stem cell therapy for Hirschsprung's disease, since such cells may need to be optimised for positional identity.     

A specific interaction of small molecule entry inhibitors with the envelope glycoprotein complex of the Junín hemorrhagic fever arenavirus

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.