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Redesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.

Background Loop ileostomies are often formed in order to defunction distal anastomoses. The aim of this study was to review the complications following closure of loop ileostomies. Methods This is a retrospective case note analysis of all loop ileostomy closures performed in the Northumbria Healthcare NHS Trust (population over 500,000) over a 5-year period between 2001 and 2005. Results A total of 123 case records were reviewed. Complications occurred in 41 patients (33.3%), with 9 patients (7.3%) requiring further intervention. There were 4 (3.3%) postoperative deaths. Complications were more common in patients with increased comorbidity ( p  = 0.0007) and postoperative death was more frequent among the elderly ( p  = 0.0006). Postoperative death was more common in those patients who had their stomas created during surgery (elective or emergency) for diverticular disease (3 patients, p  = 0.006). Patients with diverticular disease had significantly higher comorbidity and peritoneal contamination at the time of primary surgery. Ileostomy reversal after anterior resection for cancer was associated with a lower complication rate than the rest of the cohort (26%, p  = 0.0003) but there was no significant difference in mortality. Neither the grade of the surgeon, the case volume, or the anastomotic technique affected postoperative morbidity. Reoperation was more common in patients whose closure procedure took less time ( p  = 0.002) and in those who had a shorter wait from creation to reversal of the stoma ( p  < 0.0001). Conclusions Reversal of loop ileostomy may be associated with significant morbidity and mortality. Increasing the delay from creation to closure may result in fewer complications.There is an increased risk in older patients with more comorbidity, particularly when the primary procedure is for diverticular disease with significant peritoneal contamination.

Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall–specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular trafficking of endomembranes in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls.

CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

The objectives of this study were to define cell structure during pine secondary xylem development and to integrate this information with current knowledge of the biochemistry and physiology of secondary cell wall biosynthesis in gymnosperms. Lodgepole pine (Pinus contorta var. latifolia Englem.) cambium and secondary xylem were cryofixed using high pressure freezing and freeze-substitution which allowed excellent preservation of the cell structure of developing secondary xylem and enabled high-resolution transmission electron microscopic viewing of these cells for the first time. In contrast to their precursors in the adjacent cambial zone, developing tracheids were active in secondary wall deposition, with abundant cortical microtubules and developing bordered pits. These cells were also characterized by unusual Golgi structures: the trans-Golgi network was highly developed and the associated vesicles were large and darkly stained. These unusual Golgi structures persisted throughout the period of xylem maturation until programmed cell death occurred. Immuno-cytochemistry and enzyme-gold probes were used to investigate the distribution of key secretory products (mannans) and a lignification-associated enzyme (coniferin β-glucosidase) during xylogenesis. Mannans were localized to the secondary cell wall, the trans-Golgi cisternae and trans-Golgi network vesicles of developing xylem. Coniferin β-glucosidase was found only in the secondary cell wall. The cell wall localization of coniferin β-glucosidase, the enzyme responsible for cleaving glucose from coniferin to generate free coniferyl alcohol, provides a mechanism to de-glucosylate monolignols in muro. A two-step model of lignification of conifer tracheids is proposed. First, Golgi-mediated secretion deposits monolignols into the cell wall, where they polymerize in cell corners and middle lamella. Secondly, cell lysis releases stored, vacuolar monolignol glucosides into the wall where they are deglucosylated and their polymerization is influenced by the wall environment including the lignin deposited earlier.

The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 2.4.1.123), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba×grandidentata). Phylogenetic analyses of thePopulusGolS isoforms with other known GolS proteins suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. The predicted protein sequences of both isoforms (Pa×gGolSI and Pa×gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII resulted inK mvalues for UPD-galactose of 0.80 and 0.65 mM andV maxvalues of 657.5 and 1245 nM min−1, respectively. Pa×gGolSI inherently possessed a broader pH and temperature range when compared with Pa×gGolSII. Interestingly, spatial and temporal expression analyses revealed thatPa×gGolSIItranscript levels varied seasonally, whilePa×gGolSIdid not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

Key messageThe physiological responses of nine commonly planted North-American hybrid poplars to drought was evaluated, resulting in a ranking of drought resistance. Indicator genes for drought response processes were scored for differences in expression levels in the most sensitive and resistant clones, confirming levels of drought stress and differences consistent with roles in drought resistance.Poplar hybrids are cultivated in North America for environmental applications, agroforestry, and the pulp and paper industry primarily because of their fast growth and limited nutrient requirement. For the same reasons, they have been identified as suitable species for carbon sequestration and as a potential feedstock for carbon–neutral production of energy. The clones deployed on the Canadian prairies are generally regarded as drought sensitive, which poses a problem as water availability has steadily decreased in this region over the past century and a severe water crisis has been predicted. To approach this problem, we tested nine commonly deployed North-American hybrid poplars, developed for large-scale cultivation in the Canadian prairies, for their physiological responses to drought, resulting in a ranking of drought resistance. The difference between the clones showing the most and the least response of drought stress was large, and we used these clones to further examine the differences in the expression of genes known to be up-regulated in response to drought stress. This interrogation showed significant differences in transcript abundance that largely reflected the physiological status of the tested clones, but also many genes being down rather than up-regulated in response to drought stress in the drought-tolerant clone. In particular, putative positive and negative regulators of abscisic acid signaling were expressed at levels consistent with a potential role in observed differences in drought resistance.

Abstract Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.

Douglas-fir (Pseudotsuga menziesii) white-wood was supplemented with increasing concentrations of bark (0-30%) and was pretreated using SO2-catalysed steam explosion. The presence of bark in the feedstock resulted in the decreased recovery of total sugars, furfural and 5-hydroxymethylfurfural in the resultant prehydrolysate. No detrimental impact on monomer sugar recovery was observed. The concentration of lipophilic extractives present in the pre-hydrolysate increased with increasing bark loading, to a maximum of 0.43 g l(-1). The water-soluble pre-hydrolysates were fermented by Saccharomyces cerevisiae to determine the impact of bark on sugar consumption and ethanol production. Despite the inclusion of bark, fermentation of all pre-hydrolysates resulted in the complete consumption of hexose sugars within 48 h. Ethanol yields were greater than 0.43 g g(-1) for ail pre-hydrolysates regardless of bark content, indicating that, up to a content of 30%, bark had a negligible impact on the fermentation of the pre-hydrolysates to ethanol.