TRANVIA (TVA) facilitates cellulose synthase trafficking and delivery to the plasma membrane.

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

Correction to: Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes.

[This corrects the article DOI: 10.1186/1754-6834-7-20.].

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.

BACKGROUND: Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. RESULTS: The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of l-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. CONCLUSIONS: PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.

Implementing industrial-academic partnerships to advance bioenergy research: the Energy Biosciences Institute.

The Energy Bioscience Institute (EBI) is an example of an ambitious academic-industry collaboration. We present a summary of how the EBI was organized during the first eight years and examples of several major research thrusts within the Institute to develop renewable biodiesel, jet fuel and lubricants from carbohydrates. In particular, we describe how the multidisciplinary nature and management structure of the organization led to hybrid approaches in which bioconversions and chemical synthesis were combined to develop new products.

BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1.

The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.

Clinical Profile and Natural Course in a Large Cohort of Patients With Hypertriglyceridemia and Pancreatitis.

GOALS: To report the clinical profile and natural course in a large series of patients with hypertriglyceridemia (HTG) and acute pancreatitis (AP). BACKGROUND: The natural history of HTG-related pancreatitis is poorly defined. STUDY: Medical records of 121 patients with serum triglycerides (TG) levels of ≥500 mg/dL suffering 225 attacks of AP between January 2001 to August 2013 treated at the University of Pittsburgh Medical Center were retrospectively studied. Structured data were collected on initial presentation and long-term outcomes (mean follow-up 64.7±42.8 mo). AP severity was classified using Revised Atlanta Classification. RESULTS: Most patients were young-middle aged (mean 44±12.7 y), male (70%), white (78%), and had sentinel AP (63%). Peak serum TG recorded was ≥1000 mg/dL in 48%. At least 1 secondary risk factor (diabetes, high-risk drinking, obesity, offending medications) was present in the majority (78%). Sentinel AP attack varied in severity between mild (41%), moderate (26%), and severe (33%). Recurrent AP attacks occurred in 32%, often in patients with poorly controlled diabetes, alcoholism, and TG levels. A cumulative increase in prevalence of pancreatic and/or peripancreatic necrosis was observed, with 45% patients having it at some time during observation. Local complications were higher in patients with serum TG ≥1000 mg/dL. Chronic pancreatitis was noted in 16.5% patients (new-onset in 9%). CONCLUSIONS: Patients with HTG-related pancreatitis have a high prevalence of secondary risk factors. Frequent recurrences in them are usually due to poor control of secondary factors or TG. Serum TG ≥1000 mg/dL increases the risk of local complications. A subset can have or develop chronic pancreatitis.

A dual mechanism of cellulose deficiency in shv3svl1.

SHAVEN3 (SHV3) and its homolog SHAVEN3-like 1 (SVL1) encode glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) that are involved in cellulose biosynthesis and hypocotyl elongation in Arabidopsis thaliana. In a recent report, we showed that the cellulose and hypocotyl elongation defects of the shv3svl1 double mutant are greatly enhanced by exogenous sucrose in the growth medium. Further investigation of this phenomenon showed that shv3svl1 exhibits a hyperpolarized plasma membrane (PM) proton gradient that is coupled with enhanced accumulation of sucrose via the PM sucrose/proton symporter SUC1. The resulting high intracellular sucrose concentration appears to favor starch synthesis at the expense of cellulose synthesis. Here, we describe our interpretation of these results in terms of 2 potential regulators of cellulose synthesis: intracellular sucrose concentration and a putative signaling pathway that involves SHV3-like proteins.

Anisotropic Cell Expansion Is Affected through the Bidirectional Mobility of Cellulose Synthase Complexes and Phosphorylation at Two Critical Residues on CESA3.

Here we report that phosphorylation status of S211 and T212 of the CESA3 component of Arabidopsis (Arabidopsis thaliana) cellulose synthase impacts the regulation of anisotropic cell expansion as well as cellulose synthesis and deposition and microtubule-dependent bidirectional mobility of CESA complexes. Mutation of S211 to Ala caused a significant decrease in the length of etiolated hypocotyls and primary roots, while root hairs were not significantly affected. By contrast, the S211E mutation stunted the growth of root hairs, but primary roots were not significantly affected. Similarly, T212E caused a decrease in the length of root hairs but not root length. However, T212E stunted the growth of etiolated hypocotyls. Live-cell imaging of fluorescently labeled CESA showed that the rate of movement of CESA particles was directionally asymmetric in etiolated hypocotyls of S211A and T212E mutants, while similar bidirectional velocities were observed with the wild-type control and S211E and T212A mutant lines. Analysis of cell wall composition and the innermost layer of cell wall suggests a role for phosphorylation of CESA3 S211 and T212 in cellulose aggregation into fibrillar bundles. These results suggest that microtubule-guided bidirectional mobility of CESA complexes is fine-tuned by phosphorylation of CESA3 S211 and T212, which may, in turn, modulate cellulose synthesis and organization, resulting in or contributing to the observed defects of anisotropic cell expansion.

Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H+-Coupled Sucrose Symporter.

In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1 This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H(+) symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H(+) gradient that likely underlies the enhanced accumulation of Suc via Suc/H(+) symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis.

O-Glycan analysis of cellobiohydrolase I from Neurospora crassa.

We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering.

A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis.

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.

50 years of Arabidopsis research: highlights and future directions.

922 I. 922 II. 922 III. 925 IV. 925 V. 926 VI. 927 VII. 928 VIII. 929 IX. 930 X. 931 XI. 932 XII. 933 XIII. Natural variation and genome-wide association studies 934 XIV. 934 XV. 935 XVI. 936 XVII. 937 937 References 937 SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.

Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1.

We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual ß1-4-linked glucan chains with a KD of 3.2 µm. Competition assays suggests that COBRA binds individual ß1-4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging ß1-4-glucan chains by acting as a "polysaccharide chaperone."

POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis.

Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.

Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes.

BACKGROUND: Pectin-rich agricultural wastes potentially represent favorable feedstocks for the sustainable production of alternative energy and bio-products. Their efficient utilization requires the conversion of all major constituent sugars. The current inability of the popular fermentation host Saccharomyces cerevisiae to metabolize the major pectic monosaccharide D-galacturonic acid (D-GalA) significantly hampers these efforts. While it has been reasoned that the optimization of cellular D-GalA uptake will be critical for the engineering of D-GalA utilization in yeast, no dedicated eukaryotic transport protein has been biochemically described. Here we report for the first time such a eukaryotic D-GalA transporter and characterize its functionality in S. cerevisiae. RESULTS: We identified and characterized the D-GalA transporter GAT-1 out of a group of candidate genes obtained from co-expression analysis in N. crassa. The N. crassa Δgat-1 deletion strain is substantially affected in growth on pectic substrates, unable to take up D-GalA, and impaired in D-GalA-mediated signaling events. Moreover, expression of a gat-1 construct in yeast conferred the ability for strong high-affinity D-GalA accumulation rates, providing evidence for GAT-1 being a bona fide D-GalA transport protein. By recombinantly co-expressing D-galacturonate reductase or uronate dehydrogenase in yeast we furthermore demonstrated a transporter-dependent conversion of D-GalA towards more reduced (L-galactonate) or oxidized (meso-galactaric acid) downstream products, respectively, over a broad concentration range. CONCLUSIONS: By utilizing the novel D-GalA transporter GAT-1 in S. cerevisiae we successfully generated a transporter-dependent uptake and catalysis system for D-GalA into two products with high potential for utilization as platform chemicals. Our data thereby provide a considerable first step towards a more complete utilization of biomass for biofuel and value-added chemicals production.

A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations.

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.