HD-Zip Proteins GL2 and HDG11 Have Redundant Functions in Arabidopsis Trichomes, and GL2 Activates a Positive Feedback Loop via MYB23.

The class IV homeodomain leucine zipper transcription factor GLABRA2 (GL2) acts in a complex regulatory circuit that regulates the differentiation of trichomes in Arabidopsis thaliana. We describe a genetic interaction with HOMEODOMAIN GLABROUS11 (HDG11), previously identified as a negative regulator of trichome branching. gl2 hdg11 double mutants display enhanced trichome cell-type differentiation defects. Transgenic expression of HDG11 using the GL2 promoter partially suppresses gl2 trichome phenotypes. Vice versa, expression of GL2 under the control of its native promoter partially complements hdg11 ectopic branching. Since gl2 hdg11 and gl2 myb23 double mutants and the triple mutant display similar trichome differentiation defects, we investigated a connection to the R2R3-MYB transcription factor MYB23. We show that MYB23 transcript levels are significantly reduced in shoots from gl2 mutants and that GL2 can drive the expression of a MYB23-promoter fusion to green fluorescent protein. Yeast one-hybrid, chromatin immunoprecipitation, and in planta reporter gene experiments indicate that an L1-box in the MYB23 promoter acts as a GL2 binding site. Taken together, our findings reveal a functional redundancy between GL2 and HDG11, two homeodomain leucine zipper transcription factors previously thought to mediate opposing functions in trichome morphogenesis. A model is proposed in which GL2 transcript levels are maintained through a positive feedback loop involving GL2 activation of MYB23.

Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis.

Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated 'gcs-1') were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.

α-Fucosidases with different substrate specificities from two species of Fusarium.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.

Influences of pH and substrate supply on the ratio of iron to sulfate reduction.

Iron reduction and sulfate reduction often occur simultaneously in anoxic systems, and where that is the case, the molar ratio between the reactions (i.e., Fe/SO42- reduced) influences their impact on water quality and carbon storage. Previous research has shown that pH and the supply of electron donors and acceptors affect that ratio, but it is unclear how their influences compare and affect one another. This study examines impacts of pH and the supply of acetate, sulfate, and goethite on the ratio of iron to sulfate reduction in semi-continuous sediment bioreactors. We examined which parameter had the greatest impact on that ratio and whether the parameter influences depended on the state of each other. Results show that pH had a greater influence than acetate supply on the ratio of iron to sulfate reduction, and that the impact of acetate supply on the ratio depended on pH. In acidic reactors (pH 6.0 media), the ratio of iron to sulfate reduction decreased from 3:1 to 2:1 as acetate supply increased (0-1 mM). In alkaline reactors (pH 7.5 media), iron and sulfate were reduced in equal proportions, regardless of acetate supply. Secondly, a comparison of experiments with and without sulfate shows that the extent of iron reduction was greater if sulfate reduction was occurring and that the effect was larger in alkaline reactors than acidic reactors. Thus, the influence of sulfate supply on iron reduction extent also depended on pH and suggests that iron reduction grows more dependent on sulfate reduction as pH increases. Our results compare well to trends in groundwater geochemistry and provide further evidence that pH is a major control on iron and sulfate reduction in systems with crystalline (oxyhydr)oxides. pH not only affects the ratio between the reactions but also the influences of other parameters on that ratio.

Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.

Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based "shotgun" proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial "cellulase" preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.

Influence of pH on the balance between methanogenesis and iron reduction.

Methanogenesis and iron reduction play major roles in determining global fluxes of greenhouse gases. Despite their importance, environmental factors that influence their interactions are poorly known. Here, we present evidence that pH significantly influences the balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron reduction and methanogenesis occurred but the balance between them varied significantly with pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85% lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH. Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with goethite, clay minerals served as the source of ferric iron and the balance between each reaction did not vary significantly with pH. We therefore conclude that pH can regulate the relative contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial environments. We further propose that the availability of ferric (oxyhydr)oxide minerals influences the extent to which the balance between each reaction is sensitive to pH. The results of this study advance our understanding of environmental controls on microbial methane generation and provide a basis for using pH and the occurrence of ferric minerals to refine predictions of greenhouse gas fluxes.

Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants.

BACKGROUND: Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. RESULTS: Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. CONCLUSION: We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants.

Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.

High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.