Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called “isoprenoid synthase fold” that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed.

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety1

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al3+ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato1

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco1

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis1

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.

Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds1

Semipermeable cell walls or apoplastic “membranes” have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (β-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by β-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms1

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.

Purification and Characterization of a Galactose-Rich Basic Glycoprotein in Tobacco1

We found a galactose-rich basic glycoprotein (GBGP) in the cell walls of cultured tobacco (Nicotiana tabacum) cells. GBGP and extensin were isolated as the major components of basic, salt-extracted cell wall glycoproteins. GBGP and extensin were separated by gel filtration in 6 m guanidine hydrochloride as 49- and 90-kD peaks, respectively, and further purified with reverse-phase chromatography. The protein moiety of GBGP constitutes about one-half of the molecule (w/w) and contains lysine (16%), proline (12%), hydroxyproline (10%), tyrosine (4%), alanine (7%), leucine (6%), and cystine (1.4%). Galactose accounted for 72% of the sugar moiety, arabinose content was low (17%), and a significant amount of mannose (7%) was found. No immunological cross-reaction was detected between GBGP and extensin. The antibody against native GBGP with sugar chains reacted with other glycoproteins on the gel blots, whereas the antibodies against deglycosylated GBGP and native extensin were highly specific. Immunolocalization analysis in tobacco stems showed that GBGP is specific to parenchyma tissue and that extensin localizes in the epidermis. This tissue-specific and exclusive distribution suggests important functions of these basic glycoproteins.

Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously “on,” tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas

In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the α-, β-, δ-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix–loop–helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.

The Involvement of Hydrogen Peroxide in the Differentiation of Secondary Walls in Cotton Fibers1

H2O2 is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H2O2 can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H2O2 may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H2O2 generation coincided with the onset of secondary wall deposition, (b) inhibition of H2O2 production or scavenging the available H2O2 from the system prevented the wall differentiation process, and (c) exogenous addition of H2O2 prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H2O2 generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H2O2, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H2O2.