Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.     

Golgi Phosphoprotein 3 Triggers Signal-mediated Incorporation of Glycosyltransferases into Coatomer-coated (COPI) Vesicles

Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization.     

Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.     

Tillers Do Not Influence Phytotoxicity of Imazamethabenz in Wild Oat (Avena fatua)

The response of wild oat to imazamethabenz varies with the growth stage, but the role of tillers in this regard is unclear. Removal of tillers at the three-leaf stage before spraying with imazamethabenz did not significantly affect the total shoot fresh weight measured 3 weeks later. The leaf area and dry weight of intact plants at the three-leaf stage were 17–21% greater than for plants with coleoptilar and first leaf main shoot tillers (T0 and T1) removed. The greater leaf area may have increased herbicide interception per plant. Similar fresh weight reductions in main shoot, total tillers, and total shoots were found whether imazamethabenz was applied to the plant at the two-leaf without tillers or the three-leaf with two tillers stage. Imazamethabenz applied only to the main shoot reduced total shoot dry weight more than an equivalent amount of imazamethabenz applied only to tiller T1 or applied over the whole shoot. Imazamethabenz had the least inhibitory effect on whole plant growth when applied only to T1. When ¹⁴C-herbicide was applied to the first main shoot leaf of plants at the three-leaf stage with two tillers, the ¹⁴C translocated 38% to roots, 33% to the main shoot, and nearly 30% to all tillers. When ¹⁴C-herbicide was applied to the first leaf of T1 then the ¹⁴C translocated 50% to T1, 25% to the main shoot, 20% to roots, and 5% to all other tillers. The translocation pattern and fresh weight values suggested that the presence of early tillers during herbicide application neither increased nor decreased imazamethabenz efficacy in wild oat. Received June 4, 1997; accepted June 5, 1997     

Gene Cloning,Expression, and Antifungal Activities of Permatin from Naked Oat (Avena nuda)

Probiotics and Antimicrobial Proteins - Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defense. TLPs also belong to the pathogenesis-related...     

Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli)

This study presents purification, activity characterization, and (1)H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.     

Novel cyclotides and uncyclotides with highly shortened precursors from Chassalia chartacea and effects of methionine oxidation on bioactivities

Cyclotides are a new class of plant biologics that display a diverse range of bioactivities with therapeutic potentials. They possess an unusual end-to-end cyclic backbone combined with a cystine knot arrangement, making them exceptionally stable to heat, chemical and enzymatic degradation. Currently, >200 cyclotides have been discovered but only three naturally occurring linear variants (also known as uncyclotides) have been isolated. In this study, we report the discovery of 18 novel peptides, chassatides C1 to C18, composed of 14 new cyclotides and four uncyclotides from Chassalia chartacea (Rubiaceae family). Thus far, this is the largest number of uncyclotides being reported in a single species. Activity testing showed that the uncyclotides not only retain the effectiveness but also are the most potent chassatides in the assays for antimicrobial, cytotoxic, and hemolytic activities. Genetic characterization of novel chassatides revealed that they have the shortest precursors of all known cyclotides hitherto isolated, which represents a new class of cyclotide precursors. This is the first report of cyclotide genes in a second genus, the Chassalia, other than the Hedyotis (Oldenlandia) of the Rubiaceae family. In addition, we also report the characterization of two Met-oxidized derivatives of chassatides C2 and C11. The oxidation of Met residue causes loss of bioactivities, strengthening the importance of the hydrophobic patch for membrane interaction.     

Chemical constituents from the aerial parts of Meconopsis horridula (Papaveraceae)

Chemical investigation of the aerial parts of Meconopsis horridula led to the isolation and structural identification of nine flavonoids, two alkaloids and five terpenoids. Their structures were determined by comparison of ¹H and ¹³C NMR data with those reported in literature. The discovery of alkaloid cavidilinine (11) and two pentacyclic triterpenoids (15 and 16) from M. horridula provides evidence to support the view that Fumarioideae and Papaveroideae should be combined into a single family Papaveraceae.     

Regulation of Growth in Avena (Oat) Stem Segments by Gibberellic Acid and Abscisic Acid

The purpose of this study was to analyze the nature of the interaction between gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of growth in excised Avena (oat) stem segments. Growth, compared to sucrose controls, was inhibited by ABA in the range of 10^?4 to 10^?6M. GA₃-promoted growth was also inhibited by ABA in the same concentration range. A Lineweaver-Burk analysis of the interaction between GA₃ and ABA indicated that ABA acts in a non-competitive fashion with GA₃. This same result was obtained previously with GA₃-indoleacetic acid (IAA) and GA₃-kinetin interactions with Avena stem sections. Our results indicate that ABA can inhibit GA₃-promoted growth within physiological concentrations, and that it is probably acting at a different physiological site from that for GA₃.     

Uridine and the Control of Phototropism in Oat (Avena sativa L.) Coleoptiles

The short-term growth response of oat (Avena sativa L.) coleoptiles to exogenously applied uridine was studied both in excised apical segments and in the intact seedlings. In both cases growth of coleoptile tissue was inhibited by uridine. The inhibition of coleoptile growth consistently occurred 20–30 min after uridine treatment, which is within the lag period of their phototropic response. Asymmetric application of uridine to coleoptiles in the intact seedlings resulted in their bending toward the direction to which uridine was applied in the absence of light stimulus. These findings suggest that uridine or its metabolites, plays an important role in the phototropism of oat coleoptiles and provide support to the Bruinsma–Hasegawa theory as an alternative to the Cholodny–Went theory for explaining phototropism.     

植物细胞生物反应器培养的研究进展(I)

利用植物细胞大规模悬浮培养生产植物有用代谢产物在近些年来取得了很大发展,但植物细胞悬浮培养的工业化应用受到来自生物及工程技术上的限制.本文针对植物细胞培养的基本特点,详细讨论了与大规模生产有关的工程技术方面的问题,如植物细胞聚集、溶氧及气体成分、流体性能、剪切力对植物细胞培养产生的影响.     

大粒裸燕麦（莜麦）（Avena nuda L.）起源及分类问题的探讨

燕麦属（Avena L.）植物中有5个栽培种即普通栽培燕麦（A. sativa L.）、埃塞俄比亚燕麦（A. abyssinica Hochst.）、地中海燕麦（A. byzantina Koch）、砂燕麦（A. strigosa Schreb.）和大粒裸燕麦又称莜麦（A. nuda L.）,其中大粒裸燕麦的子粒不带稃皮为裸燕麦,其他物种均带稃皮为皮燕麦。国际上主要种植皮燕麦,而我国主要种植大粒裸燕麦,由此不难看出,大粒裸燕麦在世界燕麦中占有特殊的地位。然而,关于大粒裸燕麦的起源和分类地位问题,迄今学者们的意见仍不尽相同。笔者通过参阅有关文献和研究实践,对这两个问题进行探讨,认为大粒裸燕麦起源于我国山西和内蒙古一带,在植物学分类上应为一个独立的物种即A. nuda L.。     

Two new secondary metabolites isolated from Avena sativa L. (Oat) seedlings and their effects on osteoblast differentiation

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3–10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.     

Two cytotoxic triterpenes from cultures of a Kenyan Laetiporus sp. (Basidiomycota)

HPLC profiling of the mycelial culture of a poroid basidomycete collected in Mount Elgon, Kenya, which probably represents a new species of the genus Laetiporus, led to isolation of two previously undescribed lanostane type triterpenes.We propose the trivial names laetiporins A (1) and B (2). In addition, five known ones: dehydrosulphurenic acid (3), sulphurenic acid (4), eburicoic acid (5), 15α-hydroxytrametenolic acid (6) and trametenolic acid (7) were also isolated. The laetiporins (1–2) exhibited significant cytotoxic effects against various human cancer cells. The known compounds (3–5) and (7) also showed moderate cytotoxic activity, but none of the compounds showed any significant antimicrobial activity.     

Changes in Endogenous Gibberellins and the Metabolism of [H]GA(4) after Geostimulation in Shoots of the Oat Plant (Avena sativa)

The recovery from “lodging,” or bending over, by shoots of 42-day-old Avena sativa plants is controlled primarily by a negatively geotropic differential growth of the lower halves of the p-1 node-pulvinus and the base of the p-1 internode, relative to the upper halves. Although geostimulation causes a significant reduction in p-1 internode length, dry matter accumulation in the p-1 node-pulvinus is increased, apparently at the expense of the sheath. Recovery to an angle of 30° is associated with changes in endogenous gibberellin-like substances (GAs), and in differential metabolism of applied [³H]GA₄ (1.4 Curie per millimole). Although geostimulation depressed total GAs (relative to upright plant parts) to 0.40 and 0.13 for node-pulvini and sheaths, respectively, it increased them 2-fold for internodes. Within the plant part geostimulation increased GAs (relative to upper halves) 29- and 7-fold in lower halves of node-pulvini and internodes, respectively, but reduced GAs to 0.3 in lower halves of sheaths. At age 42 days a GA_4/7-like (nonpolar) substance predominates, with lesser amounts of a GA₃-like (polar) substance. Native GAs of Avena include GA₃, GA₄, and GA₇. Geostimulation enhanced the ratio of nonpolar to polar GAs for both halves of internodes, but tended to depress it for sheaths and nodepulvini.     

Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M_r 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (M_r 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.