Isolation and identification of glycosylphosphatidylinositol-anchored arabinogalactan proteins and novel beta-glucosyl Yariv-reactive proteins from seeds of rice (Oryza sativa)

Arabinogalactan proteins (AGPs) are highly glycosylated extracellular glycoproteins playing important roles in plant growth and development. We have previously reported the possibility that AGPs are involved in the induction of alpha-amylase by gibberellin (GA) in barley aleurone layers by using the beta-glucosyl Yariv reagent (beta-GlcY), which has been presumed to specifically bind AGPs. In this present study, we isolated beta-GlcY-reactive proteins from rice bran rich in aleurone cells. The N-terminal sequences of classical AGP and AG peptides were determined from hydrophilic fractions obtained by reversed phase HPLC. Interestingly, a novel non-specific lipid transfer protein-like protein (OsLTPL1) and a novel early nodulin-like protein (OsENODL1) were also identified in the more hydrophobic fractions from HPLC as beta-GlcY-reactive proteins. Expression analysis of the genes coding for these proteins was performed. While classical AGP, AG peptides and OsLTPL1 were expressed in various parts of rice, OsENODL1 showed temporally and spatially specific expression in the aleurone layers. This new beta-GlcY-reactive protein is a promising candidate for the extracellular signaling factors of GA action in cereal seeds. Furthermore, the possibility that proteins with the AG glycomodule might react with beta-GlcY may broaden the definition of AGPs.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

A new 9-lipoxygenase cDNA from developing rice seeds

We isolated a novel C9 position specific lipoxygenase (r9-LOX1) cDNA from developing rice seeds. The enzymatic features of r9-LOX1 resembled those of rice LOX-L3 known to be contained in rice germ and to have C9-specific LOX activity. However, the expression level of the r9-LOX1 gene was higher in imbibed seeds rather than developing seeds. A homology search against the rice nucleotide database revealed the r9-LOX1 gene to be on rice chromosome 3 (accession number AC093017). The restriction enzyme map of the reported genomic sequence agreed with the result of the Southern blot analysis for the r9-LOX1. The enzyme could be useful for in vitro synthesis of 9,10-ketol-octadecadienoic acid.     

The transport of prolamine RNAs to prolamine protein bodies in living rice endosperm cells

RNAs that code for the major rice storage proteins are localized to specific subdomains of the cortical endoplasmic reticulum (ER) in developing endosperm. Prolamine RNAs are localized to the ER and delimit the prolamine intracisternal inclusion granules (PB-ER), whereas glutelin RNAs are targeted to the cisternal ER. To study the transport of prolamine RNAs to the surface of the prolamine protein bodies in living endosperm cells, we adapted a two-gene system consisting of green fluorescent protein (GFP) fused to the viral RNA binding protein MS2 and a hybrid prolamine RNA containing tandem MS2 RNA binding sites. Using laser scanning confocal microscopy, we show that the GFP-labeled prolamine RNAs are transported as particles that move at an average speed of 0.3 to 0.4 microm/s. These prolamine RNA transport particles generally move unidirectionally in a stop-and-go manner, although nonlinear bidirectional, restricted, and nearly random movement patterns also were observed. Transport is dependent on intact microfilaments, because particle movement is inhibited rapidly by the actin filament-disrupting drugs cytochalasin D and latrunculin B. Direct evidence was obtained that these prolamine RNA-containing particles are transported to the prolamine protein bodies. The significance of these results with regard to protein synthesis in plants is discussed.     

Acyl carrier proteins from developing seeds of Cuphea lanceolata Ait

The structure of acyl carrier protein (ACP) may determine the fate of the acyl moieties linked to it in the course of de-novo fatty acid synthesis in higher plants. To investigate a possible correlation between the structure of ACP and the synthesis of medium-chain fatty acids, we isolated and characterized ACP from the seeds of Cuphea lanceolata Ait. (subgenus Eucuphea/Section Heterodon), an annual crop that contains up to 90% decanoic (capric) acid in seed triacylglycerols. After a cell-free extract prepared from developing seeds was treated to 65% saturation with ammonium sulfate, two ACP-isoforms (ACP 1 and ACP 2) were identified in the supernatant that could be purified to homogeneity by anion-exchange chromatography and subsequent reversed-phase high-performance liquid chromatography. The molecular mass determined by matrix-assisted ultraviolet-laser desorption ionization mass spectrometry of ACP 1 was 9315 Da, whereas further heterogeneity was observed for ACP 2 with molecular masses of 8598 and 8703 Da. Aminoterminal sequencing was performed showing a high homology in the primary structures of ACP 1 and ACP 2. Both isoforms were present in the embryo, whereas in the chloroplast-containing seed coat ACP 2 was found in minute amounts, if at all. The expression of ACP 2 correlated with the production of capric acid during the phase of storage-lipid accumulation. These data indicate that ACP 2 is part of the machinery for the synthesis of medium-chain fatty acids, whereas ACP 1 appears to be a constitutive protein.Abbreviations ACP acyl carrier protein - clACP acyl carrier protein from Cuphea lanceolata - 2D-PAOE two-dimensional polyacrylamide gel electrophoresis - DTT dithiothreitol - ecACP acyl carrier protein from Escherichia coli - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine This work was supported by a grant from the German Ministry of Research and Technology (BMFT). The authors wish to thank Professor Röbbelen, University of Göttingen, FRG, for kindly providing the plant material and A. Ingendoh, Department of Medical Physics of the University of Münster, FRG, for carrying out the mass-spectrometric analysis. Portions of this paper are part of the doctoral thesis of Markus Robers.     

Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Isolation and identification of antitumor promoters from the seeds of Cassia tora

A methanol extract of Cassia tora seeds was successively partitioned with diethyl ether, chloroform, ethyl acetate, and water, and the antitumor-promoting activity of the solvent fractions was determined by inhibition of Epstein- Barr virus early antigen (EBV-EA) activation induced by teleocidin B-4 in Raji cells. The diethyl ether (68.7%) and chloroform (91.2%) fractions and the hydrolysate (94.3%) of the ethyl acetate fraction had strong inhibitory activities. The chloroform and ethyl acetate fractions were chromatographed on silica gel and further purified by HPLC. Three active compounds, obtusifolin-2-glucoside (75.0%), chryso-obtusin-6-glucoside (56.8%), and norrubrofusarin- 6-glucoside (39.4%), were obtained from the ethyl acetate fraction, and two active compounds, questin (97.9%) and chryso-obtusin (53.8%), were isolated from the chloroform fraction.     

Nucleotide and primary sequence of a major rice prolamine

A recombinant cDNA clone encoding a major rice seed storage prolamine was isolated by antibody screening of a cDNA lambda gt 11 library. This clone contained a single open reading frame encoding a putative rice prolamine precursor (Mr 17,300). In contrast to other cereal prolamines, the primary sequence of the rice prolamine was devoid of any major tandem repetitive sequences, a feature prevalent in all cereal prolamines studied to date. No significant homology was detected between the rice prolamine and other cereal prolamines, indicating that the rice gene evolved from unique ancestral DNA segments.     

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.     

一株水稻根内生拮抗细菌SM13的分离及鉴定

【背景】作物根内生细菌具有固氮、分泌激素、产生病原真菌抗性物质等特性,根系内生菌的分离及应用成为环境友好型防控技术研究的热点之一。近年来盐碱地水稻种植面积逐年增加,而关于盐碱地水稻根内生菌的分离与应用鲜有报道。【目的】从大庆盐碱地水稻根中分离内生细菌,筛选对植物真菌病害有拮抗作用的促生菌株,初步探讨其抑菌和促生功效,为进一步研究其抑菌和促生机理提供菌种资源。【方法】对水稻根表面灭菌后研磨涂布分离内生细菌,采用对峙培养法和改良Salkowski比色法筛选具有广谱抑菌效果并有分泌吲哚-3-乙酸(Indole-3-acetic acid,IAA)能力的菌株,通过形态鉴定、革兰氏染色、生理生化测定、16S rRNA结构基因以及srfA、ituA、fenB功能基因序列系统进化分析,确定细菌的分类地位。【结果】从水稻根部分离到一株内生细菌SM13,该菌株具有广谱性抑菌作用,对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为59.38%、78.13%、53.12%、37.50%,分泌IAA的能力为5.56±0.41μg/mL (n=6)。经形态学、生理生化试验结合系统进化分析初步鉴定SM13菌株属于枯草芽孢杆菌(Bacillus subtilis),该菌株在pH 11.0、盐浓度10%的NA培养基中生长良好,具有较高的耐盐碱性。【结论】水稻根内生菌SM13菌株具有耐盐碱性、促生和生防性能,可作为微生物农药及菌肥的材料。     

Isolation of prolegumin from developing pea seeds: its binding to endomembranes and assembly into prolegumin hexamers in the protein storage vacuole

A fraction enriched in endoplasmic reticulum and Golgi membranesfrom developing cotyledons of Pisum sativum L. has proved tobe a convenient source for the isolation of prolegumin, theprecursor of the major 11S storage globulin of pea seeds. Twopro-proteins were isolated with molecular masses of 60 kDa and75 kDa, respectively. A monoclonal antibody, designated 2B1,against prolegumin was raised using the in vitro immunizationtechnique. This antibody recognizes the 60 kDa precursor polypeptide,but only the 20 kDa ß-subunit of mature legumin. Prolegumin,like the ß-subunit of the mature legumin, is a hydrophobicprotein. After import into the protein storage vacuole, andafter formation of the protein bodies trimeric 9S proleguminassembles into 12S hexamers without prior processing of theprecursor. Since prolegumin in vitro does not oligomerize intomore than 9S tnmers these results suggest that a protein-mediatedassembly of 9S prolegumin trimers into 12S prolegumin hexamersprobably occurs in the lumen of the protein storage vacuole.Prolegumin, but not mature legumin, binds very tightly to membranes.This property points to a possible way of identifying a putativeprolegumin receptor. Key words: Calcium, Endoplasmic reticulum, Golgi apparatus, legumim, monoclonal antibody, pea cotyledons     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins.

Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.     

Isolation of two novel adenosine binding proteins from bovine brain

Adenosine plays many significant roles both as a metabolic precursor and cell communicator. This report describes the preliminary characterization of two adenosine binding proteins isolated from bovine brain membranes. By using N6-9-aminononane adenosine labeled Sepharose 4B two major affinity bound proteins were purified having apparent molecular weights of 16 and 35 kDa. Either or both of the proteins could be selectively eluted from the affinity column with N6-9-aminononane adenosine, adenosine, cAMP, AMP, ADP, ATP, R-/S-phenylisopropyladenosine and NAD(H). By contrast, no proteins were eluted with caffeine, adenine, deoxyadenosine, 2',3'-AMP, inosine, IMP, xanthine, XMP, GMP, GTP or 5'-N-ethylcarboxamideadenosine. The selectivity of elution and lack of apparent enzymatic activity suggests that these proteins are novel membrane bound adenosine binding proteins.     

Expressed sequence tags and mRNA expression levels of tagged cDNAs from watermelon anthers and developing seeds

To understand the molecular events that occur during reproductive organ development and to provide genetic resources for molecular breeding, we generated 328 expressed sequence tags (ESTs) from randomly selected clones of four watermelon cDNA libraries. These libraries were prepared from young and mature anthers, as well as the seed coat and inner seed tissues. EST clones found in the young anthers and inner seed tissues showed similarity with genes related to development and signal transduction. We could deduce that, especially in the developing inner seed tissues, important morphological processes were associated exclusively with seed and embryo development In addition, seed metabolism was tailored toward the accumulation of economically valuable storage compounds such as lipids. In the seed coat, EST clones showed similarity with genes that influence the transport or conversion of nutrients such as porin, sucrose synthase, L-asparaginase, and arginine decarboxylase. We also selected two cDNA clones from each of the four classes of ESTs for studying expression levels and patterns in the various organs. Among those eight clones, three (An88, Is124, and Sc68) were expressed preferentially in their particular organ.