Differences in Protein Patterns in Suspension Cultures of Taxus cuspidata Induced by Cerium

The changes in soluble proteins induced by Ce⁴⁺ were analyzed in suspension cultures of Taxus cuspidata using two-dimensional polyacrylamide gel electrophoresis. The ultrastructure of cells obviously changed at day 4 after addition of Ce⁴⁺. Large amount of nuclear DNA fragments of about 200 bp were observed. Thirteen protein spots were different between the cultures grown with and without Ce⁴⁺ at day 4 as well as at day 6 after addition of Ce⁴⁺. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO₄ and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.Abbreviations BA 6-Benzylaminopurine - BL Brassinolide - CBB Coomassie Brilliant Blue - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA Gibberellic acid - IAA Indole-3-acetic acid - MALDI-TOF MS Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry - MMSDH Methylmalonate-semialdehyde dehydrogenase - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis - PVDF Polyvinylidene difluoride     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Proteomic analysis of bacterial blight defence signalling pathway using transgenic rice overexpressing thaumatin-like protein

Rice overexpressed thaumatin-like protein gene and the proteins from the leaf blades of 2-week-old transgenic rice seedlings were fractionated into cytosolic and membrane fractions, and separated by two-dimensional polyacrylamide gel electrophoresis and stained with Commassie brilliant blue. Among of 440 detected proteins, 5 proteins were up-regulated and 5 proteins were down-regulated by the overexpression of thaumatin-like protein. In the sense thaumatin-like protein transgenic rice and/or in rice inoculated with Xanthomonas oryzae pv. oryzae (Xo7435), 2-cys peroxiredoxin, thaumatin-like protein and glycine cleavage H protein were up-regulated, while oxygen evolving complex protein 2 was down-regulated. These results suggest that thaumatin-like protein-mediated disease resistance of rice against bacterial blight disease is the results of changes in proteins related to oxidative stress and energy metabolism in addition to changes in proteins related to defence.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

Protein profiling analysis of skeletal muscle of a pufferfish, <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

Protein profile of the skeletal muscle of Takifugu rubripes, a kind of pufferfish, was carried out with two-dimensional polyacrylamide gel electrophoresis (2-DE). Among the 112 protein spots detected in a silver-stained 2-D polyacrylamide gel, 33 were analyzed by Matrix Assisted Laser Desorption Ionisation tandem time-of-flight mass spectrometry (MALDI TOF/TOF MS), and 21 were identified by MASCOT. There were six structural proteins, such as alpha-actin, tropomyosin, and myosin heavy chain, and six with known functions such as T-cell receptor alpha chain, 4SNc-Tudor domain protein, SMC3 protein, and Translin associated factor X, as well as nine hypothetical novel proteins, including titin, andretinol dehydrogenase, and apolipoprotein A-I binding protein. These proteins were further categorized into six functional groups. This paper established a suitable technical protocol to eliminate the high abundance proteins while preserving middle abundance proteins for proteomics studies using Takifugu skeletal muscle. It is also favorable for further investigation on screening marker proteins for monitoring and controlling the quality of fish meat.     

Characterization of proteins expressed abundantly in the fruit-body ofFlammulina velutipes

Chronological changes of protein expression in the vegetative mycelium ofFlammulina velutipes and expression of these proteins in the fruit-body were investigated by two-dimensional polyacrylamide gel electrophoresis. Four proteins (FBA 1-4) expressed abundantly in the fruit-body were found to have different expression patterns in the vegetative mycelium after the fruiting treatment. FBA 1-4 had similar amino acid sequences and displayed a high similarity with the deduced amino acid sequence of theC1 cDNA, which has an Arg-Gly-Asp (RGD) cell-attachment sequence. This suggests that FBA 1-4 may have cell-to-cell attachment activity.     

Effects of ozone on the plasma membrane proteins in Arabidopsis thaliana (L.) leaves

The protein pattern of leaf plasma membranes from Arabidopsis thaliana (L.) Landsberg erecta was analysed in order to detect changes induced by acute short-term ozone treatment. Plasma membranes were isolated 0, 3 and 8 h after the end of a 2 h fumigation of the plants with 500 nmol mol^?1 of O₃. Proteins extracted from plasma membranes were separated by high-performance two-dimensional polyacrylamide gel electrophoresis. Eight hours after the end of fumigation, one new protein appeared and the amounts of two other proteins increased significantly. The reported study is a first step towards the identification of plasmalemma proteins altered by ozone and to a more detailed characterization of structural changes occurring in the plasma membrane after ozone exposure.     

Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Effect of fungal-isolate aggressivity on the biosynthesis of symbiosis-related polypeptides in differentiating eucalypt ectomycorrhizas

Changes in protein biosynthesis were examined during the early stages of differentiation of Eucalyptus grandis-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis of ³⁵S-labelled proteins. Three distinct isolates of P. tinctorius Coker & Couch were chosen based on the rate of ectomycorrhizal formation (i.e. infectivity) with E. grandis W. Hill ex Maiden. The isolate H506 was not able to induce mycorrhiza, isolate 441 showed moderate infectivity and isolate H2144 exhibited a very high infectivity. Mycorrhiza were produced in vitro in a system where seeds were germinated in the presence of fungal mycelium and exudates. The non-mycorrhizal isolate caused no changes in root protein biosynthesis as analyzed by two-dimensional polyacrylamide gel electrophoresis, whereas drastic alterations in protein biosynthesis were observed from initial contact with the aggressive mycobionts. During mycorrhizal development, there was a marked inhibition of plant polypeptides synthesis, enhanced accumulation of some fungal polypeptides and the emergence of symbiosis-specific polypeptides, the so-called ectomycorrhizins. The major changes were observed in a group of fungal acidic polypeptides (apparent molecular weight 28–32 kDa) including the ectomycorrhizin E₃₂. These polypeptides first appeared at contact and their synthesis increased during mycorrhizal formation, suggesting a role in mycorrhizal development, most likely as structural proteins. Up-regulation of the synthesis of fungal symbiosis-related polypeptides was tightly correlated to the infectivity of the strain.Abbreviations FW fresh weight - MW molecular weight - pI isoelectric point - SR-polypeptides symbiosis-related polypeptides This work was supported by a research grant from the Eureka-Eurosilva programme (Changes in Gene Expression during Ectomycorrhiza Differentiation and Function) to F.M. and a Murdoch University Special Research Grant to B.D; T.B. was a recipient of a Doctoral Fellowship from the INRA and an Australian Postgraduate Scholarship. We would like to thank Dr Denis Tagu and Dulcinéia de Carvalho (Institut National de la Recherche Agronomique, Nancy, France) for helpful discussions.     

Two-Dimensional Gel Electrophoretic Analysis of the Cerebella from Neuro-Pathologically-Mutant Weaver,Nervous and Staggerer Mice1

We have analysed the proteins of the cerebella from mutant and control mice by applying high resolution two-dimensional polyacrylamide gel electrophoresis. The tissue of each cerebellum and also the pallium cerebri were fractionated into water-soluble and particulate fractions, and these were used in gel electrophoresis. In order to augment the sensitivity for detection of protein spots, we applied silver staining. We used the cerebella from weaver (granule cell deficient), nervous (Purkinje cell deficient), and staggerer (poor dendritic arborization of Purkinje cells) mutant mice. The present technique revealed at least 700 to 800 protein spots. Among the spots detected we found 12 new significantly-changed proteins in the cerebella of the mutants. The possible significance of these proteins is discussed.     

Improved <Emphasis Type="Italic">in vitro</Emphasis> micropropagation method with adventitious corms and roots for endangered saffron

The objective of this study was to investigate development of an efficient in vitro tissue culture system for saffron (Crocus sativus L.) complete with roots and corms. In indirect organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, 100 mg L⁻¹ ascorbic acid, and the combination of 0.25 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ 6-benzylaminopurine (BAP) were best for callus initiation and growth while 1.5 mg L⁻¹ BAP was excellent for high rate of adventitious shoot formation. 1 mg L⁻¹ indole-3-butyric acid (IBA) was more preferable for adventitious corm and root initiation as well as growth. Overall, 64% rooting and 33% corm production rates were achieved in indirect organogenesis. In direct organogenesis, MS medium supplemented with 3 % sucrose, 100 mg L⁻¹ ascorbic acid and 1 mg L⁻¹ BAP was optimum for shoot growth. While 1 mg L⁻¹ IBA was best for adventitious corm formation, 2 mg L⁻¹ IBA promoted adventitious root initiation and growth. Overall, 36% and 57% of explants had corm and contractile root, respectively. The high rates suggest that efficient tissue culture system could be achieved for mass propagation and ex situ conservation of threatened saffron genetic resources.     

Effect of Auxins on Adventitious Root Development from Single Node Cuttings of Camellia sinensis (L.) Kuntze and Associated Biochemical Changes

An attempt was made to induce rooting from single node cuttings of Camellia sinensis var. TV-20 under controlled conditions and study its biochemical changes during rooting. The nodal cuttings were pretreated with different concentrations of IAA, NAA and IBA and kept in a growth chamber (25 ±2 °C, 16 h photoperiod (55 μ mol m⁻² s⁻¹) with cool, white fluorescent lamps and 65% relative humidity) for 12 h. Among the three auxins used for pretreatment, IBA showed more positive response on rooting as compared to IAA and NAA within 2 weeks of transfer to potting medium. Among four concentrations of IBA tested, 75 ppm gave maximum percentage of rooting, number of roots and root length. Therefore, IBA was used further in experiments for biochemical investigation. The adventitious rooting was obtained in three distinct phases i.e. induction (0–12 days), initiation (12–14 days) and expression (14–18 days). IAA-oxidase activity of IBA-treated cuttings increased slightly as compared to control. The activity was found to decrease during induction and initiation phases and increase during expression phase. The peroxidase activity in IBA-treated cuttings increased up to initiation phase and declined at the expression phase. Polyphenoloxidase activity increased both in IBA-treated and control cuttings during induction and initiation phase but declined slowly during expression phase. Total phenolic content was higher in IBA-treated cuttings, particularly in initiation and expression phases and it also correlated with peroxidase activity. Phenolics might be playing key role for induction of adventitious rooting, and phenolic compounds can be used as rooting enhancer in tea plant.     

Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis

Extracellular proteins of Zymomonas mobilis were analyzed by two-dimensional gel electrophoresis and protein maps drawn up. One of these proteins showed sucrose-hydrolyzing activity, as indicated by activity staining after polyacrylamide gel electrophoresis. It was purified from the extracellular extract of a glucose fermentation by polyacrylamide gel electrophoresis, using a two-step procedure. The molecular mass of the protein was 46 kDa and its isoelectric point 5.0. A rabbit antiserum was raised against this protein. As shown by immunoblotting, the same protein was present in extracellular extracts obtained from glucose, fructose and sucrose fermentations. A cross-reaction was also detected by immunoblotting, with a cellular protein of molecular mass 46 kDa present on the three carbon sources studied. However, activity staining was unsuccessful on gels after electrophoresis of these cellular extracts. The extracellular protein extract obtained from a fermentation run on glucose contained another sucrose-hydrolyzing protein of molecular mass 51 kDa and with an isoelectric point of 4.8. This protein was absent in fructose and sucrose fermentations but showed a positive reaction with the antiserum raised against the 46 kDa extracellular protein. Partially purified sucrose-hydrolyzing proteins also catalyzed transfructosylation reactions, suggesting that they could be of the levansucrase type.     

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during Pregnancy Induced Hypertension

Changes in [³⁵S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.Abbreviations dpm radioactive disintegrations per minute - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - SRP stress related protein     

Identification of neighboring protein pairs in the Escherichia coli 30 S ribosomal subunit by crosslinking with methyl-4-mercaptobutyrimidate

The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.