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1.
Stress proteomes of the cytoplasmic membrane fraction of Bacillus subtilis trp
C2-exposed to acid pH and ethanol were characterized. Although these stress factors impair the cell function in a specific manner,
they share the ability to denature proteins. Therefore, specific and general stress proteins in the membranes were investigated.
Both ethanol (3 %) and pH 5.0 increase the doubling time from 17 to 25 min. Isolated cytoplasmic membranes were subjected
to an optimized 2D PAGE analysis which permitted the separation and analysis of ≈450 distinct protein spots. Two alternative
methods of protein detection were compared, i.e. silver staining and 35S-l-methionine pulse labeling; the stress induced proteins were identified by MALDI-TOF MS. After ethanol stress, five proteins
were increased, viz. YdaP, Ctc, YfhM, YjcH and YwaC. Acid stress proteins were AcoB, YkwC, SodA, YjcH and YwaC. Proteins YjcH and YwaC were increased
after ethanol as well as acid pH treatment. 相似文献
2.
Besma Sghaier-Hammami Inmaculada Redondo-López Ana M. Maldonado-Alconada Sira Echevarría-Zomeño Jesús V. Jorrín-Novo 《Acta Physiologiae Plantarum》2012,34(3):905-922
The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol
extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the
36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative
variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins
successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h
post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post
inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following
the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common
protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent
pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins
following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic
pathways and molecular chaperones. 相似文献
3.
Eun-Young Kim Dong-Gyun Kim Yu-Ri Kim Hye-Jung Hwang Taek-Jeong Nam In-Soo Kong 《Journal of applied phycology》2011,23(1):123-130
The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused
on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield
cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of
S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution
D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte,
4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g
of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more
than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass
spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate
dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation
factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots
were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed
at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5. 相似文献
4.
Velvetbean (Mucuna pruriens) plants impede the growth of neighboring plants. One compound, 3-(3′,4′-dihydroxyphenyl)-l-alanine (l-DOPA), is responsible for the allelopathic capacity of velvetbean. This compound is an active allelochemical that decreases
root growth of several plant species. In mammals, l-DOPA is a well-known therapeutic agent for the symptomatic relief of Parkinson’s disease. However, its mode of action in
plants is still not well understood. To address such issues, gene expression in Arabidopsis thaliana plants, which had been exposed to l-DOPA, was analyzed using DNA microarrays. After 6 h of l-DOPA exposure, the expression of 110 genes was significantly upregulated, and the expression of 69 genes was significantly
downregulated. These induced genes can be divided into different functional categories, mainly on the basis of subcellular
localization, metabolism, and proteins with a binding function or cofactor requirement. Based on these results, we suggest
that l-DOPA acts by two mechanisms: it influences amino acid metabolism and deregulates metal homeostasis, especially that of iron,
which is required for the fundamental biological processes of all organisms. 相似文献
5.
Hong Zhu Ying-Dong Bi Li-Jie Yu De-Dong Guo Bai-Chen Wang 《Molecular biology reports》2009,36(8):2093-2098
Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos
genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional
electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF
MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According
to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important
biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result
may be useful for us to better understand the genetic mechanism of apomixes. 相似文献
6.
Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product
formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached
to the benzene ring and an electron donor, e.g., OH or NH2, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring.
Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including
HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K
M
values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K
cat/K
M
) were determined in the range of 430–1,110 s−1·M−1 with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to
C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan
synthase 7-DMATS from Aspergillus fumigatus. 相似文献
7.
8.
The d-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom,
the metabolism and role of d-amino acids (d-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay
in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of d-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their d-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey’s reagent and
separated by HPLC–MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied d-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of d-AAs. The addition of particular amino acids (d-Trp, d-Phe, d-Met and d-His) led to the accumulation of the corresponding l-amino acid. In almost all cases, the application of a d-AA resulted in the accumulation of d-Ala and d-Glu. The presented results indicate that soil borne d-AAs can actively be taken up and metabolized via central metabolic routes. 相似文献
9.
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11.
Konstantin V. Kiselev Anna V. Turlenko Yuri N. Zhuravlev 《Plant Cell, Tissue and Organ Culture》2009,99(2):141-149
A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium
(MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained
by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture
in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in
darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation
and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic
acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed
a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development. 相似文献
12.
Gui-Hye Hwang Jae-Yong Cho 《Journal of industrial microbiology & biotechnology》2010,37(11):1131-1136
We recently proposed a metabolic engineering strategy for l-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance l-ornithine production in a well-defined recombinant strain of Corynebacterium
glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg−1 protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted l-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during l-ornithine production in C. glutamicum. 相似文献
13.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
14.
Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences.
Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal
plots revealed K
m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N
3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate. 相似文献
15.
Christian Abel Maria Clauss Andrea Schaub Jonathan Gershenzon Dorothea Tholl 《Planta》2009,230(1):1-11
Volatile organic compounds have been reported to serve some important roles in plant communication with other organisms, but
little is known about the biological functions of most of these substances. To gain insight into this problem, we have compared
differences in floral and vegetative volatiles between two closely related plant species with different life histories. The
self-pollinating annual, Arabidopsis thaliana, and its relative, the outcrossing perennial, Arabidopsis lyrata, have markedly divergent life cycles and breeding systems. We show that these differences are in part reflected in the formation
of distinct volatile mixtures in flowers and foliage. Volatiles emitted from flowers of a German A. lyrata ssp. petraea population are dominated by benzenoid compounds in contrast to the previously described sesquiterpene-dominated emissions
of A. thaliana flowers. Flowers of A. lyrata ssp. petraea release benzenoid volatiles in a diurnal rhythm with highest emission rates at midday coinciding with observed visitations
of pollinating insects. Insect feeding on leaves of A. lyrata ssp. petraea causes a variable release of the volatiles methyl salicylate, C11- and C16-homoterpenes, nerolidol, plus the sesquiterpene (E)-β-caryophyllene, which in A. thaliana is emitted exclusively from flowers. An insect-induced gene (AlCarS) with high sequence similarity to the florally expressed (E)-β-caryophyllene synthase (AtTPS21) from A. thaliana was identified from individuals of a German A. lyrata ssp. petraea population. Recombinant AlCarS converts the sesquiterpene precursor, farnesyl diphosphate, into (E)-β-caryophyllene with α-humulene and α-copaene as minor products indicating its close functional relationship to the A. thaliana AtTPS21. Differential regulation of these genes in flowers and foliage is consistent with the different functions of volatiles
in the two Arabidopsis species. 相似文献
16.
Autolysis is an important physiological process found in fungal cultivation. However, there is hitherto no report on the autolysis
of Pleurotus tuber-regium. We have investigated the enzymes secreted by temperature-induced (40°C as treatment versus 10°C as control) autolysis of
the mycelium of P. tuber-regium grown in submerged cultivation. A comparison between the intracellular proteins (inside the mycelium) and the extracellular
proteins (in the culture medium) of the treatment and control by proteomic analysis involving 2D PAGE and MALDI–TOF–MS was
made. Twenty-two up-regulated protein spots were detected and eight proteins were identified. They included proteasome which
participates in the ubiquitin–proteasome pathway; β-1,3-glucanosyltransferase and tubulin which are involved in the renewal
and repair of cell wall; protease and endoglucanase which promote the natural degradation of cell wall and cytoplasm; 14-3-3
protein which takes part in cell signal transduction; and two putative proteins presumably relate to the autolysis process.
These identified proteins suggest partially the metabolic processes of the autolysis in the P. tuber-regium mycelium. 相似文献
17.
Muhammad Rizwan Ingrid Miller Fareeha Tasneem Josef Böhm Manfred Gemeiner Ebrahim Razzazi-Fazeli 《Mycotoxin Research》2010,26(3):171-180
Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome
profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on
2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis
via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser.
Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our
purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis
gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant
spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved
in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA
and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged
culture. 相似文献
18.
Toms C. Joseph Lawrance Anbu Rajan Nirmala Thampuran Roswin James 《Molecular biotechnology》2010,46(1):20-25
Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects
the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in
the altered amino acid sequences of the translated proteins. 相似文献
19.
Background
Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice. 相似文献20.
Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of
the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation
of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection
between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively,
under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional
symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a
peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A.
thaliana covering the entire range of its genetic diversity, as well as Arabidopsis
halleri, were found to be susceptible towards Cuscuta
reflexa. 相似文献