Proteins in the roots of the perennial weeds chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale Weber) are associated with overwintering

Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.     

Seasonal dynamics of carbohydrate and nitrogenous components in the roots of perennial weeds

Abstract Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale L.) are persistent weeds, the aerial portions of which do not survive in winter. However, subterranean tissues remain viable and facilitate the rapid resumption of growth in early spring. The source of nutrients for growth prior to the establishment of foliage is the roots. Carbohydrate and N reserves are accrued during late summer and autumn, respectively. Hydrolysis of fructans during late autumn occurs coincidentally with increments in sucrose, the latter providing a readily accessible C pool. Nitrate, free amino acids and soluble protein all play substantial roles in nitrogen storage. Asparagine is the predominant amino acid in the free pool during winter, followed by glutamine, ornithine, serine, aspartic acid and glutamic acid. Storage reserves remain at peak levels throughout winter and deeline prior to the resumption of growth. The patterns observed here provide evidence that N is an important currency of storage metabolism and, thus, a framework has been provided for the examination of regulation of N storage in perennial weeds.     

The identification of a pollen-specific LEA-like protein in Lilium longiflorum

A novel pollen‐specific LEA‐like protein, LP28, was detected in Lilium longiflorum using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N‐terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen‐specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

Identification of a novel multiple environmental factor‐responsive 1‐aminocyclopropane‐1‐carboxylate synthase gene,NT‐ACS2, from tobacco

Many abiotic environmental factors elicit the production of stress‐ethylene in higher plants. To elucidate the molecular mechanisms underlying the regulation of stress‐ethylene production and the physiological roles played by stress‐ethylene in stress responses of plants, we studied the gene expression of ACC synthase in tobacco plants that had been subjected to environmental stresses. Four new tobacco ACC synthase cDNA fragments, NT‐ACS2, NT‐ACS3, NT‐ACS4 and NT‐ACS5, were identified and sequenced. It was found that NT‐ACS2 could be induced by wounding, cold temperature and, especially, sunlight. NT‐ACS4 was induced at a faster kinetics by wounding. The multiple environmental stress‐responsive (MESR) NT‐ACS2 gene was found to contain three introns and four exons and encode a polypeptide of 484 amino acids, 54·6 kDa and pI 6·87. Computer analysis of the 3·4 kb 5 ′ flanking region upstream of the ACS coding region revealed the existence of a group of putative cis‐acting regulatory elements potentially conferring wounding, chilling, and UV light inducibility. Phylogenetic analysis of ACC synthase genes of different plant origins indicated that the chill‐inducible NT‐ACS2 gene is closely related to a chilling‐inducible citrus ACS gene.     

Brain aromatase, 5α‐reductase,and 5β‐reductase change seasonally in wild male song sparrows: Relationship to aggressive and sexual behavior

In many species, territoriality is expressed only during the breeding season, when plasma testosterone (T) is elevated. In contrast, in song sparrows (Melospiza melodia morphna), males are highly territorial during the breeding (spring) and nonbreeding (autumn) seasons, but not during molt (late summer). In autumn, plasma sex steroids are basal, and castration has no effect on aggression. However, inhibition of aromatase reduces nonbreeding aggression, suggesting that neural steroid metabolism may regulate aggressive behavior. In wild male song sparrows, we examined the neural distribution of aromatase mRNA and seasonal changes in the activities of aromatase, 5α‐, and 5β‐reductase, enzymes that convert T to 17β‐estradiol, 5α‐dihydrotestosterone (5α‐DHT, a potent androgen), or 5β‐DHT (an inactive metabolite), respectively. Enzyme activities were measured in the diencephalon, ventromedial telencephalon (vmTEL, which includes avian amygdala), caudomedial neostriatum (NCM), and the hippocampus of birds captured during spring, molt, or autumn. Aromatase and 5β‐reductase changed seasonally in a region‐specific manner. Aromatase in the diencephalon was higher in spring than in molt and autumn, similar to seasonal changes in male sexual behavior. Aromatase activity in the vmTEL was high in both spring and autumn but significantly reduced at molt, similar to seasonal changes in aggression. 5β‐Reductase was not elevated during molt, suggesting that low aggression during molt is not a result of increased inactivation of androgens. These data highlight the relevance of neural steroid metabolism to the expression of natural behaviors by free‐living animals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 209–221, 2003     

Superoxide dismutase, peroxidase, and germin-like protein activity in plasma membranes and apoplast of maize roots

Summary. The analysis of plasma membranes from maize roots by native gel electrophoresis revealed the existence of Mn-containing 120 kDa and CuZn-containing 70, 40, and 15 kDa superoxide dismutase (SOD) isoform activities. Isoelectric focusing of the plasma membranes differentiated anionic SOD isoforms with a pI of about 5 and cationic SOD isoforms at pI 8.6. Solubilization of the plasma membrane proteins further separated the cationic SOD into pI 8.6, 8.2, 8.4, and 7.2 isoforms. Double staining for both SOD and peroxidase activities showed an overlap of these activities only in the case of the high-molecular-mass (ca. 120 kDa) isoforms. High-temperature treatments demonstrated that the 120 kDa isoform was active even at 100 °C, indicating that it was a germin-like protein with superoxide-dismutating activity, different from the peroxidase with a similar molecular mass and the lower-molecular-mass CuZn-containing superoxide dismutases. These results are compared to those obtained from whole-tissue extract and apoplastic fluid. Correspondence and reprints: Maize Research Institute, POB 89-Zemun, 11081 Belgrade, Serbia and Montenegro.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

Summary Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca²⁺-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca²⁺ gradient.Abbreviations BMM butyl methyl-methacrylate - PPI polyphos-phoinositides - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Effects of the exogenous Nef protein on HIV‐1 target cells

Nef is a multifunctional gene of HIV which can increase virus replication either directly or by modulating the target cell's metabolism. Nevertheless the role of the exogenous Nef protein is not yet well understood. To investigate it, we studied the effects of the recombinant Nef protein on the expression of some antigens of lymphoid T‐cells permissive to HIV‐1 replication, and on their proliferation and on apoptosis induction. For this purpose, we utilised MT‐4 and H9 T‐cell lines. We evaluated FN (fibronectin), CD4 and CD71 expression in uninfected and acutely or chronically infected cells, both untreated and treated with Nef. Our studies showed a significant up‐regulation of FN especially in uninfected cells, with a dose of 2·5 μg ml⁻¹; in contrast, a significant down‐modulation of CD4 and CD71 both in uninfected and in acutely or chronically infected cells, was detected. The proliferation of H9 uninfected cells was significantly reduced 24 h after treatment with Nef protein in a dose‐dependent manner (ranging from 0·02 to 2·5 μg ml⁻¹); likewise a significant inhibition of proliferation of acutely and chronically infected cells was evident with 2·5 μg ml⁻¹. Moreover, we demonstrated a dose‐dependent activity of Nef on inducing apoptosis in H9 uninfected cells and no effects of this protein on modulation of INF α and γ production in peripheral blood mononucleated cells of health donors. Nef appeared to be able to increase the effect of apoptotic stimuli. In conclusion, our data suggest that in our experimental system, the exogenous Nef protein can inhibit cellular synthesis facilitating the metabolic pathway involved in virus replication. In addition it modulates the susceptibility to the HIV‐1 infection and finally, that apoptosis induction or enhancement can facilitate the release of neoformed virions. Copyright © 1999 John Wiley & Sons, Ltd.     

A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway.     

Accumulation of pathogenesis-related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter

Seasonal evaluation of total soluble protein fractions extracted from cortical parenchyma cells of mulberry (Morus bombycis Koidz.) tree identified a predominant 18 kDa protein that was directly correlated to periods of cold acclimation. The 18 kDa protein, designated as WAP18 (winter accumulating 18 kDa proteins) increased from September to December and then gradually decreased until June. The maximum levels of WAP18 were detected in mid‐winter, which corresponds to the maximum freeze tolerance in cortical parenchyma cells of mulberry tree. Two‐dimensional gel electrophoresis confirmed that WAP18 consists of at least three proteins that range between an isoelectric point of 5.0 and 6.0. All three proteins reacted with anti‐WAP18 antibodies, thereby suggesting that they represent individual isoforms. Furthermore, N‐terminal amino acid sequence analysis demonstrated that all three proteins contain high sequence similarity to each other and high homology to pathogenesis‐related (PR) ?10/Bet v 1 protein families. The purified WAP18 exhibited in vitro cryoprotective activity for the freeze labile l ‐lactate dehydrogenase (LDH) enzyme. These results suggest that WAP18 may function in the freezing tolerance mechanism of cortical parenchyma cells of mulberry tree during winter.