Ebulitins: A new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M_r 32,000, 29,000 and 29,000 for ebulitins , β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.     

Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosome-inactivating protein b-32

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn     

Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim

A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS–PAGE, with an apparent molecular mass of 13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC₅₀ of 1.6 ng/ml. Liquid chromatography–tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.     

Correct glycosylation,Golgi-processing,and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5 upstream and 1600 bp 3 downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.Abbreviations Asn asparagine - Endo H endoglycosidase H - HPLC high-performance liquid chromatography - IgG immunoglobulin G - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecylsulfate - TFMS trifluoromethanesulfonic acid     

Expression of phosphinothricin acetyltransferase from the root specific par promoter in transgenic tobacco plants is sufficient for herbicide tolerance

Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA^® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA^® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP 6-benzylaminopurine - CaMV Cauliflower Mosaic Virus - IAA indole-3-acetic acid - kb kilobases - LB Luria-Bertani - MS Murashige and Skoog - par Parasponia andersonii - PAT phosphinothricin acetyltransferase - ppt phosphinothricin - TCA trichloric acid     

Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. Theldcgene fromHafnia alvei was therefore (a) placed under the control of the 1 promoter of the bidirectional Tr promoter fromAgrobacterium tumefaciens Ti- plasmid, and (b) cloned behind therbcS promoter from potato fused to the coding region of therbcS transit peptide. Bothldc constructs, introduced intoNicotiana tabacum with the aid ofA. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of therbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3–1% of dry mass.     

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.     

Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco

Diseases of agricultural crops are caused by pathogens from several higher-order phylogenetic lineages including fungi, straminipila, eubacteria, and metazoa. These pathogens are commonly managed with pesticides due to the lack of broad-spectrum host resistance. Gastrodia anti-fungal protein (GAFP; gastrodianin) may provide a level of broad-spectrum resistance due to its documented anti-fungal activity in vitro and structural similarity to insecticidal lectins. We transformed tobacco (Nicotiana tabacum cv. Wisconsin 38) with GAFP-1 and challenged transformants with agriculturally important plant pathogens from several higher-order lineages including Rhizoctonia solani (fungus), Phytophthora nicotianae (straminipile), Ralstonia solanacearum (eubacterium), and Meloidogyne incognita (metazoan). Quantitative real-time PCR and western blotting analysis indicated that GAFP-1 was transcribed and translated in transgenic lines. When challenged by R. solani and P. nicotianae, GAFP-1 expressing lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines. These lines also exhibited reduced root galling when challenged by M. incognita. Against R. solanacearum expression of GAFP-1 neither conferred resistance, nor exacerbated disease development. These results indicate that heterologous expression of GAFP-1 can confer enhanced resistance to a diverse set of plant pathogens and may be a good candidate gene for the development of transgenic, root-disease-resistant crops.     

Antisense expression of chaperonin 60β in transgenic tobacco plants leads to abnormal phenotypes and altered distribution of photoassimilates

Chaperonins are a class of molecular chaperone, present in bacteria, mitochondria and chloroplasts, that are involved in protein folding and assembly in many organisms. Plastid α and β chaperonins have been suggested to be involved specifically in the assembly of Ribulose bisphosphate carboxylase/oxygenase. However, to date there is no direct evidence to confirm the in vivo role of plastid chaperonin 60 polypeptides as molecular chaperones. This paper reports on the production, by means of antisense technology, of transgenic tobacco plants with reduced levels of chaperonin 60β (Cpn60β). Antisense cpn 60β plants showed drastic phenotypic alterations including slow growth, delayed flowering, stunting and leaf chlorosis. The most extreme effect appeared to be lethality suggesting that cpn 60β functions are essential for viability. Cpn60β antisense plants accumulated Rubisco to specific activities equal to or higher than that of controls and had high plastid starch contents. These observations are discussed with respect to the suggestion that chaperonin is required for the assembly of active Rubisco in vivo . In addition, metabolic alterations in the antisense transgenic plants such as reduced soluble carbohydrate content as well as higher levels of starch in chloroplasts, suggest that Cpn60β may be required for import, assembly or membrane insertion of several chloroplast membrane proteins. These results are in agreements with the proposed role of Cpn60β as a molecular chaperone.     

Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally related new homodimeric lectin in fruits of Sambucus ebulus L.

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195–18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homo-dimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250 000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits. Received: 4 April 1997 / Accepted: 17 June 1997     

Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco

Several proteins are recalcitrant to expression in Escherichiacoli. To explore transgenic plants as an alternative expressionsystem, the gene encoding the potential herbicide target sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco(Nicotiana tabaccum) under the control of a duplicatedCaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle,accumulated to approximately 1.2% of total soluble protein. In order to purifyrecombinant SBPase, a sequence encoding six histidine residues was insertedC-terminally which allows a one step purification via Ni²⁺-NTAaffinity chromatography. N-terminal amino acid sequence analysis of the purifiedprotein confirmed processing of the transit peptide and revealed the previouslyunknown cleavage site. The transit peptide consists of 67 amino acids followedby the mature SBPase subunit of 342 amino acids including the C-terminalfusion. Purified SBPase was found to be enzymatically active after reduction with DTTand showed many biochemical properties of the native enzyme such as thedependence on Mg²⁺ and a pH optimum of 8.3. Subsequently, SBPaseproduced in transgenic tobacco was used in large-scale screening for thediscovery of novel herbicides.     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC₅₀ (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml⁻¹) and by HeLa, HT29 and JM cells with IC₅₀ in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml⁻¹, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Expression of heat shock protein 60 in the tissues of transported piglets

In this study, we sought to determine the distribution and expression of heat shock protein 60 (Hsp60) in the tissues of transported piglets. A total of 24 Chinese Erhualian piglets with an average body weight of 20±1 kg were assessed under both 2-h transported and normal housing conditions. Results of enzymatic analysis showed that the serum creatine kinase and aspartate aminotransferase concentrations were significantly increased in the 2-h transported piglets. Acute cellular lesions characterized by granular and vacuolar degeneration of the parenchyma cells in the tested heart, liver, and kidney were also confirmed by histopathological test after 2 h transportation. These results indicate that transport stress induces tissue damage to heart, liver, and kidney. Hsp60-positive immunostaining was consistently detected in the cytoplasm of myocardial cells, hepatocytes, renal tubular epithelial cells, and epithelial cells of fundic gland. However, results of enzyme-linked immunosorbent assay indicated that Hsp60 expression was only significantly elevated in the stomach, with lower expression in the heart and a non-significant trend of increased liver and kidney expression of Hsp60. These results indicate that different tissues had different sensitivities to transport stress, possibly resulting in varying levels of cytoprotection by Hsp60 in the different tissues. The expression of Hsp60 following 2 h transportation coincided with deterioration of cardiac cytoprotection in the heart and protection in the stomach. However, the direct role of Hsp60 in cytoprotection of heart and stomach tissues needs further investigation.     

Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance

The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum␣L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehalose-accumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance. Received: 8 July 1996 / Accepted: 10 October 1996     

The protein-body proteins phytohemagglutinin and tonoplast intrinsic protein are targeted to vacuoles in leaves of transgenic tobacco

To demonstrate the relationship between protein-bodies in seeds and vacuoles in other tissues, we expressed the coding sequences of two bean (Phaseolus vulgaris L.) protein-body proteins in tobacco (Nicotiana tabacum L.). We chose phytohemagglutinin-L (PHA-L) and tonoplast intrinsic protein (TIP) as representatives of the protein-body contents and protein-body membrane, respectively. The location of the two proteins in the leaves of transgenic tobacco was examined by immunocytochemistry and in preparations of isolated vacuoles. Tonoplast intrinsic protein accumulates primarily in tonoplasts in tobacco leaves, whereas PHA is found exclusively in the vacuolar sap, showing that the signals that target proteins to protein-bodies and their limiting membranes in seeds are correctly recognized in leaves. This observation provides further evidence that proteinbodies of dicotyledonous seeds should be considered as protein-storage vacuoles.Abbreviations TIP tonoplast intrinsic protein - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been supported by a grant from the U.S. Department of Agriculture Competitive Research Grants Office to Maarten J. Chrispeels. Herman Höfte is a recipient of a Postdoctoral Fellow-ship from the European Molecular Biology Organization. Craig Dickinson is a recipient of a National Institutes of Health Postdoctoral Fellowship. Loîc Faye was on leave from the Centre National de la Recherche Scientifique, UA203, Université de Rouen, Mont Saint Aignan France and supported by a cooperative CNRS — National S cience Foundation grant. The mention of vendor or product in this paper does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned. We thank Jürgen Denecke for providing plasmid pDE1001, Antje von Schaewen for the plant expression cassette and Marie-Theres Hauser for the modified vacuole preparation protocol.     

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants. The results of this paper have been granted US Patent No. 6,791,009.     

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)⁶⁰, and Asn⁹⁰, but the third glycosylation site (Asn²⁰²) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn asparagine - ConA concanavalin A - EndoH endoglycosidase H - Fuc fucose - GlcNAc N-acetylglucosamine - HPLC high-performance liquid chromatography - Man mannose - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulfate - Ser serine - TFMS trifluoromethanesulfonic acid - Thr threonine - Xyl xylose     

Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene. Received: 24 February 1999 / Accepted: 25 March 1999