Retention of a bean phaseolin/maize gamma-Zein fusion in the endoplasmic reticulum depends on disulfide bond formation

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like gamma-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.     

Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa

Abstract

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.     

Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers

Analysis of nectar from leek (Allium porrum) flowers by SDS-PAGE revealed the presence of two major polypeptide bands of 50 kDa and 13 kDa, respectively. Using a combination of agglutination tests, enzyme assays and N-terminal sequencing, the polypeptides have been identified as subunits of alliin lyase (alliinase, EC 4.4.1.4) and mannose-binding lectin, respectively. The latter protein is particularly abundant since it represents about 75% of the total nectar protein. Honey produced by bees foraging on flowering leek plants still contains biologically active lectin and alliinase. However, the levels of both proteins are strongly reduced as compared to those in the original nectar. It is evident, therefore, that the lectin as well as the alliinase are inactivated/degraded during the conversion of nectar into honey. Received: 24 May 1996 / Accepted: 19 August 1996     

Expression of recombinant staphylokinase,a fibrin-specific plasminogen activator of bacterial origin,in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) plants

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.     

Detection of the 3-phosphoglycerate kinase protein of Glomus mosseae

 The Glomus mosseae 3-phosphoglycerate kinase (PGK) gene encodes a polypeptide of 416 amino acids. A synthetic peptide was designed to the C-terminus of the polypeptide for the production of a polyclonal antibody. The antibody was tested against the synthetic peptide in an immuno-dot blot and was then used to investigate the asymbiotic and symbiotic accumulation of the PGK protein. Western blot analysis revealed that a polypeptide of approximately 45 kDa accumulated in G. mosseae-colonised tomato roots; this is similar to the theoretical molecular weight of 44.764 kDa. The protein was not detected in non-mycorrhizal roots. Quantitative immuno-dot blotting revealed that the polypeptide accumulated in germinating spores and hyphae of G. mosseae and also in tomato roots colonised by G. mosseae. The amount detected in the mycorrhizal root system was significantly higher than that found in germinating sporocarps. The variation in the levels of glycolytic activity in the symbiotic and asymbiotic developmental stages of G. mosseae is discussed. Accepted: 20 April 2000     

The effect of irradiance and redox-modifying reagents on the 52 kDa protein disulfide isomerase of <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts

Immunoblot analysis was used to assess the effects of light and redox-modifying chemicals on the 52 kDa protein disulfide isomerase (PDI) from chloroplasts of Arabidopsis thaliana. A monoclonal antiserum was used that preferentially cross-reacts with the 52 kDa relative to the 65 kDa isoform of PDI. The PDI-52 was most abundant in leaves, flowers, stems and seeds, but was undetected in roots. PDI-52 formed a ∼220 kDa protein complex on blue native gels, indicating that it associates with either itself or other proteins in chloroplasts. Light decreased the levels of PDI-52 by 80 %, relative to the control protein (the CF1 subunit of chloroplast ATP synthase). Treatment with dithiothreitol decreased the content of the 52 kDa protein by half. In contrast, when the reduction of plastoquinone is blocked by DCMU, or when the plants are treated with phosphate, PDI-52 contents increased by 1.5 to 2-fold relative to CF1. The effect of the chemical treatments coincided with the effect of the light/dark cycle and implied that light decreased PDI-52 protein content by way of the cellular redox environment.     

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135–160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the β-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.     

Accumulation of a lectin-like breakdown product of β-conglutin catabolism in cotyledons of germinating Lupinus albus L. seeds

During germination of Lupinus albus seeds, a 20-kDa polypeptide accumulates in the cotyledons of 4-d-old plants (Ferreira et al., 1995b, J Exp Bot 46: 211–219). Immunological, polypeptide cleavage with cyanogen bromide and amino acid sequencing experiments indicate that the 20-kDa polypeptide and ubiquitin are structurally unrelated. However there is a strong sequence homology between the 20-kDa polypeptide and the vicilin-like storage proteins from pea and soybean. Our results indicate that the 20-kDa polypeptide is an intermediate breakdown product of β-conglutin catabolism, the vicilin-like storage protein from L. albus, and that its interaction with anti-ubiquitin antibodies results from the recognition of the antibodies by the 20-kDa polypeptide rather than by the opposite. Besides rabbit anti-ubiquitin antibodies, the 20-kDa polypeptide interacts with a variety of glycoproteins, including immunoglobulin G from several animal species, peroxidase and alkaline phosphatase, suggesting that it possesses a lectin-type activity. Its activity is resistant to sodium dodecyl sulfate or methanol treatments, boiling and autoclaving. Purification of the 20-kDa polypeptide and immunological studies with anti-20-kDa-polypeptide antibodies showed that the non-glycosylated polypeptide is part of a glycoprotein with an estimated molecular mass of 210 kDa, composed of several types of structurally related subunit with molecular masses ranging from 14 to 50 kDa. Purified native protein containing the 20-kDa polypeptide self-aggregates in a calcium-dependent manner as reported for some glycosylated lectins. The possible physiological function of the 20-kDa polypeptide is discussed. Received: 28 June 1996 / Accepted: 7 February 1997     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Effect of <Emphasis Type="Italic">Medicago sativa</Emphasis> ferritin gene on stress tolerance in transgenic grapevine

The effect of Medicago sativa (alfalfa) ferritin gene (MsFer) on abiotic stress tolerance was tested using transgenic Vitis berlandieri × Vitis rupestris cv. ‘Richter 110’ grapevine rootstock lines. Leaf discs from transgenic plants maintained higher photosynthetic activity after NaCl, tert-butyl-hydroperoxide (t-BHP) or paraquat treatment than control ones. These results indicate that the increased production of ferritin significantly improved abiotic stress tolerance in transgenic grapevine plants.     

Changes in protein spectra of transgenic plants carrying differentAgrobacterium tumefaciens C58 T-DNA genes

A series of binary vector plasmids derived from the T-DNA of theAgrobacterium tumefaciens strain C58, carrying the five plant morphoregulatory genes 1, 2, 4, 5 and 6b in different combinations, was used in the transformation ofNicotiana tabacum leaf discs. Protein patterns of the transgenic tobacco analysed through SDS-PAGE have shown changes in the polypeptides with M_r: ∼120, 60, 55, 43 and 27 kDa (for tobacco with transgene 4); ∼60, 55, 43, 26–25, 21, 18 kDa (for tobacco with transgenes 1, 2 and 5); ∼70, 60, 26, 25, 18 kDa (for tobacco with transgene 5); ∼60, 55, 48, 26, 18 kDa (for tobacco with transgenes 4, 5, 6b); ∼60, 55, 22 and 18 kDa (for tobacco with transgene 6b); ∼60, 55, 43, 26 and 18 kDa (for transgenes 5, 6b); ∼60, 55, 22, 18 and 16 kDa (for transgenes 4 and 6b). All types of transgenic plants showed quantitative changes in protein content. Mendelian segregation ratio to kanamycin resistance in the progeny of transgenic tobacco clones in the R1 generation was 3∶1 except in transgenic tobacco carrying transgenes 1, 2 and 5. Communicated by T. GICHNER     

Risk of alfalfa transgene dissemination and scale-dependent effects

Pollen can function as a vehicle to disseminate introduced, genetically engineered genes throughout a plant population or into a related species. The measurement of the risk of inadvertent dispersal of transgenes must include the assessment of accidental dispersion of pollen. Factors to be considered include the rate of pollen spread, the maximal dispersion distance of pollen, and the spatial dynamics of pollen movement within seed production fields; none of which are known for alfalfa (Medicago sativa L.), an insect-pollinated crop species. Using a rare, naturally occurring molecular marker, alfalfa pollen movement was tracked from seed and hay production fields. Results indicated that leafcutter bees (Megachile spp.) used in commercial seed production show a directional, non-random bias when pollinating within fields, primarily resulting in the movement of pollen directly towards and away from the bee domicile. Within-field pollen movement was detected only over distances of 4 m or less. Dispersal of pollen from alfalfa hay and seed production fields occurs at distances up to 1000 m. By examining widely dispersed, individual escaped alfalfa plants and their progeny using RAPD markers, gene movement among escaped alfalfa plants has been confirmed for distances up to 230 m. The outcrossing frequency for large fields was nearly 10-times greater than that of research-sized plots. A minimum isolation distance of 1557 m may be required to prevent gene flow in alfalfa. Data suggest that complete containment of transgenes within alfalfa seed or hay production fields would be highly unlikely using current production practices. Received: 20 March 1999 / Accepted: 11 November 1999     

Improved methods to detect GTP-binding proteins from plants

Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn(Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (M _r 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentK _m of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (M_r 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.     

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn 1 mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn ₁ was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn ₁ mutation.