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The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum cha- perone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

The interaction between organisms and their environment generates evolutionary forces able to modify a living species. The process of gene expression, from the transcription of information encoded in a gene to the synthesis of a functional polypeptide, largely relies on evolution, which plays a significant role in determining the factors that control and regulate gene expression. Nowadays, chloroplasts are the result of a complex evolutionary history, elicited by the intracellular cohabitation of an ancient photosynthetic cyanobacterium inside a mitochondriate eukaryotic cell. In this paper, we try to describe the recent investigations on mechanisms that regulate chloroplast translation, both in Chlamydomonas reinhardtii and in land plants. After a general description of plastid translational machinery, ribosome structure with related proteins and nuclear-encoded proteins regulating plastid translation in these organisms, we focus on specific examples of chloroplast translation regulation in the green lineage. In the end, we provide a comparison between plastid translation regulation in green algae and land plants, showing that in both cases chloroplast gene expression is prevalently regulated at post-transcriptional and translational level, although with different strategies.

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of outmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories, conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

New selection systems are required to extend plastid transformation to a more significant number of plant species. After demonstrating that a bacterial mutant glutamate 1-semialdehyde aminotransferase (GSA) gene is functional in tobacco plastids and confers resistance to the phytotoxin gabaculine, in this work, we transformed the tobacco plastome with a mutated GSA gene derived from alfalfa, along with the aadA selectable marker gene conferring spectinomycin resistance. Selection exploiting gabaculine was not effective to directly regenerate transplastomic events, but some events regenerated using conventional spectinomycin selection acquired gabaculine resistance during the second or third regeneration rounds with gabaculine selection. Gabaculine selection with our plant mutant GSA gene can be further investigated for plastid transformation of species that are not transformable with the aadA-spectinomycin selection system. Resistance to the phytotoxin gabaculine was obtained in tobacco by introducing a mutant plant gene in the plastid genome; it may be implemented as a tool in plastid genetic transformation.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum.

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on "Unconventional Protein and Membrane Traffic" (UPMT) during 4-7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast.

A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of utmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories; conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

In plants, acyl carrier proteins (ACPs) are generally encoded by multigene families. We have isolated from olive (Olea europaea L.) six ACP cDNAs, and their corresponding genomic sequences, coding for plastidial ACP isoforms. Three different OeACP loci have been identified, each with a pair of alleles. The transcript abundance of OeACP1 in olive tissues is extremely low in comparison to the other OeACP genes. The OeACP2 and OeACP3 transcripts are easily detected in all tissues. OeACP2 is the most abundant in fruit and leaf tissues. In addition, OeACP3 seems to be highly expressed in flower. At the protein level, ACP is predominantly found in fruit, and to a minor extent, in flowers and leaves. The analysis of the expression pattern of ACP genes during fruit development has revealed a good correlation between the level of transcripts and the amount of protein, which indicates that ACP expression in olive pulp is mainly regulated at the transcriptional level. We suggest that OeACP2 and OeACP3 proteins are involved in the synthesis of fatty acids for the accumulation of triacylglycerols in olive fruits. © 2014 Springer-Verlag Berlin Heidelberg.

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Studies on the basic mechanisms that regulate vacuolar delivering of proteins synthesized in the endoplasmic reticulum (ER) have a great importance in plant cell biology. Indeed, many aspects of plant physiology are affected by this intracellular traffic, for example, germination or reaction to biotic stresses due to the accumulation of storage proteins in seeds or enzymes in vegetative tissues, respectively. Up to now, the Golgi complex has been considered the main hub in the sorting of vacuolar secretory proteins; those polypeptides able to reach their final destination without the aid of this organelle are regarded as exceptions to an established route. This mini-review aims to emphasize the existence of several Golgi-independent pathways involved in the trafficking of different types of vacuolar proteins.

The insertion of transgenes into the plastid genome (plastome) has proved to be an effective alternative to nuclear transformation for producing stable transformants expressing heterologous proteins. Recently, methodology, characteristics and possible fields of application of plastome transformation have been extensively reviewed (Lössl and Waheed, 2011; Scotti et al., 2012). From these reports, plastid transformation turns out to be a very promising tool for the production of recombinant proteins in plants, yet some limitations must still be overcome. Although the plastome of many species has been successfully transformed, an efficient and reproducible transformation procedure easily adoptable by any plant laboratory has been established only in tobacco, and to a minor extent in two other solanaceous species (potato and tomato), as well as in few other species (Maliga and Bock, 2011). Moreover, up to now the steady-state level of a recombinant protein coded by a gene inserted into the plastome has failed to be predictable. In spite of a huge number of reports on successful foreign protein production in plastids, above all proteins with a pharmaceutical interest, in many cases the expression level of other recombinant proteins in the transplastomic plants appears to be very low or even undetectable (e.g. Birch-Machin et al., 2004; Bellucci et al., 2005; Wirth et al., 2006). We believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of protein homeostasis, or proteostasis. This term refers to a complex network of biological pathways capable of maintaining the dynamic equilibrium of the protein pool, thus allowing the cell to get used to environmental changes (Balch et al., 2008). This concept can also be extended to subcellular level, especially for those organelles with an endosymbiotic origin like the plastids and mitochondria (Waller, 2012). These organelles retain a whole series of mechanisms for the preservation of their protein balance, including specific proteases, transcriptional and translational control, as well as molecular chaperones and enzymes useful in protein folding. Therefore, it is important to develop basic studies in factors regulating protein synthesis, stability, folding, targeting, and accumulation in plastids, including the protein quality control which contributes to the functional integrity of proteins. Our intention here is not to provide a comprehensive review of the mechanisms that regulate plastid proteostasis, but to discuss some recent insights into this field which might bring beneficial applications in plastid biotechnology for transgene expression and foreign protein accumulation.

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. 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 interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum (ER). Through interactions with chaperones such as the binding protein (BiP) and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein gamma-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

It is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome; this technology provides new opportunities for the genetic improvement of this crop and for social acceptance of genetically modified sugar beet plants.

Protein bodies (PB) are stable oligomers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. We have tried to promote the formation of Nef-containing protein bodies in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms protein bodies and is composed of the vacuolar storage protein phaseolin fused to the Nterminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Coherently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. We conclude that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.

Background Classical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae. All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered. Results We report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits. Conclusion Previous reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.

Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagines synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagines is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher that that of transgenic plants. While glutamine levels in transgenic plants are about 3-4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO2 conditions.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of γ-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type γ-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the γ-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.

We prove generic multiplicity of solutions for a scalar field equation on compact surfaces via Morse inequalities. In particular our result improves significantly the multiplicity estimate which can be deduced from the degree-counting formula in Chen and Lin (2003) [12]. Related results are derived for the prescribed Q-curvature equation.