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Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route—based on ORC3 manipulation—to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

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.

The development of plant-based protein polymers to employ in biofilm production represents the promising intersection between material science and sustainability, and allows to obtain biodegradable materials that also possess excellent physicochemical properties. A possible candidate for protein biopolymer production is phaseolin, a storage protein highly abundant in P Vulgaris beans. We previously showed that transformed tobacco chloroplasts could be employed to express a mutated phaseolin carrying a signal peptide (directing it into the thylakoids) also enriched of a cysteine residue added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as we previously demonstrated, and should promote polymerization. To verify the effect of the peptide modification and to quantify polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS): HF5 allows for the selective size-based separation of phaseolin species, whereas MALS calculates molar mass and conformation state of each population. With the use of two different HF5 separation methods we first observed the native state of P.Vulgaris phaseolin, mainly assembled into trimers, and compared it to mutated phaseolin (P*) which instead resulted highly aggregated. Then we further characterized P* using a second separation method, discriminating between two and distinct high-molecular weight (HMW) species, one averaging 0.8 × 106 Da and the second reaching the tens of million Da. Insight on the conformation of these HMW species was offered from their conformation plots, which confirmed the positive impact of the Cys modification on polymerization.

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.

In order to investigate the intraspecific diversity of wild Humulus lupulus (hop) in Central Italy, 12 populations were evaluated for their genetic polymorphism by means of 13 SSR loci together with six commercial cultivars as a reference. High levels of polymorphism were found across the populations, being 140 the number of multilocus genotypes over 159 samples analyzed. Moreover, the observed heterozygosity was higher than expected in most of the populations. High levels of gene flow were thus envisaged to occur within and among wild populations, and our sampling strategy allowed us to gain insights on the propagation modes of this species, i.e. clonal versus sexual propagation. Nevertheless, a genetic structure of populations with at least five genetically different clusters was disclosed. Private alleles were observed in both wild and cultivated hops. Chemical analysis of bittering and aromatic quality of female flowers from a subset of 8 wild populations revealed a high variability among plants, especially for essential oil components. Overall, the high variability of wild accessions here examined represent a valid source to be exploited in future breeding programs for new or improved hop cultivars development.

The indefinite growth capacity of plants is guaranteed by meristems, where cells are in continuous division and differentiation. The activity of these stem cells is under control of the negative feedback loop between the genes WUSCHEL (WUS) and CLAVATA3 (CLV3) (Brand et al. 2000). CLV3 gene encodes a small secretory protein that undergoes proteolytic maturation to release an active dodecapeptide that binds to a receptor complex (CLV1/2), but the actual maturation mechanism is still unknown (Kondo et al. 2006). The current model predicts CLV3 secretion in the extracellular space and maturation mediated by specific, but not yet identified, proteases. Conversely, a recent study suggested that CLV3-GFP maturation in transgenic tobacco plants occurred in the endoplasmic reticulum (ER) via a process mediated by ER Associated Degradation (ERAD). Moreover, the processed CLV3 dodecapeptide was biologically active because it inhibited Arabidopsis thaliana root growth (De Marchis et al. 2018). Here we show, by using a physiological assay based on root growth inhibition, that the production of the active 12-amino acid peptide is absent in transgenic algae of the genus Chlamydomonas. Our idea is that the CLV3 maturation process emerged when the first photosynthetic organism evolved into vascular plants, therefore unicellular algae should lack this process. To prove our hypothesis, we transformed cells of C. reinardtii with a CLV3-YFP fusion gene. In contrast to what is observed in transgenic tobacco, we detected secretion of the intact CLV3-YFP protein in the algal growth medium, without the production of the active dodecapeptide, therefore corroborating our hypothesis. Our future goal is to identify the plant genes involved in CLV3 maturation.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

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.

Unconventional protein secretion (UPS) in plants includes various transport routes of protein (e.g. extracellular vesicles and Endoplasmic Reticulum (ER) - derived vesicles) alternative to the conventional secretory pathway. However, an exhaustive comprehension of these unconventional pathways and of their molecular mechanisms remain elusive. Recently, secretome analysis revealed that different types of secreted proteins, including leaderless secretory proteins (LSPs), were found in the extracellular space of plant cells. LSPs may represent over 50% of the total secretome identified in plants, supporting the existence of novel mechanisms of secretion that are independent from the conventional ER-Golgi secretory pathway. Moreover, several proteins were also identified that, although containing a signal peptide, reach various compartments, such as the vacuole or endosymbiont organelles, by UPS routes. Here we report three cases study of human or plant proteins that are secreted according to UPS in plant cells: i) human lysosomal ?-mannosidase (MAN2B1), ii) human indoleamine 2,3-dioxygenase 1 (IDO1), iii) plant CLAVATA3 (CLV3). MAN2B1 has a signal peptide and in animal cells is transported to lysosomes trough the Golgi complex with a conventional pathway. Conversely, when expressed in plants, MAN2B1 is targeted to the vacuole by a UPS pathway thanks to a putative vacuolar targeting signal located in the amino-terminal domain. The immunoregulatory human IDO1 does not contain a signal peptide and it is secreted in the extracellular space according to a UPS pathway, both in mammals and in plant cells. Finally, we described the transport and maturation process of the plant stem cell regulatory protein CLV3, from which a biological active 12-14 amino acid peptide is released and extracellularly secreted. Although containing a signal peptide, the pathway of CLV3 processing deviates from the conventional secretory pathway and is subjected to proteasomal degradation, leading to secretion of the active peptide through a putative UPS mechanism.

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.

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses.

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN-6.6, later renamed PN-SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN-SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN-SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis. Fil: Bocchini, Marika. Università di Perugia; Italia Fil: Galla, Giulio. Università di Padova; Italia Fil: Pupilli, Fulvio. Consiglio Nazionale delle Ricerche; Italia Fil: Bellucci, Michele. Consiglio Nazionale delle Ricerche; Italia Fil: Barcaccia, Gianni. Università di Padova; Italia Fil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Albertini, Emidio. Università di Perugia; Italia

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Key message: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Abstract: Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30–60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types. Fil: Ochogavía, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Galla, Giulio. Università di Padova; Italia Fil: Seijo, José Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: González, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Bellucci, Michele. Consiglio Nazionale delle Ricerche; Italia Fil: Pupilli, Fulvio. Consiglio Nazionale delle Ricerche; Italia Fil: Barcaccia, Gianni. Università di Padova; Italia Fil: Albertini, Emidio. Università di Perugia; Italia Fil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina

Gli sfarinati di soia rappresentano attualmente la componente proteica più importante nei mangimi per l' alimentazione zootecnica per il loro costo relativamente basso e per l'alto tenore proteico (44-50 %). Tuttavia l'utilizzo di tale materiale comporta numerosi problemi sia di ordine ambientale che igienico sanitario. Inoltre il fatto che la soia debba essere acquistata da paesi non UE, rende gli utilizzatori del prodotto dipendenti dalle fluttuazioni dei mercati internazionali. Esiste quindi la necessità di studiare fonti proteiche alternative alla soia come integratori proteici nei mangimi per l'alimentazione zootecnica. La Vicia ervilia, una leguminosa attualmente sotto utilizzata, era usata nelle regioni temperate del bacino del Mediterraneo come fonte proteica per l'alimentazione zootecnica quando la soia non era disponibile. Tale specie si presta ad essere coltivata in aree marginali attualmente sottoposte ad una forte erosione a causa del loro abbandono. Sono stati analizzati ecotipi di V. ervilia provenienti da varie località del Mediterraneo insieme ad alcuni adattati all'areale pedoclimatico dell'Italia centrale per la loro identità genetica, per la produzione del seme, per alcune caratteristiche qualitative e per il loro effetto sulla carne di conigli di allevamento. Sulla base dei risultati ottenuti , si può ipotizzare un proficuo impiego degli sfarinati del seme di V. ervilia al posto della soia e/o dei suoi sottoprodotti delle formulazioni mangimistiche per gli animali di allevamento .

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.

International audience; AbstractTobacco, Nicotiana tabacum, is an industrial crop traditionally used for manufacturing cigarettes. However, due to restriction of European subsidies, an alternative use of tobacco is needed, such as biofuel or biomass. Tobacco is indeed an oilseed crop with an oil yield ranging from 30 to 40 % of seed dry weight. Yet, there is still little information on the cultivation of tobacco for seed oil production. Here, we tested tobacco cultivation as an oilseed crop in Central Italy, where flue-cured tobacco is traditionally cultivated, with an additional trial in Northest Italy. We used the cultivar Solaris, selected for its reduced size and elevated number of flowers, and tested its adaptability to different field management practices in 2013–2014. Results show that in Central Italy fields, at the beginning of ripening, tobacco plants showed on the average a 94–105 cm height and a stem diameter between 1.3–1.6 cm; they had 123–151 capsules and each plant produced on average from 31 to 34 g of seeds. Seed production was evaluated to be 1.1–1.8 t/ha, with an oil yield up to 0.59 t/ha. In Northest Italy, two seed harvests determined a total seed yield of 4.5 t/ha, from which 1.48 t oil/ha could be obtained. The cultivar Solaris was extremely adaptable in terms of morphological parameters and seed yield to different management practices as well as climatic conditions. This study shows for the first time the cultivation feasibility in Italy of a small-size tobacco variety selected for high seed production. With a further optimization of the cultivation protocol to increase the oil yield and to use the by-products, tobacco can really become a novel industrial crop providing renewable sources for both biofuel and biomass as well.

The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence.

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.

Bitter vetch (Vicia ervilia (L.) Willd.), a grain legume crop well adapted in marginal soils, has mainly been used for animal feed. Nowadays, bitter vetch seeds in feed formulations are replaced by other protein sources such as soybean meal. However, in the context of sustainable economic development, it may be beneficial to enhance the cultivation of bitter vetch landraces in marginal areas. Fifty-six bitter vetch accessions of different provenance were preliminarily characterised by microsatellite DNA analysis to discriminate landraces suitable for specific and restricted environments. Twenty-two landraces of two genetically different groups were then selected for further characterisation by agro-morphological analyses. Being late-flowering with a seed yield of up to 3–4 t ha–1 in experimental field conditions, with neither chemical nor water input, these plants will be valuable material for long-term study to develop new cultivars adapted for seed production under organic agricultural systems in Southern Europe. The seeds of these 22 landraces were also evaluated, with positive results, as partial replacement of soybean in rabbit diets.

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.

The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence.

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.

Plants of birdsfoot trefoil (Lotus corniculatus), alfalfa (Medicago sativa) and tobacco (Nicotiana tabacum) were transformed by Agrobacterium with binary vectors harbouring genes that code either for wild-type and gamma-zein:KDEL, or for beta-zein:KDEL. The maize seed storage proteins gamma-zein and beta-zein are rich in sulphur amino acids and our long-term goal was to improve alfalfa and birdsfoot trefoil forage quality. Significant levels of zeins were detected in leaves of birdsfoot trefoil transformants, ranging up to 0.055% and to 0.30% of total extractable protein for gamma-zein and beta-zein:KDEL, respectively. In leaves of alfalfa, gamma-zein:KDEL expression level was up to 0.026% of the total extractable protein. From a F1 population of transgenic tobacco a plant was selected in which the amount of zeins (gamma-zein:KDEL plus beta- zein:KDEL) accounted for 1.1% of total extractable protein.

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.

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO3−, NH4+ or NO3− in conjunction with NH4+, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO3− nutrition, lowest with NH4+ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO3−-fed plants than in either NH4+- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO3−-deprived than in NO3−-sufficient plants. Root malate accumulation was high in NO3−-fed plants, but decreased significantly in plants that were fed with NH4+. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO3−-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO3−- and (NO3− + NH4+)-fed plants, but was undetectable in those administered NH4+. Both the nitrate and the ammonium contents were significantly reduced in NO3−- and (NO3− + NH4+)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH4+-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.

Two gene constructs (pROK.TG1L and pROK.TG1LK) were utilized to achieve accumulation of maize γ-zein to high levels in tobacco (Nicotiana tabacum L.) leaves. Both the chimaeric genes contained the γ-zein-coding region preceded by the 5′untranslated leader from the coat protein mRNA of TMV, but one of them (pROK.TG1LK) was modified in its protein-coding region by the addition of the ER retention signal KDEL. The accumulation of γ-zein and γ-zein:KDEL in leaves was compared with heterologous protein accumulation in tobacco plants previously transformed with a γ-zein cDNA harbouring a native 5′UTR. Replacement of γ-zein 5′UTR with the TMV leader dramatically increased γ-zein production. Furthermore, γ-zein:KDEL-expressing plants, on average, accumulated twice as much foreign protein in their leaves as pROK.TG1L plants. The two-fold increase in the level of γ-zein:KDEL can probably be attributed to an improvement in the mechanism for ER retention of zeins in the transgenic cells. Transformants also showed increased production of BiP, though to a lesser extent in γ-zein:KDEL-expressing plants compared with pROK.TG1L plants. It is therefore likely that γ-zein:KDEL retention is made less dependent on the chaperone assistance of BiP by the presence of the KDEL signal on the γ-zein mutant.

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.

Lotus corniculatus plants transformed with a maize cDNA (G1L) encoding a sulphur-rich γ-zein were obtained by using two fusion genes: one with the CaMV 35S promoter, the other with the ribulose bisphosphate carboxylase small subunit (rbcS) promoter. The highest expression of G1L mRNA was found in plants transformed with G1L under the rbcS promoter. The steady state level of G1L mRNA in the leaves was generally directly correlated with the G1L copy number. However, due to a transcriptional block, no G1L mRNA was detected in some of the 35S-G1L multi-copy transformants. Analyses with methylation-sensitive restriction enzymes revealed that the T-DNA of the silenced 35S-G1L transformants was methylated. T-DNA copy number, G1L silencing activity, and the state of methylation were strictly correlated in primary transformants. A cross between two 35S-G1L transformed plants, one (S) with the T-DNA methylated and the other (NS) without, showed that: (1) the methylated state of T-DNA passed through meiosis; and (2) when T-DNA copies from the two parents were combined in the progeny, the unmethylated T-DNA copies of parent NS became methylated at different levels and G1L mRNA production was dependent on the degree of methylation.

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.

With the aim of increasing the content of sulphur amino acids (SAAs) in the aerial part of the plants, Nicotiana tabacum and two leguminous species, Medicago sativa and Lotus corniculatus, were transformed with a maize γ-zein gene (G1L), coding for a SAAs-rich protein. Gene fusions with two different promoters were constructed; one promoter was the constitutive cauliflower mosaic virus (CaMV) 35S and the other was the light-regulated ribulose bisphosphate carboxylase small subunit (rbcS) from a tobacco gene. The constructs were introduced by Agrobacterium-mediated gene transfer. All the transformants produced the G1L RNA (two differently sized transcripts were detected) and no relevant differences were found between the two promoters. Despite the presence of G1L message the γ-zein was found only in tobacco plants (up to 0.05% of the alcohol soluble proteins). Post-transcriptional mechanisms seemed to inhibit protein accumulation. The low level of G1L RNA in the transformed plants was probably the main factor that limited the production of γ-zein protein.

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.

Chloroplasts are a useful platform for the expression of recombinant proteins in higher plants. Transgenes can be introduced into the plastid genome (plastome) either by PEG transformation of plant protoplasts, or, more commonly, by the biolistic method, using leaves or suspension cells. Transgenes are integrated by double recombination events between flanking sequences in the vector and homologous sequences in the plastome. The genetic engineering of the plastome allows high-level foreign protein expression, site-specific gene integration, expression of multiple genes as operons, marker gene excision, and transgene containment. Since the first example of stable plastid transformation in higher plants, methods for DNA introduction, marker genes and selection strategies, vector types, and methods for marker excision have been improved. Although the plastids of some species remain difficult to transform, positive results have been shown for about 20 species. In this chapter, we summarize the basic structural and expression features of the plastid genome of higher plants, and discuss the development of a number of innovative enabling technologies for plastome transformation, the most recent and significant biotechnological applications, and the future perspectives of this technology.

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.

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.

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.