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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 present study aimed to investigate the enzymatic potential of Silybum marianum leaves to bioconvert phenolic acids produced in S. marianum callus into silymarin derivatives as chemopreventive agent. Here we demonstrate that despite the fact that leaves of S. marianum did not accumulate silymarin themselves, expanding leaves had the full capacity to convert di-caffeoylquinic acid to silymarin complex. This was proven by HPLC separations coupled with electrospray ionization mass spectrometry (ESI-MS) analysis. Soaking the leaf discs with S. marianum callus extract for different times revealed that silymarin derivatives had been formed at high yield after 16 h. Bioconverted products displayed the same retention time and the same mass spectra (MS or MS/MS) as standard silymarin. Bioconversion was achieved only when using leaves of a specific age, as both very young and old leaves failed to produce silymarin from callus extract. Only medium leaves had the metabolic capacity to convert callus components into silymarin. The results revealed higher activities of enzymes of the phenylpropanoid pathway in medium leaves than in young and old leaves. It is concluded that cotyledon-derived callus efficiently produces compounds that can be bio-converted to flavonolignans in leaves tissue of S. marianum.

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.

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.

There is a need to enhance the production of bioactive secondary metabolites and to establish new production systems, e.g., for liver-protective compounds of Silybum marianum seeds. Quantifying and identifying the produced phytochemicals, and examining their protective effects against genotoxic agents, is of great interest. This study established a protocol for the qualitative and quantitative production of hepatoprotective compounds in cotyledon-derived Silybum marianum callus through optimized supplementation of the MS medium with the growth regulators 2,4-D, benzylaminopurine, myoinositol, and asparagine. High-performance liquid chromatography (HPLC) coupled with electrospray ionisation mass spectrometry (ESI-MS) allowed for identification and quantification of the produced compounds. None of the growth medium combinations supported a detectable production of silymarin. Instead, the generated calli accumulated phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acid, as revealed by HPLC and mass spectrometric analysis. 4-Nitro-o-phenylenediamine (NPD) was employed in the AMES-test with Salmonella typhimurium strain TA98 because it is a potent mutagen for this strain. Results revealed that callus extract had a high anti-genotoxic activity with respect to standard silymarin but more evident with respect seed extract. The callus produced chlorogenic acid and dicaffeoylquinic acid, which revealed higher bioactivity than silymarin. Both compounds were not formed or could not be detected in the seeds of Silybum marianum Egyptian ecotype.

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.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

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.

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO2 fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment‐binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR‐Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching., The functional role of the Photosystem II monomeric antenna complexes CP29 and CP26 was investigated in Chlamydomonas reinhardtii generating single and double mutants by CRISPR‐Cas9 genome editing demonstrating their key role in non‐photochemical quenching of both Photosystem I and II.

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.

Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd. However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues.

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.

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.

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

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.

C–H and N–H tautomers of the title compound are obtained and their structural properties determined by 1H and 13C NMR spectroscopy and X-ray diffraction; solvent and base effects on the rate of the C–H to N–H transformation are investigated.