Jon Veramendi

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

A chloroplast‐derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase

As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast, this paper presents results on stress induced by continuous chlTGZ over-expression in transplastomic tobacco leaves. Thylakoid remodelling induced by chlTGZ over-expression in young leaves of tobacco chloroplasts has already been reported (Ioannidis et al. in Biochem Biophys Acta 1787:1215–1222, 2009). In the present work, we determined the induced alterations in the photosynthetic apparatus, in the chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways, regarding ageing and functionality of the tobacco transformed plants. The results revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. The decrease in pigment levels in the transformed leaves was accompanied by an increase in H2O2 and lipid peroxidation. The rise in H2O2 correlated with a decrease in catalase activity, whereas there was an increase in peroxidase activity. In addition, chlTGZ over-expression lead to a drop in reduced glutathione, while Fe-superoxide dismutase activity was higher in transformed than in wild-type leaves. Together with the induced oxidative stress, the over-expressed chlTGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections. Consequently, the electron transport between photosystems decrease in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced in vitro. These results are discussed in line with chlTGZ involvement in chloroplast functionality.

Influence of nitrogen supply on micropropagation and subsequent microtuberization of four potato cullwars

A medium containing low amounts of nitrogen (19–23 meq.l−1) produced optimum results in micropropagation as revealed by the number of nodes, internode length, chlorophyll content, and leaf area of four potato cvs. belonging each to four different maturity groups. Decreasing amounts of nitrogen also increased chlorophyll content in all four cultivars tested. The NH4+ concentration did not have an effect on micropropagation for low nitrogen supplies.In all cvs., except Baraka, there was a “carry over” effect of the nitrogen content in the micropropagation medium onto subsequent tuberization, the lower nitrogen (23 meq.l−1) advancing tuber initiation. Microtuberization of cv. Jaerla was earlier in darkness than under short days regardless of the propagation medium used.ResumenUn medio con bajo contenido en nitrógeno (19–23 meq.l−1) permitió obtener unos resultados óptimos en la micropropagación que fue evaluada midiendo el número de nudos, la longitud de entrenudos, el contenido en clorofila y el área foliar de 4 cultivares de papa pertenecientes a 4 grupos de madurez distintos. La disminución de nitrógeno en el medio producía un aumento en el contenido en clorofila en los 4 cultivares ensayados. La concentration de NH4+ no modificó la micropropagación para bajos aportes de nitrógeno.En todos los cultivares, excepto Baraka, hubo un efecto retardado del contenido de nitrógeno en el medio de micropropagación sobre la posterior tuberización, de manera que un contenido bajo de nitrógeno (23 meq.l−1) anticipé la tuberizaeión. La microtuberización del cv. Jaerla fue más precoz en oscuridad que en días cortos, independientemente del medio de propagation utilizado.

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

SummaryPhytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.

Human papillomavirus-like particles vaccine efficiently produced in a non-fermentative system based on insect larva

a Instituto de Agrobiotecnologia (Universidad Publica de Navarra–CSIC–Gobierno de Navarra), Campus Arrosadia, 31006 Pamplona, Spain Alternative Gene Expression S.L. (ALGENEX), Centro empresarial Parque Cientifico y Tecnologico de la Universidad Politecnica de Madrid Campus de Montegancedo, Pozuelo deAlarcon, 28223 Madrid, Spain Departamento de Biotecnologia, INIA, Autovia A6 Km 7, 28040 Madrid, Spain

The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts

Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance, but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.

Effect of physiological age of mother tuber and number of subcultures on in vitro tuberisation of potato ( Solanum tuberosum L.)

Abstract The physiological age of mother tubers (Solanum tuberosum L. cv. Kennebec) used as a source of material influenced kinetin-induced in vitro tuberisation. Tuberisation significantly increased with physiological age. Kinetin- or ancymidol-induced tuberisation, plantlet and microtuber dry weight decreased with increasing number of subcultures. Single-node segments obtained from tubers stored for more than 9.5 months at 4 °C showed increased kinetin-induced tuberisation rates and earlier tuberisation than those obtained from younger tubers. For any physiological age, material may be safely multiplied using node propagation until the third subculture and bioassayed for tuberisation without variation in the response.

High Stability of Recombinant Proteins Expressed in Tobacco Chloroplasts

The green fluorescent protein and the non-toxic subunit of cholera toxin were expressed in tobacco chloro- plasts. Both recombinant proteins accumulated to levels greater than 20% of the total soluble protein. We did not observe light-dependent induction of gene expression despite employing the promoter and 5´-UTR from the psbA gene. Both pro- teins were stable in young and old leaves. The estimated half-life, determined by pulse-chase labeling, was greater than 48 h. Freeze-drying of pulverized leaves supplied an easy method of post-harvest handling with no significant loss of the re- combinant protein after 7 months of storage at room temperature. These data suggest that both proteins are good candi- dates for the expression of fusion proteins (e.g. with antigens) in tobacco chloroplasts.