Ecophysiological roles of abaxial anthocyanins in a perennial understorey herb from temperate deciduous forests

Accumulation of abaxial anthocyanins is an intriguing leaf trait particularly common among deeply shaded understorey plants of tropical and temperate forests whose ecological significance is still not properly understood. To shed light on it, possible ecophysiological roles of abaxial anthocyanins were tested in the perennial understorey herb of temperate deciduous forests Saxifraga hirsuta, chosen as a model species due to the coexistence of green and anthocyanic leaves and the presence of an easily removable lower anthocyanic epidermis. Anthocyanins accumulated during autumn, which temporally matched the overstorey leaf fall. Patterns of development of abaxial anthocyanins and direct measurements of photochemical efficiency under monochromatic light were not consistent with a photoprotective hypothesis. Enhancement of light capture also seemed unlikely since the back-scattering of red light towards the lower mesophyll was negligible. Seed germination was similar under acyanic and anthocyanic leaves. A relevant consequence of abaxial anthocyanins was the dramatic reduction of light transmission through the leaf. The dark environment generated underneath the Saxifraga canopy was enhanced by the horizontal repositioning of leaves, which occurs in parallel with reddening. This might play a role in biotic interactions by inhibiting vital processes of competitors, which may be of especial importance in spring before the overstorey leaves sprout.We also thank the technicians of SGIker’s ‘Servicio Central de Analisis de Bizkaia’ Service, financed by the National Program for the Promotion of Human Resources within the National Plan of Scientific Research, Development and Innovation, ‘Ministerio de Ciencia e Innovación’, ‘Fondo Social Europeo (FSE)’ and ‘Gobierno Vasco/Eusko Jaurlaritza, Dirección de Política Científica’, for the nitrogen measurements.Peer Reviewe

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 ɛ-(γ-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 (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). 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.M. Pintó-Marijuan was the recipient of a doctoral grant from the University of Barcelona and S.M. Ortigosa was the recipient of a predoctoral fellowship from CSIC. We thank Núria Cortadellas, Eva Fernandez and Almudena García (Serveis Cientifico-Tècnics, UB) for their technical assistance and Shirley Burgess for English corrections. This research was supported by the Spanish projects MEC BFU2006-15115-01/BMC, CGL2005-03998/BOS and BIO2005-00155, and partially by CERBA (Generalitat de Catalunya).Peer reviewe

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

13 p., 6 figures, 4 tables and bibliographyAs 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.This study was supported by the Spanish projects MEC BFU2006-15115-01/BMC, BIO2005-00155 and AGL2006-07143/AGR.Peer reviewe

Over-expression of maize transglutaminase in tobacco chloroplasts

Trabajo presentado en la 5th International Meeting on Biotechnology (Biospain 2010), celebrada en Pamplona del 29 de septiembre al 1 de octubre de 2010.Transglutaminases (TGases) are intracellular and extra cellular enzymes that catalyze post-translational modification of proteins by establishing ¿-(¿-glutamyl) links and covalent conjugation of polyamines. The results of this activity are the modification of the protein conformation and the formation of high molecular weight conjugates. The interest of these enzymes is focussed on its applications in clinical trials (there are implicated in neurodegenerative diseases, blood coagulation, etc.), food additives (texturing agents), wool textiles, biopolymers, etc. As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast and the effect of TGZ heterologous over-production, this paper presents the effect of the over-expression of tgz gene from maize, the first plant transglutaminase cloned (Torne et al. 2003, Villalobos et al. 2004), in tobacco chloroplasts via plastid transformation. With this technique, the transgene is integrated in the plastid genome via homologous recombination. In the present work, the increase in TGase activity (four times in transplastomic plant leaves) induced in the tgz-transformed tobacco plants resulted in enhanced polyaminylation of thylakoid proteins and in increased thylakoid stacking (Ioannidis et al. 2009). Moreover, the over-expressed chlTGZ protein, accumulated progressively in chloroplast inclusion bodies like in E. coli cells (Carvajal et al. 2007). This accumulation was accompanied by oxidative stress symptoms, thylakoid scattering, membrane degradation and reduction of thylakoid interconnections in the old leaves (Ortigosa et al. 2010). Consequently, the electron transport between photosystems decreased. 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 and TGase production in chloroplasts.Peer Reviewe