José M. Torné

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.

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

SummaryTrehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ∘C and 35∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.

Immunogold localization of a transglutaminase related to grana development in different maize cell types

SummaryA comparative study of the subcellular localization of a plant transglutaminase (TGase; EC 2.3.2.13) in various in vivo and in vitro maize cell types was carried out with a polyclonal antibody raised against a 58 kDa TGase purified fromHelianthus tuberosus leaves. Immunocytochemical staining, followed by electron microscopy, showed that this enzyme was markedly present in the grana-appressed thylakoids of mature chloroplasts of the lightexposed cells. Moreover, during embryogénie callus chloroplast differentiation, the abundance of TGase in the grana-appressed thylakoids depended on the degree of grana development and was greater than in mature leaf chloroplasts. In addition to the 58 kDa form, two other forms of the protein (of 77 and 34 kDa) were obtained by Western blot. The 77 kDa form might correspond to the inactive form and was immunodetected in dense vesicles observed in dark-grown embryogenie callus cells. In adult leaves, the enzyme was also markedly present in the grana-appressed thylakoids of the mesophyll cell chloroplasts, though very scarce and dispersed in the bundle-sheath cell chloroplasts (which do not contain grana). The concordance of these localizations with those described for the light-harvesting antenna proteins of the photosystem II suggests that it is possible that this TGase has a functional role in photosynthesis, perhaps modulating the photosynthetic efficiency and the absorption of excess light by means of polyamine conjugation to the antenna proteins.

Polyamine and free amino acid variations in NaCL-treated embryogenic maize callus from sensitive and resistant cultivars

Summary The effect of different NaCl concentrations on maize embryogenic calluses derived from immature embryo cultures of a) the salt-sensitive inbred line W64Ao2, b) the resistant hybrid Arizona 8601 and c) the 0.4% NaCl pre-adapted W64Ao2 (WpA) calluses was studied. The effect of salt stress on growth as well as on polyamine (putrescine, spermidine and spermine) and amino acid contents of the treated calluses was determined. Enzymatic activities of the polyamine biosynthetic enzymes arginine and ornithine decarboxylase were also analyzed. A significant decrease in the growth of calluses in relation to increased salt concentrations and to the tolerance of the callus was observed. Embryogenic Arizona calluses showed the lowest growth inhibition and W64Ao2 calluses the highest inhibition after 60 days of culture in saline medium. WpA calluses showed an intermediate response. At high-salt concentrations (1.2–2.0%), calluses showed a significant increase in total polyamine content, especially caused by a rise in putrescine. This increase was proportionally higher for Arizona and WpA than for W64 calluses. Whereas the spermidine content of Arizona calluses was augmented with salt, a spermidine decrease was observed for W64 from 0.8 % NaCl. Arginine decarboxylase activity increased from 1.2 to 2.0 % NaCl for Arizona calluses, while in W64 calluses this increase was lower than in Arizona up to 2.0 %. With respect to free amino acid contents, the levels of free proline, which represent 50 % of the total free amino acid content, decreased with increasing salt in the medium. The highest amino acid increases were observed for arginine, alanine, glutamine, glutamic acid and y-aminobutyric acid. Arginine decarboxylase activity showed significant increments in relation to salt stress, which may be related to putrescine and some amino acid variations. Relationships between all the analyzed parameters and the polyamine synthesis and degradation processes under stress conditions are discussed.

Activity of maize transglutaminase overexpressed in escherichia coli inclusion bodies: An alternative to protein refolding

Transglutaminases (TGases) catalyze protein post‐translational modification by ε‐(γ‐glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS‐PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI‐TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large‐scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant‐substrate preference. Solubilization of the IB fraction with Triton X‐100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.

Trehalose and its applications in plant biotechnology

Trehalose, a nonreducing disaccharide of glucose, is one of the most effective osmoprotectants. Several strategies leading to its accumulation have been envisaged in both model and crop plants using genes of bacterial, yeast and, more recently, plant origin. Significant levels of trehalose accumulation have been shown to cause abiotic stress tolerance in transgenic plants. In this review, we describe the most biologically relevant features of trehalose: chemical and biological properties; occurrence and metabolism in organisms with special reference to plants; protective role in stabilizing molecules; physiological role in plants with special reference to carbohydrate metabolism. The emphasis of this review, however, will be on manipulation of trehalose metabolism to improve abiotic stress tolerance in plants.

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo-adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C-terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co-localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light-induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two-dimensional gel electrophoresis discriminated, for the first time, the 58-kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC-MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.

Effect of Exogenous Arginine, Ornithine, Methionine and γ-Amino Butyric Acid on Maize (Zea Mays L.) Embryogenesis, and Polyamine Content

Summary The influence of four exogenous amino acids related to polyamine metabolism (γ-aminobutyric acid, arginine, methionine and ornithine) on maize ( Zea mays L.) somatic embryogenesis was investigated. The endogenous polyamine contents of the treated calli were analyzed, and arginine and ornithine decarboxylase activities were determined. An established embryogenic callus (Type 1) of the inbred W64Ao2 was used. The endogenous polyamine content of calli was increased by addition of all four amino acids tested and the levels of spermidine plus spermine were higher than those of putrescine in all cases. Upon the addition of 2 mM arginine to the culture medium a 25% increase in embryogenesis was observed. Moreover, arginine decarboxylase (ADC) activity was significantly improved and ornithine decarboxylase (ODC) activity was also raised. The addition of 1.5 mM ornithine also increased embryogenic callus production, and ODC and ADC activities. Nevertheless, this increase was not as marked as in the case of arginine. This study indicates that the addition of amino acids, which are precursors of polyamine synthesis (especially Arg but also Orn), may be used to improve the rate of embryogenic callus production in an auxin-established maize culture system. A possible explanation of this effect is also dicussed.

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana

Summary.Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana.

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.