Mireya Santos

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.

Amyloid-like protein inclusions in tobacco transgenic plants.

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.

Methods of obtaining maize totipotent tissues. I. Seedling segments culture

Abstract From nine cultivars of Zea mays L. through in vitro culture of segments of seedlings from matured seeds, we tried to regenerate plants. The following factors were taken in consideration: (1) type of seed germination; (2) excision zone; (3) position of the inoculum in the medium. In order to obtain callus, a Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic a acid (2,4-D) was used. To induce the differentiation the 2,4-D concentration was reduced, and naphthaleneacetic acid (NAA) and 6-(γ,γ-dimethylallylamino)purine (2iP) were added. Plant regeneration took place in only one of the cultivars: hybrid H-113, and always from inoculi that contained the meristem of the seedling node. Neither the effect of the type of germination nor the position of the inoculum in the medium was conclusive for the obtention of callus and plant differentiation. A very interesting point of this work is that the totipotent maize tissue structure obtained is very different from any previously described. We have designated it ‘meristematic callus’.

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.

Methods of obtaining maize totipotent tissues. II. Atrophic tissue culture

Abstract Placing Zea mays L. immature embryos of different cultivars in modified Murashige and Skoog (MS) medium with 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and in light, anomalous germinations were produced and the so-called ‘atrophic tissues’ resulted. These cultivated in the same medium produced two types of totipotent formations: meristematic and yellowish-white callus, both proved capable of plant regeneration. The differentiation medium used contained 0.25-0 mg/l of 2,4-D plus 1 mg/l naphthaleneacetic acid (NAA) and 0.05 mg/l 6-(γ,γ-dimethylalylamino) purine (2iP). This is the method at present used in our laboratory for all in vitro maize cultures.

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.

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.

Role of plastid transglutaminase in LHCII polyamination and thylakoid electron and proton flow

Transglutaminases function as biological glues in animal cells, plant cells and microbes. In energy producing organelles such as chloroplasts the presence of transglutaminases was recently confirmed. Furthermore, a plastidial transglutaminase has been cloned from maize and the first plants overexpressing tgz are available (Nicotiana tabacum TGZ OE). Our hypothesis is that the overexpression of plastidal transglutaminase will alter photosynthesis via increased polyamination of the antenna of photosystem II. We have used standard analytical tools to separate the antenna from photosystem II in wild type and modified plants, 6 specific antibodies against LHCbs to confirm their presence and sensitive HPLC method to quantify the polyamination level of these proteins. We report that bound spermidine and spermine were significantly increased (∼80%) in overexpressors. Moreover, we used recent advances in in vivo probing to study simultaneously the proton and electron circuit of thylakoids. Under physiological conditions overexpressors show a 3-fold higher sensitivity of the antenna down regulation loop (qE) to the elicitor (luminal protons) which is estimated as the ΔpH component of thylakoidal proton motive force. In addition, photosystem (hyper-PSIIα) with an exceptionally high antenna (large absorption cross section), accumulate in transglutaminase over expressers doubling the rate constant of light energy utilization (Kα) and promoting thylakoid membrane stacking. Polyamination of antenna proteins is a previously unrecognized mechanism for the modulation of the size (antenna absorption cross section) and sensitivity of photosystem II to down regulation. Future research will reveal which peptides and which residues of the antenna are responsible for such effects.

Rice transglutaminase gene: Identification, protein expression, functionality, light dependence and specific cell location

Transglutaminases (TGases), that catalyze post-translational modification of proteins, are scarcely known in plants. As part of a project to characterize transglutaminase genes in new plant species, the identification and characterization of a TGase in rice is presented. Using differential primers, a cDNA (tgo) of 1767bp from genomic rice DNA amplification was obtained. The primers were designed from the rice DNA sequence relatively homologous to the gene encoding active maize chloroplast TGase. Amino acid sequence of the deduced rice TGase protein (TGO) indicated that it contains the enzyme catalytic triad (Cys-His-Asp), three repeats, myristoylation domains and a leucine zipper motif. The TGO recombinant protein was characterized, showing specific activity regulation, and indicating that tgo encoded for an authentic TGase. Substrate preference and Ca(2+) dependent activity were also detected. In the rice plant TGO protein was immunolocalized in the grana chloroplasts, in protein vesicles near them, and in the bulliform cells. Immunoblot analyses, tgo mRNA expression, and TGase activity indicated that TGO expression in rice was light dependent and regulated by the illumination period. This work increases significantly our plant TGase understanding. Its functional role in rice, which is a good model system for C3 plants, is discussed.

Zea mays L. transglutaminase expression in Escherichia coli

Background Transglutaminase (protein-glutamine:amine γ-glutamyltransferase, E.C. 2.3.2.13) catalyses acyl-transfer reactions between γ-carboxyamide groups of glutamine residues and the ε -amino group of lysines in proteins, leading to interor intramolecular cross-linking. Transglutaminases (TGs) have been found in mammals, plants, fish, nematodes and bacteria. Two maize cDNA clones (TGZ15 and TGZ21) that expressed active transglutaminase localized in chloroplasts were isolated [1,2].