S. Del Duca

Identification of chlorophyll-a/b proteins as substrates of transglutaminase activity in isolated chloroplasts of Helianthus tuberosus L.

Endogenous substrates of transglutaminase (TGase; EC 2.3.2.13) have been identified in choloroplasts of Helianthus tuberosus leaves. The activity of TGase is Ca2+- and light-stimulated and catalyzes the incorporation of polyamines into thylakoid and stromal proteins. These proteins were separated by two-dimensional gel electrophoresis (first dimension: Deriphat-PAGE; second dimension: SDS-urea-PAGE) and Western-blotted. The thylakoid proteins were recognized by polyclonal antibodies as apoproteins of the chlorophyll-a/b antenna complex (LHCII, CP24, CP26 and CP29); a stromal protein was recognized by antibodies as the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. A possible localization of the acyl donor site for CP26 is proposed. A comparative analysis of polyamine incorporation into trichloroacetic-acid-precipitable material indicated that spermidine was a preferential acyl-acceptor substrate with respect to putrescine, even though the above-reported substrates are the same. The nature of the substrates, together with the light stimulation, support the working hypothesis of a possible role of TGase in regulating the light-harvesting function.

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco ( Nicotiana tabacum ) flowers

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.

Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen

Abstract We investigated polyamine linkage to different structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages, indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.13). This assumption is supported by: (1) formation of a labelled TCA pellet and selective labelling of endogenous proteins by covalent binding of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ungerminated and germinated pollen. TGase activity was high at 90 min after germination and was influenced by Ca2+ supply only in the rehydrated ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular mass of 43 kDa and 52–58 kDa in both ungerminated and germinated pollen. These bands matched immunolabelled spots identified by mouse monoclonal anti-actin and anti-α-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated and germinated pollen enhanced the activity. Autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rapid cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes.

Programmed cell death: similarities and differences in animals and plants. A flower paradigm

Summary.After an overview of the criteria for the definition of cell death in the animal cell and of its different types of death, a comparative analysis of PCD in the plant cell is reported. The cytological characteristics of the plant cell undergoing PCD are described.The role of plant hormones and growth factors in the regulation of this event is discussed with particular emphasis on PCD activation or prevention by polyamine treatment (doses, timing and developmental stage of the organism) in a Developmental cell death plant model: the Nicotiana tabacum (tobacco) flower corolla. Some of the effects of polyamines might be mediated by transglutaminase catalysis. The activity of this enzyme was examined in different parts of the corolla during its life span showing an acropetal trend parallel to the cell death wave. The location of transglutaminase in some sub-cellular compartments suggests that it exerts different functions in the corolla DCD.

Comparative studies of transglutaminase activity and substrates in different organs of Helianthus tuberosus

Summary Transglutaminase activity (TGase, E.C. 2.3.2.13), which binds polyamines to proteins, was previously found in etiolated apices and activated slices of medullary parenchyma of Helianthus tuberosus L. cv. OB1 tubers; in the present paper the activity was tested in mature leaves of the same plant and additional data were acquired on the above cited tissues. Flower buds were also examined and preliminary data is presented. The following parameters were analyzed: localization in the 22,000 × g supernatant or pellet fractions, dependence on pH, light, incubation time and different concentrations of Ca 2+ , EGTA, putrescine, endogenous substrates and casein and presence of a non-enzymatic activity. More than one enzyme or different forms of the same enzyme seem to be present in the same organ and its activity can be induced. An exogenous supply of Ca 2+ is not absolutely required in any of the organs examined; however, its chelation reduces the activity. Multiple endogenous substrates seem to be present in the same organ, whereas casein is not recognized as a substrate. Results concerning the other parameters examined suggest the existence of organ-specific TGase activities. Their possible function in green and non-green tissues is discussed.

Tuber vegetative stages and cell cycle in Helianthus tuberosus : Protein pattern and their modification by spermidine

Summary This paper reports the protein amount and composition of tuber during its phases of formation, dormancy and sprouting. Parallely, the break of dormancy has been induced by excising slices of tuber parenchyma and treating them with 2,4-dichlorophenoxy acetic acid (2,4-D) to induce a new cell cycle; the protein composition, neosynthesis and post-translational modification by spermidine are reported during the phases of the cell cycle. From tuber formation to sprouting, protein content follows a bimodal trend. The protein bands were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two main bands (38 and 55 kDa) were detected. Many bands, especially of high molecular mass, changed their amount during all phases of tuber vegetative period. With the progression of the cell cycle very high molecular mass proteins increased; neosynthesis and modification by polyamines could account for this increase. Parallelly, the amount of lower molecular mass bands decreased. Many proteins are differently synthesized in the various cell cycle phases. Modification by spermidine occurred post-translationally, evident by an 18 kDa band (which in fact does not incorporate methionine), whose possible identification is discussed. A role for newly synthesized transglutaminases in the protein modification by polyamines is evaluated in the light of the immunodetection of 58 and 90 kDa bands by polyclonal anti-transglutaminase antibody.

Polyamines are common players in different facets of plant programmed cell death

Abstract Programmed cell death (PCD) is a process that occurs throughout the life span of every plant life, from initial germination of the seed to the senescence of the plant. It is a normal physiological milestone during the plant’s developmental process, but it can also be induced by external factors, including a variety of environmental stresses and as a response to pathogen infections. Changes in the morphology of the nucleus is one of the most noticeable during PCD but all the components of the plant cell (cytoplasm, cytoskeleton and organelles) are involved in this fascinating process. To date, relatively little is known about PCD in plants, but several factors, among which polyamines (PAs) and plant growth regulators, have been shown to play an important role in the initiation and regulation of the process. The role of PAs in plant PCD appears to be multifaceted acting in some instances as pro-survival molecules, whereas in others seem to be implicated in accelerating PCD. The molecular mechanism is still under study. Here we present some PCD plant models, focusing on the role of the enzyme responsible for PA conjugation to proteins: transglutaminase (TGase), an enzyme linked with the process of PCD also in some animal models. The role of PAs and plant TGase in the senescence and PCD in flowers, leaf and the self-incompatibility of pollen will be discussed and examined in depth.

Compatible and self-incompatible pollination in Pyrus communis displays different polyamine levels and transglutaminase activity

The polyamine (PA) content and the transglutaminase (TGase) activity have been investigated in Pyrus communis pollination with compatible and self-incompatible (SI) pollen in order to deepen their possible involvement in the progamic phase of plant reproduction. The PA distribution as free, perchloric acid (PCA)-soluble and PCA-insoluble fractions in ungerminated (UGP), germinating pollen (GP), styles and pollinated styles with compatible and SI pollens is discussed in the light of a possible role during pollination. Generally, the conjugated PAs both in PCA-soluble and PCA-insoluble fractions were higher than the free form. Within the conjugated PAs, the PCA-insoluble ones were the highest with the exception of the not pollinated styles. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme, never checked before in styles and pollinated styles, was examined. In the SI styles, the TGase activity is higher in comparison to style-pollinated with compatible pollen, and high molecular mass cross-linked products were formed, suggesting an involvement of TGase in SI response. This is the first evidence on the presence of this enzyme activity in not pollinated and pollinated styles.

Transgenic Rice as a Vehicle for the Production of the Industrial Enzyme Transglutaminase

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

Producing transglutaminases by molecular farming in plants: Minireview article

Summary.Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.