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First published online April 13, 2007; 10.1104/pp.106.092072 Plant Physiology 144:1211-1222 (2007) © 2007 American Society of Plant Biologists
The Acropetal Wave of Developmental Cell Death of Tobacco Corolla Is Preceded by Activation of Transglutaminase in Different Cell Compartments1,[C],[W]Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, 40126 Bologna, Italy
The activity of transglutaminase (TGase), an enzyme responsible for polyamine conjugation to proteins, was analyzed in relationship to developmental cell death (DCD) during the flower life span stages of the tobacco (Nicotiana tabacum) corolla. As the DCD exhibits an acropetal gradient, TGase was studied in corolla proximal, medial, and distal parts. TGase was immunorecognized by three TGase antibodies; the main 58-kD band decreased during corolla life, whereas a 38-kD band localized progressively from basal to distal parts. The former was present in the soluble, microsomal, plastidial (together with the 38-kD band), and cell wall fractions. The endogenous TGase activity increased during DCD reaching a maximum soon after the corolla opening. The activity maximum shifted from proximal to distal part, preceding the DCD acropetal pattern. A similar activity increase was observed by the exogenous TGase substrate (histidine6-Xpr-green fluorescent protein). Subcellular activities were detected in (1) the microsomes, where TGase activity is in general higher in the proximal part, peaking at the corolla opening; (2) the soluble fraction, where it is present only in the proximal part at senescence; (3) the plastids, where it shows an increasing trend; and (4) cell walls, prevailing in the distal part and progressively increasing. These data suggest a relationship between DCD and TGase; the latter, possibly released in the cell wall through the Golgi vesicles, could cooperate to cell wall strengthening, especially at the abscission zone and possibly during corolla shape change. The plastid TGase, stabilizing the photosystems, could sustain the energy requirements for the senescence progression.
In plants, studies on programmed cell death (PCD) are much less advanced than in animals, even though current research is addressing new aspects (Danon et al., 2000  ; van Doorn and Woltering, 2005). In plant biology there is still confusion, especially centered on the application of the terms senescence and PCD (van Doorn and Woltering, 2004), which according to the different authors, may be considered separate, partially overlapping, or even identical events (Noodén et al., 1997; Thomas et al., 2003; van Doorn and Woltering, 2005; van Doorn, 2005; Rogers, 2006).
The term PCD implies the concept genetically programmed as opposed to accidental cell death (CD), but sometimes these may be difficult to distinguish. PCD should be replaced by expressions in which the causative agent is precisely described, according to the Nomenclature Committee on Cell Death (Kroemer et al., 2005
Some features of PCD in plants are different from those in animals because of presence of specific cell compartments and for the absence of external phagocytosis events (Greenberg, 1996
In plants PCD can be either a stress-related event, as it occurs for example during hypersensitive reaction following pathogen attack (Dangl et al., 1996
TGases have been reported to be present in different plant organs including leaves, tubers, shoots, roots, and flowers (for review, see Del Duca and Serafini-Fracassini, 2005
TGases play a role in the PCD of animal cells, where the presence and the activity of TGases are considered markers of apoptosis (Fesus et al., 1987
In contrast to the relevant evidence for involvement of TGases in the mammalian PCD, only limited information is available for that in plants. In Nicotiana, petal DCD can be delayed by PA supply, with spermine (SM) being particularly efficient. The presence of intracellular free and conjugated putrescine (PU) and spermidine (SD), metabolically related to SM, increased concomitantly due to an active PA metabolism. It was observed that conjugated PAs change their relative ratios during the sequential stages of DCD (Serafini-Fracassini et al., 2002
This plant model, also used in this study, involves gradual stages (from 1–10) of development, from flower growth to senescence and death. In this study the term DCD, applied to the Nicotiana petals, is used to define the terminal process of development constituting the senescence and a CD phase. Petal cells are histologically homogenous and their senescence follows an acropetal gradient, which is completed by the death of the entire corolla at stage 10. Different morphofunctional parameters were previously analyzed to characterize the onset of corolla senescence and CD. Whereas protein and chlorophyll content decreased gradually, proteases are active from stage 6 during a short period concomitantly with the first appearance of DNA laddering, nuclear blebbing, rupture of the tonoplast membrane, pigment decrease, and modification of cell walls (Serafini-Fracassini et al., 2002 It is not known if the observed changes in TGase activity are related to changes in the amount of enzyme, particularly whether this is constitutive or expressed at a particular phase of the cell life. To evaluate the factors affecting the changes in TGase activity in Nicotiana corolla DCD, we studied, from the early differentiation stages, the presence and activity of TGase. The activity was also studied either in the presence of the endogenous substrates alone or by adding a constant amount of a specific TGase exogenous substrate; the modifications of both substrates were also studied by analyzing their changes in their electrophoretic migration and the PA glutamyl derivatives produced. Due to its acropetal senescence gradient, the corolla was sectioned in three parts and TGase activity was studied in each of these during senescence progression. TGase location and activity in the four cell compartments (microsomes, cytosol, plastids, and cell walls) were evaluated during the life span of the corolla to clarify if more TGase forms could exist and be simultaneously active in different cell compartments. In the light of the roles exerted by these compartments, some functional hypotheses are put forward to interpret the possible role of the corolla TGases in DCD.
Identification of the Tobacco Flower Corolla Developmental Stages The corolla life span was divided in 10 stages (Fig. 1 ). Stages 1 to 4: developing flower; stage 5: maximum opening of the corolla whose teeth are patent and the basal portion of the corolla does not show visible modifications (Fig. 1, detail); stage 6: transition stage in which the flower appears to be in good health, but some parameters (chlorophyll and protein decrease, water loss, DNA laddering) indicate that senescence is already primed. A ring of cells with low mechanical resistance appear at the base of the corolla, corresponding to the abscission zone (AZ; Fig. 1, detail). Rheological studies showed that until stage 5 the corolla, when subjected to traction by a dynamometer, underwent rupture by applying a weight of 300.4 ± 50.6 mg/corolla. At stage 6, the corolla became detached at the AZ by the application of a weight of 52.7 ± 13.3 mg/corolla. Stage 7: a brown ring corresponding to AZ occurred. Stages 7 to 9: senescence progression, but the corolla, even though abscised, remained in situ on the flower (supported by the calyx and the style) until stage 10; stage 10: death of the entire corolla.
Immunodetection of TGase in the Whole Corolla during Its Life Span in Proximal, Medial, and Distal Parts and in Subcellular Compartments
The immunodetection of putative TGase enzymes, in a total extract of protein from the corolla, was performed using antibodies against TGase from plant (Arabidopsis TGase; Della Mea et al., 2004b
To identify TGase expression and activity related to the acropetal progression of senescence, the corolla was divided in three parts: proximal, medial, and distal, as indicated in Figure 2D. The presence of TGase in the 58-kD band, in all stages of the three corolla parts, was confirmed by the Arabidopsis TGase antibody (Fig. 2E), more markedly so in the medial part. The 61-kD band reacted faintly. Moreover, a 38-kD band was identified in the proximal and medial parts at stages 5 and 6, but it had disappeared at stages 7 and 8. The same band appeared in the distal part at stage 6, and was maintained until stage 8. In Figure 2F, the immuno tissue print from the base of the corolla at stages 5 to 8 was positive at stages 5 and 6, especially at the vascular bundle level. Prints of the bases of the medial and distal parts were immunonegative (data not shown). The antibody against Arabidopsis TGase was used to test the presence of TGases in soluble, microsomal, plastid, and cell wall enriched fractions of the corolla (Fig. 2G). The specificity of the anti-AtPng1p polyclonal antibody is shown in Supplemental Figure S1. The 58-kD band was evident in the microsomal, (Fig. 2G, lane Mi), plastid (Fig. 2G, lane Pl), and cell wall (Fig. 2G, lane CW) fractions. A faint 61-kD band was present in plastid and cell wall fractions; in addition, in plastid a faint 38-kD band was also visible. Unexpectedly, in the soluble fraction a prominent 52-kD band was present (Fig. 2G, lanes So). This fraction was further examined to enhance band separation for all stages. In the first developmental stages (Fig. 2G, St 4), in addition to the 52 kD, there was also a faint 58-kD band, which decreased concomitantly with the gradual appearance of a positive high mass zone, which reached its maximum at stage 8 (Fig. 2G, St 8).
The TGase activity in corolla was assayed by glutamyl-derivatives analysis that identifies the cross-link products between the primary amine donor ([1,4(n)-3H] SM) and one (producing mono-derivatives) or two (producing bis-derivatives) glutamyl residues (Folk et al., 1980
Corolla extracts were incubated in the presence of His6-tagged-GFP protein (His6-Xpr-GFP), a recombinant substrate of mammalian TGases (a kind gift of S. Hirose). We examined the effect of SM on the His6-Xpr-GFP conjugation mediated by two TGases of animal origin at constant 2 mM Ca2+ concentration (Fig. 4, A and B ). Guinea pig liver TGase (GPLTGase) produced an electrophoretic shift in the His6-Xpr-GFP (Fig. 4A), possibly due to intramolecular cross-link formation, as described by Furutani et al. (2001) . The binding of SM by GPLTGase caused the formation of additional fluorescent bands of higher mass (58 and 36–40 kD) compared to the control (His6-Xpr-GFP and SM without enzyme; Fig. 4A). The use of [14C] SM confirmed these data, showing the simultaneous presence of fluorescence (due to His6-Xpr-GFP) and the radiolabeling (due to its covalent linkage with [14C] SM), which was more evident on the 36- and 58-kD bands (Fig. 4A). Radiolabeling also revealed the [14C] SM conjugation with a nonfluorescent 78-kD band, possibly due to GPLTGase autoincorporation. By contrast, some bands were fluorescent but not labeled, suggesting that  ( -glutamyl) Lys linkages were also formed, independently from PA involvement in the linkage. The incubation of His6-Xpr-GFP and GPLTGase with a higher SM concentration (10 mM) resulted in the following: (1) a decrease in monomeric His6-Xpr-GFP fluorescence, (2) the absence of any additional fluorescent bands, (3) the decrease of the Coomassie-positive TGase bands, and (4) the formation of insoluble aggregates in the sample (Fig. 4A).
The reliability of this assay with plant extracts was tested to exclude the presence of corolla autofluorescent compounds on the SDS-PAGE region corresponding to GFP migration (Fig. 4B). The enzymatic assay was performed using the corolla total extract and two different TGases of animal origin. GPLTGase, erythrocyte TGase, and corolla extracts caused the formation of the same main fluorescent bands; GPLTGase also caused the appearance of two additional higher mass faint bands. The flower extract showed a fluorescence at the dye front of the gel due to flower pigments (Fig. 4B). His6-Xpr-GFP was incubated with the corolla extracts at different developmental stages (2, 5, and 7) in the presence of 0.2 (Fig. 4C) or 10 mM SM (data not shown). The formation of three His6-Xpr-GFP fluorescent bands between 30 and 36 kD in all corolla stages agrees with a modification induced by a TGase activity in all corolla stages. The 10-mM SM caused the disappearance of chloramine T-positive bands of molecular mass higher than 90 kD. Extracts of corolla at different stages were incubated with [14C] SM and in all stages the label was located mainly in 42- to 45- and, more markedly, in 58-, 62-, and 65-kD bands (Fig. 4D). The intensity of the label along the entire lane at stage 7, and at the running-stacking gel border, was more intense in comparison to those of stage 2, and even more so in respect to stage 5. Nevertheless, the intensity of several Coomassie-positive bands (especially of 58 and 54 kD and higher than 65 kD), decreased during senescence. The same bands were also more intensely labeled when erythrocyte TGase was added to the enzyme assay, and no relevant differences in SM conjugation were visible throughout the flower stages (data not shown).
As described for the immunodetermination of the enzyme, a similar study of TGase activity was performed during all developmental stages of the entire and divided parts of the corolla. The same assays were also performed on subcellular compartments. Furthermore, to evaluate the modulation of the enzyme activity due to Ca2+ variations occurring during corolla life span, experiments were carried out in the absence of Ca2+ supply in the assay buffer. As shown in Figure 5A , the TGase activity in whole corolla, low in the first stages, rapidly increased after stage 4, doubled at stage 6, and decreased at stages 7/8. The activity profile and the amount of immunodetected polymers (Fig. 5A, insert) were strictly in agreement, confirming the hypothesis of the TGase presence in polymers. The activity in the three different corolla parts throughout flower life showed that the maxima were reached at stage 4 in the proximal part, at stage 5 in the medial, and at stage 6 in the distal part. Thus, TGase activity was progressively shifted toward the apical part of corolla (Fig. 5B).
The analysis of subcellular compartments showed that TGase was active in all of them, although with different patterns, and all these activities showed a trend change at intermediate stages (Fig. 6 ). The analysis of the microsomal enriched fraction showed that the activity increased at stage 5 in all three corolla parts (Fig. 6A). At stage 6 (coincident with the onset corolla abscission) the activity doubled in the proximal region, slowing down in the medial and distal ones. To give an overview of the microsomal TGase activity in the whole corolla, a profile of this activity was constructed using the mean values of the assays of the three parts (Fig. 6A, insert). This showed very low values at stages 2 to 4, with 10-fold increase to a maximum around stages 5/6, followed by a sharp decrease. In the soluble enriched fraction, the TGase activity was close to zero in stages 2 to 5, it increased at stage 6 and onwards, but only in the proximal region, with a marked variability (Fig. 6B). The soluble activity for the whole corolla showed an enhancement only in the late stages (Fig. 5B, insert). The analysis of plastid enriched fraction of the three corolla parts showed enhanced activity at stage 5, in the medial region, and a tendency to increase with time in the proximal and distal parts, reaching the maximum at stages 7/8 (Fig. 6C). The TGase activity in the whole corolla fraction increased at stage 4 and remained constant in the following developmental stages, with a small enhancement at stages 7/8 (Fig. 6C, insert). The maximum level of activity was comparable with that of the microsomal one, but the patterns were quite different. The activity of the cell wall enriched fraction was always prevalent in the distal region, showing a tendency to increase with time, reaching maxima at stages 5 and 7/8 (Fig. 6D). The cell wall activity in whole corolla showed a progressive increase being 5-fold that of stages 2/3 (Fig. 6D, insert).
DCD Gradient in the Corolla of Tobacco Flower
The earliest visible sign of the onset of DCD in the entire corolla of Nicotiana was the appearance at the base of the corolla of an AZ formed by a ring of cells whose physical resistance sharply decreased at stage 6. At this stage the AZ formation is concomitant with the distinct changes of several parameters (membrane integrity, protease activity, and DNA laddering, as reported by Serafini-Fracassini et al., 2002 Senescence proceeds acropetally along the Nicotiana corolla, and concludes with corolla teeth curling (stages 7–9). Senescence is followed by a gradual CD during the lifetime and along the corolla, like an acropetal death wave, which ends with the death of the entire corolla at stage 10. Thus, the corolla presents a good model to determine whether a space-temporal correlation occurs between DCD progression and TGase activity.
Data reported show that different anti-TGase antibodies recognize putative TGase bands including a 58 kD band that is always present, although slightly decreasing in intensity during the corolla life span. A protein of this molecular mass has been previously detected in leaf chloroplasts of many plants, thus in plant leaves this form of TGase seems to be the most widespread form (Del Duca and Serafini-Fracassini, 2005
The catalytic conjugation of the specific TGase recombinant substrate, His6-Xpr-GFP, by corolla TGase, further supports the identification of this enzyme in the flower corolla. In fact, the recombinant TGase substrate changes its electrophoretic apparent molecular mass in a similar manner using both plant and two different animal TGases, and by the formation of new products either fluorescent (by An excess of PAs causes the formation of very high molecular mass products that cannot be separated by electrophoresis. These might derive either from intermolecular cross-linking of the His6-Xpr-GFP and GPLTGase, separately or together, detected in lower amounts of protein by fluorescence or Coomassie staining. This also confirms that PAs act as competitive substrates for TGase activity.
The fact that the antibody raised against Arabidopsis TGase recognizes some proteins of Nicotiana, also immunodetected by two animal antibodies, and that plant and animal TGases show similar Mrs and modify GFP in a similar fashion, would suggest a similarity among these enzymes. Even though there is no sequence homology between animal TGases and that of Arabidopsis (Della Mea et al., 2004a
The enzyme activity, determined by incubating [14C] SM with the corolla extract without exogenous calcium addition, exhibits a significant increase in the second half of the corolla life span (stages 5–7/8) with a maximum at stage 6. This finding is confirmed by the increasing glutamyl-derivative formation and by the enhanced modification of the natural protein substrates, as shown by the [14C] SM conjugation observed in autoradiography. In fact, in addition to the labeling of the protein bands, having similar molecular mass of around 60 kD observed in all phases, both low and high mass labeled products appear in late phases. The former could be related to degradation of corolla proteins as a result of the elevated protease activity detected at stage 6 (Serafini-Fracassini et al., 2002
In a previous study, the corolla TGase activity was studied by incubating labeled PU under constant exogenous Ca2+ supply (Serafini-Fracassini et al., 2002
The TGase activity in the three separated parts showed that its maximum shifted progressively toward the apical part of corolla with the progression of senescence. Thus, for the TGase activity it is also possible to hypothesize an acropetal wave that seems to precede that of the senescence.
The distribution of immunodetected TGase in the three parts of the corolla clearly showed that its main 58 kD form was always prevalent in the medial green part. The distribution of a 38 kD putative enzyme appeared early in the proximal/medial parts and later in the distal one (where it was exclusively present), which is in agreement with the acropetal wave of TGase activity. On the basis of its molecular mass, this 38 kD putative enzyme could match with a TGase isolated from maize (Zea mays) thylakoids (Della Mea et al., 2004b
This data, obtained by in vitro TGase assays, suggest that some of the compartmented enzymes are presumably active forms also in vivo. Compartmentalization may be necessary as TGase is potentially a dangerous enzyme if free in the cytoplasm. The 58-kD band is the TGase more predominant form detected in the plastid, microsomal, and cell wall fractions; in all of these the enzyme was active. A 52-kD band, observed only in the soluble fraction, whose TGase activity was negligible at least up to stage 5, might derive by proteolytic cleavage or removal of nonpeptidic residues from the 58-kD enzyme. In the soluble fraction both 52- and 58-kD forms decreased with senescence progression while immunodetected polymers increased. In this fraction the activity was present only in the corolla basal starting at stage 6, when, as previously reported (Serafini-Fracassini et al., 2002
The detection of a TGase in the microsomal compartment confirms the reported presence of a TGase, provided of Golgi putative signal sequence, in the microsomal enriched fraction isolated from Arabidopsis (Della Mea et al., 2004a
Until now, the presence of TGase activity had been reported only in the cell walls of lower organisms, such as the unicellular alga Chlamydomonas reinhardtii (Waffenschmidt et al., 1999
Experimental evidence showed that in animals a significant part of the TGase cellular pool was present on the cell surface where it was involved in cell interactions with the extracellular matrix (Upchurch et al., 1987
As a working hypothesis, it is proposed that the TGase found in Nicotiana corolla might have a role in strengthening, by protein cross-linking, the walls of the entire corolla, which in fact in senescence undergoes modifications evidenced by an increased autofluorescence (Serafini-Fracassini et al., 2002 TGase activity was detected in the cell wall at the distal corolla zone, where a change occurs in the corolla teeth, which curl outwards at stage 5 and then refold at the later stages to protect the developing ovary. During these morphological events, cytoskeleton and turgor changes play a major role, but these are presumably supported by cell wall local strengthening.
The enzyme enrichment in the plastid fraction allowed the localization of the 38-kD form, in addition to the predominant 58-kD form. From the literature reported above, the 38-kD form seems to be exclusively in the plastids, but it is reported also in bacteria (Makarova et al., 1999 The increased activity of chloroplast TGase might be necessary in Nicotiana corolla senescing tissues to supply energy to sustain the morphofunctional active events of DCD (DNA laddering, protease activity, abscission ring formation, wall hardening, curl of teeth, etc.) accompanying the acropetal wave of senescence. The plastid TGase activity increases in the terminal stages particularly in the distal part. This coincides with an increase of the TGase activity in the cell wall, suggesting that this zone is particularly active, possibly to accomplish the corolla closure on top of the ovary. Moreover, these data suggest that the complex cascade of events of the tobacco corolla DCD is finalized to the protection of the developing ovary against external biological and physical-chemical factors (pathogens, dryness, temperature, mechanical injury, etc.) by a suitable envelope. To our knowledge, this is the first plant cell type in which several organelles or compartments were contemporaneously analyzed for TGase presence, whose activity was followed during senescence progression to have some information on the role of TGase in senescing cells. These roles are probably different, depending on the function and modification of the compartments in which the enzyme is located.
In conclusion, plant TGase is involved in DCD, similarly to the TGase well studied only in animal apoptosis, whose activity catalyzes the posttranslational modification of proteins, i.e. transamidation and cross-linking in which PAs may be involved. The role of PAs in the regulation of PCD is well known (Seiler and Raul, 2005
Plant System Plants of tobacco (Nicotiana tabacum; Solanaceae) ‘Samsun’ were grown in the Orto Botanico of Bologna in pots in a growth chamber at the fixed temperature of 25°C light intensity of 1015 quanta cm–2 s–1 and at photoperiod of 12 h light/dark. The developmental stages of the flowers were identified by corolla size, shape, and color by means of binocular stereoscope ZEISS Stemi SV6 (8–50x). Corollas were collected at different developmental stages. Fresh and dry weights, water, and protein content were measured. The mechanical resistance of the corolla to the detachment was measured by a dynamometer. Analyses were conducted on whole and subdivided corollas in proximal, medial, and distal parts.
Whole and subdivided corollas were homogenized in 1:3 (w/v) 50 mM Tris-HCl, pH 7.4, containing 1 mM dithiothreitol (DTT), 10 µg/mL pepstatin, 0.5 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Triton X-100 and then centrifuged for 2 min at 100g; the supernatants, after centrifugation for 10 min at 9,700g were used for protein determination, western-blotting analysis, and enzyme assays. All steps were performed at 4°C. Protein amount was determined by the method of Lowry et al. (1951)
Whole and subdivided corollas in all developmental stages were homogenized at 4°C in 1:5 (w/v) 20 mM HEPES KOH pH 7.7 extraction buffer containing 4 mM MgCl2, 330 mM sorbitol, 1 mM DTT, 5 µg/mL pepstatin, 0.5 µg/mL leupeptin, and 1 mM PMSF, filtered through two layers of Miracloth, and then centrifuged at 100g for 1 min.
Enriched Cell Wall Protein Fraction
Enriched Plastidial Protein Fraction
Enriched Microsomal and Soluble Protein Fraction
A total of 100 µg of extracted proteins were boiled in SDS loading buffer, loaded onto a denaturing 10% (w/v) SDS-PAGE, and migrated in a running buffer pH 8.3 (stock solution 5x containing 1.25 M Trizma-Base, 0.96 M glycin, and 1% SDS) at 90 V for 30 min and then at 120 V using a Bio-Rad Mini-protean III apparatus (Bio-Rad). The gel was blotted to a nitrocellulose membrane (Amersham Biosciences) using a wet Trans-Blot system (Bio-Rad). Incubation of membranes with anti-TGase antibodies has been performed as previously described by Sambrook et al. (1989) The relative densities of the bands in different lanes and their Mrs were performed with the specific software Total Lab (Raytest) on the nitrocellulose membrane scanned with a FLA3000 Laser System (Fuji).
The cut surface of transversal section of corollas at the stages 5, 6, 7, and 8 subdivided in proximal, medial, and distal parts was printed on a nitrocellulose membrane (Amersham Biosciences) as already described by Varner and Ye (1994)
A total of 50 µg of proteins from whole corollas in the three developmental stages 2, 5, and 7 were incubated for TGase assay. Recombinant fluorescent mammalian TGase substrate, His-tagged green fluorescent protein His6-Xpr-GFP (a kind gift of Professor Hirose, Tokyo Institute of Technology, Yokohama, Japan; 2 µg/mL; Furutani et al., 2001
A total of 50 µg of proteins of whole or subdivided corollas in proximal, medial, and distal parts in all developmental stages and of enriched microsomal, soluble, plastidial, or cell wall protein fraction of subdivided corollas at stages 2/3, 4, 5, 6, and 7/8 was assayed using 0.25 µCi (2.2 nmol) of [14C] SM (Amersham Biosciences), 20 mM Tris HCl pH 8.5, and 10 mM DTT. After 1 h at 30°C under light conditions assays were blocked with TCA 10% containing 2 mM unlabeled SM (5% [w/v] final concentration). The samples were incubated at 4°C overnight and then centrifuged at 12,000g for 20 min at 4°C. The TCA pellets were dissolved in NaOH 1 N for 1 h at 60°C. These steps were repeated four times. One millileter of liquid scintillation cocktail (Beckman, Ready Gel) was then added to 50 µL of the solution and the incorporation of labeled SM was subsequently counted in a liquid scintillation counter (Beckman, LS 6500).
After incubation of the corolla extracts for TGase assay in the presence of [3H] SM, TCA 10% (5% [w/v] final concentration) containing 2 mM unlabeled SM was added and the pellets washed at least three times with anhydrous diethyl ether and then proteolytically digested according to the method described by Folk et al. (1980)
Each determination was repeated at least three times. All values were means with SEs. The Student's t test was used to compare means, as reported in the figure legends. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number Q9FGY9.
The following materials are available in the online version of this article.
We are very thankful to Professor A. Serafini-Fracassini (Emeritus Professor of the University of St. Andrews, Scotland) for the restyling of the English manuscript, and to Professor P.L. Bonner (School of Biomedical and Natural Sciences, Nottingham Trent University, UK) for help in the revision. The authors are grateful to Professor S. Hirose (Department of Biological Sciences, Tokyo Institute of Technology, Yokohama) for the kind gift of His6-Xpr-GFP and to Professor K. Mehta (Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, Texas) for the kind gift of antinematode TGase antibody. We acknowledge the technical assistance of Mr. N. Mele (Dipartimento di Biologia e.s., Università di Bologna, Italy) for photographic and image assistance. Received November 14, 2006; accepted April 3, 2007; published April 13, 2007.
1 This work was supported by the Fondo per gli Investimenti della Ricerca di Base (project no. RBAU01KZ49 to D.S.F., "Proteine modificate post-traduzionalmente da transglutaminasi durante la morte cellulare programmata" of Ministero dell'Università e della Ricerca Scientifica e Tecnologica). 
2 Present address: Consorci CSIC-IRTA Laboratori de Genètica Molecular Vegetal, Jordi Girona 18–26, 08034 Barcelona, Spain. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Stefano Del Duca (stefano.delduca{at}unibo.it).
[C] Some figures in this article are displayed in color online but in black and white in print.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.092072 * Corresponding author; e-mail stefano.delduca{at}unibo.it; fax 39–051242576.
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Plant Physiol 125: 94–97 Kroemer G, El Deiry WS, Golstein P, Peter ME, Vaux D, Vandenabeele O, Zhivotovsky B, | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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of 365 nm.