1 Stefano Del Duca Dipartimento di Biologia Evoluzionistica ...

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Stefano Del Duca

Dipartimento di Biologia Evoluzionistica Sperimentale, Sede Botanica, Università di Bologna

Via Irnerio 42, 40126 Bologna, Italy.

Phone: +39-0512091292; Fax: +39-051242576. E-mail: [email protected]

(Area) Cell Biology

Plant Physiology Preview. Published on May 3, 2007, as DOI:10.1104/pp.106.092072

Copyright 2007 by the American Society of Plant Biologists

www.plantphysiol.orgon April 7, 2018 - Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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The acropetal wave of developmental cell death (DCD) of Nicotiana tabacum corolla is

preceded by activation of transglutaminase in different cell compartments

Massimiliano Della Mea, Francesca De Filippis, Valeria Genovesi, Donatella Serafini Fracassini

and Stefano Del Duca*

Addresses: Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via

Irnerio 42, 40126 Bologna, Italy.



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This research was supported by the FIRB Project No. RBAU01KZ49 to Serafini-Fracassini,

“Proteine modificate post-traduzionalmente da transglutaminasi durante la morte cellulare

programmata” of Ministero dell’Università e della Ricerca Scientifica e Tecnologica.

1 Actual address of Valeria Genovesi: Consorci CSIC-IRTA Laboratori de Genètica Molecular

Vegetal, Jordi Girona 18-26, 08034 Barcelona, Spain.

* To whom correspondence should be addressed: Stefano Del Duca, Dipartimento di Biologia

Evoluzionistica Sperimentale, Sede Botanica, Università di Bologna, via Irnerio 42, 40126 Bologna,

Italy. Fax: +39-051242576. E-mail: [email protected]



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(ABSTRACT)

The activity of transglutaminase (TGase), an enzyme responsible for polyamine conjugation to

proteins, was analysed in relationship to developmental cell death (DCD) during the flower life

span stages of the Nicotiana tabacum corolla. As the DCD exhibits an acropetal gradient, TGase

was studied in corolla proximal, medial and distal parts. TGase was immunorecognised by three

TGase antibodies; the main 58 kDa band decreased during corolla life, whereas a 38 kDa band

localised progressively from basal to distal parts. The former was present in the soluble,

microsomal, plastidial (together with the 38 kDa 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 (His6-Xpr-GFP).

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;

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, stabilising the photosystems, could

sustain the energy requirements for the senescence progression.



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(INTRODUCTION)

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 centred 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 (Nooden 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”, 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;

Pennel and Lamb, 1997), but there are events such as autophagocytosis (Liu et al., 2005) and

abscission of the entire organ. Programmed cell death in plants is accompanied by nuclear

condensation, membrane blebbing and, in some cases, DNA fragmentation and cysteine protease

activity (Pennel and Lamb, 1997; Serafini-Fracassini et al., 2002; Kusaka et al., 2004). At the

subcellular level, mitochondria may play a role in both animal and plants (Desagher and Martinou,

2000) but the molecular mechanisms may be different (Balk et al., 2003). Other organelles typical

of plants, such as the chloroplasts, vacuoles, and also possibly the cell walls, play a role in the

induction or execution of PCD, as reported during the leaf and petal senescence (Quirino et al.,

2000; Rubinstein, 2000).

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), or a developmental event, as required in

morphogenesis and sex determination (Jones, 2001; Greenberg, 1996). The term “developmental

cell death” (DCD) seems appropriate to distinguish the second form of CD. Developmental cell

death is a terminal stage of plant cell differentiation, in that the dead cells play specific functions

(e.g. vascular tissues, fiber cells, trichomes etc.), or by contrast cells die after having accomplished

their role. In reproductive organs, various tissues undergo DCD according to the type of flower,

may or may not abscise. Alternatively abscission is not always preceded by senescence (Hilioti et

al., 2000). In general once petals have completed their role they may enter senescence and

eventually remain in situ protecting the initial growth of the ovary. In long lived flowers pollination

acts as a signal for the petal senescence, while in the short lived flowers this is controlled

independently from the pollination by growth factors and hormones including: ethylene, cytokinins,



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abscisic acid and jasmonate (Mayak and Halevy, 1978; Orzaez et al., 1999; Rogers, 2006).

Senescence of different organs can be delayed by aliphatic polyamines (PAs), which have a well

established role in the plants cell division, senescence and growth (Altman and Bachrach, 1981;

Aziz, 2003; Bagni and Pistocchi, 1988; Hanzawa et al., 2000). The role of polyamines in apoptosis

of animal cells (Seiler and Raul, 2005) has not been completely clarified, even though their non

covalent interaction with nucleic acids or other molecules has been recognised for a long time.

Among the different conjugated forms, PAs can be conjugated to proteins by catalysis of

transglutaminases (E.C. 2.3.2.13) (TGases) a family of calcium-dependent enzymes.

Transglutaminases catalyse interactions between an acyl acceptor glutamyl residue and amine

donors, like lysyl residue or PAs, forming cross-links within the same or between different proteins.

Polyamines also act as physiological substrates of TGases: the terminal amino-group binds one or

two glutamyl residues giving rise either to mono-(γ-glutamyl)-PAs or bis-(γ-glutamyl)-PAs (Folk et

al., 1980). TGases may form bridges between specific proteins, including cytoskeleton proteins or

animal extracellular matrix, and is involved in the regulation of the cell growth and differentiation

(Folk et al., 1980; Ichinose et al., 1990).

Transglutaminases have been reported to be present in different plant organs including leaves,

tubers, shoots, roots and flowers (reviewed by Del Duca and Serafini-Fracassini, 2005). Plant

TGases have not yet been classified and only one enzyme has been sequenced in Arabidopsis

thaliana, which contains the typical catalytic domain of TGase superfamily and is located primarily

in the microsomes (Della Mea et al., 2004a). In plant cells, the roles of TGases are similar to those

in animal cells, in terms of several biochemical features, however their presence in particular plant

compartments and their substrates suggest that they may fulfil additional roles. Transglutaminases

were found in the chloroplasts of several higher plants and some algae, where they possibly

stabilize and protect antenna complexes and Rubisco, thus possibly being involved in the regulation

of the photosynthetic process (Margosiak et al., 1990; Della Mea et al., 2004b; reviewed by

Serafini-Fracassini and Del Duca, 2002). In some lower organisms, like Chlamydomonas

(Waffenschmidt et al., 1999) and some fungi, TGase activity has been found in the cell walls, where

the enzyme has a structural role or acts as the defence elicitors of higher plants (reviewed by Del

Duca and Serafini-Fracassini, 2005). Transglutaminases are also present in the cytoplasm where

they probably exert a structural role for example, during the dramatic cytoskeleton re-arrangement

which occurs during the rapid growth of pollen tube (Del Duca et al., 1997). The evidence that these

enzymes are located in different cell compartments was obtained in different tissues of different

plants (reviewed by Serafini-Fracassini and Del Duca, 2002). However, it is not known whether one



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cell contains more than one TGase, and if so, whether they could be differently compartmented and

simultaneously expressed.

Transglutaminases 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; Melino and Piacentini, 1998; Fesus,

1999; Griffin and Verderio, 2000). Although at present it is not possible to establish with certainty a

role of TGases in apoptosis (Griffin and Verderio, 2000; Verderio et al., 1998; Fesus and Szondy,

2005), experimental evidence confirms the expression or the accumulation of the enzyme

accompanying PCD (Candi et al., 2005); moreover, proteins modified by TGases are more

protected from protease digestion (Chen and Metha, 1998).

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 the present study, involves gradual stages (from 1 to 10) of

development, from flower growth to senescence and death. In the present paper the term DCD,

applied to the Nicotiana petals, is used to define the terminal process of development constituting

the senescence and a cell death 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 morpho-functional parameters were previously analyzed to characterise the

onset of corolla senescence and cell death. 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.



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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 forwards to interpret the possible role of the corolla TGases in DCD.

RESULTS

Identification of the tobacco flower corolla developmental stages

The corolla life span was divided in ten stages (Fig. 1). Stages from 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 from 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 sub-cellular compartments.

The immunodetection of putative TGase enzymes, in a total extract of protein from the corolla, was

performed using antibodies against TGase from plant (A. thaliana TGase) (Della Mea et al., 2004b)

mammal (CUB 7402) and nematodes (kind gift of K. Mehta). in order to confirm the partial

homology between tobacco and non-plant TGases, previously observed also for other plant TGases.

The three antibodies reacted predominantly with a 58 kDa polypetide, but also with a 61 kDa and in

some cases, with a 38 kDa band (Fig. 2A, B, C). A detailed analysis of TGase antibody positive



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bands in all the corolla stages, performed with the antibody against A. thaliana TGase, (Fig. 2C),

showed: 1) the constant presence of a 58 kDa band, decreasing with senescence progression (50% at

stage 7 in respect to stage 2); 2) a 61 kDa band, visible mainly at stages 3 and 4; 3) immunostained

polymers of 250 kDa or higher molecular mass (top of running gel). The latter markedly increased

starting from stage 5 and sharply decreased at stage 7/8.

To identify TGase expression and activity related to the acropetal progression of senescence, the

corolla was divided in three parts: a proximal, medial and distal, as indicated in Fig. 2D. The

presence of TGase in the 58 kDa band, in all stages of the three corolla parts, was confirmed by the

A. thaliana TGase antibody (Fig. 2E), more markedly so in the medial part. The 61 kDa band

reacted faintly. Moreover, a 38 kDa band was identified in the proximal and medial parts at stages

5 and 6, but it had disappeared at stage 7 and 8. The same band appeared in the distal part at stage 6,

and was maintained until stage 8. In Fig. 2F, the immuno tissue print from the base of the corolla at

stages from 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 immuno-negative (data not shown). The antibody

against A. thaliana TGase was used to test the presence of TGases in soluble, microsomal, plastid

and cell wall enriched fractions of the corolla (Fig. 2G). The 58 kDa 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 kDa band was present in Pl and CW fractions; in addition in Pl one a faint 38 kDa band

was also visible. Unexpectedly, in the soluble fraction a prominent 52 kDa 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 kDa, there was also a faint 58 kDa

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).

Transglutaminase activity in tobacco corolla: analysis of glutamyl-derivatives and of

exogenous and natural substrates

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] spermine) and one (producing

mono-derivatives) or two (producing bis-derivatives) glutamyl residues (Folk et al., 1980). The

HPLC analysis (Fig. 3) of the whole corolla protein extracts at stages 2, 5 and 7 showed an

increasing amount of product throughout corolla life. The increase was due mainly to bis-

spermidine (bis-SD), mono- and bis- putrescine (mono- and bis- PU), derived from the metabolism



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of added spermine, and mono-spermine (mono-SM).

Corolla extracts were incubated in the presence of His6-tagged-Green Fluorescent Protein (His6-

Xpr-GFP), a recombinant substrate of mammalian TGases (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. 4A 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 kDa) 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 radio

labelling (due to its covalent linkage with [14C] SM), which was more evident on the 36 and 58 kDa

bands (Fig. 4A). Radio labelling also revealed the [14C] SM conjugation with a non-fluorescent 78

kDa band, possibly due GPLTGase auto incorporation. By contrast, some bands were fluorescent

but not labelled, suggesting that ε(γ-glutamyl) lysine linkages were also formed, independently

from PA involvement in the linkage. The incubation of His6-Xpr-GFP and GPLTGase with a higher

SM concentration (10mM), 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, 4) 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 auto-

fluorescent 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, 7)

in the presence of 0.2 (Fig. 4C) or 10 mM SM (not shown). The formation of three His6-Xpr-GFP

fluorescent bands between 30 and 36 kDa 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 kDa.

Extracts of corolla at different stages were incubated with [14C] SM and in all stages the label was

located mainly in 42-45 and, more markedly, in 58, 62, 65 kDa 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



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several Coomassie-positive bands, (especially of 58 and 54 kDa and higher than 65 kDa), decreased

during senescence. The same bands were also more intensely labelled when erythrocyte TGase was

added to the enzyme assay, and no relevant differences in SM conjugation were visible throughout

the flower stages (not shown).

Analysis of TGase activity in the whole corolla, in proximal, medial and distal parts and in

sub-cellular compartments

As described for the immuno-determination 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, in order 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 showed in Fig. 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 immuno-detected 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 from 2 to 4, with ten-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 the stages from 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



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distal parts, reaching the maximum at stage 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 stage 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 five fold that of stage 2/3 (Fig. 6D insert).

DISCUSSION

DCD Gradient in the corolla of Nicotiana tabacum 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 abcission zone (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). Nevertheless earlier events of senescence

could occur in previous stages, as suggested by decrease in levels of chlorophyll and protein as

early as stages 4 and 5 respectively. In addition, a dramatic water loss was measured in the

proximal part between stage 5 and 6 (not shown). It should noted that the epidermal layer at stage 6

was still in good condition, and the DCD events may occur in the mesophyll, which underwent

nucleus blebbing and cell wall modification at stage 6 only in the proximal part (Serafini-Fracassini

et al. 2002). These data are in agreement with the observation performed in Alstroemeria by

Wagstaff et al. (2003).

Senescence proceeds acropetally along the Nicotiana corolla, and concludes with corolla “teeth

curling” (stage from 7 to 9). Senescence is followed by a gradual CD during 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.

Identification of corolla transglutaminases



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Data reported show that different anti-TGase antibodies recognise putative TGase bands

including a 58 kDa band which 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). As petals are transformed leaves

(Goethe, 1946) and also contain chloroplasts, this finding is not surprising. The formation of high

molecular mass immuno-detected polymers could be ascribed, at least in part, to TGase activity, by

auto-polymerisation (Lorand and Conrad, 1984) and/or by the enzyme entrapping or linking itself

to the polymers just formed, as also suggested by the decrease in amount of the 58 kDa protein.

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 ε−(γ-glutamyl)lysine isodipeptide bonds) or labelled (by Gln-[14C] PA-

linkages).

An excess of PAs causes the formation of very high molecular mass products that cannot be

separated by electrophoresis. These might derive either from inter-molecular 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 recognises some proteins of

Nicotiana, also immuno-detected by two animal antibodies, and that plant and animal TGases show

similar molecular weights 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 presence in the latter of the typical catalytic triad and its

structural homology with Factor XIII (Tasco et al., 2003) could be the reason for its immuno-

recognition by heterologous antibodies.

Transglutaminase activity during the corolla life span

The enzyme activity, determined by incubating [14C] spermine with the corolla extract without

exogenous calcium addition, exhibits a significant increase in the second half of the corolla life

span (stages 5 to 7/8) with a maximum at stage 6. This finding is confirmed by the increasing



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glutamyl-derivative formation and by the enhanced modification of the natural protein substrates,

as shown by the [14C] spermine conjugation observed in autoradiography. In fact, in addition to the

labelling of the protein bands, having similar molecular mass of around 60 kDa, observed in all

phases, both low and high mass labelled 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) which may make available more Gln residues for the

linkage. This is supported by the HPLC separation of increasing amounts of mono- and bis-PA

glutamyl derivatives. Several Coomassie-positive bands observed at stage 2 disappeared as ageing

progressed. However, it cannot be excluded that these bands are aggregated, as previously observed

under stress condition (Dondini et al. 2001; Del Duca et al., 2000). This is suggested by the labelled

products having elevated molecular mass, formed during the period of maximum of enzyme

activity and the simultaneous formation of high molecular mass bands immuno-detected by the

TGase antibody.

In a previous paper, the corolla TGase activity was studied by incubating labelled putrescine

under constant exogenous Ca2+ supply (Serafini-Fracassini et al., 2002). The reported decrease in

activity with the corolla age could be due to the decrease in content of the 58 kDa enzyme observed

in the present work. Moreover, a small peak of activity interrupting this decreasing activity profile

was previously observed at the onset of senescence concomitant with the maximum of activity

observed at present without Ca2+ addition. This maximum occurred before DNA laddering and

protease activation. The specific substrate His6-Xpr-GFP, assayed under a constant Ca2+

concentration was modified in a similar way by the corolla enzyme during the flower life span. It

can be concluded that Ca2+ could exert an important regulatory role of the enzyme activity. It is

known that in senescing tissues this cation increases in concentration (Huang et al., 1997),

especially because of its release from the vacuole, caused by tonoplast rupture observed to begin at

stage 6 in Nicotiana corolla (Serafini-Fracassini et al., 2002). It has been observed that Ca2+ at a

high concentration (10mM) could inhibit TGase activity (Serafini-Fracassini et al., 1988; Della

Mea et al., 2004a).

Transglutaminase in the corolla proximal, medial and distal parts

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 hypothesise an acropetal wave that seems to precede that of the senescence.



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The distribution of immuno-detected TGase in the three parts of the corolla clearly showed that

its main 58 kDa form was always prevalent in the medial green part. The distribution of a 38 kDa

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 kDa putative enzyme could match with a TGase isolated from

maize thylakoids, (Della Mea et al., 2004b) and with a 39 kDa enzyme detected in chloroplasts of

Medicago sativa, (Kuehn et al., 1991). The pattern of the 38 kDa form agrees with the chloroplast

degradation evidenced by chlorophyll decrease (Serafini-Fracassini et al., 2002).

Transglutaminase subcellular location

The present data, obtained by in vitro TGase assays, suggest that some of the compartmented

enzymes are presumably active forms also in vivo. Compartmentalisation may be necessary as

TGase is potentially a ‘dangerous’ enzyme if free in the cytoplasm. The 58 kDa 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 kDa 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 non-

peptidic residues from the 58 kDa enzyme. In the soluble fraction both 52 and 58 kDa forms

decreased with senescence progression while immuno-detected 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), cell membranes began to be degraded and proteases, activated.

This suggests that the soluble activity could be due to a possible release of TGase from microsomal

vesicles. In seedlings of Arabidopsis, vesicles containing precursors of cysteine proteinases were

delivered to the vacuole where these enzymes were activated and participated in the disassembly of

cell components during senescence (Hayashi et al., 2001). In addition, ricinosome-like vesicles are

presumed to deliver their proteases in the cytosol of the senescent daylily petals (Schmid et al.,

1999).

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 thaliana (Della Mea et al., 2004a). As the active 58 kDa TGase form was present

also in the cell wall fraction, it is possible that it was transported therein from the microsomal

vesicles, the activity of which reached a maximum at stage 6 in the corolla basal part. In this zone

where the AZ was developing, cells were reported in various plants to proceed through a series of



16

morphological changes, such as increase of rough endoplasmic reticulum associated with the Golgi

and plasma membrane, also accumulated invaginations; moreover irregular cellulose microfibril

rearrangement took place in the cell walls (Patterson, 2001). Among these cells the occurrence of a

diffusible signal in relationship to middle lamella degradation was described by Stenvik et al.

(2006).

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),

and some unicellular fungi (reviewed by Del Duca and Serafini-Fracassini, 2005). In the former,

the TGase was involved in the formation of the cell wall, which allowed the zygote to survive

desiccation. The TGase-directed formation of a soft protein envelope which organizes the self-

assembly of glycoproteins was followed by oxidative cross-linking which rendered the cell wall

insoluble. Even though no data has been published on the identification of TGase in the cell wall of

higher plants, indirect evidence of TGase presence was provided by the digestion of cell wall

polysaccharide compounds of Helianthus tuberosus parenchyma, which caused the disaggregation

of PA-conjugated proteins of high mass from polysaccharides (Dinnella et al., 1992). The presence

of PAs is well documented in the cell wall (Berta et al., 1997). The inhibition of their biosynthesis

induces modification of the structure, shape and size of the primary cell walls of Nicotiana thin

layers, with loosening of the fibrillar components, detachment of contiguous cells, lysis of wall

components. The strengthening of the links between cell wall components by PAs could be due to

their ionic interaction with pectic substances, as suggested by the authors, or/and to covalent

TGase-mediated interactions with proteins.

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; Zemskov et al., 2006), which can be considered to be analogous to the plant

cell wall, and in the repair of tissues after injury (Telci and Griffin, 2006).

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) and by the rigid/papyraceous-like aspect of the corolla. Moreover, TGase

might be involved, from stage 6 onwards, in the cell wall structural modifications of differentiating

cells located in the AZ. The increase in activity observed at stage 6 and 7 in the basal part of the

corolla is considerable especially if due only to the AZ. In this region the tissue continuity began to

be interrupted at stage 6 by the detachment of contiguous cells, due to enzymatic digestion of the

middle lamella as ultrastrucural data showed in Arabidopsis (Bleecker and Patterson, 1997). To



17

prevent the release of toxic substances, desiccation and pathogen attack after corolla abscission, the

tissues around the AZ must be protected by impermeabilisation of the scar. A possible relationship

between TGase presence and corolla AZ is suggested by the in vivo tissue-print, where only the

base of the proximal part of the corolla at the beginning of its abscission process was found to be

immuno-positive. To be detectable under these conditions, the enzyme should be released onto the

nitrocellulose support, as a consequence of membrane lysis.

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 kDa form, in

addition to the predominant 58 kDa form. From the literature reported above, the 38 kDa form

seems to be exclusively in the plastids, but it is reported also in bacteria (Makarova et al., 1999),

from which plastids are phylogenetically derived. Previous data also showed that both the Ca2+-

dependent 58 and 38/39 forms are active (Della Mea et al., 2004b; Dondini et al., 2003; Kuehn et al

1991). The 58 kDa TGase is widespread in chloroplasts of higher plants and algae and it has

probably a stabilising effect on the photosynthetic complexes (reviewed by Del Duca and Serafini-

Fracassini, 2005)

The increased activity of chloroplast TGase might be necessary in Nicotiana corolla senescing

tissues to supply energy to sustain the morpho-functional 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 finalised 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 analysed for TGase presence, whose activity was followed during

senescence progression in order 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 catalyses the post-translational modification of proteins i.e.



18

transamidation and cross-linking in which polyamines may be involved. The role of polyamines in

the regulation of programmed cell death is well known (Seiler and Raul, 2005), but their complex

molecular mechanism of action is still under evaluation and the present data can open new

perspectives in this direction.

MATERIALS AND METHODS

Plant system

Plants of Nicotiana tabacum L. (Solanaceae) cv 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 colour by means of binocular stereoscope ZEISS Stemi SV6

(8-50X). Corollas were collected at different developmental stages. Fresh and dry weights, water,

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.

Protein extraction

Whole and subdivided corollas were homogenised in 1: 3 (w/v) 50 mM Tris-HCl pH 7.4

containing 1 mM DTT, 10 ug/mL pepstatin, 0.5 ug/mL leupeptin, 1 mM PMSF and 0.1% Triton X-

100 and then centrifuged for 2 min at 100g; the supernatants, after centrifugation for 10 min at

9700g were used for protein determination, Western-blotting analysis and enzyme assays. All steps

were performed at 4°C. Proteins amount was determined by the method of Lowry et al. (1951)

using bovine serum albumin as standard. All chemicals were purchased from Sigma, Aldrich

(Milano, Italy). Samples were immediately stored at –80°C.

Preparation of enriched microsomal, soluble, plastidial and cell wall protein fractions



19

Whole and subdivided corollas in all developmental stages, were homogenised 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 ug/mL pepstatin, 0.5 ug/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. The pellet was recovered and resuspended in 1:5 (w/v) 10 mM

Hepes KOH pH 7.5 resuspension buffer containing 1% Triton X-100, 500 mM KCl, 1 mM DTT, 1

mM PMSF, 5 ug/mL pepstatin and 0.5 ug/mL leupeptin, then homogenised and incubated for 10

min at 4°C. The samples were sonicated for three times for 5 sec (medium frequency and

displacements about 9 µm) and incubated, after each sonication, in ice for 30 sec, then were

centrifuged at 1000g for 5 min. The pellets were dissolved in 500 µL of 10 mM Hepes KOH pH 7.5

resuspension buffer, then sonicated as previously described and centrifuged at 1000g for 5 min. The

pellets were dissolved in 100 µL of resuspension buffer and observed under a Zeiss Axioplan 400-

1000X microscope.

Enriched plastidial protein fraction. The steps were performed at 4°C and at low light conditions.

The 100g supernatants were centrifuged at 500g to eliminate nuclei and then the supernatant were

centrifuged at 1700g for 5 min. The plastidial pellets were resuspended, with a little paint-brush, in

500 uL of 20 mM Hepes KOH pH 7.7 extraction buffer. This step was repeated for three times to

eliminate starch and residual nuclei. Finally the pellets were resuspended in 200 uL of 50 mM Tris

HCl pH 7.4 resuspension buffer and observed under a Zeiss Axioplan 400-1000X microscope.

Enriched microsomal and soluble protein fraction. The supernatants obtained from 1700g

centrifugation were recovered and the separation of microsomal and soluble proteins was performed

as described by Terry and Williams (2002) with a 100000g centrifugation for microsomal fraction

and precipitation with ammonia sulphate for the soluble one.

Western-blot analyses

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, Italy). The gel was blotted to a nitrocellulose membrane

(Amersham Biosciences, Buckinghamshire, UK) 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). In case of chicken anti-AtPng1p polyclonal antibody the dilution was

1:2000, whereas the monoclonal CUB7402 and the polyclonal anti-nematode TGase (kind gift of



20

Prof. K Mehta) antibodies have been diluted 1: 600 and 1: 1000 respectively. Proteins were finally

detected using an anti-chicken immunoglobulins conjugated to alkaline phosphatase, an anti-mouse

immunoglobulins conjugated to peroxidase and an anti-rabbit immunoglobulins conjugated to

alkaline phosphatase, respectively. Proteins were revealed using BCIP/NBT or the

aminoethilcarbazole substrate tablets (Sigma-Aldrich).

The relative densities of the bands in different lanes and their molecular weights were performed

with the specific software Total Lab (Raytest, Milan) on the nitrocellulose membrane scanned with

a FLA3000 Laser System (Fuji).

Tissue printing

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, Buckinghamshire, UK) as already described by Varner and Ye (1994). Dried

nitrocellulose membrane was incubated over night with chicken anti-AtPng1p (antibody dilution

was 1:200) and then incubated with rabbit antibody raised against chicken immunoglobulins

conjugated to alkaline phosphatase for 1 h. Antibody dilution was 1:2000. Proteins were revealed

using Sigma Fast tablets (Sigma-Aldrich).

TGase assay with His6-Xpr-GFP

50 µg of proteins from whole corollas in three developmental stages 2, 5 and 7 were incubated for

TGase assay. Recombinant fluorescent mammalian TGase substrate, histidine-tagged green

fluorescent protein His6-Xpr-GFP (kind gift of Prof Hirose, Tokyo Institute of Technology,

Yokohama, Japan) (Furutani et al., 2001) (2 µg/mL) was added at the enzyme assay mixture

containing Tris buffer 50 mM pH 7.5, 1 mM DTT, 5 mM CaCl2. In some experiments also 0.2 mM

or 0.06 mM [14C ] SM (Amersham Biosciences, Buckinghamshire, UK) or 10 mM cold SM were

added in the assay mixture. To compare mammalian TGase activity with the TGase activity from

corolla extracts, also GPLTGase and erythrocyte TGase have been checked for TGase assay with

His6-Xpr-GFP. The mixture has been left up for 30 min at 37°C with gentle shaking. TGase

reaction has been stopped by adding cold SDS-PAGE loading buffer and let 30 min at room

temperature before to charge on the gel. The fluorescence of His6-Xpr-GFP was checked in an UV

trans-illuminator at an excitation λ of 365 nm.

Radiometric assay



21

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, Buckinghamshire, UK), 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 12000g for 20 min at 4°C. The TCA-pellets were dissolved in NaOH 1N for 1 h at

60°C. These steps were repeated four times. 1 mL of liquid scintillation cocktail (Beckman, Ready

Gel) was then added to 50 µL of the solution, and the incorporation of labelled SM was

subsequently counted in a liquid scintillation counter (Beckman, LS 6500).

Identification of glutamyl derivatives

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). Glutamyl-PAs and other derivatives in the TCA-insoluble

fractions were separated by ion-exchange chromatography using a Jasco HPLC-system (4.5 mm x

90 mm column, packed with Ultropac 8 resin, Na+ form) and the five-buffer system previously

reported (Folk et al., 1980). Conjugated PAs (γ-glutamyl-PAs) were released by acid hydrolysis of

the ion-exchange fractions and their identity determined by comparison of their retention times with

those of PA standards (Folk et al., 1980).

Statistics

Each determination was repeated at least three times. All values were means with standard errors.

The Student’s t was used to compare means, as reported in the legends.

Acknowledgments

We are very indebted to Prof. A. Serafini-Fracassini (Emeritus Professor of the University of St.

Andrews, Scotland) for the restyling of the English manuscript and to Prof. P.L. Bonner for help in

the revision. The authors are grateful to Prof. S. Hirose (Department of Biological Sciences, Tokyo

Institute of Technology, Yokohama) for the kind gift of His6-Xpr-GFP, to Prof. K. Mehta

(Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer

Center, Houston, TX, USA) for the kind gift of anti-nematode TGase antibody. We acknowledge

the technical assistance of Mr. N. Mele for photographic and image assistance.



22

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Related Ethylene and flower longevity in Alstroemeria: relationship between tepal senescence,

abscission and ethylene biosynthesis. J Exp Bot. 56: 1007-1016.

Zemskov EA, Janiak A, Hang J, Waghray A, Belkin AM (2006) The role of tissue

transglutaminase in cell-matrix interactions. Front Biosci. 11: 1057-1076.



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Legends

Figure 1. Tobacco flower corolla developmental stages. Stages 1-4: developing flower; the

corolla is changing from green colour to pink and teeth, previously closed, are opening outwards.

Stage 5: maximum corolla opening; the distal part of the corolla has an intense pink colour while

the proximal and medial portions are green and the teeth are patent; the basal part of corolla does

not show any sign of abscission, as shown in the detail. Stage 6: similar to the previous stage, but

the corolla starts to loss turgidity and presents the maximum pink pigmentation; the first signs of

loss of structural integrity at the abscission zone are shown in the detail where wilt cells, which

rapidly became brown after detachment, are visible. Stages from 7 to 9: progression of senescence;

the corolla further losses turgidity and colour; it exhibits an enlarging brown ring proceeding

acropetally from the abscission zone; teeth curl and become brown. Stage 10: massive cell death;

the corolla is dry, brown, papyraceous and de-pigmented.

Figure 2. Immunodetection of transglutaminase. Figs. A-C Entire corolla proteins were extracted

at representative flower stages and SDS-PAGE analysed, blotted and immuno-detected by

antibodies raised against TGases of: A) Mammal (CUB 7402), B) Nematode and C) A. thaliana

TGase (AtPng1p); below the relative intensity values of the main immuno-positive bands (> 250,

61, 58 kDa). D) Corolla was sectioned in three parts: proximal (Px), medial (Md) and distal (Ds). E)

Proteins extracted from these three parts of the most representative flower stages were analysed as

above by A. thaliana TGase antibody. F) Immuno-tissue printing of the base of the Px part of the

corolla at stages 5, 6, 7 and 8 with the A. thaliana TGase antibody. G) Proteins extracted from

enriched subfractions of corolla cells: Mi, microsomal, Pl, plastid, CW, cell wall and So, soluble

fractions were analysed as above by A. thaliana TGase antibodies.

Figure 3. Identification of transglutaminase activity products. Glutamyl-derivatives produced by

[3H] spermine (SM)

incubation with the whole corolla protein extracts at stages 2, 5, 7 and

separated by HPLC analysis after proteolytic degradation. The three elution regions correspond

respectively to: I. bis-putrescine (bis-PU), II. bis-spermidine (bis-SD) and mono-putrescine (mono-

PU), III. bis-spermine (bis-SM), IV. unbound spermine (SM).

Figure 4. Modification of substrates by transglutaminases. A), B) and C) Exogenous substrate:

the specific recombinant substrate of mammal TGases (2 µg ml-1) His6-Xpr-GFP (GFP) was

incubated with: A) Guinea pig liver TGase (GPLTGase) in the presence of 0.2 mM [14 C] spermine



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(SM) as alone or with 10 mM cold SM and the conjugates analysed by SDS-PAGE, stained by

Chloramine T (Cl+) or detected by autofluorescence (fl) or by autoradiography (Rad) and compared

to GFP alone as a control (C). B) Different TGases (erythrocyte, GPLTGase and corolla extract)

and the conjugates were analysed by SDS-PAGE, stained by Coomassie (C+) and detected by

autofluorescence (fl). C) Corolla extract TGase in the presence of 0.2 mM [14C] SM and the

conjugates analysed by SDS-PAGE, stained by Chloramine T (Cl+) and detected by

autofluorescence (fl). D) Endogenous substrates: TGase in corolla extract at different

developmental stages (2, 5, 7) was incubated in the presence of 0.06 mM [14 C] SM as tracer and the

conjugates analysed by SDS-PAGE, stained by Coomassie (C+) and detected by autoradiography

(Rad).

Figure 5. TGase activity during corolla life span. The activity was studied at different

developmental stages (1-8) by incubating corolla extracts in the presence of 0.2 mM [14C] spermine

(SM) as tracer and the labelled conjugates were either measured by TCA precipitation or, after

separation by SDS-PAGE, detected by autoradiography A) in whole corolla extracts, insert: detail

of >250 kDa immuno-recognised TGase polymers, B) in three parts of sectioned corolla: proximal

(Px), medial (Md) and distal (Ds). All the maxima are significant (1 % probability level, Student’s t

test).

Figure 6. TGase activity in sub-cellular compartments. The activity was assayed at different

developmental stages (2/3-7/8) by incubating enriched fractions of different sub-cellular

compartments of the three sectioned parts of corolla: proximal (Px), medial (Md) and distal (Ds), in

the presence of 0.2 mΜ [14C] spermine (SM) as tracer and the labelled conjugates measured by

TCA precipitation. A) Microsomal fraction, B) Soluble fraction, C) Plastid fraction, D) Cell wall

fraction. Only the significant maxima for each part of the corolla are indicated by * (1-5 %

probability level, Student’s t test).

In the inserts of A), B), C), D) the mathematical means of the respective sub-cellular activities of

the three corolla parts are reported. The linear coefficients of B) soluble fraction, C) plastid fraction,

D) cell wall fraction were R2 = 0.677, 0.796, 0.795.



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