Transglutaminase activity during greening and growth ofHelianthus tuberosus explants in vitro

Protoplasma (1993) 174:1-9

�9 Springer-Verlag 1993 Printed in Austria

Transglutaminase activity during greening and growth of Helianthus tuberosus explants in vitro

S. Del Duca 1, Maria Augusta Favali 2, Donatella Serafini-Fracassini 1'*, and R. Pedrazzini 2

~ Dipartimento di Biologia Evoluzionistica Sperimentale, Universit~i di Bologna, Bologna and 2 Dipartimento di Biologia Evolutiva ed Orto Botanico, UniversitA di Parma, Parma

Received November 26, 1992 Accepted February 8, 1993

Summary. Explants of dormant tubers of Helianthus tuberosus were grown in vitro, with or without 10gM 2,4-D, for 3 weeks. The 2,4- D-treated explants grew by cell enlargement and division and formed a non-photosynthetic friable callus composed of thin-walled cells. However, untreated explants, whose cells did not divide, differentiated chloroplasts and contained intercellular spaces filled with opaque material; chloroplasts were derived from non-photosynthetic plastids with tubular complexes and secondary starch grains: both disappeared when the thylakoids began to organize and form small grana. Nuclei also changed their morphology and became invagin- ated. Treated and untreated explants showed differences in their protein electrophoretic patterns and transglutaminase activity. This enzyme activity, low in dormant tubers, increased in both explants; considerably in untreated greening explants but much less in 2,4-D- treated growing ones. SDS-PAGE analysis of labelled conjugates, formed by in vitro incubation with labelled putrescine, indicated that, in addition to some apparently common substrates with M r more than 36 kDa, proteins of lower mass were also labelled in the untreated greening explants. These data are discussed in the light of the possible role of transglutaminase in plants.

Keywords: Transglutaminase; Polyamines; Plastid differentiation; Helianthus tuberosus tuber; 2,4-Dichlorophenoxyacetic acid.

Abbreviations: DAPI 4',6-diamidino-2-phenylindole; 2,4-D 2,4-dichlorophenoxyacetic acid; FM fluorescence microscopy; LM light microscopy; PA polyamines; PAGE polyacrylamide get electro- phoresis; SDS sodium dodecytsulphate; TCA trichloroacetic acid; TEM transmission electron microscopy; TGase transglutaminase; Tris-buffer tris(hydroxymethyl)aminomethane hydrochloride and tris(hydroxymethyl)aminomethane.

Introduction

Explants of medullary parenchyma of dormant tubers of Helianthus tuberosus, which contain very low

* Correspondence and reprints: Dipartimento di Biologia E.S., Universitfi di Bologna, Via Irnerio 42, 1-40126 Bologna, Italy.

amounts of growth substances and polyamines, re- spond to the presence of IAA or PA in in vitro culture by growing and differentiating collateral vascular bun- dles and a periderml anatomically similar to the stem of the mother plant (Bertossi et al. 1965, Serafini-Fra- cassi~xi and Alessandri 1983). The morphological and physiological characteristics of the explant during the first one or two cell cycles, have been described previously (Favali et al. 1984, Serafini-Fracassini 1991, Del Duca and Serafini-Fracassini 1993), In particular, it was observed that 2,4-D activation of dormant tissue grown in the dark induces ultrastructural modifications, such as changes in nuclear size and duplication of plastids which, in the dormant tubers, are charac- terized by a tubular complex (Favali etal. 1984). Due to the involvement of polyamines in the cell cycle free and bound polyamine metabolism was also studied during this early phase of explant growth (Serafini- Fracassini 1991). Results showed that the activity of transglutaminase (TGase), which catalyzes the conjugation of PA to proteins, increases during the cell cycle; the enzyme substrates were proteins of a very high molecular mass (Serafini-Fracassini et al. 1989, Grandi et al. 1992, Dinnella et al. 1992). A comparative study of the TGase activity in different organs of H. tuberosus suggested that multiple enzymes are present, but that organ-specific isoenzymes might also exist (Serafini-Fracassini etal. 1988, Del Duca etal. 1991, Falcone etal. 1993). In leaves the enzymes appeared to be particulate (Signorini et al. 1991, Fal- cone et at. 1993) and possibly to have photosyntheti- cally-related functions, as indicated by light stimulation

2 S. Del Duca et al.: Transglutaminase activity in Hetianthus tuberosus exptants

of their activity in chloroplasts (Cohen et al, 1982, Mar-

gosiak et al. 1990).

Consider ing the scanty l i terature data on the rote of

TGase in plants, we felt it could be interest ing to es-

tablish whether TGase activity can be related to a par-

ticular stage of growth or differentiat ion of the same

homogeneous tissue, avoiding the heterogeneity ac-

quired by mature p lan t organs. This could provide

more precise in fo rmat ion for further studies on TGase

funct ions in the ceil. At present we have established

that TGase activity can be induced dur ing greening of

non-photosynthe t ic medul lary pa renchyma of H, tub-

erosus, when cultured in the light for longer periods

than those previously studied by us. This tissue is no t

pre-determined to become photosynthet ical ly compe-

tent, bu t under appropria te light and ho rmone con-

dit ions it can differentiate chloroplasts (Gerola and

Dassfi 1960). TGase was also evaluated in explants

cultured under the same experimental condit ions, bu t

with 2,4-D, which induces rapid growth in cul tured

tissues and is used as an agricultural herbicide. All

explants, cul tured in the presence or absence of 2,4-D

under the same light condit ions, were analyzed

throughout the culture period. Growth and cell differ-

ent ia t ion were assessed by analyzing weight increase,

protein and chlorophyll content , the ul t ras t ructure of

plastids and nuclei, as well as the D A P I affinity of the

latter in greening and non-greening tissues.

Materials and methods

Plant material

Tubers of Helianthus tuberosus L. cw OB 1, grown in the Botanical Garden of Bologna University, were collected in November and stored in moist sand at 4 ~ Dormancy was checked by culturing tuber slices in growth medium lacking growth substances. Cylindrical explants of homogeneous medullary parenchyma (3 mm in diameter, 3 mm thick, about 50rag) were either frozen immediately after ex-

cision (non activated) and stored at - 80 ~ until use, or cultured in agarized sterile medium (Bonnet and Addicott I937), with or without 10 gM 2,4-D, under a 16 h photoperiod (light intensity 5.5 W/ m ~) at 26 ~ for 3 weeks.

Light microscopy and fluorescence

Free-hand sections of dormant and activated tuber slices were stained with 1.14ram DAPI for 20rain in the dark (Levi etal. 1986) and observed using an Axioscope LM fluorescence microscope with UV- H 365 filters. Micrographs were obtained maintaining the same pro- cedures in order to compare their staining.

Transmission electron microscopy

All the samples were fixed for 2h in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 6.9), and then postfixed for 2 h in 1% osmium tetroxide in the same buftbr. The samples were dehydrated in ethanol and embedded in Araldite. Thin and ultrathin sections were cut using an LKB III ultra microtome: the thin sections were observed under a light microscope; the ultrathin sections were stained with uranyI acetate and lead citrate and examined with a Hitachi 300 EM at 80 kV.

Transglutaminase assay

Dormant tuber explants and exptants cultured for 1 week were rap- idIy extracted in an ice-cold mortar with I/1 (w/v) 50 mM Tris-HCl pH 8, also containing freshly prepared 2 mM DTT. Following fiI- tration through four layers of cheesecloth, the protein-containing fractions of the 1,500g supernatant eluted through a Sephadex G- 25M column (PD-10, Pharmacia) were used for the enzyme assay. The enzyme assay was performed with some modifications according to the method of Haddox and Russell (198I) and optimized, The assay mixture containing 400 gl of plant extract (at least 0.4 nag of protein), 200 gM cold putrescine and 330 kBq [1.4(n)3HJputreseine (I. 11 TBq/mmol, Amersham) was taken to a volume of 600 gl with 100mM Tris-HC1. The final pH of the mixture, measured at 30 ~ was 7.8. After stopping the reaction with 10% TCA (final concentration) also containing 5 mM cold putrescine, the mixture was stored at 4~ for 24h and then centrifuged at 14,250g for 10rain. This precipitation was repeated twice. The pellet of the third centrifu- gation was solubilized in BTS450 Tissue Solubilizer (Beckman), dissolved in Ready Gel (Beckman) scintillation liquid and radioactivity counted in a Beckman LS 1800 counter.

Figs. 1 and 2. Thin sections of tuber explants of HeIianthus luberosus cultured for 3 weeks in alternating light/dark periods ( x 75)

Fig. L Explant cultured in the presence of 10 gM 2,4-D. New cell walls inside the mother cell are indicated (asterisks). Two nuclei are also evident: one is detached from the cell wall (arrow)

Fig. 2. Explant cultured on basal medium without 2,4-D. Arrows indicate chloroplasts in the superficial layer of the explant

Figs. 3--7. Free-hand sections stained with DAPI

Fig. 3. Superficial layers of explant cultured in the presence of 10 gM 2,4-D (x 75). Many highly DAPI stained nuclei are visible in the celt layer

Fig. 4. Internal portion of explant cultured in the presence of 10 ~M 2,4-D (x 75). Nuclei (arrows) are stained less than in superficial layers

Fig. g. Two nuclei per cell in an explant cultured in the presence of l0 gM 2,4-D (x 300)

Fig. 6. Nucleus with several nucleoli in the dormant parenchyma of the tuber ( x 300)

Fig. 7. Superficial layers of explant cuttmed on basal medium without 2,4-D ( • 75). Nuclei are uniformly DAPI stained

S. Del Duca et al.: Transglutaminase activity in Hetianthus tuberosus explants 3

4 S. Del Duca et al.: Transglutaminase activity in Helianthus tuberosus explants

Chlorophyll determination

Chlorophyll from cultures treated with 2,4-D for I week (and their controls) was determined spectrophotometrically according to the method of Porra et al. (1989).

Protein determination

Protein content was measured both in dormant and l-week cultured explants using the method of Lowry etal. (1951).

SDS-PAGE Proteins, extracted from the tuber explants, were freeze-dried and dissolved in sample buffer containing 2.3% SDS and 5% 2-mercap- toethanol and separated by 12% acrylamide slab gel etectrophoresis (Laemmli 1970). The gels were stained with Coomassie-blue R or with silver stain. The wet stained gels were cut and the radioactivity counted in the bands, after solubilization with 0.4 m130% HaQ and 0.2ml 60% PCA at 50~ for 24h. RuBisCo was purified from H. tuberosus leaves by Dr. C. Bergamini, according to the method of McCurry etal. (1982). All the quantitative determinations were repeated at least three times.

In untreated explants the outermost cells had suberified cell walls while the inner ones contained several chloroplasts. The cells had thick walls with intercellular spaces full o f optically dense material. No cell divisions were observed (Fig. 2). Micrographs of samples stained with DAPI showed that, in 2,4-D-treated explants (Fig. 3), a layer of cells immediately below the superficial ones underwent repeated divisions causing enlargement of the explants. In this region, many highly DAPI-stainable nuclei were visible; sometimes two nuclei per cell were observed (Fig. 5). In non-dividing cells of the more internal layers nuclei were less well-stained (Fig. 4) and usually had several nucleoli (Fig. 6). The untreated tissue appeared to be homogeneous and all the nuclei were DAPI-stained with similar fluores-

cent intensity (Fig. 7).

Results

Growth and greening o f the explants

The untreated explants became green after only one week in culture in the presence of light; chlorophyll content increased considerably, but growth was very slow. However, explants treated with 2,4-D increased considerably in fresh weight and in protein content

(Table 1), but did not turn green; the chlorophyll measurement was at the limit of detectability of the method

(even after three weeks in culture).

L M and F M microscopy observations

The thin sections prepared for T E M showed that after three weeks in culture with 2,4-D the compact medullar parenchyma of the tuber (Fig. 1), formed a friable callus: large cells, with conspicuous intercellular spaces, divided repeatedly with septa, beconfing smaller and smaller. The nuclei were still visible; some of them were

detached from the cell wall; the plastids were not easily

detected.

T E M observation

In tuber explants cultured for one and three weeks on 2,4-D-containing medium, plastids contained second- al 3' starch grains and very" electron-opaque tubular complexes (Fig. 8), like those in the cells of dormant tubers (Fig. 9). The nuclei, mainly those observed after three weeks, were irregular in shape, with deep invaginations and two or more nucleoli (Fig. 8). In one-week-old untreated explants plastids with a few thylakoids arranged in small grana were present (Figs. 10-12); the tubular complex was no longer visible. The nuclei showed some invagination of the nuclear envelope (Fig. 10). After l:hree weeks in culture the plastids contained numerous thylakoids and grana

(Fig. 13).

Transgtutaminase activity

The TGase activity (on a per mg prot basis) increased ten- and sixty-fold, respectively in 2,4-D-treated and -untreated activated explants, compared to the dor-

Table I. Fresh weight (FW), protein and chlorophyll content in dormant tubers and in explants cultured in the presence or absence of 10 gM 2,4-D for 1 week in alternating light/dark periods

Fresh weight Protein Chlorophyll

mg/expl. % mg/g FW mg/expl. ~tg/g FW pg/expl.

Dormant tuber 52 4- 4 100 3.01 0.15 n.d. n.d. Explants

+ 2,4-D 117 4- 15 225 4.17 0.48 1.84 0.21 - 2,4-D 66 + 3 126 2.64 0.17 5.93 0.39

Figs. 8 and 9. Electron micrographs of explants cultured in the presence of 2,4-D in alternating light/dark periods or of explants from dormant tubers

Fig. 8. Exptant cultured for 3 weeks in the presence of 10 gM 2,4-D. Plastids with a very electron opaque tubular complex and secondary starch grains, are more numerous (arrows). The nucleus (N) is irregular in shape with deep invaginations in both membranes of the nuclear envelope. Two nudeoli are present (• 17,000)

Fig. 9. Plastid with iarge tubular complex in dormant explants (x 45,000)

6 S. Del Duca etal,: Transglutaminase activity in Helianthus tuberosus explants

S. Del Duca et al.: Transglutaminase activity in Helianthus tuberosus explants 7

Table2. Transglutaminase activity, expressed as putrescine incor- poration in cell-free extracts of dormant tubers or in explants cultured in the presence or absence of 10 [~M 2,4-D for 1 week in alternating light/dark periods

Tissue Putrescine

nmol/expl./h nmol/mg protein/h

Dormant tuber 0.008 0.057 Dormant tuber t o 0.006 0.042 + 2,4-D 0.270 0.574 + 2,4-D to 0.021 0.039 - 2,4-D 0.510 3.013 - 2,4-D to 0.010 0.056

to Incorporations when the reaction is immediately blocked with 10% TCA

,

I I I I I I mol mass 66 45 36 29 24 20 KDa

Fig. 14. Densitometric profile of the silver-stained protein patterns in the 2,4-D-treated ( ) and untreated (- - -) explants after 1 week in culture

mant tissue (Table 2). The activity, expressed per explant, was considerably higher in greening untreated tissues than in the treated ones. In the latter, the number of cells per explant was much higher and therefore the activity per cell was much lower.

Proteins and TGase substrates

The Coomassie-positive or silver-stained protein patterns obtained from 2,4-D-treated and untreated explants, differed considerably in the various regions: in the untreated tissue, bands between 20 and 30 kDa were poorly stained, whereas the high molecular mass regions were more evident (Fig. 14). The amount of radioactivity due to putrescine conjugated to proteins along the gel was considerably higher in untreated tissue. This reflects the presence of higher amounts of total TCA-precipitable radioactivity in the latter (Table 2). The distribution of radioactivity along the gel indicates that the less stainable regions of low molecular mass were also labelled in the green tissue,

[3H] Pu dpm �9 mg prot -~

WELLS

R - S A B C D E

F G H I L

M N

O

+ 2,4 D - 2,4 D kDa

1120 ,1480

1640 4120

680

360 400 460 1000 160 400

336 292

480

116

,66

4~

36

20

L RuBisCo

120 120

Fig. 15. SDS-PAGE of the transglutaminase assay products obtained as indicated in Table 2. The radioactivity, due to [3H]putrescine covalently bound to proteins, determined after gel cutting and dis- solution, is indicated in the respective zones

Figs. 111-13. Electron micrographs of explants cultured on basal medium without 2,4-D in alternating light/dark periods

Fig. 10. Low magnification of ultrathin section of explants cultured for 1 week: the plastids have lost their tubular complex (arrow) and the nucleus shows some invaginations of the envelope (x 36,000)

Figs. 11 and 12. In the plastids of explants cultured for 1 week the tubular complex is no longer visible; very few thylakoids are present (Fig. 11, x 20,500; Fig. 12, x 25,000)

Fig. 13. Plastids of explants cultured for 3 weeks: the thylakoids are more numerous and some grana can be seen ( x 36,000)

8 s. DeI Duca et al.: Transglutaminase activity in Helianthus tuberosus explants

in addition to those of Mr above 36 kDa. The latter were labelled in 2,4-D-treated explants. Similar low amounts of radioactivity were present in the band cor- responding to the large RuBisCo subunit (Fig. 15).

Discussion

The presence or absence of 2,4-D in the culture medium induces homogeneous parenchymatous explants to ex- perience different fates. In untreated explants, the most important event during culture was that plastids began to differentiate into chloroplasts after only one week in the light. The formation of thylakoids from the tubular complex seems to be similar to that observed in differentiating plastids of several species (Gerola and Dassfi 1960, Casadoro and Rascio 1977, Rascio etal. 1979, Favali et al. 1984) and of a maize mutant (Rascio and Orsenigo 1976), Growth phenomena were negli- gible. 2,4-D greatly stimulated growth by cell prolif- eration and cell enlargement but did not allow the differentiation of new cytological structures and, in particular, the organization of thylakoid tamellae from the tubular complex. The evaluation of chlorophyll content also supports these uttrastructural observations. Nuclei of external cell layers underwent modifications (high DAPI stainability, envelope invagination, movement toward the centre of the cell) indicative of their involvement in mitosis, as also shown by the formation of new cell walls inside the mother cells. A friable callus with histological and cytological characteristics similar to those of H. tuberosus treated explants (non-green unorganized callus, thin cell walls, nuclear invagination, low chlorophyll content) is the habituated non-organogenetic sugar beet callus described by Gaspar etal. (199t) and Cr~vecoeur etal. (1987). Both tissues are under the influence of high hormone (endogenous or exogenous) concentration and exhibit rapid growth. Even the non-habituated sugar beet callus observed by these authors was very similar to our untreated explants. The possibility of inducing TGase activity was shown in parenchyma slices after only 6 h of activation in the dark, and produced only conjugates of high molecular mass over the next 24 h (Grandi et al. 1992). Thus, light and more prolonged culture conditions, such as those described in this paper, provoked the appearance of additional conjugates, possibly related to specific functions acquired by the explants. Lower TGase activity and a lower amount of high molecular mass conjugates, possibly cell wall compo- nents (Dinnella et al. 1992), were found in rapidly grow-

ing explants treated with 2,4-D, compared to untreated ones. The former consists of friable callus whose cells have thin walls and empty intercellular spaces; the latter are of compact consistency with dense material in the intercellular spaces. As a working hypothesis, it can be inferred that a relationship exists between these morphological and physiological aspects. In animal systems (data in plants are too scanty), TGase activity is gen- erally high in differentiated tissues and low in rapidly growing tumours in which the organization of intra- and extra-cellular structural proteins is rather poor. In non-tumour differentiated animal cells TGase is involved in the organization of structures responsible for cell cohesion (Folk 1980, Lorand and Conrad 1984). In addition to differences in high molecular mass conjugates, another labelled region of Mr lower than 36 kDa, was detected only in green untreated tissue, previously observed from green leaf extracts (Falcone et al. 1993) and the crude homogenate of etiolated api- ces with leaf primordia (Serafini--Fracassini et al. 1988). It is known that different forms of the enzyme and different substrates exist and they could therefore exert more than one function (Folk 1980, Serafini-Fracassini etal. 1988, Fatcone etal. 1993). TGase could be involved in photosynthesis. This is suggested by the find- ing that TGase activity is present in isolated chloroplasts, including those of H. tuberosus leaves, and can be stimulated by light (Cohen et al. 1982, Margosiak etal. 1990, Signorini etal. 1991, Del Duca etal. 1991, Falcone et al. 1991). Margosiak et al. (1990) suggested that the substrate in plastids is the large RuBisCo subunit. In the present work, a similar low amount of radioactivity was present on the SDS-PAGE slab gel at the level of the purified large subunit of RuBisCo in both explants. The discovery of differently labelled substrates and the higher activity in greening explants could perhaps be dependent on the synthesis, during the greening process, of specific substrates, or even on the induction of different enzyme isoforms, whose ex- istence has been reported above. A paper is in preparation on the TGase substrates in isolated chloroplasts. In summary, a relationship between high TGase activity and differentiated cells can exist in plants as well as in animals.

Acknowledgements This research was supported by CNR, Italy, contribution No. 91.00539.CTO4. to D. Serafini-Fracassini. The authors thank Dr. C. Bergamini, Department of Biochemistry, University of Fer- rara, for discussion and the kind gift of RuBisCo and Dr. R. Musetti for skillful technical assistance.

S. Del Duca et al.: Transglutaminase activity in HeIianthus tuberosus explants 9

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Transglutaminase activity during greening and growth ofHelianthus tuberosus explants in vitro

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