Plant Ceil Reports (1983) 2:261-264

PlantCeU Reports © Springer-Verlag 1983

Loss of Competence for Glyoxysome Formation During Somatic Embryogenesis in Anise (Pimpinella anisum L.) Suspension Cultures R. A. Kudielka and R. R. Theimer Botanisches Institut der Universit~it, Menzingerstr. 67, D-8000 Mt~nchen 19, FRG Received August 1, 1983 - Communicated by M. H. Zenk

ABSTRACT Somatic embryogenesis in anise (Pimpinella anisum L.) suspension cultures induced by t r a n ~ r ~ r e e growth medium may be synchronized by previous selection of cell aggregates with diameters between Io0-24o Nm. Around 8o-9o% of the embryoids are globular a f t e r 2-3 d, heartshaped a f t e r 5-7 d and torpedo-shaped a f t e r 9 d. In embryogenic medium without source of carbon or with 20 mmol/l acetate d i f f e r e n t i a t i o n and growth cease. But l i k e in ded i f f e r e n t i a t e d c e l l aggregates the key enzyme a c t i v i t i e s f o r glyoxysomes such as i s o c i t r a t e lyase and malate synthase are induced in globular (3 d old) and heart-shaped (5 d old) embryoids, but not in embryoids at day 7 or l a t e r . Sim i l a r l y , in explants from anise hypocotyl glyoxysomes cannot be derepressed by such treatment. I t is concluded that during d i f f e r e n t i a t i o n of heart-shaped embryoids to torpedo forms the competence of the c e l l s f o r the yet unknown inducing p r i n c i p l e f o r glyoxysomes is l o s t . ABBREVIATIONS: CAT=Catalase; CS=Citrate synthase; ICL=Isocitrate lyase; MS=Malate synthase; MDH=Malate dehydrogenase; ICDH=Isocitrate dehydrogenase (NADH); CH= Crude homogenate; CPF=Crude p a r t i c u l a t e f r a c t i o n .

INTRODUCTION Dedifferentiated anise (Pimpinella anisum L.) suspension cultures were shown to possess the competence f o r glyoxysome formation, i . e . the a b i l i t y to respond to removal of sucrose from the growth medium with the induction of the a c t i v i t i e s of f a t t y acyl-CoA oxidase, i s o c i t r a t e l y ase, and malate synthase and compartmentation of these and other glyo~ysomal enzyme a c t i v i t i e s in a newly appearing organelle (Kudielka et a l . 1981; Kudielka and Theimer 1983a; Lutzenberger and Theimer 1983). The glyoxysomes induced in the suspension cultures resemble q u a l i t a t i v e l y and q u a n t i t a t i v e l y those found in the anise endosperm which is the tissue proper f o r glyoxysome mediated f a t degradation in the anise plant (Lutzenberger and Theimet 1983). Returning the suspension cultures to sucrose medium results in the complete loss of the three inducible enzyme a c t i v i t i e s although incomplete "glyoxysomes" appear to survive (Kudielka and Theimer 1983b). Obviously, in the suspension cultures the information f o r glyoxysome production present in the anise c e l l s may be f u l l y and reversibly realized. In the present study i t was examined i f th~s depends on the stage of d i f f e r e n t i a t i o n of the anise c e l l s . Therefore, the responsiveness to changes in the exogenous source of carbon was investigated both in anise hypocotyl tissue (from which the anise cultures are o r i g i n a l l y derived - cf. Huber et a l . 1978) and during somatic embryogenesis in the suspension cultures which proceeds under appropriate conditions rather

Correspondence and offprint reques~ to: R. R. Theimer

quickly (Huber et al. Theimer 1983c). I t is mation of glyoxysomes of embryogenesis and, hypocotyl tissue.

1978) and synchronously (Kudielka and shown that the competence f o r the f o r is gradually l o s t during early stages as expected, lacks completely in the

MATERIALS AND METHODS Cell suspension cultures and anise seedlings were grown as reported previously (Kudielka et a l . 1981), Also, i n duction of the g!yoxylate cycle enzyme a c t i v i t i e s and homogenization and f r a c t i o n a t i o n of the c e l l s were performed as described elsewhere (Kudielka et a l . 1981; Kudielka and Theimer 1983a). For the induction of somat i c embryogenesis8suspension cultures grown to a c e l l density of 2-5xlo cells/ml were transferred to B-5 medium (Gamborg e t a ! . 1968) supplemented with 2o g/l sucrose but lacking 2,4-D (=embryogenic medium) and incubated on a gyratory shaker (15o/min) f o r 2h to remove adsorbed hormone. Then the cultures were f i l t e r e ~ through a s t e r i l e stainless steel net (mesh 250 pm) and the f i l t r a t e subjected to Ixg sedimentation f o r 5 min. The r e s u l t i n g p e l l e t contained cell aggregates with diameters between Ioo-24o pm which were found to d i f f e r e n t i a t e best to somatic embryoids (Huber et a l . 1978). They were suspended in ~resh 2,4-D-free growth medium to a c e l l density of Io cells/ml and incubated on a gyratory shaker (6o/min) f o r up to 14 d. The medium was renwed every 3 d. At the d i f f e r e n t times given in the t e x t the c e l l aggregates or embryoids were harvested as described, rinsed with fresh sucrose-free embryogenic medium and incubated f o r 24 or 72 h on a gyratory shaker (6o/min). To one batch 2o mmol/l acetate was added a f t e r 48 h and the cultures were incubated f o r another 24-h period. For the treatment of anise hypocotyls l-cm pieces of tissue were rinsed thoroughly and shaken f o r 24 h e i t h e r in s t e r i l e g l a s s - d i s t i l l e d water or s t e r i l e acetate medium (2o mmol/l) without sucrose. Assays f o r enzyme a c t i v i t y were carried out with a G i l ford spectrophotometer model 24oo-S as reported in the l i t e r a t u r e : Catalase (CAT), EC l . l l . l . 6 (LUck 1965), c i t r a t e synthase (CS), EC 4.1.3.7, i s o c i t r a t e lyase (ICL) EC 4.1.3.1, and malate synthase (MS), EC 4.1.3.2 (Dixon and Kornber 9 1959), malate dehydrogenase (MDH), EC l . l . 1.37 (Ochoa 1955), NADH-isocitrate dehydrogenase (ICDH), EC l . l . l . 4 1 (Wolfson and Williams-Ashman 1957). Protein contents were estimated by the Lowry procedure (Lowry et a l . 1951) and c e l l numbers were counted by the method of Huber et a l . (1978). Embryoids were defined "torpedoshaped" when the r a t i o length of l o n g i t u d i n a l axis to length of l a t e r a l axis (across the cotyledons) was at least 1.5.

262

Figure I: Micrographs of anise suspension culture cell ......... aggregates and d i f f e r e n t stages of embryoid d i f f e r e n t i a t i o n . (A) Dedifferentiated cell aggregate from a submerged culture 6 d after inoculation (late logarithmic phase of growth). (B) Globular embryoid 1 d after ini t i a t i o n of somatic embryogenesis in submerged culture. (C) Heart-shaped embryoid 5 d after start of embryogenesis. (O) Torpedoshaped embryoid 9 d after start of embryogenesis. Each bar represents o.I mm.

The removal of sucrose from the embryogenic growth medium or i t s replacement by 2o mmol/l acetate has two effects on the d i f f e r e n t i a t i n g cell aggregates. F i r s t l y , no further growth or morphological changes are observed which has also been reported for other plant species (e. g. Kochba and Button 1974). This indicates that embryogenesis is arrested at the stage reached before change of the medium. Secondly, up to day 5 after start of embryogenesis the key enzyme a c t i v i t i e s for glyoxysomes are induced in the ceil aggregates or embryoids (Fig. 3).

RESULTS AND DISCUSSION Suspension cultures of anise grow mainly as cell aggregates of variable size when cultivated in B-5 medium (Gamborg e t a l . 1968) with 5 Nmol/l 2,4-D (Fig. IA). After transfer to embryogenic B-5 medium that lacks the phytohormone most of the cell aggregates with a diameter between Ioo-25o Nm perform somatic embryogenesis in an unusually short period of time (Huber et al. 1978; Kudielka and Theimer 1983c). Both smaller and bigger cell aggregates d i f f e r e n t i a t e slower or not at a l l . Thus when the cultures are sieved to select for cell aggregates of proper size embryogenesis can be essentially synchronized, i . e . 8o-9o% of the embryoids of a given culture are in the same state of embryogenesis. After 1 d in hormone-free medium globular embryoids are predominant in the cultures (Fig. IB). Two days later the embryoids are heart-shaped (Fig. IC) and around day 9 after i n i t i a t i o n of embryogenesis essentially all embryoids have torpedo form (Fig. ID, 2). They eventually grow out into e~ongate plantlets and f i n a l l y into intact anise plants within a few weeks (Kudielka and Theimer 1983c}. This apparent synchrony in d i f f e r e n t i a t i o n observed in the cultures f a c i l i t a t e s correlations between morphological changes and cell physiological events such as the induction of enzyme a c t i v i t i e s and glyoxysome formation in the embryeids.

Figure 2: Micrograph of a submerged anise culture 7 d ......... after i n i t i a t i o n of somatic embryegenesis in sieved cultures with cell aggregates that had diameters between Ioo-24o ~m. Nearly all embryoids show morphologically the same stage of d i f f e r e n t i a t i o n . The bar represents Imm.

263

0.1 ICL "=,0.05 ~ 0

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ICDH

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DAYS OF E M B R Y O G E N E S I S Figure 3: Specific enzyme a c t i v i t i e s in the crude ho......... mogenate and in the crude p a r t i c u l a t e f r a c tion (= lo,oooxg p e l l e t ) from c e l l aggregates or embryoids at d i f f e r e n t stages of somatic embryogenesis. S ~ b o l s are: I before treatment a f t e r 48 h in medium without sucrose V f ~ and without hormone I . ' . ' I a f t e r 72 h in medium without sucrose and without hormone a f t e r 72 h in medium without sucrose I I and without hormone, but 2o mmol/l acetate added for the l a s t 24 h. Both in the crude homogenate and in the crude p a r t i c u late f r a c t i o n of the embryoids a c t i v i t i e s of i s o c i t r a t e lyase and malate synthase increase with time in the sucrose-free medium. Addition of acetate y i e l d s an a d d i t i o nal r i s e in enz~e a c t i v i t i e s as has been shown previously for d e d i f f e r e n t i a t e d suspension cultures (Kudielka and Theimer 1983a). Other glyoxysomal e n z p e a c t i v i t i e s such as c i t r a t e synthase, malate dehydrogenase or catalase that are already present in sucrose-grown cultures are j u s t as l i t t l e effected by the change in growth medium as the mitochondrial NAD-isocitrate dehydrogenase (Fig. 3). In the course of d i f f e r e n t i a t i o n from heart-shaped to torpedo-shaped embryoids the a b i l i t y to respond to chan-

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DAYSOFEMBRYOGENESIS

ges in carbon supply with the induction of glyoxysomes is obviously l o s t {Fig. 3). At day 7 when the embryoids are in an intermediate stage glyoxysomal enzyme a c t i v i t i e s can no longer be induced. During the f i r s t 5 d of embryogenesis the d i f f e r e n t i a t i n g cell aggregates e v i d e n t l y remain competent for the y e t unknown inducing p r i n c i p l e that eventually leads to the de novo formation of functional glyoxysomes (Kudielka~n~eimer 1983a; Lutzenberger and Theimer 1983), although conspicuous morphological changes s u g g e s t t h e onset of polar organization in the embryoids (Fig. l ) . The amount of inducible enzyme a c t i v i t y , however, decreases between day 3 and 5 (Fig. 3). I f t h i s is due to the loss of competence in part of the embryoids of a given culture or in certain c e l l s of each c e l l aggregate needs f u r t h e r examination. But in the course of d i f f e r e n t i a t i o n from heart-shaped to torpedo-shaped embryoids the a b i l i t y to respond to changes in carbon supply with the induction of glyoxysomes is completel y l o s t (Fig. 3).since at day 7 glyoxysomes can no longer be produced. At t h i s stage the embryoidal c e l l s have e v i d e n t l y reached the status observed f o r c e l l s in i n t a c t anise tissue such as hypocotyl explants: they do not respond to changes in the exogenously supplied carbon source (table l ) .

264 Table I: Specific a c t i v i t i e s of glyoxysomal enzymes in ........ the crude homogenate (CH) and crude particulate f r a c t i o n (CPF) of anise hypocotyl explants incubated for 24 h in d i s t i l l e d HpO or embryogenic sucrose-free B-5 medium containing 20 mmol/l acetate. Enzyme

Enzyme a c t i v i t y H20

ACKNOWLEDGEMENTS This work was in part supported by grants from the Deutsche Forschungsgemeinschaft and from the Bundesministerium fur Forschung und Technologie.

(nkat/mg protein)

REFERENCES

a c e t a t e medium

Dixon G H, Kornberg H S (1959) J Biochem 72:3

CH

CPF

CH

CPF

Gamborg OL, M i l l e r R A, Ojima K (1968) Exp Cell Res 5o:151-158

ICL

0

0

0

0

MS

0

0

0

0

CS

0.03

0.06

0.04

0.04

MDH

2.4

3.1

3.0

2.5

CAT

68

84

193

122

Since between day 5 and 7 a l t e r a t i o n s in morphology or cell mass are not nearly as dramatic as during the f i r s t 5 d of embryogenesi%intra- and i n t e r c e l l u l a r events within the embryoids are l i k e l y to account for the loss of competence to the inducing p r i n c i p l e . The finding that protein synthesis is involved in the formation of the induced glyoxysomes (Kudielka and Theimer, paper in preparation) suggests that changes in the a c c e s s i b i l i t y of genetic information for glyoxysome development may also be involved. In t h i s respect, the anise cultures p r o v i d e a v a l u a b l e system for the investigations into both the inducing p r i n c i p l e for glyexysomes and into the mechanisms that render cells competent for certain inducers.

Huber J, Constabel F, Gamborg 0 L (1978) Plant Sci Lett 12:2o9-215 Kudielka R A, Kock H, Theimer R R (1981) FEBS Lett 136:8-12 Kudielka R A, Theimer R R I1983a) Plant Sci Lett (in press) Kudielka R A, Theimer R R (1983b) Plant Sci Lett (in press~ Kudielka R A, Theimer R R (1983c) In: Longdon N, Mel i t a 0 (eds) Space Biology, ESA SP-2o6 ESA Sci Techn Public Branch, Noordwijk, pp 63-68 Lowry OH, Rosebrough NJ, Farr AL, Randall BJ (1951) J biol Chem 193:265-275 LUck H (1965) In: Bergmeyer HU (ed) Methods of enzyme analysis, Acad Press, New York-London, pp885-894 Lutzenberger A, Theimer RR (1983) Plant Cell Reports 2:16o-163 Ochoa S (1955) In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol I, Acad Press, New YorkLondon, pp735-739 Wolfson SK, Williams-Ashman HG (1957) Proc Soc Biol Med 96:231