Plant Cell Reports (1983) 2:40 - 42

Plant Cell Reports © Springer-Verlag 1983

Induction of Alcohol Dehydrogenase in Explants of Potato Tuber

(Solanum tuberosum L.) L. H. W. v a n der Plas a n d R. H. M. v a n tier Pal Biological Laboratory, Department of Plant Physiology, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Received December 15, 1982/December 21, 1982

Abstract D u r i n g callus formation a huge increase in alcoh o l d e h y d r o g e n a s e a c t i v i t y was o b s e r v e d in potato £uber tissue d i s c s . C a l l u s formation was no p r e r e q u i site for this i n c r e a s e ; s l i c i n g and s u b s e q u e n t incub a t i o n of p o t a t o tuber tissue discs always led to an increase in alcohol d e h y d r o g e n a s e a c t i v i t y , w h i c h was d e p e n d e n t on c y t o p l a s m i c p r o t e i n s y n t h e s i s . A threefold increase was o b s e r v e d d u r i n g i n c u b a t i o n in m o i s t air (periderm formation) and during i n c u b a t i o n on n u t r i e n t agar w i t h o u t carbon s o u r c e . A six- to e i g h t - f o l d increase o c c u r r e d d u r i n g i n c u b a t i o n on n u t r i e n t agar w i t h s u c r o s e , r i b o s e or p y r u v a t e as carbon source.The extra increase in alcohol dehydrogenase a c t i v i t y did not o c c u r in the p r e s e n c e of equimolar amounts of m a n n i t o l , s o r b i t o l , s u c c i n a t e or e t h a n o l . T h e extent of the a c t i v i t y increase was not d i r e c t l y c o r r e l a t e d w i t h the p r e s e n c e of a carbon source suitable for m a i n t a i n i n g growth. Abbreviation

: NAA

: naphtyl acetic acid.

Introduction Slicing and s u b s e q u e n t i n c u b a t i o n lead to p r o f o u n d changes in cell m e t a b o l i s m (Kahl 1 9 7 3 , 1 9 7 4 ) . M a n y enzymes increase in a c t i v i t y ,often due to synthesis of new enzyme m o l e c u l e s (Kahl et a l . 1 9 6 9 , L a n g e et a l . 1 9 7 0 ) . E s p e c i a l l y the enzymes linked to the degradation of starch and sugars and to the r e s p i r a t o r y p a t h w a y s have b e e n e x t e n s i v e l y studied (Kahl 1973), b u t less attention has been p a i d to the enzymes of the f e r m e n t a t i o n p a t h w a y s like lactate d e h y d r o g e n a s e and alcohol d e h y d r o g e n a s e . P r e l i m i n a r y e x p e r i m e n t s showed that during callus induction a t e n f o l d increase may o c c u r . T h i s increase is g r e a t e r than that of m a n y components linked to r e s p i r a t i o n and the r e s p i r a t o r y chain (Kahl 1973). This p a p e r describes some of the factors w h i c h influence the increase of alcohol d e h y d r o g e n a s e a c t i v i t y d u r i n g i n c u b a t i o n of sliced p o t a t o tuber t i s s u e , m e a s u r e d as increase of in v i t r o alcohol dehydrogenase activity. M a t e r i a l and Methods M a t e r i a l : Potato tubers (Solanum t u b e r o s u m L.cv. B i n t j e ) , s t o r e d at 8°C,were used t h r o u g h o u t the experiments. T r e a t m e n t of the tissue : For the i n c u b a t i o n on agar, tissue discs ( ca. 3 ram thick,6 m m in d i a m e t e r were s t e r i l i z e d with 0.5 % NaOCI for 2-3 min and subseq u e n t l y w a s h e d 3 times with sterile water. The discs were p l a c e d in sterile p e t r i dishes on a m o d i f i e d

B 5 m e d i u m (Van der Plas and W a g n e r 1981).Dependent on the e x p e r i m e n t ,various carbon sources in various c o n c e n t r a t i o n s a n d / o r 54 ~ M k i n e t i n were added. Incub a t i o n on m e d i a w i t h o u t these h o r m o n e s did not lead to callus f o r m a t i o n . T h e p e t r i dishes were i n c u b a t e d at 28°C in the dark. A s s a y of alcohol d e h y d r o g e n a s e ( EC i.i.i.I ): E x t r a c t s for the assay of a l c o h o l d e h y d r o g e n a s e were p r e p a r e d b y g r i n d i n g i-2 g of tissue in 5 m l h o m o g e nizing m e d i u m c o n t a i n i n g 0.i M K H 2 P O 4 , 0 . 0 5 M Tris and 2 mM c y s t e i n e , p H 7 . 2 . E x t r a c t i o n in other m e d i a (with Tris i n s t e a d of K H g ~ P O or B - m e r c a p t o - e t h a n o l instead of cysteine) y i e [ d e ~ less active preparations. Alcohol d e h y d r o g e n a s e was m e a s u r e d s p e c t r o D h o t o m e t r i c a l l y at 25°C in a total volume o f 3 ml against a b l a n k w i t h o u t ethanol a c c o r d i n g to B e r g m e y e r (1970). The final c o n c e n t r a t i o n s of the additions to the cuvettes were 0.05 M sodium p y r o p h o s p h a t e b u f f e r (pH + 9.0),0.08 M sodium s e m i c a r b a z i d e , a n d 0.5 m M NAD .The r e a c t i o n was started b y the a d d i t i o n of ethanol (final c o n c e n t r a t i o n 3.5 %).The g e n e r a t i o n of N A D H d u r i n g the first 5-10 m i n was c a l c u l a t e d from absorbance changes at 340 n m u s i n g . t h e a b s o r p t i o n c o e f f i c i e n t of NADH (6.22 cm- m-M-l). Results The a c t i v i t y of alcohol d e h y d r o g e n a s e in extracts from f r e s h l y sliced tuber tissue was low. Incubation of such tissue s l i c e s , h o w e v e r , l e d to a considerable increase of this in v i t r o a c t i v i t y , w h i c h was dependent on p r o t e i n synthesis.The p r e s e n c e d u r i n g incub a t i o n of the inhibitor of c y t o p l a s m i c p r o t e i n synthesis c y c l o h e x i m i d e c o m p l e t e l y p r e v e n t e d the increase of alcohol d e h y d r o g e n a s e a c t i v i t y ( f i g u r e t). D u r i n g the first 5-6 days after the start of the incubation the alcohol d e h y d r o g e n a s e a c t i v i t y gradually i n c r e a s e d to a more or less stable level w h i c h w a s m a i n t a i n e d for at least I0 days and w h i c h in the p r e s e n c e of sugar was a b o u t 2 times as h i g h as in the absence of sugar ( figure i). During incubation of tuber discs on n u t r i e n t agar m e d i u m (agar m e d i u m w i t h only mineral salts and vitamins) callus formation did not occur w h i l e the a c t i v i t y of alcohol d e h y d r o g e n a s e in the extracts p r o v e d to increase to roughly 3 times that in extracts from f r e s h l y sliced tissue (figure l).Incub a t i o n of tuber slices in m o i s t air led to p e r i d e r m formation w h i l e alcohol d e h y d r o g e n a s e a c t i v i t y i n c r e a s e d n e a r l y to the same level. The effects of v a r i o u s additions to the n u t r i e n t agar m e d i u m were tested. Increase of alcohol d e h y d r o -

41 c o m p a r a b l e w i t h or e v e n h i g h e r than that in callus f o r m i n g tissue (table l ) . A p p a r e n t l y the p r e s e n c e of an e x t e r n a l l y s u p p l i e d carbon source like sucrose was the m a i n factor g o v e r n i n g the e x t e n t of alcohol d e h y d r o g e n a s e a c t i v i t y increase. The a b i l i t y of the tuber discs to use the v a r i o u s c a r b o n sources for g r o w t h was i n v e s t i g a t e d (table 2). In d i s c s on a m e d i u m w i t h h o r m o n e s b u t w i t h e x o g e n o u s carbon source callus i n d u c t i o n was o n l y f o l l o w e d b y a short p e r i o d of callus growth. This was a c c o m p a n i e d b y a d e c r e a s e in dry w e i g h t of the discs p r e s u m a b l y due to c o n s u m p t i o n of storage starch in respiration. C a l l u s i n d u c t i o n in the p r e s e n c e of a c a r b o n source r e s u l t e d in a s m a l l e r average d e c r e a s e or an i n c r e a s e of dry w e i g h t . G r o w t h r a t e , m e a s u r e d as dry w e i g h t production was h i g h e s t w i t h 88 m M s u c r o s e , a l t h o u g h o t h e r sucrose c o n c e n t r a t i o n s gave a p p r e c i a b l e g r o w t h rates t o o . D i s c s i n c u b a t e d on m e d i a c o n t a i n i n g s o r b i t o l , m a n n i t o l or e t h a n o l showed n e a r l y the same dry w e i g h t d e c r e a s e as discs i n c u b a t e d on m e d i a

~Jrnol NADH/min/g

24j A

20"

Z~

/,,

AA /,,

1.6.

t,~

-

1.2-

/ ~

A

~

~a

A

A /-,

~" 0 0

0

0 000

0

"7 4

,

0

~

C

r

8

F i g u r e 1. Alcohol dehydro~nas~ activity (expressed as ~mol NADH produced min ~ g-~ fresh weight, with ethanol as substrate ) in extracts prepared from potato tuber tissue discs incubated on basic nutrient agar medium with various additions. o - o : no carbon source - A : 88 mM sucrose • - • : 88 m M s u ~ o s e and 10 Vg cycloheximide (CHI) ml .

genase a c t i v i t y a l w a y s o c c u r r e d w h e n slices w e r e i n c u b a t e d .As to the level ,reached and m a i n t a i n e d after 5-15 days of i n c u b a t i o n ,two types of r e a c t i o n could be d i s t i n g u i s h e d (table l).With s u c r o s e ,ribose and p y r u v a t e the a c t i v i t y was a b o u t twice as h i g h as in the c o n t r o l tissue i n c u b a t e d on a m e d i u m w i t h o u t any c a r b o n s o u r c e . W i t h s u g a r a l e o h o l s (mannit o l , s o r b i t o l ) , e t h a n o l a n d s u c c i n a t e as the c a r b o n s o u r c e , t h e a c t i v i t y i n c r e a s e w a s s o m e w h a t lower than in the c o n t r o l . A n i n c r e a s e of the s u c r o s e c o n c e n t r a tion in the range of 44-175 m M d i d n o t lead to appreciable e x t r a i n c r e a s e of e n z y m e activity. D o u b l i n g the m a n n i t o l c o n c e n t r a t i o n f r o m 88 to 175 m_M a l s o h a d little e f f e c t . T h e same h e l d for the a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of NaC1. E s p e c i a l l y i n t e r e s t i n g was the e f f e c t of v a r i o u s c o n c e n t r a t i o n s o r g a n i c a c i d s . T h e a c t i v i t y increase w i t h p y r u v a t e was about three times as h i g h as w i t h s u c c i n a t e . A d d i t i o n of c i t r a t e or a c e t a t e in the same c o n c e n t r a t i o n s led to a r a p i d d e c l i n e in a l c o h o l d e h y d r o g e n a s e a c t i v i t y a n d d e a t h of the tissue w i t h i n a week. In discs on b a s i c n u t r i e n t agar m e d i u m s u p p l i e d w i t h the a p p r o p r i a t e h o r m o n e s and w i t h sucrose as carbon source b o t h callus i n d u c t i o n and s u b s e q u e n t callus g r o w t h o c c u r r e d w i t h a s i x f o l d i n c r e a s e of enzyme a c t i v i t y . F u r t h e r e x p e r i m e n t s s h o w e d t h a t the level to w h i c h the a l c o h o l d e h y d r o g e n a s e a c t i v i t y i n c r e a s e d did n o t p r i m a r i l y d e p e n d on the type of m o r p h o g e n e t i c response. O m i s s i o n of sugar f r o m the a g a r m e d i u m s u p p l i e d w i t h h o r m o n e s d i d lead to callus i n d u c t i o n a l t h o u g h callus g r o w t h s t o p p e d a f t e r about two w e e k s . T h e n the i n c r e a s e of a l c o h o l d e h y d r o g e n a s e a c t i v i t y w a s low as in d i s c s i n c u b a t e d on b a s i c n u t r i e n t a g a r m e d i u m (table 1).On the o t h e r h a n d discs ,on a m e d i u m w i t h o u t h o r m o n e s b u t w i t h sugar d i d n o t f o r m any callus .Yet ,a h i g h i n c r e a s e of alcohol dehydrogenase activity activity occurred

Table l.Activity of alcohol dehydrogenase in extracts from potato tuber tissue discs, incubated on a basic nutrient agar medium with various additions or in moist air. Activity was measured 5-15 days after the start of the incubation and is expressed as percentage ( + SD ) of the activity of extracts from tissue incubated on basic nutrient agar medium without carbon source and hormones. Between brackets :number of independent determinations.

Incubation

A. Slices

Alcohol dehydrogenase activity

in m o i s t

air

84 + 25

(5)

%

(22) (21)

% %

B. Slices on b a s i c m e d i u m w i t h the f o l l o w i n g a d d i t i o n s i. N o n e

100 a

2. H o r m o n e s ~ sugar N A A and k i n e t i n N A A , k i n e t i n and 88 mM sucrose

99 + 26 185 ~ 55

3. Sugars 44 m M 88 m M 175 mM 88 m M

248 260 289 189

sucrose sucrose sucrose ribose

4. S u g a r a l c o h o l s / e t h a n o l 88 m M m a n n i t o l 175 m M m a n n i t o l 88 m M s o r b i t o l 88 m M e t h a n o l 5. O r g a n i c acids 88 m M sodium 88 m M s o d i u m 88 m M s o d i u m 88 mM s o d i u m

succinate pyruvate citrate acetate

6. S o d i u m c h l o r i d e 257 m M N a C l 513 m M N a C l

56 48 82 22

9) 8) 8) 6)

+ 12 + 26 + 9 T II

15) 5) 8) 7)

56 + 33 1 8 4 b ~ 16 0 -0b

8) 6)

73 + 19 97 + 18

3) 3)

76 87 82 79

+ ~ ~ ~

aThe activity of alcohol dehydrogenase in extracts from slices incubated on basic nutrient agar medium without additions was 0.71 + 0.1~ ¢~_~1 NADH produced (with ethanol as substrate) min g (n=49).The activity of freshly sliced tissue was + 30 % of the activity of slices incubated on basic ~utrient agar ~edium without further additions. Tissue died within a week.

42 w i t h o u t carbon source.Discs ,incubated on ribose, succinate or p y r u v a t e were able to compensate the r e s p i r a t o r y losses of dry weight. Discussion The increase in the alcohol d e h y d r o g e n a s e activity during i n c u b a t i o n of p o t a t o tuber tissue discs on sugar c o n t a i n i n g m e d i a either w i t h or w i t h o u t horm o n e s is about twice as h i g h as w i t h o u t sugar.The p r e s e n c e of h o r m o n e s (NAA and kinetin) and the induction of callus caused b y these h o r m o n e s does not a p p r e c i a b l y affect this enzyme a c t i v i t y increase (table l).Apparently the p r e s e n c e of e x t e r n a l l y s u p p l i e d sugar is an i m p o r t a n t factor g o v e r n i n g the

Growth rate of potato tuber callus incubated on basic nutrient agar medium supplied with NAA and kinetin and with various carbon sources. Dry weight at the start of the incubation was 165 mg.Tissues incubated on basic medium in the absence of external carbon source lost 2.1 mg dry weight per day. Growth rate is expressed as the difference between this dry weight loss and the average dry weight changes per day during incubation on various carbon sources. Each value resulted from 4 separate determinations during the first month of callus growth. Table 2.

C a r b o n source

Growth rate (dry w e i g h t production) m g per day

88 175 44 88 88 88 88 88 88

m M sucrose mM sucrose m M sucrose m M ribese m M sodium succinate m M sodium p y r u v a t e m M sorbitol mM mannitol m M ethanol none

+ + + + + + + + +

8.8 5.3 5.0 2.2 1.5 1.6 0.5 0.4 0.4 0

% of growth rate on 88 mM sucrose i00 60 57 25 17 18 6 ~ 4 4 0

extent of the increase of the alcohol d e h y d r o g e n a s e activity.The results of table i show that the observed d i f f e r e n c e s in alcohol d e h y d r o g e n a s e activity are not caused b y the d i f f e r e n c e s in o s m o l a r i t y of the i n c u b a t i o n m e d i u m . O n l y a d d i t i o n of sugars like sucrose and ribose and of p y r u v a t e gives rise to e x t r a increase of the alcohol d e h y d r o g e n a s e a c t i v i t y , w h i l e e q u i m o l a r amounts of NaCI have no effect. A direct c o r r e l a t i o n b e t w e e n the extent of the a c t i v i t y increase and the p r e s e n c e of a carbon source suitable for m a i n t a i n i n g growth is absent. Sucrose in v a r i o u s c o n c e n t r a t i o n s gives rise to a substantial increase in dry w e i g h t , w h i l e a d d i t i o n of ribose and p y r u v a t e o n l y l e a d to compensation of the dry w e i g h t loss o b s e r v e d in the controls w i t h o u t s u g a r , w h i c h is p r o b a b l y caused b y d e g r a d a t i o n of storage starch for r e s p i r a t i o n (table 2).Yet ,all these additions result in h i g h alcohol d e h y d r o g e n a s e activities (table l).On the c o n t r a r y a d d i t i o n of succinate has the same effect on the course of dry w e i g h t during inculbation as p y r u v a t e w i t h o u t causing h i g h alcohol d e h y d r o g e n a s e activities. A common c h a r a c t e r i s t i c of the carbon sources that lead to e x t r a enzyme a c t i v i t y (sucrose,ribose and p y r u v a t e ) is that they all can be converted to ethanol v i a the i n d u c e d alcohol d e h y d r o g e n a s e a c t i v i t y . H a g e m a n n and F l e s h e r (1960) and C r a w f o r d and M c M a n n o n (1968) suggest that acetaldehyde ,a p r e c u r s o r of ethanol in the c o n v e r s i o n of p y r u v a t e to ethanol plays a role as inducer of alcohol d e h y d r o g e n a s e s y n t h e s i s . T h i s m e c h a n i s m m a y function here too : the p r e s e n c e of compounds w h i c h can be easily c o n v e r t e d to ethanol via a c e t a l d e h y d e m a y lead to extra alcohol d e h y d r o g e n a s e induction. References B e r g m e y e r H U (1970) M e t h o d e n der E n z y m a t i s c h e n A n a l y s e ,Verlag Chemie ,Weinheim C r a w f o r d R M M , M c M a n n o n M (1968) J Exp Bot 19 : 435-441 H a g e m a n n R H , F l e s h e r D (1960) Arch B i o c h e m Biophys 87 : 203-209 Kahl G , L a n g e H ,Rosenstock G (1970) Z N a t u r f o r s c h 24b : 911-918 Kahl G (1973) Bot R e v 39 : 274-299 Kahl G (1974) Bot Rev 40 : 263-314 Lange H,Kahl G , R o s e n s t o c k G (1970) P l a n t a 91 : 18-31 V a n der Plas L H W , W a g n e r M J (1981) P l a n t Cell P h y s i o l 22 : 1075-1084