Plant Cell Reports

Plant Cell Reports (1988) 7:495-498

© Springer-Verlag 1988

Cold induced gene expression in Arabidopsis thaliana L. Sirpa Kurkela 1, Marianne Franck 1, Pekka Heino 2, Viola Lfing 2, and E. Tapio Palva 2 Molecular Genetics Laboratory, Department of Genetics, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki, Finland 2 Department of Molecular Genetics, Swedish University of Agricultural Sciences, Box 7003, S-75007 Uppsala, Sweden Received February 8, 1988/Revised version received March 9, 1988 - Communicated by H. LSrz

ABSTRACT

MATERIALS

E x p o s u r e of Arabidopsis thaliana L. to an a c c l i m a t i o n t e m p e r a t u r e (+4°C) results in a rapid increase of frost t o l e r a n c e from -3°C to -7°C. This increase could be c o r r e l a t e d to changes in soluble p r o t e i n pattern. Analysis of in vitro t r a n s l a t i o n p r o d u c t s from isolated mRNA suggests that i n d u c t i o n acts at the t r a n s c r i p t i o n a l level.

The (Columbia wild type), was o b t a i n e d from Chris Sommerville. A x e n i c cultures were p r e p a r e d by surface s t e r i l i z a t i o n of seeds in 6% (w/v) sodium h y p o c h l o r i t e for 10 m i n u t e s f o l l o w e d by five rinses in an excess of sterile water. The seeds w e r e g e r m i n a t e d and grown on M u r a s h i g e ' s M i n i m a l Organic M e d i u m (Flow Laboratories) c o n t a i n i n g 9.6% agar in a c o n t r o l l e d e n v i r o n m e n t chamber with an air t e m p e r a t u r e of 20°C day, 18°C night using an 16h l i g h t / 8 h dark cycle. Cold a c c l i m a t i o n was p e r f o r m e d by exposing 3 week old p l a n t l e t s to +4°C for the time periods i n d i c a t e d under o t h e r w i s e similar conditions. The heat shock t r e a t m e n t was done at +40°C for 4h.

INTRODUCTION F r e e z i n g t o l e r a n c e in several plant species is an i n d u c i b l e c h a r a c t e r i s t i c that develops when plants are e x p o s e d to low but nonf r e e z i n g t e m p e r a t u r e s (Levitt 1980). Plants respond s i m i l a r l y also to other e n v i r o n m e n t a l factors such as o s m o t i c stress, d e s i c c a t i o n or high temperature. Generally, exposure to m o d e r a t e stress allows the plant to adapt and survive a s u b s e q u e n t stronger stress. Cold a c c l i m a t i o n is a c c o m p a n i e d by m a n y changes in p l a n t p h y s i o l o g y and metabolism, for instance in m e m b r a n e lipid composition, c a r b o h y d r a t e content, osmotic concentration, h o r m o n a l b a l a n c e and p r o t e i n q u a l i t y and q u a n t i t y (Sakai and L a r c h e r 1987). However, the causal r e l a t i o n s h i p s b e t w e e n the changes are far from clear. N e i t h e r is it k n o w n h o w such changes may s p e c i f i c a l l y lead to adaptation of the plant and w h e t h e r there are d i f f e r e n c e s in m e c h a n i s m s of cold a d a p t a t i o n b e t w e e n d i f f e r e n t plant species. It has been shown that p r o t e i n synthesis is e s s e n t i a l for cold a c c l i m a t i o n (Chen and Li 1982). F u r t h e r more, in several species cold a c c l i m a t i o n has been c o r r e l a t e d to a p p e a r a n c e of new p r o t e i n s (Lewitt 1980; M e z a - B a s s o et al. 1986; Guy et al. 1986; Guy and Haskell 1987).

Jrabidopsis

thaliana is an ideal model plant for both p h y s i o l o g i c a l and m o l e c u l a r studies (Estelle and S o m e r v i l l e 1986; Pang and M e y e r o w i t z 1987). In this study we show that A. thaliana can cold acclimate, and that this involves i n d u c t i o n of new p r o t e i n s and mRNAs.

Offprint requests to. E.T. Palva

AND M E T H O D S

Plant m a t e r i a l

Arab£dopsis

and g r o w t h

conditions.

thal{ana L. strain used

F r o s t t o l e r a n c e assessment. Frost t o l e r a n c e was d e t e r m i n e d by freezing p l a n t l e t s (without roots) in a c o n t r o l l e d t e m p e r a t u r e bath (Sukumaran and W e i s e r 1972). A f t e r e q u i l i b r a t i o n the t e m p e r a t u r e was lowered at a rate of 2°C/h. E x t r a c e l l u l a r freezing was initiated by adding small amounts of ice at o a t e m p e r a t u r e of -1.5 C. Samples were r e m o v e d at I C intervals and thawed on ice overnlght. F r e e z i n g injury was d e t e r m i n e d by measuring the e l e c t r o l y t e loss by changes of c o n d u c t i vity. Tissue showing 50% or m o r e e l e c t r o l y t e leakage was c o n s i d e r e d killed. O

,

.

in vivo l a b e l l i n g and e x t r a c t i o n of proteins. R a d i o a c t i v e l a b e l l i n q ~ Q f proteins was p e r f o r m e d by adding [ a D S ] - m e t h i o n i n e d i r e c t l y to the growth m e d i u m (100 uCi/ml) . After 6-12h of l a b e l l i n g 100 mg of plant tissue (without roots; c o r r e s p o n d i n g to 2-4 plants) was rapidly h o m o g e n i z e d w i t h a glass rod in 100 ul of ice cold h o m o g e n i z i n g b u f f e r (50mM Tris-HCl, pH 6.8, I% ~ - m e r c a p t o e t h a n o l , 9.13 mg/ml leupeptin) in a m i c r o c e n t r i f n g e tube. The c e l l u l a r debris were removed by c e n t r i f u g a t i o n for 3 m i n u t e s at 13.990 xg, the s u p e r n a t a n t was collected, m i x e d with an equal a m o u n t of e l e c t r o p h o r e s i s sample b u f f e r (Lae~mli 1970~, b o i l e d for five m i n u t e s and stored at -20 C.

496 E x t r a c t i o n of total RNA and p o l y ( A + ) R N A ,, in vitro translation. P l a n t l e t s exposed to the cold a c c l i m a t i o n temperature for 24h were h a r v e s t e d (without roots) and rapidly frozen in liquid nitrogen. The RNA was isolated as d e s c r i b e d ( C h i r g w i n et al. 1979; Teeri et al. 1987). The p o l y ( ~ + ) - f r a c t i o n was separated by c h r o m a t o g r a p h y through oligo-dT cellulose (BRL) as d e s c r i b e d by M a n i a t i s et al. (1982). The in vitro t r a n s l a t i o n of p o l y ( A ~ ) R N A was p e r f o r m e d using rabbit r e t i c u l o c y t e lysate (Amersham International) according to the instructions p r o v i d e d by the manufacturer. E l e c t r o p h o r e s i s @nd f l u 0 r o g r a p h y . One dimensional d e n a t u r i n g p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was carried out according to Laemmli (1970). The a p p a r e n t m o l e c u l a r weights of proteins were e s t i m a t e d using m o l e c u l a r weight markers from A m e r s h a m International. After e l e c t r o p h o r e s i s gels were stained with Coomassie blue, treated with A m p l i f y (Amersham International), dried and f l u o r o g r a p h e d using X-ray films (Kodak).

m°

~ -8 ss

~-6,

T DAYS

RESULTS AND D I S C U S S I O N Induction of frost tolerance Exposure of A. thaliana plantlets to +4°C initiates cold acclimation, and the freezing tolerance of the tissue is increased (Fig. I). The plantlets are almost fully a c c l i m a t e d within 2-3 days. A f t e r this the frost tolerance is increased only slowly during a period of 7 to 10 days to reach a m a x i m u m level of about -7 ° to -8°C. It thus appears that most of the key p h y s i o l o g i c a l changes required for cold acclimation must take place within 2 to 3 days, the following slow increase in frost tolerance could be due to more secondary effects. In contrast, many of the other plant species studied show c o n s i d e r a b l y slower rates of cold a c c l i m a t i o n (Kacperska-Palacz 1978; C l o u t i e r and S i m i n o v i t c h 1982). Induction of proteins In vivo labelling of plant proteins w i t h [ 3 5 ~ m e t h i o n i n e allowed the a s s e s s m e n t of protein induction during cold acclimation. A set of new protein species was induced by the low t e m p e r a t u r e exposure (Fig. 2). Some of these proteins a p p e a r e d to be present already at +~.o zu C but were s u b s t a n t ± a l l y increased during acclimation, others appeared as de novo induced bands. Proteins with apparent m o l e c u l a r weights of 150, 85, 69, 69, 45, 30 and 24 kDa were m o s t clearly and r e p r o d u c i b l y induced. In addition, synthesis of some proteins was severely reduced by the cold t r e a t m e n t (Fig. 2). However, in this case p r o l o n g e d a c c l i m a t i o n a p p e a r e d to restore this synthesis, at least partially. .

,

The changes o b s e r v e d in protein synthesis b e t w e e n plants grown at +20°C and at +4°C seemed to persist t h r o u g h o u t the a c c l i m a t i o n treatment. Thus, proteins induced early in a c c l i m a t i o n were s y n t h e s i z e d during the whole a c c l i m a t i o n period. This is in contrast to another type of temperature stress, heat shock, where the heat shock proteins are induced but their synthesis will d e c r e a s e during p r o l o n g e d stress (Cooper and Ho 1983).

Fig. 1. Cold a c c l i m a t i o n of Arabidopsis thaliana L. A x e n i c a l l y grown 3 week old A. thaliana p l a n t l e t s were exposed to +4°C for the time periods indicated. Frost tolerance was assayed as d e s c r i b e d in M a t e r i a l s and Methods. The frost killing temperature indicates the temperature where the plants showed 50% or more electrolyte leakage. Each sample c o n t a i n e d 4-5 plantlets and each value is an average of three i n d e p e n d e n t experiments.

A n o t h e r feature of the cold induced p r o t e i n s is that most of them are induced early in the acclimation. This induction is already evident after I day of cold a c c l i m a t i o n and thus appears to precede the onset of increased frost tolerance. This lends support to the hypothesis that these proteins could a c t u a l l y be involved in the increase of frost tolerance (Weiser 1970; Guy and Haskell 1987) To rule out that the cold induced p r o t e i n s could be some kind of common stress proteins, the p l a n t l e t s were heat shocked and the c o r r e s p o n d i n g protein induction pattern c o m p a r e d to that o b t a i n e d by cold treatment (Fig. 2). The heat shock t r e a t m e n t r e s u l t e d in synthesis of heat shock proteins of 100, 90, 83, 73, 23 and 16-18 kDa. The two types of stress p r o t e i n s appear to c o n s t i t u t e clearly d i s t i n c t and separate classes as no common protein species were induced by these stress treatments. Furthermore, another d i s t i n g u i s h i n g feature b e t w e e n cold stress and heat shock is that heat shock treatment results in overall inhibition of the synthesis of normal plant proteins (Nagao et al. 1986). This b e h a v i o u r was not evident during cold a c c l i m a t i o n as most of the proteins P r e s e n t at +20°C w e r e also synthesized at +4°C. Only a limited number of protein species a p p e a r e d to be p e r m a n e n t l y labelled at a reduced level.

497

1 1

2

3

4

5

6

2

3

4

7

2q 200-

Fig. 2. A n a l y s i s of cold induced proteins. P l a n t l e t s were s u b j e c t e d to cold a c c l i m a t i o n t r e a t m e n t for I (lane 3), 2 (lane 4), 3 (lane 5), 5 (lane 6) or 7 days (lane 7). Proteins from control plants grown at +20°C are shown in lane 2 and from those e x p o s e d to heat shock t r e a t m e n t for 4h at +4O°C -,in:lane 1. The p r o t e i n s were l a b e l l e d by ~ 5 a S J - m e t h i o n i n e extracted, a n d c h a r a c t e r i z e d by electrop h o r e t i c a n a l y s i s and fluorography. A r r o w h e a d s on the right point to cold induced proteins, and those on the left to heat shock proteins. M i g r a t i o n of m o l e c u lar w e i g h t standards is i n d i c a t e d on the left.

In vitro

translation

of m R N A

In vitro t r a n s l a t i o n of poly(A+)RI]A from control and cold a c c l i m a t e d plants (24h at +4°C) i n d i c a t e d that new mRNAs were p r e s e n t (Fig. 3). A set of three new p o l y p e p t i d e s were apparent. They could c o r r e s p o n d to three d o m i n a n t new p r o t e i n s d e t e c t e d also by in v i v o l a b e l l i n g (lane 4, Mr ]50, 45 and 24 kDa). In conclusion, A. t h a l i a n a is capable of cold a c c l i m a t i o n and the r e s u l t i n g increase in frost t o l e r a n c e is c o r r e l a t e d to synthesis of new proteins. W h e t h e r any of these cold induced p r o t e i n s has a role in plant cold a c c l i m a t i o n remains to be a n a l y z e d by gene cloning and transfer.

Eig. 3. In vitro t r a n s l a t i o n p r o d u c t s of m R N A isolated from n o n - a c c l i m a t e d (lane ]) and 24h a c c l i m a t e d (lane 2) plants. Lane 3 and 4 show in vivo l a b e l l e d controls from nona c c l i m a t e d and ] day a c c l i m a t e d plants, respectively. A r r o w h e a d s indicate cold induced proteins.

Acknowledgements We thank R o s e - M a r i e A n d e r s s o n for secretarial assistance. This research was s u p p o r t e d by The F i n n i s h National Fund for R e s e a r c h and D e v e l o p m e n t (SITRA) and the Swedish N a t u r a l Sciences Research Council.

REFERENCES Chen HH, Li PH (1982) In: Li PH, Sakai A (eds) Plant cold h a r d i n e s s and f r e e z i n g stress, vol II, A c a d e m i c Press, L o n d o n and N e w York, pp 5-22 C h i r g w i n JM, P r z y b y l a AE, Mac D o n a l d RJ, R u t t e r WJ (1979) B i o c h e m i s t r y 18:5294-5299 C l o u t i e r J, S i m i n o v i t c h D (1982) Plant P h y s i o l 69:256-258 C o o p e r P, Ho T-HD (1983) Plant Physiol 71: 215-222 E s t e l l e MA, S o m e r v i l l e CR (7986) Trends in Genetics: 89-93 Guy CL, Niemi KJ, Brambl R (]986) Proc Natl A c a d Sci USA 8 2 : 3 6 7 3 - 3 6 7 7 Guy CL, Haskell D (1987) Plant Physiol 84: 872-878 K a c k p e r s k a - P a l a c z A (1978) In: Li PH, Sakai, A (eds) Plant cold h a r d i n e s s and f r e e z i n g stress, vol I, A c a d e m i c Press, L o n d o n and N e w York, pp 139-]52

498 Laemmli UK (1970) Nature 227:680-685 Levitt J (1980) Responses of plants to environmental stresses, vol I. Chilling, freezing and high temperature stresses. 2nd edn. Academic Press, London and New York Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Meza-Basso L, Alberdi M, Raynal M, FerreroCadinaros M-L, Delseny M (1986) Plant Physiol 82:733-738 Nagao RT, Kimpel JA, Vierling E, Key JL (1956) In: Miflin B (ed) Oxford surveys

of plant molecular and cellular biology, vol III, Oxford University Press, Oxford, pp 384-438 Pang PP, Meyerowitz EM (1987) Biotechnology 5:1177-1181 Sakai A, Larcher N (1987) Frost survival of plants. Responses and adaptations to freezing stress. Springer, Berlin Heidelberg New York Sukumaran NP, Weiser CJ (1972) Hortscience 7:467-468 Teeri TT, Kumar V, Lehtovaara P, Knowles J (1987) Anal Biochem 164:60-67 Weiser CJ (1970) Science 169:1269-1278