Plant Cell Reports

Plant Cell Reports (1988) 7:178-181

© Springer-Verlag 1988

Modulation of soluble auxin-binding proteins in soybean cell suspensions Birgit Herber 1 Bernhard Ulbrich 2 and Hans-J~rg Jacobsen 1 i Institut f/Jr Genetik, UniversiQit Bonn, Kirschallee 1, D-5300 Bonn 1, Federal Republic of Germany 2 Nattermann Research Laboratories, Postfach 35 0120, D-5000 K61n 30, Federal Republic of Germany Received December 11, 1987/Revised version received March 8, 1988 - Communicated by E. Weiler

were found by van der Linde et al. (1984), Sakai et a1.

ABSTRACT Rapidly growing c e l l suspensions of soybean were

1986, Sakai, pers. comm., Libbenga et al. 1987 and

analyzed f o r the presence of cytoplasmic h i g h - a f f i n i t y

Mayerbacher and Jacobsen-in preparation) by demonstra-

binding sites for auxin. Cytosol preparations were stu-

ting the stimulation of in v i t r o - t r a n s c r i p t i o n in iso-

died in lag , log

lated nuclei by p a r t i a l l y purified sABP~fractions with

and early stationary phase of the

growth cycle. Two binding sites were detected, which

IAA added at physiological concentrations (Io-8M).

showsome s i m i l a r i t i e s with binding sites previously

Further indication can be derived from data showing

reported from etiolated pea epicotyls. While the number

growth stage and time course specific occurrence of

of both sites declined in the cytoplasm during the

sABP in pea (Jacobsen and Hajek 1987). The present con-

growth cycle, the number of one of the two sites in-

t r i b u t i o n gives evidence for the modulation of both me

creased at the onset of rapid c e l l divisions. In par-

number of binding sites as well as the binding a f f i n i t y

a l l e l , both sites exhibited an increase in binding

in correlation to the increase of c e l l numbers of soy-

a f f i n i t y during the growth cycle.

The data w i l l be

bean c e l l suspensions.

discussed in relation to other reports on soluble

MATERIAL AND METHODS

auxin binding as well as to possible physiological functions.

AG (Monheim, FRG). The stock culture was maintained on

ABBREVIATIONS

B5-medium (Gamborg et al. 1968) with I mg/l 2,4-D at

A soybean c e l l suspension was obtained from Bayer

CF: Chromatofocusing. TCA: t r i c h l o r o a c e t i c acid. sABP:

25% in the dark on a rotary shaker at 12o rpm. Subcul-

soluble auxin-binding protein. DIECA:diethyldithiocar-

turing and propagation of c e l l material for fermentor

bamate. PEI: polyethylen-imine ( f i l t e r

assay for ligand

inoculation was every 7 days in G-medium (Ulbrich 1986)

binding). ASP:ammonium sulphate precipitation assay.

supplemented with 2 mg/l 2,4-D,o.5 mg/l NAA, o.5 mg/l

RT:number of binding sites. KD:dissociation constant.

IAA and o.4 mg/l kinetin under the same growth con-

TSBS/DMSDP:TRIS (o.1M)-sucrose(35%)-bromopheno1

d i t i o n s . Mass production of c e i l s was performed in a

blue

30 1 fermentor, equipped with a helical s t i r r i n g device

( o. o 1%)-SDS-d i th i othre i to i - 2-merc apto eth ano i -SDS-DMSOPMSF.

at Nattermann Research Laboratories, Cologne (FRG).

INTRODUCTION

Fermentation was started with an i n i t i a l

Soluble high a f f i n i t y auxin-binding proteins (sABP)

c e l l density of

2xlo5cells/ml. Samples of 5 1 each were harvested at

with putative receptor functions have been reported in

d a i l y intervals over a period of 5 days ( s t a r t of sta-

cytoplasm and nuclei of tobacco tissues (Oostrom et aI.

tionary phase), and several growth parameters were

1975, 198o, Bailey et aI. 1985, Libbenga et al. 1987),

estimated routinely by the s t a f f of Nattermann Res.

in etiolated pea epicotyls (Jacobsen 1982, 1984, Jacob-

Labs. ( c e l l number, dry weight, fresh weight and suc-

sen and Hajek 1985, Jacobsen et a l . 1487), mung bean

rose content of the medium). Medium was removed by

seedlings (Sakai and Hanagata 1983, Sakai 1984, 1985). F i r s t clues for receptor functions in some analogy to

filtration,

the model of steroid action (O'MalIey and Schrader 1976) Offprint requests to.i H.-J. Jacobsen

and the c e l l s were immediately frozen at

-142°C and stored at -25% u n t i l preparation of cytosolic proteins.

179 Extraction and preparation of cytosol

SDS-PAGE

8o g freshweight from each harvested sample were ground

Aliquots of each protein f r a c t i o n were p r e c i p i t a t e d by

in 280 ml Tris-HCI (50 mM, pH 7.8, adjusted in thecold)

20% TCA ( f i n a l volume), redissolved in TSBS/DMSDP,

using a Waring blender with Io strokes of Io s each

boiled and separated on a Io-15% SDS-gradient PAGE

and 60 s i n t e r v a l s to prevent warming up of the c e l l

according to Murray and Key (1978)

material. The r e s u l t i n g brei was f i l t e r e d through 3 layers of nylon cloth with d i f f e r e n t pore sizes (leo, 6o and Io ~m, r e s p e c t i v e l y ) . The f i l t r a t e d

homogenate

was centrifuged at 142,ooo x g f o r 2 h at 4°C inaBeck-

RESULTS The growth of soybean c e l l s in our fermenter system showed the t y p i c a l time course with respect to the parameters determined (Fig. I ) .

man L8-M u l t r a c e n t r i f u g e using the fixed-angle rotor 5o.2 T i . The supernatant (=crude cytosol) was concentrated by u l t r a f i l t r a t i o n ,

2.4 ~

and low molecular weight

!80

• & 0 0

--~ ,m -

compounds were removed by Sephadex G-25 gel f i l t r a t i o n .

cellnumber freshweioht dry#eight

5 / /

1;'

/

sucrose content

3 § m

The pooled protein f r a c t i o n s were again concentrated by ultrafiltration

and centrifuged at 7,ooo x g f o r 5 min

in a refrigerated table-top centrifuge (Sigma MK-2)

~0

1.6

/////

p r i o r to chromatofocusing. 4-5 extractions were performed from each harvested sample. 1.2

Chromatofocusing

2O

Crude cytosol proteins were separated in a preparative way by a modified chromatofocusing system (Jacobsen

0.8

$ .........

_

"~.

•""

80

5

1984). The supernatant from the f i n a l c e n t r i f u g a t i o n step Was loaded on a preparative column f i l l e d

with

PBE 94 (Pharmacia) in a pressure-resistant glasscolumn (Io9o mm x 12.7 mm, Latek, Heidelberg,FRG), e q u i l i b r a -

o.

o

ted with 25 mM imidazol-HCl, pH 7.3, and eluted with

i 0

i 1

i 2

doys of

i 3

i 4

~! 5

fermentotion

leo mM Na-cacodylate, pH 5.o. Strongly bound acidic proteins were removed from the matrix by I M NaCl in

Fig. I: Growth curve of a soybean c e l l suspension cul-

e l u t i o n buffer, pH of each f r a c t i o n was determined and

ture in a 3o I fermenter. The growth c h a r a c t e r i s t i c s

f r a c t i o n s were pooled according to s i m i l a r pH and ab-

are indicated by the increase in c e l l number, fresh

sorbance readings at 28o nm.

weight, dryweight and the decrease in sucrose content

Binding assays

of the medium.

IAA-binding a c t i v i t y was determined both by ammonium-

Separation of cytosol proteins by chromatofocusing ren-

sulphate p r e c i p i t a t i o n (ASP, Wardrop and Polya 1977)

dered f i v e main protein f r a c t i o n s (Fig. 2) according

and the P E l - f i l t e r method f i r s t

to t h e i r respective pH-ranges. Corresponding polypep-

described by Bruns et

a1. (1983) f o r steroid binding. The assays were perfor-

t i d e patterns exhibited s i g n i f i c a n t differences bet-

med with 3H-IAA from CEA, France, with s p e c i f i c a c t i v i -

ween a11 5 f r a c t i o n s (Fig. 3).

t i e s of I.o73 TBq/mmol (batch I) or o.77 TBq/mmo1(batch

.o.

2), respectively, and unlabelled auxin at d i f f e r e n t ratios.

~b

Io

'

In both assays the incubation of binding pro-

"t

7.5

teins with hormones was 6 min at O°C. Kinetic data

7,O

were calculated according to Scatchard (1949) using a computer program k i n d l y provided by A.Maan(Leiden) on

k

o

:i

o

L6,o 'o

an Apple computer. A l l data were related to I mg of protein assayed according to Bradford (1976) with lyo-

is.s

p h i l i z e d soybean cytosol proteins as a standard. The 5,0

use of homologous proteins as standard was necessary, because BSA gave completely false estimations due to a higher absorbance of the BSA-coomassie complexes at 595 nm as compared to soybean proteins.

I

~.5

fractions

Fig.2:Typica] e l u t i o n p r o f i l e of c y t o s o l i c extracts prepared at day 2 of the growth cycle a f t e r f r a c t i o n a t i o n by preparative chromatofocusing

180 b) when we calculated the number of binding sites on a per cell - level, the absolute number of sABP2_soy de-

la Ea,b lit ~a

clines during the growth cycle, while sABP1_soy declines f i r s t , but shows an increase just at the onset of rapid cell divisions at day 2 (Fig. 4). This increase in the number of binding sites is paralleled by the increase of binding a f f i n i t y by one order of magnitude.

67.000--I

Table I: Dissociation constants (KD) for h i g h - a f f i n i t y auxin-binding in fractions I l l (sABP2_soy) and IVb (sABP1_soy) during the growth cycle

25.300-days of culture

sABP1-soy S.E.M.

sABP2-soy

S.E.M.

...................................................................

14.

Fig. 3: Polypeptide patterns of fractions I-V of Fig.2

o

1.99xlo-8M *o.37xio-8 4.o25xlo'8M ~o.66xIo-8

I 2 3 4

6.1oxlo-SM 1.76xlo-9M 1.42xlo-9M 2.65xlo-9M

"1.22xio -9 ~o.52xI0-9 {o.24xIo-9 {o.89xIo-9

4.18xlo-SM 5.66 xlo'8M 1.12 xlo'8M 4.63 xlo-9M

~o.83xIo-8 ~.38xIo -8 ~o.13xIo-8 {2.96xIo -9

Both the elution p r o f i l e and the polypeptide patterns were found to be reproducible in various replications of comparable samples, but minor differences were detecteG between samples from d i f f e r e n t stages of cell suspension growth mainly between days 3 and 4, respec' t i v e l y . Only minor differences were found to the elution profiles of cytosolic extracts from pea epicotyl,

? 6

!

I s.E...

6- 0.6

o -2.8

but, as could be expected, gross differences in the polypeptide compositions of pea and soybean cells (Jacobsen 1984, Jacobsen and Hajek 1985) were observed.

0.5

~_. -2.t,

Each of the fractions I-V was assayed for the presence of h i g h - a f f i n i t y

auxin-binding proteins. In some of

-2.0

~

the experiments, onlyone of the assays was positive, but since binding assays under certain circumstances may be doubtful (Venis 1984), only those data were in-

-1.6

3 0.3

cluded, in which both independent binding assays gave clear cut results. Unequivocally, only fractions I l l (sABP2_soy; pl-range:6.4-6.3) and IVb (sABP2_soy; plrange: 5.5-5.3) showed h i g h - a f f i n i t y auxin-binding(the numeration of the binding sites is in accordance to the binding sites found in pea epicotyls, where in fraction IVb the binding appears f i r s t during germina-

-1.2

2 ,0.2 J t

-0~

I. -0.6

tion, Jacobsen et a l . , 1987). In some experiments, indications for the presence of h i g h - a f f i n i t y sites were detected in fraction IVa, but this was probably due to incomplete separation between fractions IVa and IVb. A comparison of kinetic parameters of binding in fractions I l l and IVb with the increase in cell numbers during the growth curve revealed some interesting features: a) We observed an increase in binding a f f i n i t y during the growth of the ceils for both binding sites (Tab.l),

T

T

T

3

4

days of ferrnenlotlon

Fig.4: Change in the number of binding sites (RT) present in fractions I l l (sABP2_soy) and IVb (sABP1_soy) in comparison to the growth curve of soybean cell suspensions

181 DISCUSSION

appear in the cytoplasm during stationary phase, i n d i -

A protocol s i m i l a r to that used f o r the detection of

cating a receptor function of t h i s protein in analogy

soluble h i g h - a f f i n i t y auxin-binding sites in e t i o l a t e d

to the steroid action model from animal c e l l s . Whether

pea epicotyls (Jacobsen 1984) was applied to soybean

the same phenomenon can be proved f o r soybean c e l l

c e l l suspensions. This system was chosen, because c e l l

suspensions is c u r r e n t l y under i n v e s t i g a t i o n . Also i t

suspensions represent a more homogenous c e l l population

is an open Question, whether the increase in sABP1_soy

than tissues from seedlings or mature plants. Moreover,

binding sites at the onset of c e l l d i v i s i o n s is causal

r e l a t i n g growth phenomena to changes in putative recep-

or only temporarily correlated. For an understanding of the functions of the binding s i t e s , i t is also im-

t o r c h a r a c t e r i s t i c s . We decided to grow c e l l s in a fer-

portant to know, i f there are homologies between the

mentor rather than in Erlenmeyer flasks mainly f o r

sites found in pea and in soybean c e l l suspensions in

c e l l suspensions permit a more s u i t a b l e system f o r cor-

three reasons:

rather s i m i l a r protein f r a c t i o n s . To answer t h i s and

-a minimum of 8o g of c e l l s (fresh weight) is required

other relevant questions, we c u r r e n t l y are r a i s i n g

f o r t h i s type of studies, - s t a r t i n g a fermentor with s u f f i c i e n t and constant initial

c e l l densities is less error prone than star-

t i n g various i n d i v i d u a l cultures, -control and determination of growth parameters can be performed continously and more r e l i a b l y in a fen~entor than in i n d i v i d u a l cultures. High a f f i n i t y

auxin-binding was detected in two protein

f r a c t i o n s characterized by s i m i l a r pl-ranges as i t was found in pea e p i c o t y l s (Jacobsen 1984, Jacobsen and Hajek 1985). In pea i t was observed that the occurrence of sABP appears to be growth-stage dependent and tissue s p e c i f i c (Jacobsen and Hajek 1987): Binding s i t e sABPI in pea is predcminantly localized in in internodes of e t i o l a t e d epicotyls from young seedlings, while a second s i t e (sABP2), which is not detectable in binding assays p r i o r to day nine of germination, seems to be r e s t r i c t e d to nodal tissues of the seedlings. I n soybean c e l l suspensions, we found two binding sites expressed throughout the growth cycle, which however exh i b i t e d some q u a n t i t a t i v e modulation (Tab.l, Fig.4). The f i r s t

s t r i k i n g feature is the decline of the num-

bers of both binding sites during the growth of the c e l l s : Whether t h i s decline is due to the degradation of the binding s i t e s , remains open, but one possible explanantion can be derived from data provided by Bailey et al. (1985) with tobacco c e l l suspensions: In t h i s paper i t was shown that cytoplasmic auxin-binding sites disappear from the cytoplasm during the lag phase of growth, but can be detected in the nucleus, and re-

monoclonal antibodies against the putative auxin receptors from pea. REFERENCES Bailey HM,Barker RDJ, Libbenga KR, van der Linde PCG, Mennes AM, E l l i o t t MC.(1985) Biol.P1ant. 27: Io6Io9 Bradford MM (1976) Anal.Biochem. 72:248-251 Bruns RF, Lawson-Wendling K, Pugsley TA ~1983) Anal. Biochem. 132:74-81 Gamborg OL, M i l l e r RL, Ojima K (1968) Exptl. Cell Res. 5o: 151-158 Jacobsen H-J (1982) Physiol.Plant. 56:161-167 Jacobsen H-J (1984) Plant Cell Physiol. 25:867-873 Jacobsen H-J, Hajek K (1985) B i o l . P1ant.27:11o-113 Jacobsen H-J, Hajek K (1987) Molec.Biol. of Plant Growth Control, Fox, JE, Jacobs M (eds), Alan R. Liss, I n c . , New York: 257-266 Jacobsen H-J, Hajek K, Mayerbacher R, Herber B (1987) NATO-ASI Series: Plant Hormone Receptors, Kl~mbt D (ed), Springer Verlag, Heidelberg:63-69 Libbenga KR, van Telgen HJ, Mennes AM, van der Linde PCG, van der Zaal B (1987) Molec. B i o l . of Plant Growth Control, Fox JE, Jacobs ME (eds) Alan R. Liss, Inc., New York 1987:229-243 Murray MG, Key JL (1978) Plant Physiol. 61:19o-197 O'Malley BW, Schrader WT (1976) Sci. American 234:32-43 Oostrom H, van Loopik-Detmers MA, Libbenga KR (198o) FEBS-Lett. 59:194-197 Oostrom H, Kulescha Z, van V l i e t ThB, Libbenga KR(198o) Planta 149:44-47 Sakai S, Hanagata T (1983) Plant Cell Physiol 26:685693 Sakai S (1984) Agric.Biol.Chem. 48:257-259 Sakai S, Seki J, Imaseki H (1986) P]ant Cell Physiol. 27:635-643 Scatchard G (1949) Ann. New York Acad. Sci. 51:66o-672 Ulbrich B (t986) Proc. 7th Conf. Global Impacts of Applied Microbiol. He]sinki 1985, Found. f . Biotech. and I n d u s t r i a l Ferment. Res. 4:147-164 van der Linde PCG, Bouman H, Mennes AM, Libbenga KR (1984) Planta 16o:1o2-1o8 Venis MA (1984) Planta 162:5o2-505 Wardrop AJ, Polya GM (1977) Plant Sci. Lett.8:155-163