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