Planta (Berl.) 107, 325--340 (1972) 9 by Springer-Verlag 1972

In-vitro Auxin Binding to Particulate

Cell Fractions from Corn Coleoptiles R. Hertel, K . - S t . Thomson, a n d V. E. A. Russo Institut fiir Biologie III, Universiti~t Freiburg i. Br., BRD Received January 31/June 6, 1972 Summary. When low concentrations (e. g. 10-6 M) of labelled 3-indoleacetic acid (14C-IAA)or a-naphthaleneacetic acid (14C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent K M between 10-6 M and 10-~ M and a concentration of specific sites of 10-7--10-6 ~r per tissue volume. The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growthactive n-dichlorophenoxyisopropionic acid at 10-4 ~/~inhibits laC-NAA binding more than the less active L-isomer. The auxin-binding fractions are practically free of DlqA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis--derived mainly from physiological d a t a - that auxin receptors are localized at the plasma membrane.

Introduction

P r i m a r y h o r m o n e a c t i o n can be s t u d i e d b y a n a l y z i n g b o t h t h e e a r l y physiological effects a n d t h e p r i m a r y b i n d i n g sites (receptors). I n this p a p e r , studies on t h e in-vitro binding of t h e p l a n t h o r m o n e a u x i n are r e p o r t e d . I n t h e search for t h e a u x i n receptor, criteria d e r i v e d f r o m physiological d a t a on a u x i n effects a n d t r a n s p o r t m u s t be fulfilled. The b i n d i n g is p r o b a b l y reversible a n d n o n - c o v a l e n t (Hertel a n d F l o r y , 1968). The in-vitro binding site should be specific; i.e., o n l y g r o w t h - a c t i v e t r a n s p o r t a b l e auxins, a n d c o m p e t i t i v e a n t i a u x i n s or t r a n s p o r t i n h i b i t o r s b u t no i n a c t i v e analogues (see t t e r t e l et al., 1969; Joensson, 1961) should i n t e r a c t w i t h t h e receptor. F r o m t h e s a t u r a t i o n kinetics of a physiological a u x i n effect, e.g. g r o w t h (see t t o u s l e y , 1961), or of t h e t r a n s p o r t s y s t e m one m a y r o u g h l y e s t i m a t e a m a x i m u m affinity of a u x i n to its b i n d i n g site. E x c l u d i n g special c o m p a r t m e n t a t i o n , t h e K M of t h e binding can n o t be lower t h a n t h a t of t h e physiological effect, or, more precisely, the K M of t h e in.vitro b i n d i n g should n o t be m u c h lower t h a n t h e u p p e r range where t h e physio-

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logical effect still responds to e o n e n t r a t i o n differences. F o r the a u x i n s y s t e m a K M b e t w e e n 10 -6 a n d 5 • 10 -s M is suggested. U n f o r t u n a t e l y , no well defined zero controls are available such as m u t a n t s t h a t do n o t respond to a u x i n s a n d should, therefore, n o t b i n d auxin. The following work was based on the hypothesis t h a t a u x i n i n t e r a c t s with the p l a s m a m e m b r a n e a t least d u r i n g its t r a n s p o r t a n d p r o b a b l y also d u r i n g its action (e.g. H e r t e l a n d Flory, 1968). E v i d e n c e will be provided for specific in-vitro a u x i n b i n d i n g sites i n p a r t i c u l a t e fractions from corn coleoptiles. Materials and Methods a) Plant Material. Corn coleoptiles (Zea maya L., "Hybridmais Prior" from Badische Landwirtsehaftliche Zentralgenossenschaft, Freiburg, Germany, or, when indicated, WF9 >