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Plant Molecular Biology 15: 513-516, 1990. © 1990 Kluwer Academic Publishers. Printed in Belgium. 513 Plant Molecular Biology Update RFLP mapping of the abpl locus in maize (Zea mays L.) Marian L6bler and Ann M. Hirsch University of California Los Angeles, Department of Biology, 405 Hilgard Ave, Los Angeles, CA 90024, USA Received and accepted 12 June 1990 The plant hormone auxin controls a wide variety of growth and differentiation processes in higher plants. The molecular mechanism by which this control is exerted is essentially unknown. Auxin binds specifically to an auxin-binding protein (ABP) which is thought to be the first element in a signal transduction pathway [7]. However, the other components of an auxin-activated signal transduction pathway, which lead to the biological response, have not been identified. If mutations in the presumed signal transduction pathway could be obtained, they would be extremely useful in elucidating the molecular mechanism of auxin action. Auxin biosynthesis and auxin response mutants have been described in some dicotyledonous plants [6, 9]. That such mutants are found at frequencies 10-100 times lower than null mutants of other loci [3, 6], e.g. starch or lipid biosynthesis [6], strongly suggests that null mutants related to auxin physiology are lethal [8]. The mutants that have been characterized show pleiotropic effects and hence a variety ofphenotypes. By determining the genetic locus of at least one component of the auxin signal transduction pathway, we should be able to characterize auxin response phenotypes better. This will allow us to look for similar phenotypes with the goal of finding mutants in the signal transduction pathway. We have used RFLP mapping (restriction fragment length polymorphism mapping) to assign a locus to the ABP, the putative first element in the auxin signal transduction pathway. Maize recombinant inbred lines from the F2 populations of TXCM as well as the parental lines T232 and CM37 [ 1] were used to prepare genomic D N A from leaves. The isolation of an ABP c D N A clone (pBS.ABP) is described elsewhere [ 10]. All restriction enzymes and the nick translation kit were purchased from Bethesda Research Laboratory, Bethesda, MD. D N A was extracted from young leaves (approx. 1 g of tissue) following a procedure of Cone [2] with some modifications. In brief, leaf tissue frozen on dry ice at the time of harvest was homogenized in liquid nitrogen with pestle and mortar. The resulting fine powder was added to 5 ml homogenization buffer (7 M urea, 0.35 M NaC1, 0.05 M EDTA, 0.01 M TrisHC1 pH 7.6, 2% sodium lauroylsarcosine) in a glass scintillation vial and rocked on a shaker until it appeared homogeneous (ca 15 min). An equal volume of phenol, equilibrated with homogenization buffer, was added and again shaken until apparent homogeneity (ca 15 min). The highly viscous slurry was centrifuged and the aqueous phase was extracted with an equal volume of chloroform : isoamyl alcohol (24 : 1). D N A was precipitated by adding sodium acetate (final concentration 0.2 M) and 0.55 volumes of isopropanol. High molecular weight D N A was spooled out with a bent pasteur pipette and resuspended in 4 ml TE (10 m M Tris-HCl pH 8, 1 mM EDTA). A final ethanol precipitation in the presence of 2 M ammonium acetate yielded a nucleic acid preparation that could be digested with restriction enzymes. Restriction digests were performed according to manufacturer's protocols using 1.5 ~g of D N A and 20 units of enzyme for a 3 h incubation time at the appropriate temperature. Restriction fragments were separated on 0.8~ agarose gels and blotted onto nitrocellulose (Schleicher & Schuell) 514 Fig. 1. Southern blot of maize genomic D N A from the parental lines T232 and CM37 indicated by T and CM respectively. The restriction enzymes used are indicated above the lanes. Nick translated ABP c D N A was used as a hybridization probe. Fig. 2. Southern blot of maize genomic Sst I D N A fragments from the recombinant inbred F2 families (TXCM) and the parental lines T232 and CM37. Nick translated ABP c D N A was used as a hybridization probe. Note that the observed hybridization pattern of the recombinant inbreds matches either one of the parental lines (T232 or CM37). 515 or GeneScreen (BioRad) membranes according to manufacturers' protocols. Blots were hybridized to a nick-translated probe of pB S.ABP insert overnight at 37 °C in the presence of 50~o formamide. After the blots had been washed with 0.2 × SSC (30 mM NaCI, 3 mM sodium citrate) containing sodium lauroylsarcosine (0.05 ~o) and pyrophosphate (0.01 ~o) at 55 °C, they were exposed to X-ray film for 16 h. Nick translation was performed according to the protocol provided by BRL. For the Southern hybridizations we followed the protocols recommended by the manufacturers of the transfer membranes. B. Burr analyzed the RFLP data by comparison to the RFLP database for maize [ 1]. In the homozygous parental lines T232 and CM37, one or two genomic restriction fragments hybridize to the ABP cDNA (Fig. 1), suggesting that there is only a single gene for the auxinbinding protein in maize. On a Southern blot developed under low stringency conditions, no additional bands specific for the ABP can be detected (data not shown). Of the restriction enzymes used to digest the genomic DNA, only Eco RI cuts the ABP cDNA, yielding two fragments similar in size [10]. When genomic DNA is probed, we detect only one Eco RI restriction fragment which hybridizes to the ABP cDNA (Fig. 1). The signal intensity of this particular band is only half of the signal intensity of other restriction fragments (Hind III and Kpn I). We therefore conclude that one half of the ABP gene is digested by Eco RI to very short fragments which are not contained on the blot. Because only one Eco RI fragment and two restriction fragments of the Barn HI, Bgl II, Sca I, Sph I and Sst I digests of the genomic DNA hybridize to the ABP cDNA (Fig. 1) we conclude that introns are present in the abpl gene. A restriction fragment length polymorphism (RFLP) was found between the parental lines T232 and CM37 in the restriction digests Hind III, Sca I, Sph I, and Sst I (Fig. 1). Because the Sst I RFLP could be scored most easily, Sst I was chosen to digest DNA from the recombinant inbreds derived from the F2 generation. Southern hybridization with the ABP cDNA probe revealed the RFLP pattern shown in Fig. 2. A locus was assigned to the ABP cDNA clone by computer analysis of the data presented in Fig. 2. It maps to the long arm of chromosome 3 at position 70. We name this locus abpl for several reasons. First, the analysis of the cDNA clones isolated by different groups [4, 5, 10] reveals that the ABP is a soluble protein and lacks transmembrane domains as one would expect for a receptor protein. Second, the publications concerning the ABP cDNA have used ABP to name the isolated clones [5, 10], with the exception of Hesse et al. [4] who used axr-1. Third, we wish to avoid confusion with auxin resistance genes (,4xr-1) described by Estelle and Sommerville [3]. The abpl locus lies within a chromosomal region that is rich in genetically characterized develop- m 20 e8 8,15 . 8.35 61 - 71 -- ¢% p d g 2 ~ -- tpi4 6.06 abpl 5,37 8.01 10.24A -"" 138 141 15.20 6.16 3.18 1.297 mdh3 --- al -- npi425 Fig. 3. Maize c h r o m o s o m e 3 with R F L P marker locations and the abpl locus (triangle). The c h r o m o s o m e m a p was r e d r a w n from reference 1. 516 mental mutants [ 11]. However, we do not know if the abpl locus is identical to any of the known genetic marker loci. 4. Acknowledgements We would like to express our gratitude to R.J. Schmidt from UC San Diego who let us pick leaf samples from the maize genotypes used in this study, to B. Burr from Brookhaven Natl. Laboratory who compared the RFLP data with the database, and to B.O. Phinney and S. Kirchanski for critically reading the manuscript. This work was supported by a N S F grant to A.M.H. (DCB 8703297). 5. 6. 7. 8. References 1. Burr B, Burr FA, Thompson KH, Albertsen MC, Stuber CW: Gene mapping with recombinant inbreds in maize. Genetics 118:519-526 (1988). 2. Cone K: Yet another rapid plant DNA prep. Maize Genetics Cooperation News Letter 63:68 (1989). 3. Estelle MA, Sommerville C: Auxin-resistant mutants of 9. 10. 11. Arabidopsis thaliana with an altered morphology. Mol Gen Genet 206:200-206 (1987). Hesse T, Feldwisch J, Balsh~isemann D, Bauw G, Puype M, Vandekerckhove J, L6bler M, Kl~tmbt D, Schell J, Palme K: Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin. EMBO J 8:2453-2461 (1989). Inohara N, Shimomura S, Fukui T, Futai M: Auxinbinding protein located in the endoplasmic reticulum of maize shoots: Molecular cloning and complete primary structure. Proc Natl Acad Sci USA 86:3564-3568 (1989). Last RL, Fink GR: Tryptophan-requiringmutants of the plant Arabidopsis thaliana. Science 240:305-310 (1988). L6bler M, Kl~imbt D: Auxin-binding protein from coleoptile membranes of corn (Zea mays L.) II. Localization of a putative auxin receptor. J Biol Chem 260: 9854-9859 (1985). Mirza JI, Maher EP: More 2,4-D resistant mutants. Arabidopsis Inf Serv 17:103-107 (1980). Scott IM: Plant hormone response mutants. Physiol Plant 78:147-152 (1990). Tillmann U, Viola G, Kayser B, Siemeister G, Hesse T, Palme K, L6bler M, Kl~mbt D: cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods. EMBO J 8:2463-2467 (1989). Maize Genetics Cooperation Newsletter 64 (1990).