The Plant Cell, Vol. 7, 75-84. January 1995 zyxwvutsrq S 1995 American Society of Plant Physiologists Cloning and Characterization of the Maize zyx Anl Gene Robert J. Bensen,' Gurmukh zyxwvutsr S. Johal,a3' Virginia C. Crane,' John T. Tossberg,' Patrick S. Schnable,b Robert 6. Meeley,' and Steven P. Briggsav2 Pioneer Hi-Bred International, Inc., P.O. Box 1004, Johnston. lowa 50131 Department of Agronomy, lowa State University. Ames, lowa 50011 The Anther earl (Anl) gene product is involved in the synthesis of ent-kaurene, the first tetracyclic intermediate in the gibberellin (GA) biosynthetic pathway. Mutations causing the loss of Aní function result in a GA-responsive phenotype that includes reduced plant height, delayed maturity, and development of perfect flowers on normally pistillate ears. The anl::Mu2-891339 allele was recovered from a Mulator (Mu) F2 family. Using Mu elements as molecular probes, an Anl-containing restriction fragment was identified and cloned. The identity of the cloned gene as Anl was confirmed by using a reverse genetics screen for maize families that contain a Mu element inserted into the cloned gene and then by demonstrating that the insertion causes an a n l phenotype. The predicted amino acid sequence of the Anl cDNA shares homology with plant cyclases and contains a basic N-terminal sequence that may target the Aní gene product to the chloroplast. The sequence is consistent with the predicted subcellular localization of AN1 in the chloroplast and with its biochemical role in the cyclization of geranylgeranyl pyrophosphate, a 20-carbon isoprenoid, to ent-kaurene. The semidwarfed stature of a n l mutants is in contrast with the more severely dwarfed stature of GA-responsive mutants at other loci in maize and may be caused by redundancy in this step of the GA biosynthetic pathway. DNA gel blot analysis indicated that Anl is a single-copy gene that lies entirely within the deletion of the anl-bz2-6923 mutant. However, homozy- gous deletion mutants accumulated ent-kaurene to 20Oh of the wild-type level, suggesting that the function of Anl is supplemented by an additional activity. INTRODUCTION The morphological consequences of gibberellin (GA) deficien- cies vary among plant genera but typically include reduced cell elongation and ,aberr,antfloral development (Reid, 1986). The phenotype of GA-responsive mutants of maize includes reduced plant stature due to shorter internode lengths, shorter broader leaves, anda reduced number of branches in the tas- sels. In addition, anthers develop on the pistillate ear, resulting in a perfect flower in the normally pistillate dista1 floret and a staminate flower in the normally aborted proximal floret of each spikelet on the ear (Emerson and Emerson. 1922). These phenotypes are illustrated in Figures 1A to 1D. GAs are synthesized from the 20-carbon isoprenoidgeranyl- geranyl pyrophosphate(GGPP), beginning with the cyclization of GGPP to copalyl pyrophosphate (CPP) and then of CPP to enf-kaurene. This two-step process is,catalyzed by kaurene synthases A and B (previously kaurene synthetases A and zyxwvut 6). respectively (Duncan and West. 1981). In maize, and likely in most higher plants, ent-kaurene is oxidized stepwise to enf- 7\~hydroxykauren,oic,acid, which is converted to the first GA in the pathway, GAI2-aldehyde (Suzuki et al., 1992). This is then oxidized to an active GA by,o,ne of thjee parallel pathways. zyxwvuts I Current address: Department of Agronomy. University of Missouri. Columbia. MO 65211. .. To whom correspondence.should be addressed. , ,.. In maize, the major pathway appears to be the early 13- hydroxylation pathway (Hedden et al., 1982): with GA, being the primary bioactive product (Phinney and Spray, 1982). The biosynthetic block in two of the GA-responsivemutants of maize, d7 and d5, has been assigned by measuring accumu- lation of endogenous GA-biosynthetic intermediates and by observing growth responses to and metabolic tates of applied intermediates(Hedden and Phinney, 1979; Phinney and Spray. 1982; Spray et al., 1984; Fujioka et al., 1988). The biosynthetic role of a third gene, Antherearl (Anl) is less well defined. Mu- tations in An7 result in a GA-responsive phenotype that is reversiblewith applied enf-kaurene(Katsumi, 1964), suggest- ing that the An7 gene product functions in ent-kaurene synthesis. Cloning and'characterizationof Anl may clarify its function. The Mufafor (Mu) transposable.elementsystem has been used to clone many genes based only upon the phenotype of a mu- tant allele (Walbot, 1992). One drawback of transposontagging with Mu is that revertant alleles are rare. Therefore. confirm- ing that a tagged gene has been isolated typically requires characterizationof additional alleles. We have addressed this drawback by using a reverse genetics technology that permits the rapid recovery of new alleles containing Mu insertions. For Anl, this was accomplished by using a pair of polymerasechain reaction (PCR) primers, one from the terminal inverted repeat