New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields. Here we show that the erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers.

Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.strigolactone | biosynthesis | cytochrome P450 | Arabidopsis | rice S trigolactones (SLs) are allelochemicals, exuded from plant roots, that stimulate seed germination of root parasitic plants, Striga spp., Orobanche spp., and Phelipanche spp. (1). The hyphal branching of the biotrophic arbuscular mycorrhizal (AM) fungi is also induced by SLs in the vicinity of host roots to ensure symbiosis with host plants (2). SLs are not only host recognition signals in the rhizosphere but also play important roles in the SLproducing plants themselves. Since the mid-1990s, the existence of novel hormone-like signals involved in shoot branching inhibition of plants had been proposed following the isolation and analysis of mutants with increased shoot branching, ramosus (rms) of pea (Pisum sativum), decreased apical dominance (dad) of petunia (Petunia hybrida), more axillary growth (max) of Arabidopsis (Arabidopsis thaliana), and dwarf (d) and high tillering dwarf (htd) of rice (Oryza sativa) (3-6). Recently, these mutants have been identified as SL-deficient or -insensitive mutants, providing decisive evidence that SLs function as shoot branchinhibiting hormones (7,8). In addition, further characterization of these mutants has shown that SLs affect root growth and development, leaf shape and senescence, internode elongation, secondary growth, and drought and salinity stress responses (9-11).Despit...

Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene ␤-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (P450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profilingbased selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of ␤-amyrin at C-11 to produce 11-oxo-␤-amyrin, a possible biosynthetic intermediate between ␤-amyrin and glycyrrhizin. CYP88D6 coexpressed with ␤-amyrin synthase in yeast also catalyzed in vivo oxidation of ␤-amyrin to 11-oxo-␤-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a ␤-amyrin 11-oxidase in the glycyrrhizin pathway.expressed sequence tag ͉ medicinal plant ͉ secondary metabolite ͉ monooxygenase ͉ isoprenoid

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton b-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding b-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-b-amyrin (a possible biosynthetic intermediate between b-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-b-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of b-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.