SUMMARYThe 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes -DOXA, DOXB and DOXC -based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4-hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC-class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm-specific clades were found to be involved in various lineage-specific specialized metabolisms, but 31 of the 57 DOXC-class clades were only found in a single species. Therefore, the evolution and diversification of DOXC-class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.

Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H 2 O 2 production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice.Cell death is important in the development and defense of multicellular organisms (1). Cell death occurs in normal plant development and during infection by avirulent pathogens (2-5). It plays a key role in plant defense and shares several features with apoptosis in mammalian cells (3,5,6). Rapid production of reactive oxygen species (ROS) often is associated with cell death during resistance reactions to pathogens, and a plasma membrane NADPH oxidase similar to the neutrophil enzyme is suggested to be responsible for ROS production (5-7). However, the molecular mechanisms for ROS production and cell death in plants are largely unknown.In phagocytic cells, activation of NADPH oxidase leads to production of superoxide, which effectively kills invading microorganisms (8, 9). The neutrophil NADPH oxidase is a multicomplex enzyme consisting of two membrane proteins, gp91 phox and p22 phox , and three cytosolic factors, p47 phox , p67 phox , and Rac (8, 9), and the plant enzyme is thought to be similar to the neutrophil enzyme (10-13). Genes whose deduced amino acid sequences are similar to those of the Rho͞Rac family of the small GTP-binding proteins have been isolated in plants (14-18), and some were shown to play a role in the control of pollen tube growth (19). However, functions of most of these genes are not known. Genes whose amino acid sequences are similar to that of the animal gp91 phox also have been isolated from plants (20, 21), but their functions have not been investigated. In this study we analyzed functions of Rac in rice by expressing constitutively active and dominant negative Rac genes in transgenic cell cultures and plants and found that Rac plays an important role in ROS production as well as cell death in rice. MATERIALS AND METHODSBiochemical Analysis of Recombinant OsRac1. OsRac1 cDNA was cloned in an expression vector, pGEX-4T-1 (Pharmacia), and transformed into Escherichia coli. The glutathione S-transferase (GST)-fusion protein was purified by glutathioneSepharose beads (Pharmacia) and used for assays of GTPbinding and GTPase activities according to published protocol (22). For the assay of the GTP-binding activity, the binding of Rice Transformation. OsRac1-G19V was made by substitution of the glycine corresponding to G12 of human Rac1 by valine by the use of an LA PCR in vitro Mutagenesis kit (Takara Shuzo, Kyoto). OsRac1-T24N similarly ...

Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and β-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to β-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a β-amyrin 28-oxidase able to modify β-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce β-amyrin. In addition, CYP716A12 was evaluated for its potential α-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with β-amyrin 28-oxidase, α-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.

Production of reactive oxygen intermediates (ROI) and a form of programmed cell death called hypersensitive response (HR) are often associated with disease resistance of plants. We have previously shown that the Rac homolog of rice, OsRac1, is a regulator of ROI production and cell death in rice. Here we show that the constitutively active OsRac1 (i) causes HR-like responses and greatly reduces disease lesions against a virulent race of the rice blast fungus; (ii) causes resistance against a virulent race of bacterial blight; and (iii) causes enhanced production of a phytoalexin and alters expression of defense-related genes. The dominant-negative OsRac1 suppresses elicitor-induced ROI production in transgenic cell cultures, and in plants suppresses the HR induced by the avirulent race of the fungus. Taken together, our findings strongly suggest that OsRac1 has a general role in disease resistance of rice.