Bipyridinium herbicides, including paraquat and diquat, are believed to act by generating highly reactive, oxygen-centered free radicals within chloroplasts when treated plants are exposed to sunlight. This hypothesis has not yet been confirmed by direct chemical measurements of specific free radicals. We studied paraquat-treated plants using a new method able to detect and quantify formation of highly reactive and deleterious hydroxyl radicals (HO*), in which dimethyl sulfoxide (DMSO) is used as a molecular probe. DMSO is oxidized by HO* to form the stable, nonradical compound, methane sulfinic acid, which can be easily extracted from plant ffssue and measured spectrophotometrically. Initial experiments revealed formation of extraordinary numbers of hydroxyl radicals in light-exposed, paraquat + DMSOtreated plants, equivalent at least to the cumulative number of HO* radicals per gram of fresh tissue that would be produced by 10,000 rads of gamma irradiation. This appears to be the greatest production of hydroxyl radicals yet observed in a biological system and is quite sufficient to explain the rapid death of top growth in paraquat-treated plants.

Summary• The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable.• Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the Nterminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2.• Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein.• This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

a-Cyclopropyl-a-Ip-methoxyphenyll-5-pyrimidine methyl alcohol (ancymidol) is an inhibitor of ent-kaur-16-ene oxidation in microsomal preparations from the liquid endosperm of immature Marab macrocarpus seeds. The Ki for this inhibitor is about 2 x 10-9 M. Ancymidol also blocks entkaur-16-en-19-ol and ent-kaur-16-en-19-al oxidation by the same preparations with a similar efficiency, but does not significantly inhibit ent-kaur-16-en-19-oic acid oxidation. Ancymidol appears to be specific for this series of oxidations in higher plant tissues. It does not inhibit the oxidation of kaurene nor kaurenoic acid in rat lver microsomes and has no significant effect on the oxidation of cinnamic acid in microsomal preparations from Sorghum bicolor seedlings. Ancymidol Ancymidol' is a substituted pyrimidine with potent growth regulatory activity in higher plants (1,10,19,22,23). The inhibition of normal growth by ancymidol can be overcome by applications of gibberellic acid (GA3) (1,10,19,23 other oxidative reactions to determine if ancymidol is a general mixed function oxidase inhibitor, or if it has some degree of specificity for the reactions of this pathway. This paper describes the effects of ancymidol on the oxidation of kaurene and some of its closely related metabolic derivatives in microsomal preparations from immature seed of Marah macrocarpus, the fungus Fusarium moniliforme, and rat liver. The effects of ancymidol on the hydroxylation ofcinnamic acid in microsomal preparations from sorghum seedlings are also reported. In addition, some evidence is presented which indicates that ancymidol interacts directly with Cyt P-450. MATERIALS AND METHODSEnzyme Sources. Immature fruits of M. macrocarpus (Greene) Greene, wild cucumber, were collected in the spring of 1976 in the Santa Monica Mountains. Immature seeds were removed, rinsed, and stored at -20 C until used. Liquid endosperm was removed from the seeds, ground in a Teflon to glass homogenizer (Thomas), and centrifuged 15 min at 10,000g. The resulting supernatant was centrifuged 90 min at 150,000g in a Beckman model Ti-60 rotor. The 150,000g pellet was then resuspended in 0.1 M Tris-HCl buffer (pH 7.5) containing 25% (v/v) glycerol. The soluble and microsomal preparations were used either directly as an enzyme source or frozen and stored in liquid N2 for future use.Sorghum bicolor (Northrup King) seeds were disinfected with 1% (v/v) NaOCl for 1 hr, thoroughly rinsed, and germinated in moist Vermiculite in the dark. The top 2 cm were harvested on the 5th day after planting and used to prepare microsomes. Approximately 20 g of leaf tissue were ground in a chilled mortar and pestle with 2 volumes of 0.15 M K-phosphate (pH 8) containing 0.35 M NaCl and 1 g of insoluble PVP (Polyclar AT). The homogenate was filtered through Miracloth and centrifuged as above for M. macrocarpus microsomes. The microsomal pellets were resuspended in 4 ml of 75 mm Tricine (pH 8), frozen, and stored in liquid N2 until used. F. moniliforme (ACC 917, M419) stock cultures were maintai...

Recent use of Sorghum bicolor as a target for grass genomics has presented new resources for gene discovery in novel metabolic pathways in Poaceae. Sorghum synthesizes a unique class of flavonoid phytoalexins, the 3-deoxyanthocyanidins, in response to fungal infection. The biosynthetic pathways for 3-deoxyflavonoids are largely uncharacterized but are known to involve transcriptional activation of chalcone synthase (CHS). CHS, or naringenin CHS, catalyzes the formation of naringenin, the precursor for different flavonoids. We have isolated seven sorghum CHS genes, CHS1 to 7, from a genomic library on high-density filters. CHS1 to 7 are highly conserved and closely related to the maize C2 and Whp genes. Several of them are also linked in the genome. These findings suggest that they are the result of recent gene duplication events. Expression of the individual CHS genes was studied in silico by examination of expressed sequence tag (EST) data available in the public domain. Our analyses suggested that CHS1-7 were not differentially expressed in the various growth and developmental conditions represented by the cDNA libraries used to generate the EST data. However, we identified a CHS-like gene, CHS8, with significantly higher EST abundance in the pathogen-induced library. CHS8 shows only 81-82% identity to CHS1 to 7 and forms a distinct subgroup in our phylogenetic analysis. In addition, the active site region contains substitutions that distinguish CHS8 from naringenin CHS. We propose that CHS8 has evolved new enzymatic functions that are involved in the synthesis of defense-related flavonoids, such as the 3-deoxyanthocyanidins, during fungal infection.

Growth of Alaska peas (Pisum satvum) is inhibited more than 60% by a-cyclopropyl-a-(p-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) treatment. This growth inhibition can be reversed completely by gibbereflic acid application. CeU-free enzyme preparations from pea shoot tips and wild cucumber (Marah oreganus) endosperm were used to test the effects of this substituted pyrimidine on the incorporation of mevalonic acid-'4C into ent-kaurene and ent-kaurenol, respectively. Ancymidol (10O M) completely blocks the conversion of ent-kaurene to ent-kaurenol. This result was confirmed with the wild cucumber endosperm system by testing the direct conversion of labeled ent-kaurene to ent-kaurenol. Ancymidol at higher concentrations (10-3 M) inhibits the incorporation of mevalonic add-'4C into ent-kaurene to a lesser extent. It is conduded that one mode of action of this growth regulator is the inhibition of gibbereilin biosynthesis.