Besides the cytochrome c pathway, plant mitochondria have an alternative respiratory pathway that is comprised of a single homodimeric protein, alternative oxidase (AOX). Transgenic cultured tobacco cells with altered levels of AOX were used to test the hypothesis that the alternative pathway in plant mitochondria functions as a mechanism to decrease the formation of reactive oxygen species (ROS) produced during respiratory electron transport. Using the ROS-sensitive probe 2,7-dichlorof luorescein diacetate, we found that antisense suppression of AOX resulted in cells with a significantly higher level of ROS compared with wild-type cells, whereas the overexpression of AOX resulted in cells with lower ROS abundance. Laser-scanning confocal microscopy showed that the difference in ROS abundance among wild-type and AOX transgenic cells was caused by changes in mitochondrial-specific ROS formation. Mitochondrial ROS production was exacerbated by the use of antimycin A, which inhibited normal cytochrome electron transport. In addition, cells overexpressing AOX were found to have consistently lower expression of genes encoding ROSscavenging enzymes, including the superoxide dismutase genes SodA and SodB, as well as glutathione peroxidase. Also, the abundance of mRNAs encoding salicylic acid-binding catalase and a pathogenesis-related protein were significantly higher in cells deficient in AOX. These results are evidence that AOX plays a role in lowering mitochondrial ROS formation in plant cells.

Plants, some fungi, and protists contain a cyanide-resistant, alternative mitochondrial respiratory pathway. This pathway branches at the ubiquinone pool and consists of an alternative oxidase encoded by the nuclear gene Aox1. Alternative pathway respiration is only linked to proton translocation at Complex 1 (NADH dehydrogenase). Alternative oxidase expression is influenced by stress stimuli-cold, oxidative stress, pathogen attack-and by factors constricting electron flow through the cytochrome pathway of respiration. Control is exerted at the levels of gene expression and in response to the availability of carbon and reducing potential. Posttranslational control involves reversible covalent modification of the alternative oxidase and activation by specific carbon metabolites. This dynamic system of coarse and fine control may function to balance upstream respiratory carbon metabolism and downstream electron transport when these coupled processes become imbalanced as a result of changes in the supply of, or demand for, carbon, reducing power, and ATP.

94305-41 O1 (S.S., C.S.)High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative fundion to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequence similarity to previously charaderized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing projeds is provided.

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an altemative pathway that terminates with an altemative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M, of 37, 36, and 35 kilodaltons (kD).Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective altemative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit altemative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of altemative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the altemative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified altemative oxidase proteins, along with the inhibition of altemative oxidase activity by these monoclonals, supports the role of these proteins in constituting the altemative oxidase.The plant mitochondrial electron transport chain consists in part of several substrate dehydrogenase complexes that reduce a common pool of the membrane lipid ubiquinone. This reduced pool of ubiquinone is then oxidized by either the classical cytochrome pathway that terminates with cytochrome oxidase or by the alternative pathway that terminates with the alternative oxidase. Both oxidases reduce oxygen to form water as a product (23). When electrons flow through the cytochrome pathway, energy is conserved in the form of an electrochemical gradient across the inner mitochondrial membrane. This gradient is then used to drive membraneassociated energy-dependent processes such as metabolite transport and ATP synthesis. In contrast, when electrons flow through the alternative pathway per se, no gradient is formed 'Supported in part by National Science Foundation postdoctoral fellowship (T. E. E.) grant DMB-8508782 and Department of Energy contract DE-AC02-76ERO-1 338.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P,, while D t gives rise to the dark-stable EPR spectrum known as signal II. D t has recently been shown to be a tyrosine radical.Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by sitedirected mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P6. to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.In plants, algae, and cyanobacteria, light initiates electron transfer reactions that generate the chemical free energy and reducing equivalents required for biosynthesis and CO2 fixation. These reactions take place in the thylakoid membrane and involve several multisubunit protein complexes including photosystems I and II, the cytochrome b6f complex, and ATP synthase (for review, see refs. 1 and 2). Photosystem II (PSII) couples light-induced charge separation with the reduction of plastoquinone and the oxidation of water (for review, see refs. 3 and 4). Molecular oxygen is released as a waste product of the water-splitting reactions. Of the seven polypeptides that comprise the minimal unit of PSII, attention has focused on two of these, D1 and D2, as binding key elements of the photochemical apparatus (5-7). These include P680, a specialized monomer or dimer of chlorophyll (Chl) that serves as the light-induced electron donor, and the pheophytin and quinone electron acceptors. The D1 and D2 polypeptides have molecular masses in the 30-kDa range and exhibit sequence similarity with each other and with the L and M subunits of reaction centers from purple nonsulfur bacteria. These similarities and the C2 symmetry present in the crystallographic structure of the reaction centers of Rhodopseudomonas viridis (8) and Rhodobacter sphaeroides (9) have led to the suggestion that a similar symmetry is present in the D1/D2 core (8,(10)(11)(12)(13).Although the precise mechanism of oxygen evolution by PSII is unknown, a cluster of four Mn atoms accumulates the oxidizing equivalents necessary for water splitting by donating electrons to the oxidized primary donor P " . These electron transfers proceed through an intermediate charge carrier usually designated as Z. The ZT species is EPR d...

Resistance of different species of weeds to s-triazines, a commonly used class of herbicides, has been shown to involve a change in the binding affinity of the herbicide to a chloroplast polypeptide of 32,000 daltons. A single amino acid difference in this 32,000-dalton protein appears to be responsible for resistance to the herbicide in Amaranthus hybridus.

Photosystem II contains two redox-active tyrosines. One of these, YZ, reduces the reaction center chlorophyll, P680, and transfers the oxidizing equivalent to the oxygen-evolving complex. The second, YD, has a long-lived free radical state of unknown function. We recently established that YD is Tyr-160 of the D2 polypeptide by site-directed mutagenesis of a psbD gene in the unicellular cyanobacterium Synechocystis 6803 [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430]. YZ is most likely the symmetry-related Tyr-161 of the D1 polypeptide. To test this hypothesis, we have changed Tyr-161 to phenylalanine by site-directed mutagenesis of a psbA gene in Synechocystis. The resulting mutant assembles PSII, as judged by its ability to produce the stable Y+D radical, but is unable to grow photosynthetically and exhibits altered fluorescence properties. The nature of the fluorescence change indicates that forward electron transfer to P+680 is disrupted in the mutant. These results provide strong support for our identification of Tyr-161 in the D1 polypeptide with YZ.

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