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Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues 1-4 . H 2 O 2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H 2 O 2 also exist on the cell surface remains unknown. In plant cells, H 2 O 2 triggers an influx of Ca 2+ ions, which is thought to be involved in H 2 O 2 sensing and signalling. Here, by using forward genetic screens based on Ca 2+ imaging, we isolated hydrogen-peroxide-induced Ca 2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H 2 O 2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H 2 O 2 -induced activation of Ca 2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H 2 O 2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca 2+ concentration, which activate Ca 2+ -binding proteins and upregulate the Na + /H + antiporter in order to remove Na + . Salt-induced increases in Ca 2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca 2+ -imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca 2+] i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca 2+ spikes and waves, Na + /H + antiporter activation, and regulation of growth. Na + binds to GIPCs to gate Ca 2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Salinity is one of the formidable environmental factors that affect plant growth and development and constrain agricultural productivity. Experimentally imposed short-term NaCl treatment triggers a transient increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) via Ca 2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS), such as H 2 O 2 . It is well established that short-term H 2 O 2 treatment also triggers a transient increase in [Ca 2+ ] i . However, whether and how long-term NaCl and H 2 O 2 treatments affect the basal levels of [Ca 2+ ] i as well as plant responses to additional NaCl and H 2 O 2 stresses remain poorly understood. Using an aequorin-based Ca 2+ imaging assay, we found that the long-term treatment of Arabidopsis seedlings with both moderate NaCl and H 2 O 2 in the growth media reduced the basal [Ca 2+ ] i levels. Interestingly, we found that the long-term treatment with NaCl, but not H 2 O 2, affected the responses of plants to additional NaCl stress, and remarkably the roots displayed enhanced responses while the leaves showed reduced responses. These findings suggest that plants adapt to the long-term NaCl stress, while H 2 O 2 might be an integrator of many stresses.

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca(2+)]i). Considering that in bacteria and animals osmosensing Ca(2+) channels serve as osmosensors, hyperosmolality-induced [Ca(2+)]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca(2+) channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca(2+)]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca(2+)]i increase 1 (osca1), displays impaired osmotic Ca(2+) signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca(2+)]i increases induced by a stimulus in plants, opening up new avenues for studying Ca(2+) machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2',5')pA and A(3',5')pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition., (© 2011 Blackwell Publishing Ltd.)

Two Cys residues, Cys(I) and Cys(II), are present in most plant alternative oxidases (AOXs). Cys(I) inactivates AOX by forming a disulfide bond with the corresponding Cys(I) residue on the adjacent subunit of the AOX homodimer. When reduced, Cys(I) associates with alpha-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys(I) site. Cys(II) may also be a site of AOX activity regulation, through interaction with the small alpha-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys(I) and Cys(II) confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys(I)) was limited to Cys(I). A variety of Cys(II) substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys(I), charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys(II) substitution interference with pyruvate stimulation at Cys(I), and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys(I) must be in the reduced state for activation at Cys(II) to occur.

The alternative oxidase (AOX) pathway of plant mitochondria uncouples respiration from mitochondrial ATP production and may ameliorate plant performance under stressful environmental conditions, such as cold temperatures, by preventing excess accumulation of reactive oxygen species. We tested this model in whole tissues by growing AtAOX1a-transformed Arabidopsis (Arabidopsis thaliana) plants at 12 degrees C. For the first time, to our knowledge, in plants genetically engineered for AOX, we identified a vegetative shoot growth phenotype. Compared with wild type at day 21 after sowing, anti-sense and overexpressing lines showed, on average, 27% reduced leaf area and 25% smaller rosettes versus 30% increased leaf area and 33% larger rosette size, respectively. Lines overexpressing a mutated, constitutively active AOX1a showed smaller phenotypic effects. These phenotypic differences were not the result of a major alteration of the tissue redox state because the changes in levels of lipid peroxidation products, reflecting oxidative damage, and the expression of genes encoding antioxidant and electron transfer chain redox enzymes did not correspond with the shoot phenotypes. However, the observed phenotypes were correlated with the amount of total shoot anthocyanin at low temperature and with the transcription of the flavonoid pathway genes PAL1 and CHS. These results demonstrate that (1) AOX activity plays a role in shoot acclimation to low temperature in Arabidopsis, and that (2) AOX not only functions to prevent excess reactive oxygen species formation in whole tissues under stressful environmental conditions but also affects metabolism through more pervasive effects, including some that are extramitochondrial.

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinone pool to oxygen without energy conservation. AOX can use reductant in excess of cytochrome pathway capacity, preventing reactive oxygen species (ROS) formation from an over-reduced ubiquinone pool, and thus may be involved in acclimation to oxidative stresses. The AOX connection with mitochondrial ROS has been investigated only in isolated mitochondria and suspension culture cells. To study ROS and AOX in whole plants, transformed lines of Arabidopsis (Arabidopsis thaliana) were generated: AtAOX1a overexpressors, AtAOX1a anti-sense plants, and overexpressors of a mutated, constitutively active AtAOX1a. In the presence of KCN, leaf tissue of either mutant or wild-type AOX overexpressors showed no increase in oxidative damage, whereas anti-sense lines had levels of damage greater than those observed for untransformed leaves. Similarly, ROS production increased markedly in anti-sense and untransformed, but not overexpressor, roots with KCN treatment. Thus, AOX functions in leaves and roots, as in suspension cells, to ameliorate ROS production when the cytochrome pathway is chemically inhibited. However, in contrast with suspension culture cells, no changes in leaf transcript levels of selected electron transport components or oxidative stress-related enzymes were detected under nonlimiting growth conditions, regardless of transformation type. Further, a microarray study using an anti-sense line showed AOX influences outside mitochondria, particularly in chloroplasts and on several carbon metabolism pathways. These results illustrate the value of expanding AOX transformant studies to whole tissues.

Detached roots of Poa annua were used to study alternative oxidase protein expression upon the addition of sucrose, glucose, fructose, inositol, mannitol, citrate or malate, at a concentration of 1 or 10 mM for 24 h. After 24 h the capacity of cytochrome c oxidase was decreased equally in all treatments. Only citrate induced the expression of the alternative oxidase, especially at a concentration of 1 mM (15-fold). The activity of the alternative pathway (measured with the (18)O-fractionation technique) was not affected by the addition of sucrose for 24 h as compared with time zero. However, after the addition of citrate or mannitol the activity of the alternative pathway decreased to almost zero. The discrepancy between the large increase in alternative oxidase protein concentration when citrate was applied and the concomitant decrease in alternative pathway activity is discussed.

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.

Charles F. Yocum has been a leader in the applications of biochemical techniques to the resolution and reconstitution of Photosystem II. His formal science education began as an undergraduate in biochemistry at Iowa State University and continued with graduate work in photosynthesis, first at the Illinois Institute of Technology and later at Indiana University. Following postdoctoral work at Cornell University, he joined the faculty of the University of Michigan where he has remained throughout his academic career. Charlie's contributions to a biochemical understanding of photosynthesis, particularly Photosystem II have been considerable, but most notably include his initial isolation of the first highly active oxygen-evolving particle from higher plant chloroplasts, the well-known and widely utilized 'BBY particles'. In the aftermath of that isolation, Charlie's research further resolved these particles into ever finer and simpler, but active, Photosystem II complexes. In addition, Charlie's research has provided significant insight into the roles of both Cl(-) and Ca(2+) as required cofactors in photosynthetic oxygen evolution.

The activity of the alternative pathway is affected by a number of factors, including the level and reduction state of the alternative oxidase (AOX) protein, and the reduction state of the ubiquinone pool. To investigate the significance of these factors for the rate of alternative respiration in vivo, we studied root respiration of six wild monocotyledonous grass species that were grown under identical controlled conditions. The activity of the alternative pathway was determined using the oxygen isotope fractionation technique. In all species, the AOX protein was invariably in its reduced (high activity) state. There was no correlation between AOX activity and AOX protein concentration, ubiquinone (total, reduced, or oxidized) concentration, or the reduction state of the ubiquinone pool. However, when some of these factors are combined in a linear regression model, a good fit to AOX activity is obtained. The function of the AOX is still not fully understood. It is interesting that we found a positive correlation between the activity of the alternative pathway and relative growth rate; a possible explanation for this correlation is discussed. Inhibition of the AOX (with salicylhydroxamic acid) decreases respiration rates less than the activity present before inhibition (i.e. measured with the 18O-fractionation technique).

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.

Mitochondria from all plants, many fungi and some protozoa contain a cyanide-resistant, alternative oxidase that functions in parallel with cytochrome c oxidase as the terminal oxidase on the electron transfer chain. Characterization of the structural and potential regulatory features of the alternative oxidase has advanced considerably in recent years. The active site is proposed to contain a di-iron center belonging to the ribonucleotide reductase R2 family and modeling of a four-helix bundle to accommodate this active site within the C-terminal two-thirds of the protein has been carried out. The structural features of this active site are conserved among all known alternative oxidases. The post-translational regulatory features of the alternative oxidase are more variable among organisms. The plant oxidase is dimeric and can be stimulated by either alpha-keto acids or succinate, depending upon the presence or absence, respectively, of a critical cysteine residue found in a conserved block of amino acids in the N-terminal region of the plant protein. The fungal and protozoan alternative oxidases generally exist as monomers and are not subject to organic acid stimulation but can be stimulated by purine nucleotides. The origins of these diverse regulatory features remain unknown but are correlated with sequence differences in the N-terminal third of the protein.

Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis. The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. When this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids. A Cys in the N-terminal section of the protein is responsible for both of these features. We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to alpha-keto acids. Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely. Alternative oxidase activity in fungal mitochondria was not stimulated by the alpha-keto acids pyruvate and glyoxylate. Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase. In contrast, added GMP was a strong activator of fungal alternative oxidase activity. Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences. This domain may be where dimerization of the plant enzymes occurs. In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of alpha-keto acids., (Copyright 2000 Academic Press.)

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19 degrees C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28 degrees C but was unchanged in soybean cotyledons grown at 14 degrees C compared with plants grown at 28 degrees C. When the short-term response of tissue respiration was measured over the temperature range of 35 degrees C to 9 degrees C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19 degrees C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.

On average, a doubling in current atmospheric [CO(2)] results in a 15 to 20% direct inhibition of respiration, although the variability associated with this value is large within and among species. Direct effects of CO(2) on respiration may also be relevant to tree canopies because of dynamic fluctuations between nighttime and daytime [CO(2)] throughout the growing season. The mechanism by which CO(2) inhibits respiration is not known. A doubling of ambient [CO(2)] inhibits the activity of the mitochondrial enzymes, cytochrome c oxidase and succinate dehydrogenase. If inhibition of these enzymes is the only factor involved in the direct inhibition of respiration, the overall inhibition of specific respiration will be proportional to the control that such enzymes exert on the overall respiratory rate. We analyzed the effects of [CO(2)] on respiration in an attempt to scale the direct effects of CO(2) on respiratory enzymes to the whole-plant level. Sensitivity analysis showed that inhibition of mitochondrial enzymes by doubling current atmospheric [CO(2)] does not explain entirely the CO(2) inhibition of tissue or whole-plant respiration. We conclude that CO(2)-dependent suppression of respiratory enzymatic activity will be minimal when cytochrome c oxidase inhibition is scaled up from the mitochondria to the whole tree and that the primary mechanism for the direct inhibitory effect remains to be identified.

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system.

URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea mays L.) carrying the cms-T cytoplasm. This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 contains three transmembrane alpha-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers in cms-T maize mitochondria and when expressed in Escherichia coli cells. To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking. We introduced Cys residues individually into consecutive positions 78-82, believed to be in helix III. We expressed these proteins in E. coli cells and tested for cross-linking through disulfide bond formation or by using Cys-Cys cross-linkers. URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys-Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules. Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers. Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers. Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys-Cys cross-linker) and N,N'-dicyclohexylcarbodiimide (Lys-Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.

The cyanide-resistant alternative oxidase of plant mitochondria is known to be activated by alpha-keto acids, such as pyruvate, and by the reduction of a disulfide bond that bridges the two subunits of the enzyme homodimer. When the regulatory cysteines are oxidized, the inactivated enzyme is much less responsive to pyruvate than when these groups are reduced. When soybean cotyledon mitochondria were isolated in the presence of iodoacetate or N-ethylmaleimide, the intermolecular disulfide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternative oxidase activity, but iodoacetate was found to stimulate activity much like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocked inhibition of the enzyme by N-ethylmaleimide, indicating that all three compounds acted at the same sulfhydryl group on the alternative oxidase protein. The site of pyruvate and iodoacetate action was shown to be a different sulfhydryl than that involved in the redox-active regulatory disulfide bond, because iodoacetate bound to the alternative oxidase at the activating site even when the redox-active regulatory sulfhydryls were oxidized. Given the nature of the covalent adduct formed by the reaction of iodoacetate with sulfhydryls, the activation of the alternative oxidase by alpha-keto acids appears to involve the formation of a thiohemiacetal.

Two soybean [Glycine max (L.) Merr.] lipoxygenase cDNA clones were isolated that represent lipoxygenase genes (designated LOX7 and LOX8) that display increased expression in leaves following wounding. LOX7 and LOX8 were found to be differentially expressed in soybean leaves after wounding. Increased transcript levels of both genes were detected in wounded leaves within 8 h after wounding, but only the expression of LOX7 displayed a systemic wound response. Additionally, the elevated expression of LOX7 in wounded leaves was transient. Twenty-four hours postwounding, LOX7 transcripts were no longer detectable in leaves. In contrast, LOX8 transcript levels were elevated in wounded leaves from 8 to 72 h after wounding. In addition, treatment of soybean plants with methyl jasmonate resulted in higher levels of both LOX7 and LOX8 transcripts in leaves. High levels of expression of both genes were also detected in young leaves, flowers, and immature seed pods, and increases in LOX7 and LOX8 transcripts were observed in leaves following the removal of reproductive sink tissues. The expression of LOX7 and LOX8 in unwounded soybean tissues and increased expression following wounding suggest that the lipoxygenases encoded by these genes may participate in general physiological processes that are enhanced following physical damage.

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.

A major characteristic of plant mitochondria is the presence of a cyanide-insensitive alternative oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the alternative oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the alternative oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the alternative oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.

URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis. Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation. Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E. coli cells. The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria.

Mitochondria isolated from maize containing cms-T cytoplasm are specifically sensitive to pathotoxins (T-toxins) produced by the fungi Bipolaris maydis race T and Phyllosticta maydis. T-toxins interact with a 13 kDa membrane-bound toxin receptor protein, URF13, to produce hydrophillic pores in the membrane. Expression of URF13 in Escherichia coli produces bacterial cells that form hydrophillic pores in the plasma membrane when exposed to T-toxin or methomyl. Topological studies have established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and may contribute to pore formation. URF13 specifically binds T-toxin in a cooperative manner. Oligonucleotide-directed mutagenesis of URF13 led to the isolation of methomyl/T-toxin-resistant mutations at 39 separate positions throughout the URF13 primary sequence. Chemical cross-linking of URF13 demonstrated the presence of URF13 oligomers and established that the pore-forming species is oligomeric. The ability of the carboxylate-specific reagent, dicyclohexycarbodiimide to cross-link URF13 has been used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle that may undergo a conformational change after T-toxin or methomyl binding. Experimental evidence indicates that URF13 acts as a ligand-gated, pore-forming T-toxin receptor.

The cyanide-resistant, alternative oxidase of plant mitochondria catalyzes the four-electron reduction of oxygen to water, but the nature of the catalytic center associated with this oxidase has yet to be elucidated. We have identified conserved amino acids, including two copies of the iron-binding motif Glu-X-X-His, in the carboxy-terminal hydrophilic domain of the alternative oxidase that suggest the presence of a hydroxo-bridged binuclear iron center, analogous to that found in the enzyme methane monooxygenase. Using the known three-dimensional structures of other binuclear iron proteins, we have developed a structural model for the proposed catalytic site of the alternative oxidase based on these amino acid sequence similarities.

The redox poise of the ubiquinone pool during electron transfer in isolated soybean mitochondria has been compared using two different procedures: the rapid organic extraction of ubiquinone followed by quantification of the oxidized and reduced forms using high-pressure liquid chromatography and an electrochemical technique that measures ubiquinone reduction voltametrically. The goal of these studies was to rigorously test the use of the voltametric technique to monitor the redox status of ubiquinone during the course of mitochondrial electron transfer. The linear relationship between the two methods confirms the reliability of the data obtained with the voltametric technique; however, redox inactive pools of ubiquinone were detected with the HPLC technique. We also quantified the absolute amounts of the ubiquinone homologues ubiquinone-9 and ubiquinone-10 in mitochondria isolated from different soybean tissues and compared their behavior during electron transfer in the presence and absence of pyruvate, an allosteric effector of the cyanide-resistant electron transfer pathway. Both homologues belong to a common redox-active pool in the inner mitochondrial membrane. The results indicate that ubiquinone can be a limiting component of electron transfer through the cyanide-resistant pathway, particularly in roots where its concentration is much lower than in cotyledons.

The levels of individual lipoxygenase isozymes in soybean [Glycine max (L.) Merr.] leaves were assessed during leaf development, after mechanical wounding, and in response to reproductive sink removal. Native isoelectric focusing followed by immunoblotting was employed to examine individual lipoxygenase isozymes. In leaves of all ages, two distinct classes of lipoxygenase isozymes were detected. One class of lipoxygenase isozymes had nearly neutral isoelectric points (pls) ranging from pH 6.8 to 7.2. The other class of lipoxygenase isozymes had acidic pls ranging from pH 4.7 to 5.6. During leaf development, all of the neutral lipoxygenase isozymes and most of the acidic isozymes were present in greatest abundance in the youngest leaves examined and declined in amount as leaf age increased. However, four acidic lipoxygenase isozymes (pl = 4.70, 4.80, 4.90, 4.95) were more abundant in intermediateage leaves than in either the youngest or oldest leaves examined. Following mechanical wounding of leaves, these same four acidic isozymes also increased in abundance both locally and systemically in leaves from wounded plants. Unlike the specific effects of wounding on the lipoxygenase isozymes in leaves, reproductive sink removal stimulated a general increase in most of the acidic lipoxygenase isozymes in leaves.

URF13 is a mitochondrially encoded, integral membrane protein found only in maize carrying the cms-T cytoplasm. URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B. maydis race T and P. maydis. Binding of T-toxin to URF13 leads to membrane permeability. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl. Using mutated forms of URF13 expressed in E. coli cells, we determined the molecular mechanism of DCCD protection. We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13). DCCD treatment of K37I-URF13-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E. coli or cms-T mitochondria. We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization. Our results also indicate that helices II form a central core in URF13 oligomers.

Two factors known to regulate plant mitochondrial cyanide-resistant alternative oxidase activity, pyruvate and the redox status of the enzyme's intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated alternative oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced alternative oxidase protein, a wide range of enzyme activity is possible in vivo.

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.

URF13 is a membrane protein unique to mitochondria from maize having the Texas male-sterile cytoplasm (cms-T), which is capable of permeabilizing biological membranes in the presence of a family of pathotoxins (T-toxins) produced by certain fungi or the insecticide methomyl. The carboxylate-specific reagent dicyclohexylcarbodiimide has been shown previously to protect URF13-containing membranes against the permeabilizing effects of added T-toxin or methomyl. Dicyclohexylcarbodiimide was found to covalently cross-link URF13 into higher order oligomers, including dimers, trimers, and tetramers, in isolated cms-T mitochondria and Escherichia coli cells expressing URF13. In intact E. coli cells and isolated spheroplasts, the observed protection against the effects of methomyl was not associated with the appearance of dimers but was correlated with the appearance of cross-linked trimers and tetramers. Following treatment of E. coli cells expressing URF13 with dicyclohexylcarbodiimide, the specific binding of tritiated T-toxin was reduced by 50% and all binding cooperativity was lost. A similar decrease in the level of T-toxin binding and loss of binding cooperativity were observed with site-directed, T-toxin-insensitive URF13 mutants at aspartate 39, the residue known to undergo reaction with dicyclohexylcarbodiimide. When coupled with a postulated three membrane-spanning helical model of URF13, these results provide initial insights into the intermolecular interactions involved in URF13 oligomer formation.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.

Discrimination against (18)O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%(o) for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%(o)) compared with nongreen tissues (25.3-25.9%(o)). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.