A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography-tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered. Nearly 20% of the experimentally identified proteins are of unknown function, suggesting a wealth of undiscovered mitochondrial functions in plants. Only approximately half of the experimental set is predicted to be mitochondrial by targeting prediction programs, allowing an assessment of the benefits and limitations of these programs in determining plant mitochondrial proteomes. Maps of putative orthology networks between yeast, human, and Arabidopsis mitochondrial proteomes and the Rickettsia prowazekii proteome provide detailed insights into the divergence of the plant mitochondrial proteome from those of other eukaryotes. These show a clear set of putative cross-species orthologs in the core metabolic functions of mitochondria, whereas considerable diversity exists in many signaling and regulatory functions.

SummaryTreatment of Arabidopsis cell culture for 16 h with H 2 O 2 , menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H 2 O 2 /menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H 2 O 2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O 2 consumption by mitochondria was significantly decreased by H 2 O 2 treatment.Abbreviations: DCF-DA, 2 0 ,7 0 -dichlorofluorescin diacetate; FDA, fluorescein diacetate; MDA, malondialdehyde; TCA, tricarboxylic acid; SOD, superoxide dismutase; ROS, reactive oxygen species; PDI, protein disulphide isomerase.

Mitochondrial respiration in plants provides energy for biosynthesis, and its balance with photosynthesis determines the rate of plant biomass accumulation. We describe recent advances in our understanding of the mitochondrial respiratory machinery of cells, including the presence of a classical oxidative phosphorylation system linked to the cytosol by transporters, discussed alongside nonphosphorylating (and, therefore, non-energy conserving) bypasses that alter the efficiency of ATP synthesis and play a role in oxidative stress responses in plants. We consider respiratory regulation in the context of the contrasting roles mitochondria play in different tissues, from photosynthetic leaves to nutrient-acquiring roots. We focus on the molecular nature of this regulation at transcriptional and post-transcriptional levels that allow the respiratory apparatus of plants to help shape organ development and the response of plants to environmental stress. We highlight the challenges for future research considering spatial and temporal changes of respiration in response to changing climatic conditions.

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.

Infection of legume roots or stems with soil bacteria of the Rhizobiaceae results in the formation of nodules that become symbiotic nitrogen-fixing organs. Within the infected cells of these nodules, bacteria are enveloped in a membrane of plant origin, called the peribacteroid membrane (PBM), and divide and differentiate to form nitrogen-fixing bacteroids. The organelle-like structure comprised of PBM and bacteroids is termed the symbiosome, and is the basic nitrogen-fixing unit of the nodule. The major exchange of nutrients between the symbiotic partners is reduced carbon from the plant, to fuel nitrogenase activity in the bacteroid, and fixed nitrogen from the bacteroid, which is assimilated in the plant cytoplasm. However, many other metabolites are also exchanged. The metabolic interaction between the plant and the bacteroids is regulated by a series of transporters and channels on the PBM and the bacteroid membrane, and these form the focus of this review.

Agricultureisexpandingintoregionsthatareaffectedbysalinity.Thisreviewconsiderstheenergetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H +-ATPase also is a critical component. One proposed leak, that of Na + influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na + and Cl À concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will informexperimentationandallowaquantitativeassessmentoftheenergycostsofNaCltoleranceto guide breeding and engineering of molecular components.

l h e claim that succinate and malate can directly stimulate the activity of the alternative oxidase i n plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1 042) was reinvestigated using sweet potato (Ipomoea batatas 1.) mitochondria. I n whole mitochondria, succinate (in the presence of malonate) and both i-and D-malate stimulated respiration via alternative oxidase in a pH-(and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both i-and D-malate, although the latter at only a low rate and only at acid pH. I n submitochondrial particle preparations with negligible malic enzyme activity, neither i-nor o-malate stimulated alternative oxidase activity. However, even i n the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither i-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. I n contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 p~ in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion.We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intramitochondrial generation of pyruvate via malic enzyme.

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts ll6) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.The development of N-fixing nodules on legume roots upon invasion of Rhizobium (or Bradyrhizobium) bacteria is subject to regulation by factors both external and internal to the plant host. In particular, the extent of nodulation is restricted by a process termed autoregulation, in which the formation ofnodules on one part of the root systemically inhibits subsequent nodule formation in other root regions (3,15). Nodulation is also severely restricted by the presence of nitrate in the soil (7). Our laboratory has recently isolated several soybean mutants with altered symbiotic features, including some tolerant to nitrate (nts3) which also supernodulate (3, 4), and others which do not form any nodules (nod-) (1, 8).Clearly nodulation is subject to control by plant factors; the sites of this control are unknown, although some experimentation has implicated shoot-root interactions (1 1-13). By means of grafts between these mutant and wild-type plants, we Table I were inoculated with B. japonicum strain USDA110 (approximately 107 bacteria per plant) and were cultured in 25 cm pots filled with vermiculite:sand mixture (1:2 ratio). Glasshouse temperatures were held between 14 and 30°C and incandescent 100 W bulbs extended the photoperiod to 16 h near summer conditions). The pots received 1.2 L of nutrient solution as described by Herridge (10) three times a week (Table I