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We investigate the mechanical response of 50-600 nm epitaxial Ge films on a Si substrate using nanoindentation with a nominally spherical (R≈4.3 µm) diamond tip. The inelastic deformation mechanism is found to depend critically on the film thickness. Sub-100 nm Ge films deform by pressure-induced phase transformation, whereas thicker films deform only by shear-induced dislocation slip and twinning. Nanoindentation fracture response is similarly dependent on film thickness. Elastic stress modelling shows that differing stress modes vary in their spatial distribution, and consequently the film thickness governs the stress state in the film, in conjunction with the radius of the nanoindenter tip. This opens the prospect of tailoring the contact response of Ge and related materials in thin film form by varying film thickness and indenter radius.

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the gamma-glutamyl cycle, where GSH is degraded by the sequential reaction of gamma-glutamyl transpeptidase (GGT), gamma-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that gamma-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of gamma-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

Here we report on a functional gene-mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene-mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium-mediated gene transfer. A transgenic seed library consisting of >125,000 independent lines was generated and screened for salt-tolerant lines via a high-throughput genetic screen. A number of salt-tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein-encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6-66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain- and loss-of-function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene-mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous (14)C-acetate and (14)C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

Biochemical and genetic studies of xenobiotic metabolism in the model plant Arabidopsis have significant potential in providing information for phytoremediation. This paper presents the toxicity of 2,6-dinitrotoluene (2,6-DNT) to Arabidopsis under axenic conditions, the fate and transformation of 2,6-DNT after uptake by the plant, and the effect of a putative glutathione S-transferase (GST), which is highly induced by 2,4,6-trinitrotoluene (TNT) in the previous study, on the detoxification of 2,6-DNT. 2,6-DNT had toxic effects on the growth of Arabidopsis based on whole seedling as well as root growth assays. Using [U- 14C]2,6-DNT, the recovery was over 87% and less than 2% accounted for the mineralization of 2,6-DNT in axenic liquid cultures during the 14d of exposure. About half (48.3%) of the intracellular radioactivity was located in the root tissues in non-sterile hydroponic cultures. 2-Amino-6-nitrotoluene (2A6NT) and two unknown metabolites were produced as transformation products of 2,6-DNT in the liquid media. The metabolites were further characterized by proton NMR spectra and the UV-chromatograms when the plant was fed with either 2,6-DNT or 2A6NT. In addition, polar unknown metabolites were detected at short retention times from radiochromatograms of plant tissue extracts. The GST gene of the wild-type of Arabidopsis in response to 2,6-DNT was induced by 4.7-fold. However, the uptake rates and the tolerance at different concentrations of 2,6-DNT and TNT were not significantly different between the wild-type and the gst mutant indicating that induction of the GST gene is not related to the detoxification of 2,6-DNT.

gamma-Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the gamma-peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid-rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild-type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.

gamma-Glutamyl transpeptidase (GGT) is the enzyme responsible for breaking the gamma-glutamyl bond between Glu and Cys in glutathione (GSH). We are using this gene family to study GSH degradation in plants. There are four putative GGT genes in Arabidopsis, and one of them, GGT3 (At4g29210), is analyzed in this study. GGT3 is localized to the vacuole based on organelle-targeting programs, subcellular distribution of GFP fusion proteins during transient expression in onion (Allium cepa) epidermal tissues, and its ability to metabolize vacuolar substrates in Arabidopsis plants. While Northern blots and promoter:GUS expression patterns have suggested that GGT3 is transcribed at relatively high levels in all parts of the plant, a comparison of enzyme activities in different organs of wild-type and a ggt3 knockout mutant showed that GGT3 was a major contributor to total GGT activity in roots, but a relatively minor contributor in other tissues. Wild-type Arabidopsis plants treated with monobromobimane (mBB) form a fluorescent GSH-mBB conjugate that is moved into the vacuole and then metabolized to Cys-Gly-mBB and Cys-mBB in that order. The first step is catalyzed by GGT3, and GSH-mBB metabolism is completely blocked in the roots of ggt3 knockout plants. In ggt3 leaves, some GSH-mBB metabolism still proceeds using the apoplastic GGT1. This identifies GGT3 as catalyzing the obligate initial step in GSH conjugate metabolism, and suggests that it has an important role in protecting plants from some xenobiotic chemicals.

Basic knowledge of the plant transformation pathways and toxicity of 2,4-dinitrotoluene (2,4-DNT) will assist in the design and assessment of a phytoremediation strategy. This study presents the toxicity and fate of 2,4-DNT and gene expression in response to 2,4-DNT exposure using the model plant Arabidopsis thaliana, an increasingly popular system for genetic and biochemical studies of phytotransformation of explosives. From the results of biomass and root growth assays for toxicity, 2,4-DNT was toxic to the plants at concentrations as low as 1 mg/L. In the uptake study, 95% of the initial 2,4-DNT was removed by 15-day-old seedlings from liquid media regardless of the initial 2,4-DNT concentrations while 30% accounted for the adsorption to the autoclaved plant materials. The mass balance was over 86% using [U-14C]2,4-DNT, and the mineralization by the plants was less than 1% under sterile conditions during 14 days of exposure. The percentage of the bound radioactivity increased from 49% to 72% of the radioactivity in the plants, suggesting transformed products of 2,4-DNT may be incorporated into plant tissues such as lignin and cellulose. Monoaminonitrotoluene isomers and unknown metabolites with short retention times were detected as transformed products of 2,4-DNT by the plants. Most (68%) of the radioactivity taken up by the plants was in the root tissues in nonsterile hydroponic cultures. Glutathione and expression of related genes (GSH1 and GSH2) in plants exposed to 2,4-DNT were 1.7-fold increased compared to untreated plants. Genes of a glutathione S-transferase and a cytochrome P450, which were induced by 2,4,6-trinitrotoluene exposure in previous studies, were upregulated by 10- and 8-fold, respectively. The application of phytoremediation and the development of transgenic plants for 2,4-DNT may be based on TNT phytotransformation pathway characteristics because of the similar fate and gene expression in plants.

One of the key steps in developing transgenic plants for the phytoremediation of metal containing soils is to develop plants that accumulate metals in the aerial tissues. With the goal of changing the distribution of phytochelatin (PC)-dependent cadmium accumulation from roots to the leaves, the phytochelatin synthase (PCS) deficient cad1-3 mutant and wild type (Col-0) Arabidopsis plants were transformed with an Arabidopsis phytochelatin synthase (AtPCS1) under the control of a leaf-specific promoter. Three independent transformant lines from each genetic background were chosen for further analysis and designated cad-PCS and WT-PCS. PCS activity in the cadPCS lines was restored in the leaves, but not in the roots. Additionally, when whole plants were treated with cadmium, PCs were found only in the leaves of cad-PCS plants. Although the inserted AtPCS1 gene was leaf-specific, cad-PCS lines showed an overall decrease in cadmium toxicity evidenced by a partial amelioration of the "brown-root" phenotype and root growth was restored to wild type levels when treated with cadmium and arsenate. WT-PCS lines showed an increase in leaf PCS activity but had only wild type PC levels. In addition, cadmium uptake studies indicated that there was no difference in cadmium accumulation among all types tested. So, while we were able to protect the plants against cadmium by expressing PC synthase only in the leaves, we were not able to limit cadmium accumulation to aerial tissues.

Activation sequence-1 (as-1) cognate promoter elements are widespread in the promoters of plant defense-related genes as well as in plant pathogen promoters, and may play important roles in the activation of defense-related genes. The as-1-type elements are highly responsive to multiple stress stimuli such as jasmonic acid (JA), salicylic acid (SA), H(2)O(2), xenobiotics and heavy metals, and therefore provide a unique opportunity for identifying additional signaling components and cross-talk points in the various signaling networks. A single as-1-type cis-element-driven GUS reporter Arabidopsis line responsive to JA, SA, H(2)O(2), xenobiotics and heavy metals was constructed for mutagenesis. A large-scale T-DNA mutagenesis has been conducted in the reporter background, and an efficient high-throughput mutant screen was established for isolating mutants with altered responses to the stress chemicals. A number of mutants with altered stress responses were obtained, some of which appear to identify new components in the as-1-based signal transduction pathways. We characterized a mutant (Delta8L4) with a T-DNA insertion in the coding sequence of the gene At4g24275. The as-1-regulated gene expression and GUS reporter gene expression were altered in the Delta8L4 mutant, but there was no change in the expression of genes lacking as-1 elements in their promoters. The phenotype observed with the Delta8L4 mutant was further verified using RNAi plants for At4g24275 (8L4-RNAi), suggesting the feasibility of use of this high-throughput mutant screening in isolating stress-signaling mutants.

Basic knowledge of the plant transformation pathways of TNT will aid phytoremediation design and assessment. While TNT transformation by plant metabolism has been demonstrated in previous studies, the presence and role of hydroxylamines in the transformation pathway has not been sufficiently understood. Hydroxylamines are unequivocally shown to be formed by plant transformation of TNT by two axenic plant systems (Arabidopsis thaliana and Catharanthusroseus). In addition, confirmation was obtained for conversion of these hydroxylamines to previously identified conjugates. Further characteristics of TNT transformation in Arabidopsis, an increasingly popular model system for genetic and biochemical studies of TNT transformation, were elucidated by [U-14C]TNT mass balance studies and metabolite feeding studies. These studies showed the rapid conversion of TNT to unextractable bound compounds by Arabidopsis seedlings in agreement with the green-liver model. Arabidopsis seedlings formed and transformed 4-substituted metabolites more efficiently than the 2-substituted metabolites. A qualitative kinetic rate analysis of the pathway was performed to propose rate-limiting steps in the pathway and theoretical schemes for improved rates are suggested.

Reactive oxygen species (ROS), such as O2- and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2- and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Background: A new device, Intrabeam, is available for intraoperative radiotherapy. We have prospectively examined its feasibility and tolerability in delivering adjuvant breast cancer treatment., Methods: Thirty-five patients undergoing breast-conserving surgery received targeted tumour bed irradiation consisting of 5 Gy (at 10 mm) in a single fraction. This single intraoperative treatment was used to replace the external beam radiotherapy "boost" that would usually be given in 10 daily treatments following 5 weeks of whole breast irradiation. Patients later completed external beam radiotherapy as usual. Potential toxicities were prospectively assessed fortnightly prior to external beam radiotherapy, weekly during it, and 3 monthly subsequently., Results: The intraoperative radiotherapy was able to be delivered without difficulty, either at time of initial cancer surgery or as a second procedure. When performed as a separate procedure the median operating time was 56 min. The treatment was well tolerated, with only one patient experiencing any grade 3 or 4 toxicities--this was acute grade three itch. There was an overall early breast infection rate of 17%. No unexpected toxicities were seen., Conclusions: This simple and well-tolerated treatment delivers a useful radiation dose to the area of highest risk of tumour recurrence. The early infection rate is similar to that reported in the literature, for treatments without intraoperative radiotherapy. Whether such a treatment may adequately replace the entire adjuvant radiation therapy treatment for low-risk patients is now being studied in a randomized trial.

The pyruvate dehydrogenase multi-enzyme complex is the main source of acetyl-CoA formation in the plastids of plants and is composed of multiple copies of four different subunits, E1alpha, E1beta, E2, and E3. A T-DNA insertion into the gene for the plastidic E2 (dihydrolipoyl acetyltransferase) subunit, plE2, of the complex in Arabidopsis destroys the expression of that gene. The resulting mutation has no apparent phenotype in the heterozygous state, but the homozygous mutation is lethal. Haploid sperm and eggs that contain only the disrupted plE2 gene function normally resulting in the formation of an embryo that is homozygous for the mutation. This embryo only develops to an early stage before the development arrests resulting in an early embryo-lethal phenotype. While the mutation could not be complemented with the cDNA for the plE2 gene under control of the 35S, the AtSERK1, or the napin promoter, it could be complemented using the endogenous plE2 promoter to drive expression of the plE2 cDNA. This verifies the essential nature of the plastidic pyruvate dehydrogenase complex and its role in embryo formation.

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1-14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.

A tremendous amount of DNA sequence information is now available to scientists and engineers. These DNA sequences provide the foundation for studying how the genome of an organism is functioning and they are particularly useful for metabolic engineers interested in manipulating plants for the production of chemicals and enzymes. Functional genomics relies on high-throughput techniques for measuring the mRNA (the transcriptome), protein (the proteome), and metabolite (the metabolome) components of plants as well as their organs and tissues. Microarray technologies, recent advances in protein mass spectrometry, and high-throughput metabolite analyses are beginning to provide detailed information on the total mRNA, protein, and metabolite components of plants. This knowledge will allow scientists to monitor changes in proteins and metabolites in plants. Ultimately, it may allow them to discover new metabolic pathways and to model metabolic and regulatory networks in plants.

In contrast to peas (Pisum sativum), where mitochondrial lipoamide dehydrogenase is encoded by a single gene and shared between the alpha-ketoacid dehydrogenase complexes and the Gly decarboxylase complex, Arabidopsis has two genes encoding for two mitochondrial lipoamide dehydrogenases. Northern-blot analysis revealed different levels of RNA expression for the two genes in different organs; mtLPD1 had higher RNA levels in green leaves compared with the much lower level in roots. The mRNA for mtLPD2 shows the inverse pattern. The other organs examined showed nearly equal RNA expressions for both genes. Analysis of etiolated seedlings transferred to light showed a strong induction of RNA expression for mtLPD1 but only a moderate induction of mtLPD2. Based on the organ and light-dependent expression patterns, we hypothesize that mtLPD1 encodes the protein most often associated with the Gly decarboxylase complex, and mtLPD2 encodes the protein incorporated into alpha-ketoacid dehydrogenase complexes. Due to the high level of sequence conservation between the two mtLPDs, we assume that the proteins, once in the mitochondrial matrix, are interchangeable among the different multienzyme complexes. If present at high levels, one mtLPD might substitute for the other. Supporting this hypothesis are results obtained with a T-DNA knockout mutant, mtlpd2, which shows no apparent phenotypic change under laboratory growth conditions. This indicates that mtLPD1 can substitute for mtLPD2 and associate with all these multienzyme complexes.

A functional analysis of the role of glutathione in protecting plants from environmental stress was undertaken by studying Arabidopsis that had been genetically modified to have altered glutathione levels. The steady-state glutathione concentration in Arabidopsis plants was modified by expressing the cDNA for gamma-glutamyl-cysteine synthetase (GSH1) in both the sense and antisense orientation. The resulting plants had glutathione levels that ranged between 3% and 200% of the level in wild-type plants. Arabidopsis plants with low glutathione levels were hypersensitive to Cd due to the limited capacity of these plants to make phytochelatins. Plants with the lowest levels of reduced glutathione (10% of wild type) were sensitive to as little as 5 microM Cd, whereas those with 50% wild-type levels required higher Cd concentrations to inhibit growth. Elevating glutathione levels did not increase metal resistance. It is interesting that the plants with low glutathione levels were also less able to accumulate anthocyanins supporting a role for glutathione S-transferases for anthocyanin formation or for the vacuolar localization and therefore accumulation of these compounds. Plants with less than 5% of wild-type glutathione levels were smaller and more sensitive to environmental stress but otherwise grew normally.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.

We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.

Lipoamide dehydrogenase is a subunit of the alpha-ketoacid dehydrogenases and the glycine decarboxylase complex in mitochondria, and the pyruvate dehydrogenase complex in plastids. We report here the unexpected finding of two plastidic isoforms of lipoamide dehydrogenase from Arabidopsis thaliana that are different from the mitochondrial form of the enzyme. The cDNA clones were confirmed by sequence alignment analysis and their location verified by chloroplast import assay. They are single copy genes that appear to be expressed in parallel in different tissues with highest level in developing siliques. Phylogenetic analysis gives further exemplary evidence for the plastidic evolution derived from cyanobacteria.

Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.

A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.

Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H2O2, nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels.

Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.

Two cDNA clones which appear to encode different subunits of NAD(+)-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD(+)-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.

A cDNA EST clone encoding the C-terminal portion of Arabidopsis thaliana fumarase was identified by homology analysis. A fragment of cDNA encoding the N-terminal region of fumarase was amplified from a cDNA library using PCR and cloned. Genomic DNA corresponding to the coding region of fumarase was amplified and cloned. Arabidopsis fumarase was expressed as a chimeric fusion protein and polyclonal antibodies were generated. Fumarase was purified to near-homogeneity (over 600-fold) from etiolated Pisum sativum mitochondria. The identification of fumarase was confirmed by a combination of immunoblot and N-terminal amino acid sequencing. Kinetic analysis of highly purified fumarase yielded a KM(malate) of 0.45 mM and a Vmax(malate) of 650 mumol of fumarate/min/ mg. The pea fumarase was inhibited by the alpha-keto acids pyruvate and alpha-ketoglutarate at low millimolar concentrations. Adenylates were highly inhibitory; the degree of this inhibition was reduced in the presence of Mg2+, suggesting that uncomplexed adenylates are the inhibitory species.

Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S. pombe mutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombe and the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.

Glutathione synthetase catalyses the ATP-dependent ligation of gamma-glutamylcystene with glycine to form glutathione. Amino acid sequence comparisons between the Arabidopsis and the Escherichia coli proteins suggested that a region, identified as a small flexible loop that covers the active site of the E. coli protein, might be conserved in the eukaryotic protein. Three site-directed mutations in the Arabidopsis protein were generated to test this hypothesis. Two mutations within the conserved region (Lys367/ Pro368-->Asn/Ser and Gly374-->Val) inactivated the enzyme in an in vivo assay based on cadmium resistance in S. pombe, and in an in vitro assay of the activity of the enzyme expressed in E. coli. A third mutation outside of this conserved region (Leu363-->Glu) had a smaller effect in both assays. These results are consistent with the idea that this glycine-rich loop in the Arabidopsis and E. coli proteins might serve the same function in covering the active site of the enzyme.

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated from Arabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast, Schizosaccharomyces pombe, and shared only a small region of similarity with the Escherichia coli protein. A 4.3 kb SstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons. When the Arabidopsis cDNA cloned in a special shuttle vector was expressed in a S. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in the gsh2- mutant, and restored to substantial levels by the expression of the Arabidopsis cDNA. The S. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols, 109Cd2+ binding activity, and cadmium resistance. Since the Arabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.

The four component proteins of the glycine decarboxylase multienzyme complex (the P-, H-, T-, and L-proteins) comprise over one-third of the soluble proteins in mitochondria isolated from the leaves of C3 plants. Together with serine hydroxymethyltransferase, glycine decarboxylase converts glycine to serine and is the site of photorespiratory CO2 and NH3 release. The component proteins of the complex are encoded on nuclear genes with N-terminal presequences that target them to the mitochondria. The isolated complex readily dissociates into its component proteins and reassociates into the intact complex in vitro. Because of the intimate association between photosynthesis and photorespiration, the proteins of the complex are present at higher levels in leaves in the light. The expression of these genes is controlled at the transcriptional level and the kinetics of expression are closely related to those of the small subunit of Rubisco. Deletion analysis of fusions between the promoter of the H-protein of the complex and the reporter gene beta-glucuronidase in transgenic tobacco has identified a region responsible for the tissue specificity and light dependence of gene expression. Gel shift experiments show that a nuclear protein in leaves binds to this region. Glycine decarboxylase has proven to be an excellent system for studying problems in plant biochemistry ranging from protein-protein interactions to control of gene expression.

Premenopausal patients undergoing surgery for breast cancer were prospectively studied. Data regarding menstrual history, pathological parameters and hormone receptor status were collected. Serum oestradiol, prolactin and progesterone levels, tumour epidermal growth factor receptor (EGFR) levels, tumour epidermal growth factor (EGF) levels and flow cytometry were measured. Patients were allocated to the follicular or luteal phase of their cycle both by history and progesterone level. No significant differences were seen in hormone receptor levels, pathological parameters or EGF levels between the two groups. EGFR levels were significantly higher in women undergoing surgery during the follicular phase of their cycle, when classified by menstrual history. Patients operated on during this phase have previously been found to have a poorer prognosis, and these results may provide a basis for this finding. This may have implications for prognosis and timing of surgery, and further investigation is warranted.

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [(14)C]citrate for internal citrate or malate but not for preloaded d,l-isocitrate. Similarly, although external malate, succinate, and citrate competed with [(14)C]citrate in the exchange reaction, d,l-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less active than the transporter isolated from animal tissues.

The NAD(+)-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 +/- 200, 690 +/- 90, and 300 +/- 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 +/- 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP(+)-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 +/- 10 kD. About half of the NAD(+) and NADP(+)-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD(+)-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S(0.5) = 0.3 mm). When the enzyme was aged at 4 degrees C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD(+) isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD(+) (K(m) = 0.2 mm). NADH was a competitive inhibitor (K(i) = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K(i) = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.

The experience of the use of the TRC-Mammotest machine for stereotactic fine needle biopsy (SFNB) of the breast in Perth is presented. During the period 20 October 1988 to 10 January 1990, 404 SFNB were performed on 389 women with impalpable, mammographically detected lesions of the breast. Surgical biopsy was performed in 73 cases, of which 38 were malignant, giving a benign to malignant ratio in less than 1:1. The sensitivity for detecting cancer was 95%, with a positive predictive value of 100%. Using a combination of the mammographic and cytologic rating for the likelihood of cancer, all the cancers were detected and no cancers have developed in those considered mammographically and cytologically benign. The importance of a combined assessment of mammography and cytology in the management of patients with mammographically detected abnormalities is stressed.

We report two cases of villous adenoma of the duodenum, one arising from the main papilla and the other from the accessory papilla. Both were managed by local resection. In one case endoscopic biopsies and intraoperative frozen sections were negative for carcinoma but histology of the locally resected specimen revealed a focus of invasive adenocarcinoma. Villous adenomas of the duodenum have a high risk of malignant change and foci of carcinoma can be missed on endoscopic biopsy. The literature is reviewed and the clinical, diagnostic, pathological and therapeutic aspects of villous adenomas of the duodenum are discussed.

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. When the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex.

The glycine decarboxylase multienzyme complex is located in the mitochondrial matrix and catalyzes a key reaction of the photorespiratory C-2 cycle of C3 plants. The cDNA encoding the smallest subunit, the 13,900-dalton H-protein, of the glycine decarboxylase complex from pea (Pisum sativum) leaves was cloned, identified, characterized, and sequenced. The 678-nucleotide sequence contained a 495-nucleotide open reading frame capable of encoding the 165-amino-acid H-protein precursor. The N terminus of this protein contains a 34-amino-acid sequence which does not appear in the mature protein. This presequence resembles the amphiphilic helices observed with mitochondrial leader sequences in yeast. The 131-amino-acid mature protein from peas shares substantial homology with the enzyme isolated from chicken liver. The abundance of the H-protein mRNA was about 5-fold greater in light-grown pea seedlings compared to dark-grown seedlings. The amount of H-protein transcript increased within 4 h after the plants were transferred to white light and continued to increase up to 24 h. The time course for the accumulation of the H-protein mRNA was similar to that for the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase.

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic (14)CO(2) fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O(2) level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O(2) concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO(2) per gram fresh weight per hour (a 60% increase) when the O(2) level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO(2) per gram fresh weight per hour (a 22% increase). The decrease in the O(2) sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O(2) concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO(2) fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

Monoclonal antibodies specific for the dicarboxylate transporter of pea mitochondria were prepared and used to identify this substrate carrier. The hybridoma library was derived from mice that had been immunized with total mitochondrial membranes. The monoclonal antibodies specific for the dicarboxylate transporter were selected by screening hybridoma supernatants for their ability to inhibit malate- and succinate-dependent oxalacetate reduction by osmotically shocked pea leaf mitochondria. Three monoclonal antibodies were shown to be specific for the dicarboxylate transporter by their ability to 1) inhibit malate and succinate metabolism without affecting alpha-ketoglutarate, citrate, pyruvate, glycine, glutamate, or aspartate metabolism by mitochondria and 2) inhibit malate uptake by a partially purified transporter fraction reconstituted into asolectin vesicles. The dicarboxylate transporter was identified by Western blotting and immunoprecipitation and had an apparent molecular mass of 26,000 Da. The techniques described should prove useful for identifying a number of membrane proteins that can be assayed in situ but are difficult to assay following dissolution of the membrane.