Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber.

We have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development. In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes. Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media. The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate. Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL. Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase. However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL. It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.

Induction of Malate Synthase Gene Expression in Senescent and Detached Organs of Cucumber.

Expression of the malate synthase (MS) gene is activated in cotyledons of cucumber seedlings during postgerminative growth and then repressed as the cotyledons become photosynthetic. MS gene expression is subsequently reactivated in the cotyledons as they senesce a few weeks later. In situ hybridization revealed that MS RNA is distributed throughout the organ during postgerminative growth and senescence, showing that the same cells express the gene at different stages of development. MS RNA also appears in senescing leaves and petals of cucumber plants. In addition, we found that MS RNA appears in mature expanded leaves and roots when they are removed from the plant and incubated in darkness for several days, thus providing a potential experimental system for the manipulation of MS gene expression. Leaves from transgenic Nicotiana plumbaginifolia containing the cucumber MS promoter fused to the [beta]-glucuronidase (GUS) reporter gene accumulated GUS activity when detached, demonstrating an activation of transcription from the MS promoter following leaf excision. These results are discussed in terms of the metabolic regulation of MS gene expression.

Classification of normal and male-sterile cytoplasms in maize. I. Electrophoretic analysis of variation in mitochondrially synthesized proteins.

Male-sterile cytoplasms of maize have previously been classified into three groups (T, S and C) according to their fertility ratings in various inbred backgrounds. In earlier studies, mitochondria from three male-sterile cytoplasms, representing each of these three groups, have been found to synthesize characteristic variant polypeptides that distinguish them from each other and from those of normal (N) cytoplasm. In order to determine the extent of cytoplasmic variation, we have now analyzed the translation products of mitochondria from 28 additional cytoplasmic sources. The results show that on this basis 18 of the cytoplasms are identical to the USDA (S) cytoplasm, three are identical to the Texas (T) cytoplasm and two are identical to the C cytoplasm. The five remaining cytoplasms are indistinguishable from normal, male-fertile (N) cytoplasm. Our classification of the cytoplasms is in general agreement with those based on fertility restoration. However, of three cytoplasms that have previously remained unclassified, two (B and D) have now been assigned to the S group and one (LF) to the N group. No heterogeneity in mitochondrial translation products was detected within the normal or any of the three male-sterile groups. The usefulness of the analysis of mitochondrial translation products as a method for classifying normal and male-sterile cytoplasms is discussed.

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures.

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of [gamma]-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 [mu]M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 [mu]M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.

Mitochondria Increase Three-Fold and Mitochondrial Proteins and Lipid Change Dramatically in Postmeristematic Cells in Young Wheat Leaves Grown in Elevated CO2.

A dramatic stimulation in mitochondrial biogenesis during the very early stages of leaf development was observed in young wheat plants (Triticum aestivum cv Hereward) grown in elevated CO2 (650 [mu]L L-1). An almost 3-fold increase in the number of mitochondria was observed in the very young leaf cells at the base of the first leaf of a 7-d-old wheat plant. In the same cells large increases in the accumulation of a mitochondrial chaperonin protein and the mitochondrial 2-oxoglutarate dehydrogenase complex and pyruvate dehydrogenase complex were detected by immunolabeling. Furthermore, the basal segment also shows a large increase in the rate of radiolabeling of diphosphatidylglycerol, a lipid confined to the inner mitochondrial membrane. This dramatic response in very young leaf cells to elevated CO2 suggests that the numerous documented positive effects of elevated CO2 on wheat leaf development are initiated as early as 12 h postmitosis.

A cDNA encoding a putative SPF1-type DNA-binding protein from cucumber.

A cDNA clone encoding a polypeptide with homology to the novel SPF1 DNA-binding protein of sweet potato has been isolated from a cDNA library from RNA of senescing cucumber (Cucumis sativus, L.) cotyledons. Comparison of the two sequences reveals similar features which may be important in the evolution and function of this protein, including a duplicated region of about 56 amino acids (aa). The first half of the duplicated region is enriched in basic aa and is very highly conserved, both within and between each polypeptide. In contrast, the second half of the duplicated region is poorly conserved within each polypeptide, but highly conserved when cucumber and sweet potato sequences are compared. Southern blot analysis with cucumber DNA shows a simple hybridisation pattern indicating one or very few genes. Northern blot analysis shows that the expression of the cucumber gene increases in cotyledons as they expand and become photosynthetic and remains high in senescence. The possibility that the cucumber SPF1-type protein may be involved in carbohydrate regulation of gene expression is discussed.

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria.

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.

Identification of a chaperonin-10 homologue in plant mitochondria.

Chaperonin-60 and chaperonin-10 form stable binary complexes in the presence of ATP. This phenomenon has been used as the basis for the identification of a chaperonin-10 homologue in potato (Solanum tuberosum L.) mitochondria. ATP-dependent binary complexes formed between chaperonin-60 and chaperonin-10 in potato mitochondrial extracts were isolated by affinity chromatography on a column of immobilised chaperonin-60 antibodies. Partial amino acid sequence analysis of the chaperonin-10 protein indicates that it is related in primary structure to rat mitochondrial chaperonin-10.

Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants.

In mammals mitochondria play a critical role in the activation of programmed cell death (PCD). One mechanism by which mitochondria can commit a cell to death is by translocating cytochrome c into the cytosol where it activates cell death caspases. However, release of cytochrome c does not appear to be a feature of caspase activation in nematodes or insects, similarly, there is no evidence for cytochrome c release during the caspase-independent PCD that can occur in Dictyostelium cells. In an attempt to understand the underlying regulation of PCD in plants we investigated if mitochondrial components were released into the cytosol when plant cells are induced to undergo PCD. PCD was triggered in cucumber cotyledons by subjecting them to a short 55 degrees C heat treatment. This heat treatment has previously been shown to trigger PCD in other plant species and cell death was confirmed in cucumber using morphological (cellular condensation) and molecular (DNA 'laddering') markers of PCD. We present evidence that, unlike Dictyostelium and invertebrate PCDs, cytochrome c release is an early event in plant PCD. The mitochondrial release of cytochrome c following a PCD-inducing stimulus in both plants and mammals suggests the pathways have been conserved during evolution, having been derived from ancestral unicellular death programmes.

Characterisation of an Arabidopsis thaliana cDNA encoding glutathione synthetase.

An Arabidopsis thaliana cDNA (AtGSHS) encoding a protein with high primary sequence identity to cDNAs previously isolated from Xenopus laevis (42%), Schizosaccharomyces pombe (40%), Rattus norvegicus (40%) and Homo sapiens (37%) encoding glutathione synthetase (EC 6.3.2.3) has been isolated by functional complementation of an Escherichia coli mutant deficient in this enzyme. AtGSHS is encoded by a single gene, GSHB, as determined by Southern blot analysis and the corresponding mRNA is abundant in both roots and leaves of Arabidopsis.

An RNA helicase (AtSUV3) is present in Arabidopsis thaliana mitochondria.

The proteins involved in mitochondrial mRNA processing and degradation in higher plants have yet to be identified. As a first step towards this aim, we report here the characterisation of a nuclear-encoded DExH box RNA helicase (AtSUV3) localised in Arabidopsis thaliana mitochondria. The AtSUV3 mRNA is assembled from the 16 exons of a weakly expressed unique gene and the predicted protein has a calculated molecular weight of 63.6 kDa. Subcellular fractionation of transgenic plants expressing AtSUV3/GUS fusion proteins localises this protein in mitochondria. The N-terminal domain of AtSUV3 containing the motifs characteristic of DExH box RNA helicases exhibits a low endogenous ATPase activity in vitro which can be stimulated by the presence of mitochondrial RNA, confirming that AtSUV3 is an RNA helicase.

Nucleoside diphosphate kinase III is localized to the inter-membrane space in plant mitochondria.

Three types of nucleoside diphosphate kinases (NDPKs) are found in plants but the intra-cellular compartmentation of these proteins is not certain, especially the location of the recently identified type III proteins. Through the fractionation of plant mitochondria from potato and Arabidopsis, display of protein profiles by 2D gel electrophoresis, and identification by mass spectrometry, we present the first direct evidence that type III proteins are localized in the inter-membrane space of plant mitochondria. The possible metabolic functions of NDPK III are discussed in light of its sub-cellular localization.

Molecular integrity of chloroplast ribosomal ribonucleic acid.

The majority of chloroplast 1.1x10(6)-mol.wt. rRNA molecules are nicked at specific points in the polynucleotide chain, the molecules being kept intact at low temperatures by their secondary structure. Conditions that break hydrogen bonds and lead to loss of secondary structure cause dissociation of the molecule.

Programmed cell death in cell cultures.

In plants most instances of programmed cell death (PCD) occur in a number of related, or neighbouring, cells in specific tissues. However, recent research with plant cell cultures has demonstrated that PCD can be induced in single cells. The uniformity, accessibility and reduced complexity of cell cultures make them ideal research tools to investigate the regulation of PCD in plants. PCD has now been induced in cell cultures from a wide range of species including many of the so-called model species. We will discuss the establishment of cell cultures, the fractionation of single cells and isolation of protoplasts, and consider the characteristic features of PCD in cultured cells. We will review the wide range of methods to induce cell death in cell cultures ranging from abiotic stress, absence of survival signals, manipulation of signal pathway intermediates, through the induction of defence-related PCD and developmentally induced cell death.

Synthesis of a dicyclohexylcarbodiimide-binding proteolipid by cucumber (Cucumis sativus L.) mitochondria.

When isolated cucumber (Cucumis sativus L.) mitochondria were treated with (14)C-labelled dicyclohexylcarbodiimide (DCCD), a single polypeptide was predominantly labelled. This polypeptide was soluble in 1-butanol or chloroform: methanol (2: 1, v/v) and had an apparent molecular mass of approximately 7 kDa; it therefore had the characteristic properties of the DCCD-binding proteolipid subunit of the ATP synthase complexes of mitochondria, chloroplasts, and prokaryotes.When isolated cucumber mitochondria were allowed to synthesize protein in the presence of [(35)S]methionine and then extracted with 1-butanol or chloroform: methanol (2: l, v/v), a (35)S-labelled proteolipid that migrated more rapidly on SDS-polyacrylamide gels than the pro-teolipid labelled by [(14)C]DCCD was solubilized. Treatment of mitochondria with unlabelled DCCD after they had been allowed to synthesize protein, specifically converted some of the [(35)S]methionine-labelled proteolipid to a form that comigrated with the [(14)C]DCCD-labelled proteolipid. We therefore conclude that a DCCD-binding proteolipid is synthesized by isolated cucumber mitochondria.