Level Of mRNA Accumulation Research Articles

Expression pattern of Aux/IAA genes in the iaa3/shy2-1D mutant of Arabidopsis thaliana (L.).

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.

Tumor necrosis factor expressed by primary hippocampal neurons and SH-SY5Y cells is regulated by alpha(2)-adrenergic receptor activation.

Neuron expression of the cytokine tumor necrosis factor-alpha (TNF), and the regulation of the levels of TNF by alpha(2)-adrenergic receptor activation were investigated. Adult rat hippocampal neurons and phorbol ester (PMA)-differentiated SH-SY5Y cells were examined. Intracellular levels of TNF mRNA accumulation, as well as TNF protein and that released into the supernatant were quantified by in situ hybridization, immunocytochemistry and bioanalysis, respectively. Both neuron cultures demonstrated constitutive production of TNF. Activation of the alpha(2)-adrenergic receptor increased intracellular levels of TNF mRNA and protein in SH-SY5Y cells after addition of graded concentrations of the selective agonist, Brimonidine (UK-14304) to parallel cultures. Intracellular levels of mRNA were increased in a concentration-dependent fashion within 15 min of UK-14304 addition and were sustained during 24 hr of receptor activation. In addition, the levels of TNF in the supernatant were increased in both types of neuron cultures within 15 min of alpha(2)-adrenergic receptor activation. Furthermore, levels of TNF significantly increased in the supernatants of both neuron cultures after potassium-induced depolarization. A reduction in this depolarization-induced release occurred in hippocampal neuron cultures after exposure to the sympathomimetic tyramine with media replacement to deplete endogenous catecholamines. This finding reveals a role for endogenous catecholamines in the regulation of TNF production. Potassium-induced depolarization resulted in the release of TNF in hippocampal neuron cultures within 15 min but not until 24 hr in SH-SY5Y cultures demonstrating a temporally mediated event dependent upon cell type. Neuron expression of TNF, regulated by alpha(2)-adrenergic receptor activation demonstrates not only how a neuron controls its own production of this pleiotropic cytokine, but also displays a normal role for neurons in directing the many functions of TNF.

Circadian Clock-Regulated Expression of Phytochrome and Cryptochrome Genes in Arabidopsis

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA toPHYE) and cryptochrome (CRY1 andCRY2) promoters fused to the luciferase (LUC +) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC + constructs, with the exception ofPHYC::LUC +, display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY andCRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.

Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis.

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.

Expression of a cold-responsive Lt-Cor gene and development of freezing tolerance during cold acclimation in wheat (Triticum aestivum L.).

Time-courses of the development of freezing tolerance and the expression of a cold-responsive gene wlt10 were monitored during cold acclimation in wheat (Triticum aestivum L.). Bioassay showed that cold acclimation conferred much higher freezing tolerance on a winter cultivar than a spring cultivar. Northern blot analysis showed that the expression of wlt10 encoding a novel wheat member of a cereal-specific LT-COR protein family was specifically induced by low temperature. A freezing-tolerant winter cultivar accumulated the mRNA more rapidly and for a longer period than a susceptible spring cultivar. The increase in the amount of mRNA was temporary but the peak occurred at the time when the maximum level of freezing tolerance was attained. The mRNA accumulated more in the leaves than in the roots, and different light/dark regimes modulated the level of mRNA accumulation. Genomic Southern blot analyses using the nulli-tetrasomic series showed that the wlt10 homologues were located on the homologous group 2 chromosomes.

Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis.

The Arabidopsis KNOLLE gene encodes a cytokinesis-specific syntaxin that localises to the plane of division and mediates cell-plate formation. KNOLLE mRNA and protein expression is tightly regulated during the cell cycle. To explore the significance of this regulation, we expressed KNOLLE protein under the control of two constitutive promoters, the flower-specific AP3 and the cauliflower mosaic virus 35S promoter. The transgenic plants developed normally, although KNOLLE mRNA and protein accumulated to high levels in non-proliferating cells and protein was incorporated into membranes. Immunolocalisation studies in transgenic seedling roots revealed mistargeting of KNOLLE protein to the plasma membrane in tip-growing root hairs and in expanding root cells, whereas no mislocalisation was observed in proliferating cells. By comparative in situ hybridisation to embryo sections, the 35S promoter yielded, relative to the endogenous KNOLLE promoter, low levels of KNOLLE mRNA accumulation in proliferating cells that were insufficient to rescue cytokinesis-defective knolle mutant embryos. Our results suggest that in wild type, strong expression of KNOLLE protein during M phase is necessary to ensure efficient vesicle fusion during cytokinesis.

Origins of phytochrome-modulated Lhcb mRNA expression in seed plants.

The levels of Lhcb mRNA in higher plants are regulated by phytochrome, cryptochrome, and an endogenous circadian oscillator. To determine whether similar regulatory mechanisms operate in the ancient gymnosperm Ginkgo biloba, we measured Lhcb mRNA levels in seedlings in response to different light conditions. Removal of a diurnally oscillating light stimulus caused dampening of maximal Lhcb mRNA accumulation levels, with little change in periodicity. Although low fluence pulses of both red and blue light given to etiolated seedlings caused maximal accumulation of Lhcb mRNAs characteristic of the phasic/circadian response seen in flowering plants, the additional initial acute response seen in flowering plants was absent. The induction of Lhcb gene expression in both cases was at least partially reversible by far-red light, and appeared biphasic over a range of red fluences. Together, these data indicate that Lhcb genes in G. biloba appear to be regulated in a manner similar to that of flowering plants, whereas signaling and attenuation of mRNA levels through the photoreceptor systems and circadian clock show features distinct from those characterized to date. The implications for these findings are discussed in light of the evolution of circadian clock input signaling.

MIP genes are down-regulated under drought stress in Nicotiana glauca.

Water flux across cell membranes has been shown to occur not only through the lipid bilayer, but also through aquaporins, which are members of the major intrinsic protein (MIP) super-family of channel proteins. Aquaporins greatly increase the membrane permeability for water, but may also be regulated, allowing cellular control over the rate of water influx/efflux. Water flux is crucial for stomatal opening and closing, but little is known about the role that aquaporins play in stomatal physiology. Our initial goal was to isolate and characterize the MIP genes expressed in guard cells of the model plant, Nicotiana glauca. Degenerate oligonucleotides corresponding to amino acid sequences conserved in tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs) were used to amplify portions of MIP genes by RT-PCR. These PCR products were used as probes in screening a N. glauca guard cell cDNA library. We isolated three clones (NgMIP1, NgMIP2 and NgMIP3) homologous to TIPs and two clones (NgMIP4 and NgMIP5) homologous to PIPs. All of the MIP genes we characterized displayed highest levels of mRNA accumulation in roots or stems, with lower levels of expression in mesophyll cells and whole leaves, and lowest transcript accumulation in guard cell RNA. Interestingly, the accumulation of transcripts arising from NgMIP2, NgMIP3 and NgMIP4 diminished dramatically in drought-stressed plants. This down-regulation of MIP gene expression may result in reduced membrane water permeability and may encourage cellular water conservation during periods of dehydration stress.

Legionella pneumophila suppresses interleukin-12 production by macrophages.

In vitro infection of macrophages with Legionella pneumophila induced interleukin-1alpha (IL-1alpha), IL-10, monocyte chemotactic protein 1 (MCP-1), and MCP-3 but not IL-12. The lipopolysaccharide (LPS)-induced production of IL-12 was down-regulated by infection with virulent L. pneumophila, but other cytokines were not affected. In contrast, avirulent L. pneumophila or UV-killed, virulent L. pneumophila did not induce any suppression of IL-12. The IL-12 suppression occurred at the level of mRNA accumulation for IL-12 genes in response to LPS stimulation, but the infection induced a marked accumulation of mRNA for both MCP-1 and MCP-3, which are known to suppress IL-12 production in LPS-stimulated macrophages. However, pretreatment of macrophages with MCP-1 did not suppress LPS-induced IL-12 production at the concentrations induced by L. pneumophila infection. These results suggest that L. pneumophila selectively suppresses IL-12 production induced by LPS from macrophages in vitro by an MCP-independent mechanism.

Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities.

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.

P53-Independent Elevation of p21 Expression by PMA Results from PKC-Mediated mRNA Stabilization

The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within ∼4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.

Relation between chloroplast genome copy number, transcription, transcript abundance and translation rate in Chlamydomonas reinhardtii

In order to unveil possible relations between chloroplast genome copy number, transcription, transcript abundance and protein synthesis rates in Chlamydomonas reinhardtii, we conducted two sets of experiments. We first tried to establish a possible correlation between the genome copy number and the protein synthesis rates in the chloroplast. Using the drug 5-fluoro-deoxyuridine, we showed that a twentyfold reduction in chloroplast genome copy number, although differentially affecting mRNA accumulation levels, was not echoed by differences in chloroplast protein synthesis rates. This result implies that ploidy is not limiting for the expression of the chloroplast genome, at least for the duration of our experiment. We then tried to unveil the possible relation between overall chloroplast mRNA accumulation levels and protein synthesis rates for cells grown either under heterotrophic or phototrophic growth conditions. After six hours of treatment with rifampicin (an inhibitor of chloroplast transcription) chloroplast transcripts displayed various behaviors in regard to their accumulation levels. It also appeared that rifampicin had a more pronounced effect on chloroplast transcript accumulation under phototrophic conditions than under heterotrophic conditions. Anyhow, the changes in mRNA abundance in either growth condition were not reflected in changes in chloroplast protein synthesis rates. Our results thus show that no direct correlation between genome copy number, transcription, transcript accumulation levels and protein synthesis rates could be found in the chloroplast of Chlamydomonas reinhardtii, pointing to the existence of a multitude of posttranscriptional regulatory steps, that are essential for the expression of the chloroplast genome.

Co-suppression mediated virus resistance in transgenic tobacco plants harboring the 3'-untranslated region of Andean potato mottle virus.

Nicotiana tabacum plants were transformed with the 3'-untranslated region of the Andean Potato Mottle Virus (APMoV) genome RNA-2. Three strategies were used: the introduction of this region in sense and in antisense orientation and of a fragment comprising the entire 3'-untranslated region from RNA-2 and part of the CP22 coat protein sequence. The transgenic lines were inoculated with the virus and different responses were observed, ranging from susceptibility to APMoV to complete immunity to virus infection, in which neither the virion nor viral RNA was detected in the inoculated leaf and leaves that emerged after inoculation. The R1 progeny from different R0 lines also showed an array of virus resistance phenotypes, which was not correlated with the zygotic state of the transgene. Resistance was positively correlated with low levels of transgene mRNA accumulation, indicating a co-suppression-mediated mechanism towards the transgenic transcripts and APMoV viral RNA.

Expression of a membrane-anchored endo-1,4-beta-glucanase from Brassica napus, orthologous to KOR from Arabidopsis thaliana, is inversely correlated to elongation in light-grown plants.

A PCR fragment derived from a membrane-anchored endo-1,4-beta-glucanase cDNA was amplified using degenerated oligonucleotides and mRNA from oilseed rape (Brassica napus L.) siliques. Sequence analysis of the corresponding gene, Cel16, showed that the predicted Cel16 protein has high identity with the Arabidopsis KOR protein (94%). High-stringency genomic Southern analysis further revealed that Cel16 and KOR are most likely orthologous genes performing a similar function in both species. Northern blot and GUS analysis of transgenic Arabidopsis containing a fusion between a 2.0 kb Cel16 promoter fragment and the GUS reporter gene showed that Cel16 was expressed at a low level in the primary raceme, the young lateral stems, the elongation zone of the primary root and the older root base. By contrast, a high level of Cel16 mRNA accumulation was found in the young root and in the main stem carrying flowers and young siliques. Cel16 transcripts were localized to the apical meristem, cambium, primary xylem and cortex of oilseed rape stem tissue by in situ RT-PCR. A similar pattern of activity was found in the GUS analysis of transgenic Arabidopsis. Cel16 mRNA accumulation in the main stem was lower in the zone of most rapid cell elongation than in the subjacent, fully elongated internodes. Similarly, Cel16 transcripts accumulated to a higher level in leaves as they reached full size than during early leaf expansion. Analysis of the expression pattern in elongating, light-grown seedlings showed that Cel16 mRNA accumulated at a lower level in the elongating upper third than elsewhere in the hypocotyl. This is contrary to etiolated hypocotyls, where we found a higher expression level in the rapidly elongating upper part. This difference in expression most probably reflects a difference in cell wall assembly between light- and dark-grown seedlings.

Nonsense mutations in the human β-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation

Generally, nonsense codons 50 bp or more upstream of the 3′-most intron of the human β-globin gene reduce mRNA abundance. In contrast, dominantly inherited β-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human β-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG→TGA), 39 (C→T), or 127 (C→T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C→T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C→T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG→TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3′-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

Nonsense mutations in the human β-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation

AbstractGenerally, nonsense codons 50 bp or more upstream of the 3′-most intron of the human β-globin gene reduce mRNA abundance. In contrast, dominantly inherited β-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human β-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG→TGA), 39 (C→T), or 127 (C→T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C→T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C→T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG→TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3′-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.).

We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.

Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides.

The carB gene, encoding the phytoene dehydrogenase of Mucor circinelloides, was isolated by heterologous hybridisation with a probe derived from the corresponding gene of Phycomyces blakesleeanus. The cDNA and genomic copies complemented phytoene dehydrogenase defects in Escherichia coli and in carB mutants of M. circinelloides, respectively. Fluence-response curves for transcript accumulation were constructed after different blue-light pulses. The level of carB mRNA accumulation reached values up to 150-fold higher than basal levels in darkness. Several elements in the promoter of this gene resemble a consensus sequence identified in Neurospora crassa (APE) which is essential for blue-light regulation. Comparison of the available phytoene dehydrogenase sequences from plants, fungi, algae and bacteria suggests that the two known types of phytoene dehydrogenase are more closely related to each other than previously thought.

Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions

Cyclophilins (CyPs) are ubiquitous proteins with an intrinsic enzymatic activity of peptidyl-prolyl cis- trans isomerase that catalyzes the rotation of X-Pro peptide bonds. These enzymes are believed to play a role in the folding of certain proteins. In addition, CyPs might be important in signal transduction processes. A cDNA library was prepared from potato ( Solanum tuberosusm) tubers infected with the fungus Fusarium solani f. sp eumartii. Using a PCR-amplified subtracted cDNA probe, a clone encoding a cytosolic form of CyP, called StCyP ( S̱ olanum ṯ uberosum CyP), was isolated. Except in tubers, StCyP is expressed at high levels in tissues of healthy potato plants. Northern blot analyses revealed that both wounding and fungal infection increased the level of StCyP mRNA in tubers. However, whereas wounding causes a transient accumulation of StCyP mRNA, fungal infection results in a maintained accumulation of this transcript. StCyP mRNA accumulation is also stimulated by the application of absicic acid (ABA) and methyl jasmonate (MeJA) in tubers. Treatment with fungal elicitor or salicilic acid (SA) has no effect on the level of StCyP mRNA accumulation. Together these results indicate that the observed accumulation of StCyP mRNAs in fungal-infected potato tubers might be a response to the wound produced by the penetration and colonization of the tissue by the pathogen. Furthermore, accumulation of StCyP transcripts was also detected when the potato tubers were exposed to heat-shock treatment. These findings support a role for cyclophilins in the plant response to environmental stresses.

ADAPTATION OF SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) TO COPPER‐DEFICIENCY: TRANSCRIPTIONAL REGULATION OF PCY1 BUT NOT CPX1

In contrast to the situation in Chlamydomonas reinhardtii, plastocyanin expression in the green alga Scenedesmus obliquus (Turp.) Kütz. (Chlorophyceae) is regulated by copper ion availability at the level of both protein and mRNA accumulation. To study the mechanism of copper‐dependent mRNA accumulation, plastocyanin‐encoding genomic and cDNA clones have been isolated and sequenced. The single nuclear S. obliquus Pcy1 gene contains two introns and a bipartite transit peptide sequence typical of plastid lumen‐localized proteins. Despite the similarity of the S. obliquus and Chlamydomonas reinhardtii Pcy1 genes, the S. obliquus Pcy1 gene was not expressed from its own promoter, nor could the S. obliquus Pcy1 promoter drive expression of a C. reinhardtii gene when it was introduced into the genome of C. reinhardtii cells. Using the cDNA clone as a probe, copper‐responsive regulation of Pcy1 expression was further characterized in S. obliquus. Experiments examining the time course of Pcy1 degradation on depletion of copper argue against a specific mechanism for Pcy1 mRNA decay in S. obliquus, suggesting that the copper‐responsive change in abundance occurs through transcriptional regulation. The Cpx1 gene (encoding coproporphyrinogen III oxidase) also responds differently to changes in cellular copper status in S. obliquus compared to C. reinhardtii. Whereas Cpx1 is highly induced in copper‐deficient cultures of C. reinhardtii, in S. obliquus the Cpx1 gene is not copper responsive.