A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide.A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein.The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots.The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.     

Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).     

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.     

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.     

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.     

Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX.

The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis. Two previously isolated hemY mutations were sequenced. Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly. HemY was found to be a 53-kDa peripheral membrane-bound protein. In agreement with recent findings by Dailey et al. (J. Biol. Chem. 269:813-815, 1994) B. subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively. The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III. The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate. The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway. However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.     

Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes Involved in Tetrapyrrole Biosynthesis and Leads to Light-Dependent Necrosis

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterized by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(ogen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at high-light intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes in the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.     

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.     

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.