Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria. Received: 28 January 1999 / Accepted: 29 April 1999     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts – partial purification and biochemical properties

 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn²⁺ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K _m values for Mn²⁺, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors

The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1,5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response. Received: 22 April 1999 / Accepted: 6 June 1999     

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane. Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger

The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360–440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 °C. The fraction containing the enzyme activity contained at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0. Received: 9 May 1999 / Accepted: 30 November 1999     

Identification of a signal peptide for oryzacystatin-I

 A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Received: 29 July 1999 / Accepted: 25 August 1999     

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth

Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg⁻¹ dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO₂ atmosphere (4,000 μl CO₂ l⁻¹), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO₂ at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape. Received: 14 July 2000 / Accepted: 27 August 2000     

A β-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae

 A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999     

Clp protease complexes and their diversity in chloroplasts

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95–00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing “intermediates” and the mature nClpP are found in assembled form. Received: 11 March 1998 / Accepted: 8 July 1998     

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The K _m values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg²⁺, p-chloromercuribenzoate, and diisopropylfluorophosphate. Received: 23 May 1999 / Received revision: 27 October 1999 / Accepted: 5 November 1999     

Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing

High-pressure freezing and freeze substitution were used to prepare leaves of rice (Oryza sativa L.) for ultrastructural analysis. Under these preparative conditions, plastid-derived stroma-containing protuberances were preserved and described with the electron microscope for the first time. Similar protuberances were observed previously only in living cells examined with the light microscope. Infoldings of the chloroplast inner envelope were a prominent ultrastructural feature of protuberances. Infoldings were also observed in the main body of the chloroplasts and sometimes appeared contiguous with thylakoid membranes. Protuberances also contained infoldings in the form of bifurcated tubules. Apparent interconnection between protuberances of adjacent plastids was observed only in one instance. A distinct gradient in the staining density of thylakoid lumina appeared to be a function of grana position and orientation relative to the cell wall. Immunocytochemistry was used to determine that the stroma within protuberances contained 1,5-bisphosphate carboxylase/oxygenase enzyme. Received: 21 December 1998 / Accepted: 29 January 1999     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

A simple and rapid method to gain high amounts of manganese peroxidase with immobilized mycelium of the agaric white-rot fungus Clitocybula dusenii

The agaric basidiomycete Clitocybula dusenii was used for the production of the extracellular ligninolytic enzyme, manganese (Mn) peroxidase. An immobilization technique is described using cellulose and polypropylene as carrier for the fungal mycelium. High amounts of Mn peroxidase were obtained with agitated cultures of immobilized fungus (up to 3,000 U l⁻¹) while the biomass was recovered and used for further production cycles. Purification of Mn peroxidase revealed the existence of two forms: MnP1 (molecular mass 43 kDa, pI 4.5) and MnP2 (42 kDa, pI 3.8). Received: 30 July 1999 / Received revision: 1 December 1999 / Accepted: 3 December 1999     

Cytokinin affects nuclear- and plastome-encoded energy-converting plastid enzymes

Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition of cytokinin (N⁶-Δ²-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa) were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate kinase (EC 2.7.2.3), and one was identified as the β-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed. Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts. Received: 4 July 1996 / Accepted: 4 October 1996     

Effects of Nano-anatase TiO<Subscript>2</Subscript> on Absorption,Distribution of Light,and Photoreduction Activities of Chloroplast Membrane of Spinach

The effects of nano-anatase TiO₂ on light absorption, distribution, and conversion, and photoreduction activities of spinach chloroplast were studied by spectroscopy. Several effects of nano-anatase TiO₂ were observed: (1) the absorption peak intensity of the chloroplast was obviously increased in red and blue region, the ratio of the Soret band and Q band was higher than that of the control; (2) the great enhancement of fluorescence quantum yield near 680 nm of the chloroplast was observed, the quantum yield under excitation wavelength of 480 nm was higher than the excitation wavelength of 440 nm; (3) the excitation peak intensity near 440 and 480 nm of the chloroplast significantly rose under emission wavelength of 680 nm, and F ₄₈₀ / F ₄₄₀ ratio was reduced; (4) when emission wavelength was at 720 nm, the excitation peaks near 650 and 680 nm were obviously raised, and F ₆₅₀ / F ₆₈₀ ratio rose; (5) the rate of whole chain electron transport, photochemical activities of PSII DCPIP photoreduction and oxygen evolution were greatly improved, but the photoreduction activities of PSI were a little changed. Together, the studies of the experiments showed that nano-anatase TiO₂ could increase absorption of light on spinach chloroplast and promote excitation energy to be absorbed by LHCII and transferred to PSII and improve excitation energy from PSI to be transferred to PSII, thus, promote the conversion from light energy to electron energy and accelerate electron transport, water photolysis, and oxygen evolution.     

The mus308 locus of Drosophila melanogaster is implicated in the bypass of ENU-induced O-alkylpyrimidine adducts

The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC → AT and AT → GC transitions, and AT → TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O-alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions. Received: 22 March 1999 / Accepted: 17 November 1999     

pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves

After opening the stomata in CO₂-free air, darkened leaves of several plant species were titrated with CO₂ at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO₂ for times ranging between 4.4 and 300 s, fast CO₂ release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO₂ was released with time constants of about 5, 15 and approximately 300 s. With CO₂ as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit⁻¹ in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit⁻¹, i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO₂ reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO₂-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO₂ minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO₂-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO₂ titration with and without added buffer was somewhat below 4 mM pH-unit⁻¹. Thus the apoplastic and cytosolic pH responses to additions of CO₂ indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast. Received: 27 November 1998 / Accepted: 22 March 1999