Chloroplast nucleoids in large number and large DNA amount with regard to maternal inheritance inChlamydomonas reinhardtii

Summary We studied the maternal chloroplast inheritance ofChlamydomonas reinhardtii by epifluorescence microscopy after staining with DNA specific fluorochrome DAPI and by genetic methods, using wild type cells and cells containing previously isolated mutation of cond-1 and cond-2. Wild type cells contained about 7 chloroplast (cp) nucleoids, while mutants, cond-1(+) and cond-2(+), contained about 14 and 23 cp nucleoids, respectively, after one week culture on agar plates. The total cpDNA contents were almost proportional to the numbers of cp nucleoids. When cells containing cond-1 or cond-2 mutation were used as a parental source to cross with wild type cells of the other parent, preferential digestion of cp nucleoids from male parent (mt^–) origin occurred in the zygotes, although the frequencies of the digestion were slightly lower than that in the zygotes from the cross between wild type cells. Western blot analysis of the protein ofzyslB gene, which has been found related to preferential digestion of mt^– origin cp-nucleoids DNA, showed that a high amount of this protein was detected with the initiation of preferential digestion of mt^– cp nucleoids and disappeared with the completion of the digestion. Cp genetic markers for antibiotic resistance were maternally inherited in all crosses. These results showed that although the preferential digestion of cp nucleoids consisting of large number and large cpDNA amount requires a slightly longer period to complete, this high ploidy of the cp nucleoids does not disturb maternal inheritance.     

Low Density Membranes Are Associated with RNA-binding Proteins and Thylakoids in the Chloroplast of Chlamydomonas reinhardtii

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thylakoids.     

Aluminum-Induced Changes in Cell-Wall Glycoproteins in the Root Tips of Al-Tolerant and Al-Sensitive Wheat Lines

To investigate wheat (Triticum aestivumL.) responses to Al stress, KCl- and SDS-extracted glycoproteins (covalently bound proteins isolated by cell-wall digestion by cellulysine–pectolase mixture) and extensins (hydroxyproline-containing glycoproteins, HRGPs) were isolated from cell-wall preparations purified from the root apices of Al-sensitive and Al-tolerant near-isogenic lines ES8 and ET8. Under Al stress conditions, two lines differed mostly in their extensins. The untreated plants of two lines were low in covalently bound extensins, although the content of this protein fraction in ES8 was higher than in ET8. When the seedlings were treated with Al, the extensin content increased in both wheat lines and especially in the Al-tolerant ET8 plants. Using two-dimensional electrophoresis, the authors demonstrated the accumulation of polypeptides with mol wts of 22.2 kD (pI 5.5–6.5), 24.5 kD (pI 5.8–6.0), and 33.1 kD (pI 5.25) and polypeptides of 22.2 kD (pI 6.8–7.6) and 40.5 kD (pI 7.6) in the extensin fraction from the cell walls of the Al-sensitive plants. The regulation of cell responses to Al stress may involve extensin expression.     

Association of dehydrins with wheat mitochondria during low-temperature adaptation

In mitochondria from the crowns of field-grown winter wheat plants or their seedlings hardened in the laboratory, thermostable proteins immunologically related to dehydrins were detected. It was found that two dehydrins with mol wts of 63 and 52 kD bound with the outer mitochondrial membrane during autumnal hardening or during adaptation to low temperature in the laboratory. Dehydrins of similar mol wts were detected among proteins in the total membrane fraction from low-temperature-adapted wheat plants. In addition, dehydrins with mol wts of 209 and 196 kD were present in this fraction as well. Dehydrins of similar mol wts were bound with mitochondria from seedlings adapted to low temperature and those from the crowns of plants after autumnal hardening. In spring, the amount of dehydrins associated with mitochondria from the crowns declined to the level characteristic of early autumn. Dehydrin association with mitochondria is evidently an important defense mechanism of frost-resistant plants.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 221–226.Original Russian Text Copyright © 2005 by Borovskii, Stupnikova, Antipina, Anuchina, Voinikov.This revised version was published online in April 2005 with a corrected cover date.     

Cytoplasmic ribosomal proteins from Chlamydomonas reinhardtii: characterization and immunological comparisons

Summary Experiments were undertaken to characterize the cytoplasmic ribosomal proteins (r-proteins) in Chlamydomonas reinhardtii and to compare immunologically several cytoplasmic r-proteins with those of chloroplast ribosomes of this alga, Escherichia coli, and yeast. The large and small subunits of the C. reinhardtii cytoplasmic ribosomes were shown to contain, respectively, 48 and 45 r-proteins, with apparent molecular weights of 12,000–59,000. No cross-reactivity was seen between antisera made against cytoplasmic r-proteins of Chlamydomonas and chloroplast r-proteins, except in one case where an antiserum made against a large subunit r-protein cross-reacted with an r-protein of the small subunit of the chloroplast ribosome. Antisera made against one out of five small subunit r-proteins and three large subunit r-proteins recognized r-proteins from the yeast large subunit. Each of the yeast r-proteins has been previously identified as an rRNA binding protein. The antiserum to one large subunit r-protein cross-reacted with specific large subunit r-proteins from yeast and E. coli.     

Accumulation of Dehydrins and ABA-Inducible Proteins in Wheat Seedlings during Low-Temperature Acclimation

Using one-dimensional SDS-PAGE and immunochemical methods, we detected the presence and estimated the content of dehydrins and ABA-inducible (RAB) polypeptides in etiolated seedlings of four spring and three winter wheat (Triticum aestivum L.) cultivars differing in frost hardiness. We hardened three-day-old seedlings at 4°C for nine days or grew them at 22°C for a day (control seedlings). We established that heat-stable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD, which are characteristic of hardened wheat seedlings, were homologous to polypeptides from a dehydrin family and polypeptides with mol wts of 209, 196, 66, 50, and 41 kD were immunologically related to RAB-proteins. We supposed that these COR polypeptides were involved in the prevention of local protein dehydration and denaturation during hypothermia. Analysis of the relative content of COR proteins revealed a close correlation between the cultivar frost hardiness and the concentration of these proteins. It seems evident that different accumulation of dehydrins and RAB polypeptides in different cultivars of a single species is one of the causes for different plant frost hardiness.     

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic α-helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic β-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-tenninal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.     

Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved

Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,³⁵S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when³⁵S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.     

Light activates binding of membrane proteins to chloroplast RNAs in Chlamydomonas reinhardtii

Several membrane proteins were previously shown to bind to the 5 leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13–15-fold by light, and a 46 kDa protein is activated within 1–10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.     

Effect of Ca<Superscript>2+</Superscript> and cAMP on protein phosphorylation in mitochondria of maize seedlings

The effect of common intracellular signals (Ca²⁺ and cAMP) on the activity of protein phosphorylation in mitochondria was investigated in coleoptiles of maize (Zea mays L.). Treatment of isolated mitochondria with 2 mM CaCl₂ brought about an increase in the level of phosphorylation of proteins with mol ws of 74, 60, and 33 kD but considerably reduced phosphorylation of the protein with a mol wt of 51.5 kD. In the presence of Ca²⁺, phosphorylation of polypeptides with mol wts of 59 and 66 kD was also detected. cAMP considerably reduced phosphorylation of essentially all the investigated proteins in isolated mitochondria, which could be explained by activation of their dephosphorylation. Phosphorylation of mitochondrial proteins involves a polypeptide of about 94 kD showing kinase activity, which may be proper protein kinase or one of the subunits of a compound structure. In maize mitochondria, PP1A phosphatases were found. A hypothesis was advanced that redox-dependent phosphorylation/dephosphorylation of mitochondrial proteins plays an important role in mitochondrial signaling in higher plants.     

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.     

The effect of light on the number of chloroplast nucleoids in daughter cells of the algaScenedesmus quadricauda

Summary Synchronous cultures of the green algaScenedesmus quadricauda (Turp.) Bréb. grown at mean irradiances 25Wm^–2, 75Wm^–2, and 130Wm^–2 PhAR were exposed to different illumination regimes (ratio of light to dark interval varied from 2:22 to 24:0 hours). The populations of daughter cells released under these conditions differed markedly in their progress in the cell cycle. The cells from these populations were stained with DAPI and the shape, localization and number of chloroplast nucleoids were examined. The nucleoids were of spherical shape, divided asynchronously having dumbbell shape during fission. In the chloroplast, nucleoids were located symmetrically about the transverse axis of the cells. The mean number of nucleoids varied from two in the least developed daughter cells to 16 in the daughter cells of the highest developmental stage. The progress of these cells and thus also the number of nucleoids were proportional to the portion of the light energy amount which these daughter cells shared from the total light energy amount obtained by their mother cells.Abbreviations DAPI 4, 6-diamidino-2-diphenylindole - PhAR photosynthetically active radiation (400–700 nm)     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Woody plants of Yakutia and low-temperature stress

Physiological and biochemical features of woody plants (Pinus sylvestris L. and Betula platyphylla Sukacz.) during transition from vegetative to frost-resistant state under conditions of extremely severe climate of Yakutia were studied. In P. sylvestris such transition was accompanied by a decrease in the content of chlorophylls long before first frosts and by an increase in the proportion of Xanth and simultaneous decrease in the content of β-carotene in needles during the first and second phases of hardening. In the period of preparation to dormancy, overwintering organs of both P. sylvestris (needles) and B. platyphylla (buds) accumulated the two groups of major dehydrins, with low mol wts of 15–21 kD and middle mol wts of 66–141 kD. Simultaneously, low temperature led to a great increase in the content of polyunsaturated fatty acids (FAs) in lipids of P. sylvestris needles and B. platyphylla buds, primarily linoleic acid and also eicosenoic FAs differing in the extent of desaturation. Observed qualitative and quantitative changes in pigments, total proteins, dehydrins, and FAs during autumn hardening of P. sylvestris and B. platyphylla plants presume their important role in the development of resistance of these tree species to low-temperature stress (down to −60°C) in the cryolithic zone of Yakutia.     

Protective Effect of Arachidonic Acid during Viral Infection: Synthesis of New Proteins by in vitroPotato Plants

The mechanisms of the protective, immunostimulating effects of arachidonic acid (AA) were studied, and its efficiency in the induction of defense reactions in the moderately virus-resistant potato cultivar Nevskii (Solanum tuberosumL.) was determined. Virus-free in vitropotato plants treated with AA and inoculated with phytopathogenic viruses were used as a model. The data on the X virus accumulation obtained by the enzyme-linked immunosorbent assay confirmed the immunizing effect of AA; the optimum concentration of the compound was 10^–8M. The antiviral effect of AA was maintained in infected in vitropotato plants for at least two or three weeks. The electrophoretic analysis of leaf proteins revealed a 33-kD polypeptide induced by the potato virus Y. Two weeks after inoculation with virus X, a 40-kD protein was identified in potato plants pretreated with AA. In addition, the relative content of the two groups of proteins consisting of two or three components with mol wts about 50 kD and above70 kD changed both upon viral infection and pretreatment with AA. Only small changes in the isozyme patterns of peroxidase in potato plants were observed during the development of systemic acquired resistance; they were manifested in some treatments in the band intensities. The existence of the alternative pathways of systemic acquired resistance in potato plants specifically activated by viral infection and AA was suggested.     

Lectin activity and composition in the wheat cell walls under the effects of low temperature and inhibitors of calcium signaling pathway

In the roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings, the effects of neomycin (100 μM), an inhibitor of phospholipase C, and dilthiazem (250 μM), a blocker of calcium channels on lectin activity and composition at low-temperature treatment (2–3°C) were studied. Hypothermia induced the appearance of two peaks of cell wall-bound lectin activity, e.g., in 0.5 and 6 h. Under these conditions, the inhibitors suppressed lectin activity. In 0.5 h of hypothermia, substantial changes in total profile of proteins were observed: lectins with mol wts of 85, 78, and 54 kD disappeared, and novel lectins with mol wts of 110, 105, 70, 50, and 34.5 kD appeared. In the presence of dilthiazem, the set of lectin proteins remained similar to that in unhardened plants, and the increase in the lectin content and activity was observed only after 3-h exposure to low temperature. This indicates that blocking dilthiazem-sensitive calcium channels slowed plant response to stress and did not permitt the cell to start rapidly the development of defense mechanisms. The important role of lectins with mol wts of 110 and 60 kD in the formation of freeze tolerance is supposed because these lectins did not appear in the presence of dilthiazem.     

Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast

A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark. The CTBVP1 fusion protein was expressed in C. reinhardtii chloroplast and accounted for up to 3% of the total soluble protein. The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins. These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.     

Nucleotide sequence of cDNA clones encoding the β subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: The precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension

cDNA and genomic clones encoding the subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79–83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165–168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.     

Chloroplast DNA base substitutions: an experimental assessment

An experimental assessment was carried out to determine directly the frequency and types of spontaneous base substitutions that occur in chloroplast DNA. A target site within the chloroplast 16S rRNA gene of the green alga Chlamydomonas reinhardtii was chosen for the assay. Mutations at this site were known to confer spectinomycin resistance and simultaneously result in the loss of an AatII cleavage site. In the experiments reported here, base substitutions at any individual base occurred at a frequency in the range of 0.9–11 per 10⁹ viable cells plated. Four new mutations that confer resistance to spectinomycin were identified at the target site in the Chlamydomonas chloroplast 16S rRNA gene. When the relative rates of transition and transversion mutations were quantified, a bias toward transversions was observed. The prominence of A/T C/G transversions in the observed mutation spectrum suggests that oxidative damage may be the major cause of base substitution mutations within the chloroplast.