Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.     

Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non-vascular plants

A single general import pathway in vascular plants mediates the transport of precursor proteins across the two membranes of the chloroplast envelope, and at least four pathways are responsible for thylakoid protein targeting. While the transport systems in the thylakoid are related to bacterial secretion systems, the envelope machinery is thought to have arisen with the endosymbiotic event and to be derived, at least in part, from proteins present in the original endosymbiont. Recently the moss Physcomitrella patens has gained worldwide attention for its ability to undergo homologous recombination in the nuclear genome at rates unseen in any other land plants. Because of this, we were interested to know whether it would be a useful model system for studying chloroplast protein transport. We searched the large database of P. patens expressed sequence tags for chloroplast transport components and found many putative homologues. We obtained full-length sequences for homologues of three Toc components from moss. To our knowledge, this is the first sequence information for these proteins from non-vascular plants. In addition to identifying components of the transport machinery from moss, we isolated plastids and tested their activity in protein import assays. Our data indicate that moss and pea (Pisum sativum) plastid transport systems are functionally similar. These findings identify P. patens as a potentially useful tool for combining genetic and biochemical approaches for the study of chloroplast protein targeting. Abbreviations: EST, expressed sequence tag; LHCP, light-harvesting chlorophyll-binding protein; NIBB, National Institute for Basic Biology; OE17, 17 kDa subunit of the oxygen-evolving complex; PC, plastocyanin; PEP, Physcomitrella EST Programme; SPP, stromal processing peptidase; SRP, signal recognition particle; Tat, twin-arginine translocation; Tic, translocon at the inner membrane of the chloroplast envelope; Toc, translocon at the outer membrane of the chloroplast envelope; TPP, thylakoid processing peptidase; TPR, tetratricopeptide repeatSupplementary material to this paper is available in electronic form at .This revised version was opublished online in July 2005 with corrected page numbers.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Zea mays Fe deficiency‐related 4 (ZmFDR4) functions as an iron transporter in the plastids of monocots

Iron (Fe)‐homeostasis in the plastids is closely associated with Fe transport proteins that prevent Fe from occurring in its toxic free ionic forms. However, the number of known protein families related to Fe transport in the plastids (about five) and the function of iron in non‐green plastids is limited. In the present study, we report the functional characterization of Zea mays Fe deficiency‐related 4 (ZmFDR4), which was isolated from a differentially expressed clone of a cDNA library of Fe deficiency‐induced maize roots. ZmFDR4 is homologous to the bacterial FliP superfamily, coexisted in both algae and terrestrial plants, and capable of restoring the normal growth of the yeast mutant fet3fet4, which possesses defective Fe uptake systems. ZmFDR4 mRNA is ubiquitous in maize and is inducible by iron deficiency in wheat. Transient expression of the 35S:ZmFDR4–eGFP fusion protein in rice protoplasts indicated that ZmFDR4 maybe localizes to the plastids envelope and thylakoid. In 35S:c‐Myc‐ZmFDR4 transgenic tobacco, immunohistochemistry and immunoblotting confirmed that ZmFDR4 is targeted to both the chloroplast envelope and thylakoid. Meanwhile, ultrastructure analysis indicates that ZmFDR4 promotes the density of plastids and accumulation of starch grains. Moreover, Bathophenanthroline disulfonate (BPDS) colorimetry and inductively coupled plasma mass spectrometry (ICP‐MS) indicate that ZmFDR4 is related to Fe uptake by plastids and increases seed Fe content. Finally, 35S:c‐Myc‐ZmFDR4 transgenic tobacco show enhanced photosynthetic efficiency. Therefore, the results of the present study demonstrate that ZmFDR4 functions as an iron transporter in monocot plastids and provide insight into the process of Fe uptake by plastids.     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic instability of carotenoid biosynthesis in the tomato ghost mutant and its effect on plastid development

The tomato (Lycopersicon esculentum (L.) Mill.) ghost plant is a mutant of the San Marzano cultivar affected in carotenoid biosynthesis. ghost plants exhibit a variable pattern of pigment biosynthesis during development. Cotyledons are green but true leaves are white. Green sectors, which appear to be clonal in origin, are frequently observed in the white tissue. Because of the lack of photosynthesis ghost plants have a very low viability in soil. We have developed a strategy for propagating ghost plants that employs organ culture to generate variegated green-white plants which, supported by the photosynthetic green areas, develop in soil to almost wild-type size. These plants were used to analyze the pigment content of the different tissues observed during development and plastid ultrastructure. Cotyledons and green leaves contain both colored carotenoids and chlorophyll but only the colorless carotenoid phytoene accumulates in white leaves. the plastids in the white tissue of ghost leaves lack internal membrane structures but normal chloroplasts can be observed in the green areas. The chromoplasts of white fruits are also impaired in their ability to form thylakoid membranes.     

Ultrastructural characterization of the reversible differentiation of chloroplasts in cucumber fruit

The changes in plastid ultrastructure in the pericarp of cucumber (Cucumis sativus L) fruit were studied during fruit yellowing (which accompanied maturation) and regreening. In the course of fruit maturation, the thylakoid system was progressively reduced, and only a small number of membranes remained in the plastids of mature fruit. At the same time, the plastoglobules increased in size, often remaining in close proximity to the degrading thylakoids. In pericarp tissue which turned green again, the thylakoid network in the plastids was gradually reconstituted. Morphological similarities between the plastids in mature and regreening fruit indicated that the chloroplasts in regreened tissue were redifferentiated from the plastids of mature fruit. Reconstitution of the thylakoid system appeared to start from two morphologically distinct types of membranes: from double membranes which resembled thylakoids and from membrane-bound bodies (MBBs). The latter appeared to form thylakoids by two mechanisms: by detachment of extensions from their surfaces and by fragmentation. The plastoglobules remained in the plastids during thylakoid system reconstitution and were often observed in close proximity to developing thylakoids. In the course of chloroplast redifferentiation, several types of membraneous structures were found to be associated with the plastid envelope: (i) vesicles which appeared to separate from the envelope and to fuse subsequently with the developing thylakoids, (ii) tubules, and (iii) double-membrane sheets which appeared asde novo forming thylakoids.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

Some unusual features of the ultrastructure of the chloroplasts of the green algal orderCaulerpales and their development

Summary The ultrastructure of developing and mature chloroplasts of members of the green algal orderCaulerpales is described. The mature chloroplasts develop from small starch containing plastids. These small starch containing plastids may also develop into the large amyloplasts characteristic of this order. The thylakoid organizing body (TOB), a system of concentric lamellae found at one end of the plastid, appears to be involved in initial thylakoid membrane synthesis. During early plastid development the first formed thylakoids, the plastid DNA and lipid are closely associated with this body. Many developing plastids also have a number of microfilaments near the chloroplast envelope. These microfilaments extend from the TOB towards the opposite end of the plastid.The size and structure of the mature caulerpalean chloroplast varies greatly between species, as does the size and structure of the TOB. The simplest type of TOB occurs inAvrainvillea erecta and the most complex inCaulerpa cactoides. The membranes of the TOB are connected by crossbridges and they are also connected with the inner chloroplast envelope membrane. The structure of the TOB, its relation to the chloroplast envelope, its association with the thylakoids and its possible functions are described.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

Characterization of a cytoplasmically inherited yellow foliar mutant (cyt-Y 3) in soybean

Summary Genetic analysis of a yellow foliar mutant in soybean (Glycine max L. Merr.) showed maternal inheritance of the mutant phenotype designatedcyt-Y ₃. The mutant was grown beside normal green sibs (cyt-G ₃) under three different photosynthetic photon flux densities (PPFD), and samples were collected to determine pigment content and for electron microscopy analyses of plastid ultrastructure. The plastid ultrastructure ofcyt-Y ₃ appeared normal at low PPFD and the carotenoid level ofcyt-Y ₃ was also normal, but the chlorophyll content was only approximately one-third that ofcyt-G ₃. Under medium and high PPFD,cyt-Y ₃ plastids lacked a structured thylakoid, and total chlorophyll content was only 28% and 1% of normal, respectively; the carotenoid levels ofcyt-Y ₃ also dropped to 33% and 2% of normal, respectively. These data indicate that the effect of high PPFD oncyt-Y ₃ might result from a deficiency in a plastid membrane protein. The resulting changes in membrane configuration could then interfere with the accumulation or stabilization of chlorophylls and carotenoids, thereby resulting in the subsequent photooxidation of both at medium and high PPFD. This mutant could be useful in the study of thylakoid biosynthesis and pigment stabilization, or could provide a source of conditionally identifiable plastids for organelle segregation studies.This is a joint contribution of North Central Region, USDA-ARS., and Journal Paper No. J-11124 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, Project 2471. Mention of a trademark of proprietary product by Iowa State University or the USDA does not imply its approval to the exclusion of other products that may also be suitable     

Morphology and ultrastructure of Mammillaria gracillis (Cactaceae) in in vitro culture

Morphogenetic status of cactus Mammillaria gracillis Pfeiff. tissue culture was studied by light and electron microscopy. In vitro propagated shoots spontaneously developed callus. This callus regenerated normal and hyperhydric shoots without exogenous hormones. Tumour tissue induced by wild or rooty strains of Agrobacterium tumefaciens never expressed any morphogenetic potential. Light microscopy showed cellular characteristics of morphologically different tissues. Ultrastructural studies revealed changes in plastids: secondary dedifferentiation of mature chloroplasts, thylakoid swelling and disruption, phytoferritin accumulation, plastid elongation and increase in size. Changes in chlorophyll and carotenoid content were in accordance with degradation of the thylakoid system. Plastids were confirmed as very sensitive organelles to an artificial hyperhydric environment as well as to Agrobacteria-mediated cell transformation.     

The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal–dependent manner.     

Plastid ultrastructure and photosynthesis in greening petaloid hypsophylls

The ultrastructural and biochemicalphysiological aspects of postfloral greening have been studied in hypsophylls of Heliconia aurantiaca Ghiesbr., Guzmania cf. x magnifica Richter and Spathiphyllum wallisii Regel. In all three species the greening of the hypsophylls is due to plastid transformation, chloroplast formation proceeding from the initially different types of plastids. The degradation process of the original plastid structures and the mode of thylakoid formation are distinct in each case. In none of the species do the transformed plastids look identical to the chloroplasts of the corresponding foliage leaves. On a chlorophyll basis, the rate of photosynthesis of the greened hypsophylls surpasses the rate of the leaves considerably in Spathiphyllum, but is much lower in Heliconia (no data for Guzmania). In all species, anatomy, plastid structure, pigments, 77° K-fluorescence emission, ribulose-1,5-bis-phosphate carboxylase activities and short-term photosynthesis ¹⁴CO₂-assimilation patterns prove the greened hypsophylls to be capable of providing additional carbon to the developing fruits, thus supplementing the import of organic matter from the foliage leaves.Abbreviations MDH malate dehydrogenase (EC 1.1.1.37) - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Conservative sorting in the muroplasts of Cyanophora paradoxa: a reevaluation based on the completed genome sequence

After primary endosymbiosis, massive gene transfer occurred from the genome of the cyanobacterial endosymbiont to the nucleus of the protist host cell. In parallel, a specific protein import apparatus arose for reimport of many, but not all products of the genes moved to the nuclear genome. Presequences evolved to allow recognition of plastid proteins at the envelope and their translocation to the stroma. However, plastids (and cyanobacteria) also comprise five other subcompartments. Protein sorting to the cyanobacterial thylakoid membrane, the thylakoid lumen, the inner envelope membrane, the periplasmic space, and the outer envelope membrane is achieved by prokaryotic protein translocases recognizing, e.g., signal sequences. The “conservative sorting” hypothesis postulates that these translocases remained functional in endosymbiotic organelles and obtained their passengers not only from imported proteins but also from proteins synthesized in organello. For proteins synthesized in the cytosol, a collaboration of the general import apparatus and the former prokaryotic translocase is necessary which is often reflected by the use of bipartite presequences, e.g., stroma targeting peptide and signal peptide. For plants, this concept has been experimentally proven and verified. The muroplasts from Cyanophora paradoxa, that have several features more in common with cyanobacteria than with plastids, were analyzed with the availability of the recently completed nuclear genome sequence. Interesting findings include the absence of the post-translational signal recognition particle pathway, dual Sec translocases in thylakoid and inner envelope membranes that are produced from a single set of genes, and a co-translational signal recognition pathway operating without a 4.5S RNA component.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710