Esra Galun

Evidence for in vivo trans splicing of pre-mRNAs in tobacco chloroplasts

The rps12 gene codes for chloroplast ribosomal protein S12. In the tobacco chloroplast genome (156 kbp circular DNA), exons II and III of this gene are separated by an intron of 536 bases and are present in two copies in the inverted repeat region, while exon I is located in the large single copy region at a distance of 90 kb and 126 kb from the two copies of exons II and III. These three exons were artificially combined in cloned DNA fragments and hybridized with tobacco chloroplast RNA. Electron microscopic analysis of RNA-DNA hybrids showed that exon I is transcribed as part of a polycistronic RNA containing upstream and downstream sequences; the same is true for exons II and III. Exon I is shown to be transcribed separately from exons II and III. In the most abundant class of the hybridized RNA molecules, exon I was covalently linked to exon II. In these molecules the sequences downstream of exon I and upstream of exon II are not present. These data indicate that maturation of rps12 pre-mRNAs in chloroplasts of tobacco involves trans splicing.

Ethylene and in vitro culture of potato: suppression of ethylene generation vastly improves protoplast yield, plating efficiency and transient expression of an alien gene

Ethylene release by potato shoots cultured in closed boxes was suppressed by the addition of silver thiosulfate to the culture medium. Shoots cultured in the presence of silver thiosulfate produced appreciably more tissue and the yield of protoplasts per unit tissue mass was vastly increased, resulting in an 8 fold increase of protoplast yield per shoot. Exposure of pricked leaves to macerating enzymes facilitated ethylene generation. Leaves of shoots which were previously cultured in silver thiosulfate containing medium generated much less ethylene than leaves from control shoots and this generation could be further reduced by the addition of acetylsalicylic acid during maceration. The capability of polyethylene glycol treated potato protoplasts to produce microcalli was vastly increased by the addition of silver thiosulfate during exposure of protoplasts to Ca(NO3)2 following the polyethylene glycol treatment. Similarly, when a plasmid (pCAP212) containing an expressible gene for chloramphenicol acetyltransferase was introduced into potato protoplasts through a polyethylene glycol treatment, the transient expression of acetyltransferase was very much increased by the addition of a short incubation of the protoplasts with silver thiosulfate.

The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts.

The chloroplast genes coding for the 16S ribosomal RNA from several spectinomycin‐resistant Nicotiana mutants were analyzed. Two classes of mutants were identified. In one class, a G to A base transition is found at position 1140 of the tobacco‐chloroplast 16S rRNA gene, which eliminates an AatII restriction endonuclease site. This base transition is proximal to a mutation previously described for spectinomycin resistance in Escherichia coli. In the other class, a novel G to A transition is found at position 1012 of the 16S rRNA gene. Although the mutations in the two classes are 128 nucleotides apart, the secondary structure model for 16S rRNA suggests that the two mutated nucleotides are in spatial proximity on opposite sides of a conserved stem structure in the 3′ region of the molecule. Phylogenetic evidence is presented linking this conserved stem with spectinomycin resistance in chloroplasts. Perturbation of the stem is proposed to be the molecular‐genetic basis for rRNA‐dependent spectinomycin resistance.

Tissue-Specific Expression of the Tobacco Mosaic Virus Movement Protein in Transgenic Potato Plants Alters Plasmodesmal Function and Carbohydrate Partitioning

Transgenic potato (Solanum tuberosum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) under the control of the promoters from the class I patatin gene (B33) or the nuclear photosynthesis gene (ST-LS1) were employed to further explore the mode by which this viral protein interacts with cellular metabolism to change carbohydrate allocation. Dye-coupling experiments established that expression of the TMV-MP alters plasmodesmal function in both potato leaves and tubers when expressed in the respective tissues. However, whereas the size-exclusion limit of mesophyll plasmodesmata was increased to a value greater than 9.4 kD, this size limit was smaller for plasmodesmata interconnecting tuber parenchyma cells. Starch and sugars accumulated in potato leaves to significantly lower levels in plants expressing the TMV-MP under the ST-LS1 promoter, and rate of sucrose efflux from petioles of the latter was higher compared to controls. It is interesting that this effect was expressed only in mature plants after tuber initiation. No effect on carbohydrate levels was found in plants expressing this protein under the B33 promoter. These results are discussed in terms of the mode by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation, and the possible role of plasmodesmal function in controlling these processes.

In vitro tuberization in transgenic potatoes harboring ß-glucoronidase linked to a patatin promoter: effects of sucrose levels and photoperiods

Abstract Sections of axenic potato shoots containing one leaf were found to synchronically produce a microtuber in their single leaf axil 7 days after transfer to inductive medium containing 8% sucrose, kinetin and ancymidol (a gibberellin-biosynthesis inhibitor). Shoots of transgenic Solanum tuberosum harboring a chimeric gene containing a patatin promoter sequence followed by the gene coding for s-glucuronidase as a reporter protein, were utilized. Microtubers were initiated in inductive medium under either long-day (LD) or short-day (SD) photoperiods. The minimal duration of culture in 8% (w/v) sucrose (followed by culture in 2% (w/v) sucrose) is 1 or 3 days, when the shoots were subsequently maintained (for 6 or 4 days) in LD or SD, respectively. Changes in patatin transcription were evaluated by following s-glucuronidase activity. Strong increase in s-glucuronidase activity was observed in tuber initials 5 days after transfer to tuber-induction medium. This increase paralleled the accumulation of radioactivity in the tuber initials of shoots cultured in radioactive sucrose. s-Glucuronidase activity analyses at various periods after induction and in different tissues of the in vitro cultured shoot-sections as well as the elimination of one of the obligatory induction conditions, suggested that the requirement for high sucrose levels does not represent an energy demand but rather an induction signal. Day-length has apparently a decisive role, after exposure to high-levels of sugar, in maintaining the tuberization signal.

Pleiotropic effects of tobacco-mosaic-virus movement protein on carbon metabolism in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing wild-type or mutant forms of the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to study the effects of the TMV-MP on carbon metabolism in source leaves. Fully expanded source leaves of transgenic plants expressing the TMV-MP were found to retain more newly fixed 14C compared with control plants. Analysis of 14C-export from young leaves of TMV-MP plants, where the MP is yet to influence plasmodesmal size exclusion limit, indicated a similar pattern, in that daytime 14C export was slower in TMV-MP plants as compared to equivalent-aged leaves on control plants. Pulse-chase experiments were used to monitor radioactivity present in the different carbohydrate fractions, at specified intervals following 14CO2 labeling. These studies established that the-TMV-MP can cause a significant adjustment in short-term 14-C-photosynthate storage and export. That these effects of the TMV-MP on carbon metabolism and phloem function were not attributable to the effect of this protein on plasmodesmal size exclusion limits, per se, was established using transgenic tobacco plants expressing temperature-sensitive and C-terminal deletion mutant forms of the TMV-MP. Collectively, these studies establish the pleiotropic nature of the TMV-MP in transgenic tobacco, and the results are discussed in terms of potential sites of interaction between the TMV-MP and endogenous processes involved in regulating carbon metabolism and export.

Novel alloplasmic Nicotiana plants by “donor-recipient” protoplast fusion: cybrids having N. tabacum or N. sylvestris nuclear genomes and either or both plastomes and chondriomes from alien species

SummaryThe “donor-recipient” fusion method was utilized to investigate interspecific transfer of organelles and organelle-controlled traits from donor to recipient Nicotiana species.To follow organelle transfer and sorting-out under conditions which will be either selective or non-selective for chloroplast transfer we used, respectively, albino (N. tabacum VBW) or normally pigmented (N. sylvestris) plants as protoplast-recipients. N. alata, N. bigelovii and N. undulata served as donors. Organelle composition of the cybrid plants was investigated by the analysis of chloroplast DNA restriction pattern, tentoxin sensitivity, chloroplast pigmentation, mitochondrial DNA restriction pattern and alloplasmic male sterility. In certain cybrids the analysis of restriction patterns of chloroplast and mitochondrial DNA was augmented by Southern hybridization with the respective organelle-DNA probes. A total of 341 fusion-derived plants (from 109 calli) were analysed and 102 of these were found to be cybrids with novel nuclear/organelle compositions. The incidence of chloroplast and mitochondrial transfer was strongly facilitated when iodoacetate-treated protoplasts of N. tabacum VBW (albino) rather than N. sylvestris (normal chloroplasts) protoplasts served as recipient. Mitochondrial DNA restriction patterns in cybrids commonly differed from those of either fusion partner. All fusion combinations resulted in at least some male-sterile cybrids, having either donor or recipient plastomes thus indicating that interspecific “donor-recipient” protoplast fusion is an efficient mean to produce plants with alloplasmic male sterility.

Recent advances in protoplast culture of horticultural crops: Citrus

Abstract The full sequence from in vitro cultured ovules, namely nucellar callus, isolated protoplasts, protoplast derived embryogenic callus and the establishment of protoplast-derived normal fruiting Citrus trees is described. Somatic hybridization and cybridization were performed and respective cybrid trees were verified by their mitochondrial DNA restriction profiles. Future trends and possibilities for integrating molecular methodologies with tissue culture and protoplast manipulation, in order to produce novel transgenic Citrus trees with favorable breeding traits, are discussed.

Nuclear-mitochondrial interrelation in angiosperms

Abstract In this review we wish to draw attention to aspects of nuclear-genome/mitochondrial genome interactions in plants where additional information could improve our understanding of this interrelationship. The core of the review should thus be regarded as a prologue to the ‘Open Vistas’ (section at the end of this review). Obviously such aspects cannot be appreciated without due background on the structure and function of the mitochondrial genome, on nuclear-coded components and their routing to the mitochondrion and on dysfunctions and malformations attributed to nuclear-mitochondrial incompatibility. In each of these topics we shall summarize the information for angiosperms but also provide relevant information from some other organisms (e.g. fungi, mammals), mostly by referring to reviews. One topic, the dysfunction and malformation of the stamens (i.e. ‘male-sterility’) unique to angiosperms is treated rather comprehensively; thus, alloplasmic male sterility in Nicotiana , in Petunia and in maize will serve to illustrate the consequences of nuclear/mitochondrial incompatibility.

A protoplast-to-tree system in Microcitrus based on protoplasts derived from a sustained embryogenic callus

Adventitious embryos derived from a zygotic embryo in an in vitro cultured ovule of Microcitrus were transferred several times on solidified medium containing benzyladenine and 3-indoleacetyl-L-alanine to induce embryogenic callus. This callus was maintained for several years on medium devoid of growth regulators without losing its embryogenic capacity. Exposure of this callus to maceration enzymes led to protoplast suspensions. Purified protoplasts were plated in solid medium devoid of growth regulators. Somatic embryos were derived efficiently from individual protoplasts and most of these could be regenerated into mature trees bearing normal flowers and typical fruits. This system differs from the Citrus protoplast-to-tree system. In the latter embryogenic callus was derived from the nucellus of polyembryonic species while Microcitrus is monoembryonic and required hormone-induced callus formation from proliferating zygotic embryos.