Comparison of proteins synthesized in vivo and in vitro by mRNA from isolated protoplasts

Studies of proteins synthesized in vitro by messenger RNA (mRNA) extracted from tobacco protoplasts showed that the changes in protein synthesis and especially the lack of certain proteins observed previously in isolated protoplasts did not result from a failure of translation.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Auxin-binding by particulate fractions from tobacco leaf protoplasts

In vitro binding of 1-naphthaleneacetic acid (NAA) to particulate fractions from tobacco leaf protoplasts was studied. In freshly isolated protoplasts no specific binding could be detected, whereas it was present in particulate fractions from tobacco leaves. It is concluded that the NAA-binding-sites are probably located at the external face of the plasma membrane; they are destroyed during protoplast isolation by proteolytic enzymes in the cellulase and macerozyme preparations. After culturing the protoplasts for 3–4 d, the first cell divisions were observed and at the same time specific NAA-binding became detectable. The affinity constant for NAA was approx. 2·10⁶ mol^-1 and the number of binding sites increased during further culture.Abbreviations MES 4-morpholinoethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Enrichment of heterokaryocytes between mesophyll and epidermis protoplasts by density gradient centrifugation after electric fusion

Summary Mesophyll protoplasts from Nicotiana glauca were fused with epidermal protoplasts from N. langsdorffii by an electric pulse. After the fusion products were centrifuged on stepwise density gradient centrifugation using Percoll and sea water, somatic hybrids were observed at 70%–80% in the fraction recovered from the intermediate specific gravity fraction between epidermis and mesophyll protoplasts. From offsprings of these somatic hybrids, teratomatous plants were regenerated. Since the difference of specific gravity between mesophyll and epidermis protoplasts is inherent, this procedure can be essentially applied to obtain somatic hybrids between any combination of plants. The significance of this study is discussed in relation to obtaining somatic hybrids between plant materials without any appropriate genetic markers.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40–50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kbHind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells ofEscherichia coli, and colonies were recovered that carried pGEM withHind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transformE. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning intoE. coli.     

Time course of hormonal control of the first mitosis in tobacco mesophyll protoplasts cultivated in vitro

The presence of auxin and cytokinin is necessary for the induction of mitosis in tobacco mesophyll protoplasts cultivated in vitro. In their absence, protoplasts firstly accumulate inhibitors of mitosis in the culture medium, possibly because of non-coordinated cell-wall synthesis, and secondly evolve a nonmitotic and degenerative metabolism. By changing the intoxified medium, it is possible to show that auxin is necessary from the beginning of culture, while cytokinin is only required later to allow a step in the development of the mitotic apparatus.Abbreviations 2.4-D 2.4 dichlorophenoxyacetic acid - 6-BA 6-benzyladenine - NAA naphthaleneacetic acid - IAA indoleacetic acid     

Changes in protein synthesis during the initial stage of life of tobacco protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (in vitro labeling) and into leaves, from which the protoplasts were isolated later (in situ labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.     

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts

Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the -glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low -glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.Abbreviations CaMV cauliflower mosaic virus - PCR polymerase chain reaction - PEG polyethylene glycol - Mu 4-methyl umbelliferone     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Transfer of cytoplasmic organelles from an oligomycin-resistant Nicotiana cell suspension into tobacco protoplasts yielding oligomycin-resistant cybrid plants

Summary Oligomycin-resistant lines were derived from a Nicotiana sylvestris cell suspension, after N-nitroso-N-methylurea mutagenesis followed by selection in the presence of 0.4 g/ml oligomycin, a specific mitochondrial ATPase inhibitor. One of the lines, oli ^R38 was further analyzed to investigate the role of mitochondria in this resistance. The oli ^R38 line proved to be also highly resistant to venturicidin, another specific inhibitor of mitochondrial ATPase. By the donor-recipient protoplast-fusion procedure the cytoplasmic organelles of oli ^R38 were transferred to protoplasts of Line 92, a line of tobacco plants which contain the cytoplasmic organelles of N. undulata. Cell suspensions prepared from several cybrid plants, containing the cytoplasmic organelles of oli ^R38, exhibited the same level of oligomycin resistance as the oli ^R38 line.     

The fission yeast homologue of Glel is essential for growth and involved in mRNA export

We have isolated Glel homologue (named as spglel) as a partial multicopy suppressor of the synthetic lethality of rael-167 elfl-21 in fission yeast Schizosaccharomyces pombe. The spglel is also able to complement partially temperature-sensitive phenotype of rael-167 only at a lower restrictive temperature. The spglel gene contains one intron and encodes a 480 amino-acid protein with predicted molecular weight of 56.2 kDa. We showed that spglel gene is essential for vegetative growth and functional Glel-GFP protein is localized mainly in NPC. The accumulation of poly(A)(+) RNA in the nucleus is exhibited when expression of spglel is repressed or over-expressed. These results suggest that the spGle1 protein is also involved in mRNA export in fission yeast.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

Ribosomal proteins of Thermus thermophilus fused to beta-galactosidase are imported into the nucleus of eukaryotic cells

Archaea, Bacteria, and Eukarya have 34 homologous ribosomal protein (RP) families in common. Comparisons of published amino acid sequences prompted us to question whether RPs of the prokaryote Thermus thermophilus contain nuclear localization signals (NLSs), which are recognized by the nuclear import machinery of eukaryotic cells and are thereby translocated into the nucleoplasm ultimately accumulating in the nucleolus. Several RPs of T. thermophilus - specifically S12, S17, and L2 - were selected for this study since their three-dimensional structures as well as rRNA interaction patterns are precisely known at the molecular level. Fusion proteins of these RPs were constructed and subsequently expressed in COS cells. N-terminally tagged fusions with dimeric EGFP and C-terminally tagged hybrids with beta-galactosidase of prokaryotic RP S17 (S17p) were targeted to the nucleoplasm where they were visualized by direct fluorescence and by indirect immune staining, respectively. A region containing the classical monopartite NLS KRKR, which is known to physically interact with karyopherin alpha2, was delineated by tagging specific S17p fragments with beta-galactosidase. Unexpectedly, S12p and L2p hybrids accumulated in the nucleolus. Due to their size, RPs tagged with beta-galactosidase can only be imported into the nucleus when NLS-recognition is mediated by karyopherins since they are otherwise excluded from entry into the nucleoplasm of eukaryotic cells. Our results indicate that after the formation of the nuclear compartment during evolution, the newly established eukaryotic cell relied on the pre-existing basic amino acid clusters of the prokaryotic RPs for use as NLSs.     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation

Summary Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.     

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10⁷/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO₄ (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.     

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

Various factors affecting the culture of Brassica napus and B. juncea mesophyll protoplasts were examined in order to develop suitable culture media for these species. The basic components (salts and vitamins) of culture media K3 and Kao best supported cell division and colony development in protoplast culture of both species. The addition of casamino acids to Kao's medium resulted in colony browning in B. napus genotypes. B. napus protoplasts grew well with glucose as the osmotic stabilizer, whereas B. juncea protoplasts responded better to sucrose. High NAA and low 2,4-D combinations were effective in stimulating colony growth. Colony development was rapid for a range of genotypes cultured with these recommendations in these media and plant regeneration was obtained from protoplast-derived calli in both species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-Benzylaminopurine - MES 2(N-Morpholino)ethane sulfonic acid Contribution No. 931.