Wild-type Protoplasts Research Articles

Isolation of an Arabidopsis thaliana mutant in which the multiplication of both cucumber mosaic virus and turnip crinkle virus is affected.

During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designated cum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with the cum2-1 mutation as far as we examined. Therefore, the cum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses.

Dynamics of Cell Wall Formation in Fission Yeast,Schizosaccharomyces pombe

Studies on the dynamics of surface and intracellular structures during cell wall formation from the reverting protoplast ofSchizosaccharomyces pombewere reviewed, and the correlation between cell wall formation and actin cytoskeleton, which is the most important conductor of the mechanism, is described in this paper. A close spatial and temporal relationship between actin cytoskeleton and cell wall formation was found by using wild type and actin point-mutantcps8ofS. pombe.Concomitant with the cell wall formation, dynamic behavior of the intracellular secretion machinery, especially the Golgi apparatus and secretory vesicles, was analyzed by three-dimensional reconstruction of 40 to 80 serial sections at five reverting stages. Total reverting protoplast volume increased by 3.8 and 4.3 times at 3 and 5 h, respectively, and the volume of the Golgi apparatus in the corresponding stages increased 2.3- and 2.5-fold over the same periods. The number of secretory vesicles also markedly increased by 3.4 and 5.8 times over that of the corresponding reverting protoplasts. Actin point-mutantcps8cells have abnormal structure in the cell wall and septum, and the distribution pattern of the actin cytoskeleton during the reversion process was different from wild-type protoplasts. The profiles of actin showed one or two thick cables and patches in the cytoplasm which remained throughout reversion. The development of crosslinkage of the glucan fibrils which are β-1,3-glucan in nature on the reverting protoplast surface was defective; the glucan networks consisted of thin, rope-shaped fibrils up to 30 nm in width which formed a ribbon-shape 200 nm wide in wild-type reverting protoplasts. The intrafibrillar space is not filled with amorphous particles of α-galactomannan in nature. The secretion machinery was seen to have a similar profile as the wild type. The above results suggest that actin cytoskeleton may control secretion of β-1,6-glucan and other cell wall substances such as α-glucan and α-galactomannan rather than β-1,3-glucan. Study of the role of actin cytoskeleton in the cell wall formation is contributing to the development of antifungal agents together with basic cell biology.

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.

Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed

Cucumber mosaic virus (CMV) is known to systemically infect Arabidopsis thaliana ecotype Columbia plants. In order to identify the host factors involved in the multiplication of CMV, we isolated an A. thaliana mutant in which the accumulation of the coat protein (CP) of CMV in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in the mutant plants was caused by a single, nuclear and recessive mutation designated cum1-1, which was located on chromosome IV. The cum1-1 mutation did not affect the multiplication of tobacco mosaic virus, turnip crinkle virus or turnip yellow mosaic virus, which belong to different taxonomic groups from CMV. Accumulation of CMV CP in the inoculated leaves of cum1-1 plants was also delayed either when CMV virion or CMV virion RNA was inoculated. On the other hand, when cum1-1 and the wild-type Col-0 protoplasts were inoculated with CMV virion RNA by electroporation, the accumulations of CMV-related RNAs and the coat protein were similar. These results suggest that the cum1-1 mutation did not affect the uncoating of CMV virion and subsequent replication in an initially infected cell but affected the spreading of CMV within an infected leaf, possibly the cell-to-cell movement of CMV in a virus-specific manner.

Molecular and functional characterization of Slide, an Ac-like autonomous transposable element from tobacco.

A new transposable element of tobacco, Slide, was isolated from the tl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases of Ac, Tam3 and hobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left by Ac and Tam3. Tobacco lines derived from the tl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomous Ac-like element characterized in Solanaceae species.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts.

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82-120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33-78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.

Expression Variability of Marker Genes during Plant Regeneration from Protoplasts of R1 Transgenic Mustard (Brassica juncea) Via Somatic Embryogenesis

Summary Expression of marker genes, neomycin phosphotransferase (NPT) II and s-glucuronidase (GUS), in the R1 progeny originating from a transgenic mustard plant line was investigated. The R1 shoots grown in the absence of kanamycin for 6 months showed reduced GUS activity in various tissues and an inability to form roots in the presence of kanamycin. Protoplasts isolated from these shoots expressed GUS at a frequency of 1-5% after 3, 10, 14 and 28d of culture, whereas wild type protoplasts did not show GUS activity throughout the process of somatic embryogenesis. Exogenous application of 20 μM 5-aza-cytidine greatly enhanced the frequency of GUS expressing cells, ranging from 26 to 34%, but not for the wild type during somatic embryogenesis. In protoplast-derived plants, GUS activity was detected in 4 out of 9 plants analysed. Southern analysis revealed that the NPTII gene was present in all GUS positive plants, but absent in GUS negative plants. The pattern of GUS expression in leaves was age-dependent irrespective of the plant developmental stage. GUS was strongly expressed in mature leaves, which was concomitant with an accumulation of a 1.8 kb GUS transcript. In contrast, GUS activity was either absent or occurred only at serrated tips of the young leaves, in which the GUS transcript levels were barely detectable. This study shows transgene instability in R1 mustard plants, which may be due to DNA methylation and/or deletion of the transgenes.

Effects of the tom1 mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus RNA in protoplasts

For the multiplication of RNA viruses, specific host factors are considered essential, but as of yet little is known about this aspect of virus multiplication. To identify such host factors, we previously isolated PD114, a mutant of Arabidopsis thaliana, in which the accumulation of the coat protein of tobacco mosaic virus (TMV) in uninoculated leaves of an infected plant was reduced to low levels. The causal mutation, designated tom1, was single, nuclear, and recessive. Here, we demonstrate that the tom1 mutation affects the amplification of TMV-related RNAs in a single cell. When protoplasts were inoculated with TMV RNA by electroporation, the percentage of TMV-positive protoplasts (detected by indirect immunofluorescence staining with anti-TMV antibodies) was lower (about 1/5 to 1/10) among PD114 protoplasts than among wild-type protoplasts. In TMV-positive PD114 protoplasts, the amounts of the positive-strand RNAs (the genomic RNA and subgenomic mRNAs) and coat protein reached levels similar to, or slightly lower than, those reached in TMV-positive wild-type protoplasts, but the accumulation of the positive-strand RNAs and coat protein occurred more slowly than with the wild-type protoplasts. The parallel decrease in the amounts of the coat protein and its mRNA suggests that the coat protein is translated from its mRNA with normal efficiency. These observations support the idea that the TOM1 gene encodes a host factor necessary for the efficient amplification of TMV RNA in an infected cell. Furthermore, we show that TMV multiplication in PD114 protoplasts is severely affected by the coinoculation of cucumber mosaic virus (CMV) RNA. When PD114 protoplasts were inoculated with a mixture of TMV and CMV RNAs by electroporation, the accumulation of TMV-related molecules was approximately one-fifth of that in PD114 protoplasts inoculated with TMV RNA alone. No such reduction in the accumulation of TMV-related molecules was observed when wild-type protoplasts were inoculated with a mixture of TMV and CMV RNAs or when wild-type and PD114 protoplasts were inoculated with a mixture of TMV and turnip crinkle virus RNAs. These observations are compatible with a hypothetical model in which a gene(s) that is distinct from the TOM1 gene is involved in both TMV and CMV multiplication.

Compartmentation of indole-3-acetic acid metabolism in protoplasts isolated from leaves of wild-type and IAA-overproducing transgenic tobacco plants

Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [15N1]IAA synthesised from [15N1]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SRI plants, and in IAA-overproducing plants expressing the Agrobacterium tumefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was located exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conversion into IAA by action of the iaaH-encoded hydrolase. The free-IAA level, however, was raised only 8% in iaaM/iaaH compared to iaaM protoplasts, whereas the level of IAA-conjugates was increased more than fivefold. For both genotypes, the location of IAA conjugates was restricted to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion to the strong cauliflower mosaic virus (CaMV) 35S promoter. Compared with the wildtype, this led to an 18-fold higher conversion of [15N1]tryptophan to [15N1]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protoplasts. Also in these genotypes, IAA conjugates were exclusively cytosolic. There was no major difference between transgenic and wildtype protoplasts in the proportion of chloroplastic to total cellular IAA, although the chloroplastic IAA and [15N1IAA pools in the transformant were threefold and eightfold higher, respectively. Since the IAM pool in transgenic plants is exclusively cytosolic, these findings suggest that the increased chloroplastic [15N1IAA pool in 35S-iaaM/iaaH protoplasts is synthesised in the cytosol but rapidly transported into the chloroplast. Furthermore, the presence of IAA in the chloroplast together with the exclusively cytosolic location of IAA conjugates, suggests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxylative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 562–568), whereas the second is located in the chloroplast and is seemingly not directly regulated inside the organelle by either of these two processes. The cytosolic IAA control mechanisms, however, also affect the chloroplastic pool size due to the rapid transport of IAA between the two compartments.

Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene.

We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt(60),100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via "gamma"-fusion.

NicotJanamine and the Distribution of Iron into Apoplast and Symplast of Tomato (Lycopersicon esculentumMill.): II. UPTAKE OF IRON BY PROTOPLASTS FROM THE VARIETY BONNER BESTE AND ITS NICOTIANAMINE-LESS MUTANT CHLORONERVA AND THE COMPARTMENTATION OF IRON IN LEAVES1

The influence of nicotianamine (NA) on the distribution of iron into apoplast and symplast of a NA-containing tomato wild-type and its NA-less mutant was investigated. Isolated protoplasts from wild-type and mutant leaves are able to reduce exogenous iron(III)citrate at equal rates. In spite of this, protoplasts from mutant leaves take up more iron from iron(III)citrate than wild type protoplasts. The mutant leaves accumulate higher amounts of iron in apoplast and symplast than wild-type leaves with an iron supply of 10 fiM FeEDTA in nutrient solution. NA treatment of the mutant leaves decreases both apoplasmic and symplasmic iron in the direction of wild-type values. It is concluded that NA is not essential for iron transport through the plasmalemma of protoplasts, but that endogenous NA decreases the high amount of iron in protoplasts by affecting the feed-back regulation of iron uptake by leaf cells.

En/Spm encoded tnpA protein requires a specific target sequence for suppression.

The En/Spm encoded suppressor function has been reconstituted in transgenic tobacco protoplasts. The suppressor affects genes which contain an En/Spm responsive transposable element in the transcribed sequences. The En/Spm encoded protein tnpA binds a defined cis element in the inserted transposon, repressing expression of the adjacent gene. This was shown by monitoring transient expression of a bacterial marker gene (GUS) expressed from a strong plant viral promoter. Suppressible variants of the marker gene were produced by inserting I element sequences into the untranslated sequences of the GUS transcript. Comparison of transient expression of these variants in wildtype tobacco protoplasts with their expression in protoplasts transgenic for tnpA protein demonstrates that tnpA is the suppressor. In addition, the minimal cis element required for suppression has been defined as a dimer consisting of two 12 bp tnpA binding sequences in a particular inverted orientation. One of these dimers occurs in each En/Spm end close to the characteristic 13 bp terminal inverted repeat. TnpA binding sites in different arrangements do not respond as well to tnpA. The implications of this observation are discussed. This system can be used to analyse tnpA-DNA interactions involved in gene regulation further.

Microculture and Electrofusion of Defined Protoplasts of the Moss Funaria hygrometrica

AbstractProtoplasts isolated from protonemata of Funaria hygrometrica wild type and developmentally abnormal mutants were individually selected and cultured in microdroplets of defined, unconditioned culture medium. Up to 60 % of the microcultured wild type protoplasts regenerated whole plants after 4 weeks. Electrofusion in different combinations of defined protoplast pairs of wild type and mutated forms was performed. The products derived from one‐to‐one electric field induced fusion were efficiently microcultured and somatic hybrids were regenerated.

Auxin Effect on the Transmembrane Potential Difference of Wild-Type and Mutant Tobacco Protoplasts Exhibiting a Differential Sensitiity to Auxin

The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity.

Production and Secretion of Saccharomyces cerevisiae -Glucanases: Differences between Protoplast and Periplasmic Enzymes

Cell-free extracts and culture medium from Saccharomyces cerevisiae S288C contained the two glucan hydrolases that are known to be periplasmically located in vegetative cells of this yeast. These were an endo-1,3-β-glucanase and a much more abundant exo-1,3-β-glucanase. Cell-free extracts of strains carrying two different mutant alleles of the EXB1 gene were totally deficient in the latter enzyme, whereas the former appeared in multiple heterogeneous forms, probably due to incomplete glycosylation. In contrast, protoplast lysates of wild-type and exb1 mutant strains were identical in their complement of glucanases, which consisted of two enzymes clearly distinguishable from the periplasmic glucanases. One was an exo-1,3-β-glucanase, which was active on pustulan, p-nitrophenyl β-d-glucoside, salicin and cellobiose, and was of higher M r than periplasmic exoglucanase. The other was a hydrolase acting on 1,3-β-glucan and 1,6-β-glucan but not the simple glucosides, which was not retained by DEAE-Biogel. Wild-type and exb1 mutant protoplasts secreted portions of the glucanases detected in protoplast lysates, when cultured in osmotically stabilized regeneration medium; the former also secreted the periplasmic exo-1,3-β-glucanase but the latter consistently failed to secrete it. It is concluded that the classically known glucanases of S. cerevisiae, located in the periplasmic space, must be formed as active enzymes, upon secretion, from inactive cytoplasmic precursors. On the other hand, the two new protoplast glucanases could be secreted by an alternative route that assures their incorporation into the wall structure, thus making their release into the culture medium more difficult.

Expression of nuclear-cytoplasmic genomic incompatibility in interspecific Petunia somatic hybrid plants

Somatic hybrid plants were regenerated following calcium-high pH fusion of the unidirectional, sexually incompatible cross of Petunia parodii wild-type leaf mesophyll protoplasts with protoplasts from a cytoplasmic determined chlorophyll-deficient mutant of P. inflata. Genic complementation to chlorophyll synthesis and sustained growth in the selective medium was used to visually identify hybrid calluses. Hybrid calluses were subsequently regenerated to shoots, rooted, and confirmed as somatic hybrids by their intermediate floral and leaf morphology based on comparison to the 2 n = 4 x = 28 sexual counterpart, dominant anthocyanin expression in the corolla, chromosome number, and peroxidase and maleic dehydrogenase isozyme patterns. Certain cytologically stable somatic hybrids displayed aberrant reproductive and floral morphologies including subtle to moderate corolla and leaf pigment variegation, floral dimension changes and reduced pollen viability. In contrast, cytologically unstable somatic hybrids showed various degrees of aneuploidy coupled with corolla splitting, and irregularities in reproductive organs such as double stigmas and styles in addition to reduced pollen viability. Postulated mechanisms to account for these phenotypic changes in stable and unstable somatic hybrids include nuclear-cytoplasmic genomic incompatibility, chromosome loss in a biparental cytoplasm, or a phenomenon similar to hybrid dysgenesis occurring as a result of somatic fusion.