Cell Suspension Protoplasts Research Articles

Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A.thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

Electroporation and morphogenic potential of Gentiana kurroo (Royle) embryogenic cell suspension protoplasts

This article presents our further in vitro studies into the morphogenic potential of gentian cells, organs, and tissues after modification of their genome. The objective was to study the effect of electroporation and the introduction of foreign genes on the morphogenic potential of Gentiana kurroo embryogenic cell suspension protoplasts. Protoplasts were electroporated with DNA plasmids carrying nptII and bar genes. The stability of cell membranes, the contents of electroporation buffer, the length of electric pulse, the number of pulses and the strength of the electric field were studied. We determined the highest electroporation efficiency by evaluating the highest protoplast survival rate under specific physical conditions. The best results were achieved in the presence of EB1 electroporation buffer where the viability of protoplasts was 70.1%. Protoplast survival at this higher level required culture temperatures near 0EC, and a 20 μs electric pulse with an electric field of 1.0 kV/cm. After seven days of agarose embedded protoplast culture, a selective agent – kanamycin – was introduced to the medium. The cell transformation effect was improved by a long term culture of callus, regenerated somatic embryos and transformants in the presence of 50 mg/l kanamycin.

Bimolecular fluorescent complementation (BiFC) by MAP kinases and MAPK phosphatases.

The adaptation of plants to the environment is a key property for survival. Adaptation responses to environmental cues are generated in cells by signaling initiated from cell receptors. Signal transduction is based on protein phosphorylation that is employed in mitogen-activated protein kinase (MAPK) cascades to integrate signals from receptors to cellular responses. MAPK activity is determined by phosphorylation of amino acid residues within the kinase activation loop and their dephosphorylation by phosphatases is essential to control signal duration and intensity.Monitoring protein-protein interactions (PPIs) of MAPKs with MAPK phosphatases in vivo provides valuable information about specificity and intracellular localization of the protein complex. Here, we report studying PPIs between Arabidopsis MAPKs and PP2C-type MAPK phosphatases using bimolecular fluorescent complementation (BiFC) in suspension cell protoplasts. The interactions of the MAPKs MPK3, MKP4 and MPK6 with the phosphatases AP2C1 and AP2C3 have been tested.

Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film

Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots

The rice ( Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5′-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB’s 5′-flanking DNA fragments (nucleotides –1066 to +298) had about 20 and 3–4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (–56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (–56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5′-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from –329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (–329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.

Small-Scale Isolation of Viral RNA-Dependent RNA Polymerase from Protoplasts Inoculated with In Vitro Transcripts

Cowpea chlorotic mottle virus (CCMV) replicated in tobacco suspension cell protoplasts inoculated with in vitro transcripts of CCMV RNA1, 2, and 3. CCMV RNA-dependent RNA polymerase (RdRp) isolated from these protoplasts specifically recognized CCMV and Brome mosaic virus (BMV) subgenomic RNA promoters and directed in vitro RNA synthesis in a manner indistinguishable from CCMV RdRp more laboriously isolated from systemically infected cowpea leaves. Omission of CCMV RNA3 from the protoplast inoculum or replacement with in vitro transcripts of BMV RNA3 reduced CCMV (+)-strand RNA1 and 2 accumulation to approximately 1/40 and approximately 1/10, respectively, of the level attained when CCMV RNA3 was present. The absence of CCMV RNA3 did not prevent assembly and isolation of highly active, template-dependent and template-specific CCMV RdRp, which directed synthesis of products identical in size to those of RdRp isolated from protoplasts inoculated with all three CCMV genomic RNAs. These results demonstrate that CCMV RNA1 and 2 are sufficient for CCMV replication and RdRp assembly in tobacco protoplasts. This approach for isolation of functional viral RdRp will be especially useful for viruses for which large quantities of infected tissue are unavailable, such as those with specific tissue tropisms or mutants incapable of systemic movement.

An Arabidopsis cyclin promoter region is active in transgenic maize plants

We have produced transgenic maize carrying Arabidopsis cyclin promoter and beta-glucuronidase chimeric fusion (cycB1;1At::GUS) by direct DNA uptake into embryogenic cell suspension protoplasts to study the activity of this cyclin promoter in a heterologous cell system. The transgenic maize lines exhibiting different levels of reporter gene expression show that regulatory elements from cycB1;1At gene as a dicot promoter can function in maize. Expression patterns revealed by fluorimetric, as well as, histochemical GUS reporter enzymatic assays indicated cell division-dependent expression of the GUS gene driven by this cycB1;At;1 promoter with elevated activity in proliferating cells and meristems. Treatment of tissues with inhibitors of mitosis increased the activity of cycB1;1At promoter, that suggests a link between mitotic phase of cell division and activity of cycB1;1At regulatory elements in transgenic maize.

Meristem, cell division and S phase-dependent activity of wheat histone H4 promoter in transgenic maize plants

We have studied the activity of a wheat histone H4 promoter in transgenic maize plants produced by direct DNA uptake into embryogenic cell suspension protoplasts. Expression patterns revealed by fluorimetric as well as histochemical GUS reporter enzyme assays and Northern hybridisation indicated the cell division-dependent expression of the GUS gene driven by the histone H4 promoter in proliferating cells and meristems. Changes in gene expression associated with the progression through the cell cycle in maize cell-suspension cultures were also investigated after partial synchronisation of cell division by aphidicolin or hydroxyurea. The transgene expression regulated by the wheat histone H4 promoter coincided with elevated levels of mRNAs from other S phase-associated genes such as maize histone H4, histone H2B and proliferating cell nuclear antigen ( PCNA) . The reporter gene expression was low during mitosis when accumulation of the transcripts of a B-type mitotic cyclin CycB1;zm;1 could be detected. The presented data indicate that the used 720-bp-long promoter region can provide replication-dependent expression for the GUS reporter gene in transgenic maize. In these maize plants, external hormone stimuli generated by a synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D) or abscisic acid (ABA) have modified the histone H4 promoter-driven GUS gene transcription. These findings support the usefulness of the H4::GUS transgenic plants for further studies on cell cycle activation/inactivation by mitogenic or stress related stimuli in maize.

Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solanum tuberosum L

Freshly isolated mesophyll and suspensions-cell protoplasts of S. tuberosum cvs. Desiree and Maris Piper were cultured in different media i.e. modified MS, V-KM and MS-KM. Protoplast plating efficiencies were higher in MS-KM medium. Resulting protoplast-derived calluses were transferred either onto the medium of Bokelmann and Roest (1983) or that of Lam (1977) for shoot regeneration. Calluses derived from mesophyll cell protoplasts differentiated about 2 weeks earlier than calluses derived from suspension-cell protoplasts. Shoot initiation was also about 2 weeks earlier from calluses subcultured onto the former medium as compared to the latter.

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus.

Variation among plants regenerated from microspore-derived cell suspension protoplasts of rice

Variation among plants regenerated from microspore-derived cell suspension protoplasts of rice

Mutations That Alter a Conserved Element Upstream of the Potato Virus X Triple Block and Coat Protein Genes Affect Subgenomic RNA Accumulation

The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation ofNicotiana tabacumNT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

Plant regeneration from highly embryogenic callus, cell suspension and protoplast cultures of Trifolium fragiferum

Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum (2n=16). (L.). The best media for induction of embryogenic cultures were based on Kao (1977) or Kao and Michayluk (1975). Somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants, somatic embryo protoplasts and cell suspension protoplasts, leaflets and various explants of immature zygotic embryos. The process of somatic embryogenesis was maintained for over two years on Murashige and Skoog's (1962) medium supplemented with 0.5 mg l-1 benzyladenine and 0.05 mg l-1 naphthaleneacetic acid. These long term cultures were capable of regenerating plants that were fertile and produced seeds. These results were compared with those from protoplast, tissue and organ culture of other species of the Trifolium genus.

Production of Intergeneric Hybrid Calli from C3 and C4 Species of Amaranthaceae through Protoplast Fusion.

Intergeneric somatic hybrid calli were obtained through electrofusion between protoplasts of Celosia cristata L. (C3 species) cell suspension and Amaranthus tricolor L. (C4 species) cotyledon. The highest binary fusion frequency, 4∼5%, was attained when the protoplasts were suspended in fusion solution containing 0.4 M sorbitol and 2.0 mM CaCl2, aligned by an AC field (200 V/cm, 0.5 MHz) for 30 s and fused by a single DC square pulse at 1.6 kV/cm for 40 μs. Fusion products were identified by characteristics of both parents in the hybrid cells : they possessed green chloroplasts or pink plastids from cotyledonary protoplasts as well as a cytoplasm-rich colorless sector derived from cell suspension protoplasts. The occurrence of two types of calli, compact and friable, obtained after culturing on proliferation medium lead to the isolation of putative hybrid calli without any artificial selection because C. cristata protoplasts regenerated only friable calli and A. tricolor protoplasts did not produce calli. A total of 14 lines of putative hybrid compact calli were obtained by screening on the basis of callus morphology. An isozyme analysis of acid phosphatase confirmed the hybridity of 14 lines of compact calli. Callus morphology was also studied by scanning electron microscopy.

Transformation of haploid, microspore-derived cell suspension protoplasts of rice ( Oryza sativa L.)

Transformation of haploid, microspore-derived cell suspension protoplasts of rice ( Oryza sativa L.)

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 2∶1 or 1∶1 ratio, led to 0.8 × 10(-5) and 1.6 × 10(-5) resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and β-glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.

A Subgenomic RNA Associated with Cherry Leafroll Virus Infections

Cherry leafroll nepovirus (CLRV) genomic RNA 1 (8 kb) and genomic RNA 2 (7 kb) have 3′ polyadenlyate tracts and, extending 5′ from the polyadenylate, nearly identical sequences of 1.6 kb termed the 3′ common region. We observed RNAs 1 and 2 and a third RNA of 1.5 kb in nucleic acid extracts of CLRV-infected Nicotiana tabacum suspension cell protoplasts and Chenopodium quinoa plants, using a hybridization probe complementary to 1 kb of the 3′ common region. The third RNA was partially purified by preparative gel electrophoresis and chromatography on an oligodeoxythymidylate column. Analyses of transcripts primed by a complementary oligodeoxyribonucleotide and of cDNA clones revealed that the third RNA corresponds to the 3′ 1500 nucleotide residues of RNA 1. Hence we designate the newly characterized RNA as RNA 1A. RNA 1A was not detected as encapsidated RNA in extracts of either protoplasts or C. quinoa plants. The amount of accumulated RNA 1A declined between 24 and 48 hr after inoculation of protoplasts with CLRV virions, although CLRV RNAs 1 and 2 continued to accumulate. Other results were not consistent with cleaved RNA 1 being the origin of RNA 1A. RNA 1A has the properties of a subgenomic RNA, presumably synthesized from negative-sense RNA 1 as template.

Transient Gene Expression of Antisense RNA and Coat Protein-Encoding Sequences Reduced Accumulation of Cherry Leafroll Virus in Tobacco Protoplasts

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA. Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3′ terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electropotation of plasmids intended to express coat protein or 3′ antisense RNA, but not by electropotation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.