Suspension Culture Protoplasts Research Articles

A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

A Kunitz-type protease inhibitor co-purified from cauliflower florets with a granulin domain cysteine protease that cleaved barley proaleurain to yield a molecular form the same size as that for mature aleurain. The purified cauliflower protease required treatment with SDS detergent to become active. This observation raised the question of whether the protease inhibitor might have the ability to interact with the granulin domain protease. Here we express an Arabidopsis homolog of the protease inhibitor as a recombinant protein and demonstrate that it is a potent inhibitor of the recombinant proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts, it colocalized with BP-80, a vacuolar sorting receptor that interacts with proaleurain and traffics to prevacuolar compartments for lytic vacuoles. Our results indicate that the cauliflower and Arabidopsis protease inhibitors would traffic through cellular compartments where proaleurain also traffics. Their ability to inhibit a cysteine protease implicated in maturation of proaleurain to active form at the acidic pH found in vacuoles raises the possibility that they could participate in regulating activation of aleurain.

Factors influencing in vitro regeneration from protoplasts of wild cotton (G. klotzschianum A) and RAPD analysis of regenerated plantlets

Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×105 ml−1, and the medium was k3 inorganic salts with modified KM8P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA3 on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity.

New green fluorescent protein genes for plant transformation: Intron-containing, ER-localized, and soluble-modified

We describe several new modifications of theAequorea victoria green fluorescent protein (GFP) gene. TheerGFP5INTreporter gene combines the PIV2 intron fromgusINTandLUCINTwith the ER-localizedmGFP4-ER gene. TheerGFP6INT, erGFP7INT, anderBFP8INTgenes also include the fluorophore and solubility modifications of smGFP, smRS-GFP, and smBFP, respectively. A parallel set of reporter genes (erGFP5, erGFP6, erGFP7, anderBFP8) is otherwise identical to the respectiveerGFPINTgenes but lacks the PIV2 intron. The intron-containing genes are expressed in plant cells but not in bacteria, allowing detection of plant cell expression in the presence ofAgrobacterium during the early stages of transformation. Transient expression of theerGFPINTanderGFP genes is comparable in tobacco and maize suspension culture protoplasts, indicating that the PIV2 intron is spliced effectively in both monocotyledonous and dicotyledonous plant species.

Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells.

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleurone RNase revealed that, like the pattern for alpha-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase alpha-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling

it is not always easy to prepare healthy, viable ones, and also it is not easy to measure the kinetics of their changing A quick and accurate method of monitoring changes dimensions in response to an osmotic gradient. Recent of volume of mesophyll cell protoplasts (MCP) of pea studies used vesicles prepared from root cells of wheat was devised using the difference in absorbance of the (Niemietz and Tyerman, 1997) or from suspension-cultured protoplasts at 440 nm and 750 nm (OD 440‐750); when tobacco cells (Maurel et al., 1997) and indirectly measured protoplasts expanded in response to changing the changes in volume by monitoring light scattering of a external medium from 0.4 M sorbitol to 0.3 M sorbitol suspension of vesicles with stopped-flow spectrofluorimetry. OD 440‐750 values increased and, conversely, when Such a system is diYcult to use with protoplasts since they protoplasts were transferred from 0.4 to 0.5 M sorbitol, sediment relatively quickly. In this study, however, a protoplasts contracted. The kinetics of expansion or method of indirectly measuring volume changes of protocontraction of the protoplasts could also be monitored plasts is described and the eVects of mercuric chloride and using this method and the half-time for water exchange mercaptoethanol on the volume changes induced by for expanding protoplasts (about 10 s) was slightly osmotic gradients are reported in order to investigate the higher than that for contracting protoplasts. A study involvement of water channels in such phenomena. of the effects of the water channel blocker, mercuric Mercury ions are reported to inhibit water movement chloride, on swelling protoplasts showed that 500 mM through water channels (aquaporins) while sulphydryl irreversibly damaged protoplasts, 5‐10 mM had a neg- reagents, such as mercaptoethanol, are reported to reverse ligible inhibitory effect on swelling while 100 mM had the inhibition (Steudle and Henzler, 1995). a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercaptoethanol reversed the inhibitory effect of mercuric Materials and methods chloride on protoplast swelling. Plant material and protoplast preparation

Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways.

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of alpha-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of gamma-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of gamma-TIP did not alter this traffic. In contrast, the alpha-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and alpha-TIP protein colocalized in them with the alpha-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.

Molecular cloning and further characterization of a probable plant vacuolar sorting receptor.

BP-80 is a type I integral membrane protein abundant in pea (Pisum sativum) clathrin-coated vesicles (CCVs) that binds with high affinity to vacuole-targeting determinants containing asparagine-proline-isoleucine-arginine. Here we present results from cDNA cloning and studies of its intracellular localization. Its sequence and sequences of homologs from Arabidopsis, rice (Oryza sativa), and maize (Zea mays) define a novel family of proteins unique to plants that is highly conserved in both monocotyledons and dicotyledons. The BP-80 protein is present in dilated ends of Golgi cisternae and in "prevacuoles," which are small vacuoles separate from but capable of fusing with lytic vacuoles. Its cytoplasmic tail contains a Tyr-X-X-hydrophobic residue motif associated with transmembrane proteins incorporated into CCVs. When transiently expressed in tobacco (Nicotiana tabacum) suspension-culture protoplasts, a truncated form lacking transmembrane and cytoplasmic domains was secreted. These results, coupled with previous studies of ligand-binding specificity and pH dependence, strongly support our hypothesis that BP-80 is a vacuolar sorting receptor that trafficks in CCVs between Golgi and a newly described prevacuolar compartment.

A Novel Method of Plant Regeneration from Suspension Culture Protoplasts of Sugarcane

Summary Protoplasts were released from cell suspension cultures of two genotypes (87-588 and F177) of sugarcane ( Saccharum spp. hybrids). They were embedded in an agarose block with PRM medium, over which 5 mL nurse cell culture was flooded. The nurse cell culture was changed at weekly intervals to reduce osmotic pressure and to readjust the cell density. Genotype 87-588 micro-colonies formed on the agarose block within 1 month. After having grown to a larger size, they were transferred to a solid MS based medium where they took on a rectangular shape (1.2x2.8cm). This proto-callus (from 87-588) was surrounded by a 0.8-cm thick wall composed ofF 177 callus covered with newly differentiated green shoots. By using the nurse green shoot's differentiating factor carry-over idea, the enclosed 87-588 callus was induced to undergo meristematic growth. After 30 days, the treated-callus was placed on a differentiation medium where many green buds were regenerated within a 1-month incubation. Using a similar method, plant regeneration from protoplasts of F177 was also successful.

Stably transformed cell lines from protoplasts of maize endosperm suspension cultures

Protoplasts were isolated from Zea mays (L.) A69Y endosperm suspension cultures and transformed by polyethylene glycol mediated DNA uptake with chimaeric gene constructs containing β-glucuronidase (GUS) or neomycin phosphotransferase II (NPTII); GUS-expressing and Kanamycin-resistant cultures were recovered. The transformed cells showed integration of the introduced foreign genes into genomic DNA and maintained their ability to synthesize endosperm-specific reserve proteins (zeins). No deletion or rearrangement of zein genes were observed in transformed cultures. Stable transformation of cultured maize endosperm cells may therefore represent a new methodological approach for the study of the transcriptional regulation of endosperm-expressed genes.

Metabolism of glucose monophosphates by leucoplasts and amyloplasts from soybean suspension cultures

Leucoplasts and amyloplasts were isolated on Nycodenz gradients from lysates of protoplasts of suspension cultures of soybean ( Glycine max). The labelling of CO 2 and starch was determined after incubating intact and lysed preparations of plastids with 14C-labelled substrates. The substrates were said to have been metabolized by the plastids if the labelling of the product was greater in the intact than in the lysed preparations. Leucoplasts and amyloplasts metabolized both [ 14C]glucose 1-phosphate and [ 14C]glucose 6-phosphate to 14CO 2. In both types of plastid [ 14C]glucose 6-phosphate was the better precursor. Neither substrate was converted to starch by intact leucoplasts. Amyloplasts converted [ 14C]glucose 1-phosphate, but not [ 14C]glucose 6-phosphate, to starch in the presence of exogeneous ATP. It is suggested that in these plastids the reaction catalysed by phosphoglucomutase is not at equilibrium.

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10(6)/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R1 plants were self-pollinated and immature R2 embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R2-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.

A modified β-glucuronidase gene: Sensitive detection of plant promoter activities in suspension-cultured cells of tobacco and rice

A translational initiation sequence, conserved among soybean lipoxygenase genes, was fused with the β-glucuronidase (GUS) gene, placed under 35S cauliflower mosaic virus promoter control, then electroporated into suspension-cultured protoplasts of tobacco and rice. These constructs produced 11- and 7-fold increases in GUS activity, respectively, compared with the original GUS construct. The transcriptional activity of the modified GUS gene fused with the TATA box from a soybean lipoxygenase gene was also detected with great sensitivity. The fusion construct is useful for detectingcis-regulatory element effects on transcription, in particular, silencers.

Intracellular location of NADP+-linked malic enzyme in C3 plants

The aim of this work was to determine the maximum catalytic activity and intracellular location of NADP(+)-linked malic enzyme (EC 1.1.1.40) in C3 plants. Appreciable activities, ranging from 80 to 712 nmol · (gFW)(-1) · min(-1), were found in a wide range of tissues (roots and leaves of Pisum sativum L., cotyledons of Cucurbit a pepo Alef., developing seeds of Brassica napus L., mesocarp of Persea americana Gaertn., and suspension cultures of Glycine max L.). Overall, activity showed a rough positive correlation with biosynthesis. Differential and density-gradient fractionation of extracts of the cotyledons of germinating marrow (C. pepo) and lysates of protoplasts of suspension cultures of G. showed that the enzyme had the same distribution as the plastid marker enzymes. It is suggested that in C3 plants NADP(+)-linked malic enzyme is confined to the plastids and involved in biosynthesis.

Improved electrofusion of protoplasts of varied fusibility by selective pairing: application of asymmetric breakdown of plasma membranes

Asymmetric membrane breakdown was used for electrofusion of plant protoplasts with different physical properties. The model systems, in which parental cell types differ in fusibility, were (1) mesophyll protoplasts of Pennisetum americanum and suspension culture protoplasts of Panicum maximum and (2) vacuolated and evacuolated tobacco mesophyll protoplasts. The inherent or artificially induced differences in specific densities of the fusion partners were utilized to achieve an ordered pair formation. In a suitable sedimentation medium placed between horizontal electrode plates the mixed protoplasts separated to form two successive monolayers (‘differential sedimentation’). Under these conditions high rates of heterokaryotic fusion products were obtained. Fusion yields depended on the direction of the applied field pulse: if the contact zone was established at the anodic face of the more susceptible partner, fusion rates were up to two times higher, compared to the opposite polarity. The application of these results is discussed.

Regeneration of fertile plants from embryogenic suspension culture protoplasts of Sorghum vulgare

Protoplasts were isolated from immature inflorescence-derived embryogenic suspension cultures of two cultivars of Sorghum vulgare. The protoplasts were cultured in a modified K8P liquid medium. They started to divide after 4-5 days of culture, and achieved 16.8% division frequency by 10 days. Protocalli proliferated further upon transfer to C1 solid medium. After that, they were moved to C1 differentiation medium to induce shoot formation, followed by whole plant regeneration. So far, 60 plants have been obtained, with only two albinos. Some of these have been transplanted to soil in pots and grown to flowering and have set seeds.

Regeneration of Plants from Embryogenic Suspension Culture Protoplasts of Wheat (Triticum aestivum L.)

We describe the regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus. Proto-colonies were also obtained from a non-regenerable cell line established from friable callus that arose spontaneously on the aged callus, but did not give rise to any plants. Plating efficiencies well in excess of twenty percent were routinely obtained from protoplasts isolated from the regenerable as well as the non-regenerable cell lines in Kao and Michyaluk, as well as Murashige and Skoog based, nutrient media. Four to five week old protocolonies, derived from regenerable suspension culture protoplasts, formed distinct somatic embryos and shoots upon transfer to various regeneration media. Shoots were rooted on transfer to rooting media. Successive transfer of the plantlets to MS or half-strength MS medium, with or without activated charcoal, produced healthy plants that were successfully transferred to soil and grown in the greenhouse.

Callus regeneration from Trifolium subterraneum protoplasts and enhanced protoplast division by low-voltage treatment and nurse cells

Seedling and suspension culture protoplasts of subterranean clover (Trifolium subterraneum L.) were successfully cultured in semi-solid drops of calcium alginate and ultrafiltered liquid medium. Protoplast-derived subterranean-clover colonies developed as the osmolality was lowered over three steps. Callus was established from these colonies. Calli derived from protoplasts have failed to regenerate on a range of media. The frequency of dividing subterranean-clover protoplasts was increased in the presence of lucerne (Medicago sativa L.) nurse cells. Low-voltage treatments (200 mV) for the first 16–132 hours of culture also resulted in a 100% increase in the frequency of dividing protoplasts.

Inhibition of transgene expression in plant protoplasts by the presence in cis of an opposing 3′-promoter

The possibility that gene expression could be negatively modulated by RNA polymerases transcribing a gene from its 3′ end towards its 5′ end concurrent with normal 5′ to 3′ transcription has been investigated. The effect of 3′ promoter presence and orientation on 5′ to 3′ gene transcription was determined by comparing transient chloramphenicol acetyltransferase (CAT) activity in Nicotiana tabacum suspension culture protoplasts electroporated with various cat gene containing plasmids. Initial constructs contained the CaMV 35S promoter 5′ to the Tn9 cat gene- nos terminator and in some cases the CaMV 35S promoter 3′ to the nos terminator. Using these constructs in linearized form a statistically significant reduction, about two-fold, in CAT activity as compared to normal single 5′ promoter configuration driven expression, was seen only in the presence of a 3′ promoter inverted with respect to the 5′ promoter. The presence of a 3′ promoter in the same orientation as the 5′ promoter resulted in no significant reduction in CAT activity. Possible mechanisms include antisense RNA production, RNA polymerase ‘colllision’, transcriptionally induced topological constraints and/or destabilization of positive transcriptional factors. The effects of decreasing the 5′ promoter strength and providing a strong polyA-signal/terminator for the antisense strand are also reported.

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus).

Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues. Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15%. After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol. Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol. Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent. However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol. Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.