Plasmalemma hyperpolarity and culture of sense and antisense abp transgenic tobacco protoplasts

The relationship among transfer and expression of auxin binding protein gene (abp), auxin (NAA)-induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisenseabp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of senseabp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisenseabp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there. Project supported by the State Key Laboratory of Plant Molecular Genetics and National Natural Science Foundation of China (Grant No. 39670078).     

Plasmalemma hyperpolarity and culture of sense and antisense abp transgenic tobacco protoplasts

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In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of β-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of β-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating β-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced β-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 × 10⁻⁵ molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco β-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.     

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.     

Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.     

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     

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.     

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.     

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     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Rol genes alter hormonal requirements for protoplast growth and modify the expression of an auxin responsive promoter

Summary Growth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.Abbreviations T-DNA transferred DNA - TL-DNA left transferred DNA - NAA naphthalene acetic acid - PEG polyethylene glycol - GUS glucuronidase - CaMV cauliflower mosaic virus     

Nutritional requirements of protoplast-derived,haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l^-1 NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1–4 cells ml^-1, provided the concentration of NAA in the medium was lowered to below 0.3 mg l^-1. The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30–40%, independent of the cell density in the range of 1–4 to 3·10⁴ cells ml^-1, has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l^-1 NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [¹⁴C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.Abbreviations BA 6-benzyladenine - EDTA ethylene diaminetetraacetic acid - NAA naphtaleneacetic acid     

Controlled cell wall regeneration for efficient microinjections of Nicotiana tabacum var. Carlson protoplasts

Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection. Furthermore, the duration of protoplast microinjection was extended for up to 3–4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.Abbreviations AMV Alfalfa Mosaic virus - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - DB 2,6-dichlorobenzonitrile - LR lissamine rhodamine - NAA 1-naphthalene-acetic acid     

Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants

Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the -glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid     

Behavior and viability of tobacco protoplasts in response to electrofusion parameters

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

     

紫云英叶片及叶肉原生质体的培养

本工作研究了豆科植物紫云英的叶片及叶肉原生质体的培养。叶片培养实验表明,诱导愈伤组织的最适培养基为MS加1.0-2.0毫克/升2,4-D和0.25毫克/升KT;诱导根分化需加1.0—5.0毫克/升NAA和0.5毫克/升BA;而苗分化则以0—0.5毫克/升IAA和0.5毫克/升BA为好。高浓度的NAA有利于根分化而抑制茎芽形成;高浓度的IAA对根和芽分化都有抑制作用。叶肉原生质体分离和培养试验表明,紫云英叶肉原生质体的释放及其培养活力受叶龄、植株生理状态和酶浓度的影响。叶肉原生质体在改良的KM8P培养基中能分裂。用改良KM8细胞培养基定期稀释,可使分裂持续进行而得到细胞团。BA和2,4-D为诱导紫云英叶肉原生质体分裂所必需。其最佳组合激素为BA 0.21毫克/升和2,4-D 1.13毫克/升。葡萄糖作为渗透压稳定剂时,其浓度明显影响原生质体的存活率。弱光条件下培养比黑暗培养有利于叶肉原生质体分裂。由叶肉原生质体形成的愈伤组织能形成瘤状结构和根。     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid