Amino-acid transport in suspension-cultured plant cells

The carrier system which transports L-leucine (L-leu) into suspension-culturedNicotiana tabacum L. cv. Wisconsin 38 cells appeared to be constitutive since it was always present and was not induced by L-leu even in nitrogen-starved cells. However, L-leu uptake rates for cells grown in medium containing L-leu were transiently reduced as a result of either transinhibition or repression. Growth-phase cells appeared to have more L-leu carriers per unit area of membrane than stationary-phase cells, and for this reason growing-phase cells exhibit higher L-leu uptake rates. These higher rates reflect a physiological or developmental condition since growth-phase cells did not dramatically change their L-leu uptake rates when subcultured, while stationary-phase cells doubled their rates within 6 h after being subcultured. Cells grown in a medium lacking a useable carbon souce had uptake rates higher than control rates for several days. These higher rates peaked after about 1 d and then decreased over the next several days. Cells grown in a medium lacking a nitrogen souce responded similarly except that the increased rates peaked after about 3 d and persisted longer. Kinetic analysis of uptake rates in cells grown without a carbon souce for 1 d or without a nitrogen souce for 3 d indicated that the L-leu carrier had K_ms similar to those of untreated cells. These results indicate that cultured tobacco cells respond to their environment by increasing or decreasing the number or activity of kinetically similar L-leu carriers.Abbreviations L and S medium Linsmaier and Skoog (1965) medium with additions - L-leu L-leucineIV=McDaniel et al. 1981     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Preferential synthesis of plastid DNA and increased replication of plastids in cultured tobacco cells following medium renewal

During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 46-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [³H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.Abbreviations pt plastid - DAPI 4,6-diamidino-2-phenylindole     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Movement of elicitins,necrosis-inducing proteins secreted by Phytophthora sp., in tobacco

In culture, Phytophthora fungi — except P. nicotianae — secrete proteins, called elicitins, which cause necrosis on the leaf of the non-host tobacco (Nicotiana tabacum L.) at a distance from the inoculation site, and are responsible for the incompatible reaction. Cryptogein and capsicein are elicitins secreted by P. cryptogea and P. capsici, respectively, and form part of a novel family of 10-kDa holoproteins. On tobacco, the necrotic activity of cryptogein is approx. 100-fold higher than that of capsicein. Using elicitins radioactively labelled in vivo, we have demonstrated that cryptogein and capsicein (i) move from a wound in the stem towards the leaves where they interact directly, (ii) reach their target without undergoing any molecular alteration, (iii) are carried in, and at the same rate as, the sap flow in the xylem, (iv) do not alter the rate of the xylem flow although they are able to provoke drastic damage to the lamina. Consequently, the remote necrotic activity of elicitins does not require any transportable secondary plant elicitor, so the differences in necrotic properties should be due to structural features involved in the interaction of elicitins with the leaf target cells.Abbreviations M_r relative molecular mass - RPLC reversephase liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresisThe authors are indebted to Mauricette Sallé-Tourné, Marc Sallantin and Christian Ouali for their skilful technical assistance.     

Effect of virus infection on symplastic transport of fluorescent tracers in Nicotiana clevelandii leaf epidermis

The molecular weight exclusion limit of plasmodesmata in subveinal epidermal cells of Nicotiana clevelandii (Gray) leaves was estimated by microinjection and fluorescence microscopy using fluorescein isothiocyanate-peptide conjugates, carboxyfluorescein and Lucifer Yellow CH. The largest fluorochrome which moved symplastically between cells had a molecular weight of 749, although movement did not appear to depend purely on molecular weight parameters. Systemic infection of plants by tobacco rattle tobravirus, tomato black ring nepovirus or potato Y potyvirus did not alter the limits of plasmodesmatal conductance of the fluorochromes. However, carrot mottle umbravirus and groundnut rosette umbravirus diminished the symplastic mobility of some fluorescent tracers. These results imply that intercellular movement of these viruses does not involve a long-lasting increase in the plasmodesmatal molecular size exclusion limit.Abbreviations CMotV carrot mottle umbravirus - GRV groundnut rosette umbravirus - Glu l-glutamate - GluGlu -glutamyl glutamate - FITC fluorescein isothiocyanate - Ala₆ hexa-l-alanine - Gly₆ hexa-l-glycine - PVY potato Y potyvirus - TBRV tomato black ring nepovirus - TRY tobacco rattle tobravirus - TyrGlyGly tyrosylglycylglycine     

Quantitative studies of bud initiation in cultured tobacco tissues

After transferring leaf, pith, and stemcortex tissues ofNicotiana tabacum L. cv. Havana 425 from a complete medium containing auxin and cytokinin to an inductive medium with auxin deleted, there is lag phase of approx. 14d followed by a linear phase in which the rate of bud initiation is constant. The incidence of buds formed is very low, approx. one bud per 10³ or 10⁴ cells. Statistical analysis of the distribution of buds among explants and subcloning experiments provide evidence that the paucity of buds results from neither negative interactions among bud forming centers nor a paucity of cells with the potential for organogenesis. Our results are consistent with the hypothesis that the frequency of bud initiation is determined by the availability of competent cells, by position effects, or by a combination of both mechanisms.     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

Mechanism of arginine transport in Chlorella

The incubation of Chlorella cells with glucose causes the induction of an uptake system, which is specific for the basic amino acids arginine and lysine. Both amino acids are taken up in the positively charged form and with high affinity (K _m values 2 M and 7 M, respectively). The transport of arginine depolarizes the membrane by 20–30 mV. The charge compensation is achieved within a few seconds after arginine addition by the proton pump, later on K⁺ efflux serves for charge compensation. No evidence for arginine-proton symport was found, neither by inhibitor studies nor by use of other Chlorella strains which have a slower-responding proton pump. The accumulation of arginine is appreciably higher than it should be according to the thermodynamic force of the membrane potential. There is, however, some evidence that a large proportion of arginine is trapped by intracellular compartments and is therefore not in equilibrium with the outside arginine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP p-trifluoromethoxycarbonylcyanide phenylhydrazone     

Changes in the electrical polarity of tobacco cells following the application of weak external currents

Weak externally applied electric currents changed the natural electrical pattern surrounding cells from tobacco (Nicotiana tabacum L.) suspension cultures. The artificial currents were applied transversely to short filaments of cells placed between a microelectrode lose to the filament surface and a large platinum electrode some distance away. The natural current patterns before and after electrical treatment were measured with a vibrating probe. Significant effects were confined to the cell adjacent to the microelectrode. Currents with densities of 100 A · cm^–2 at the cell surface applied for 10 min or 3 A · cm^–2 for several hours caused a localized increase in the natural current entering the part of the cell which had been nearest the positive electrode. There was no corresponding local increase in current leaving from the opposite side of the cell. Instead, the extra current appeared to leave over a relatively large area. The overall effect was a tendency for the cell to repolarize transversely with a greater proportion of its transcellular currents flowing in the direction of the current applied. The effect was measurable for several hours after the external current was discontinued and may be evidence for a natural mechanism by which neighbouring cells entrain one another's polarities during differentiation. The effect of external currents on cells growing in a 2,4-dichlorophenoxyacetic acid (2,4-D) medium (which suppresses differentiation) was qualitatively the same as on cells in an indole-3-acetic acid medium (which promotes differentiation). If anything, the response was greater in 2,4-D, implying that the disruptive effect of 2,4-D on cell and tissue polarization is not a consequence of it preventing cells sensing the transcellular currents of their neighbours.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid The authors are indebted to the Agricultural and Food Research Council of the U.K. for financial support and to the Royal Society for the provision of the vibrating probe.     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

Electrical patterns of tobacco cells in media containing indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid

A simple, inexpensive, and stable drive-unit for a vibrating probe is described. It was used to measure transcellular electrical currents and their stability in cells from suspension cultures of Nicotiana tabacum L. var. virginica. The cells were highly variable in size, morphology and current-pattern. The magnitude and pattern of the currents depended on the age of the culture, the morphology of the cells and the auxin in the culture medium. Currents in small cell clusters were weakest during the lag-phase of growth and strongest when the cultures were actively growing. The shape of the cells was related to the electrical pattern surrounding them, electrically polar cells tending to be elongated. The proportion of polar cells depended on the auxin composition of the culture medium. About 75% of the cells from suspensions grown in the presence of indole-3-acetic acid (IAA) were electrically polar. These cells normally divided at right angles to their electrical axes to form filaments. Only around 20% of the cells grown in medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) were electrically polar, the remainder had randomly oriented currents and divided in random directions to form irregular clusters rather than filaments. The electrical patterns of cells in 2,4-D were much less stable than those of cells in IAA. When currents were measured repeatedly at fixed locations on cells, those in 2,4-D were about twice as likely to disappear, arise de novo, or change direction as those in IAA. When cells were transferred from 2,4-D to IAA media, the percentage of polar cells increased from 25 to 40 within 1 d, but when they were transferred from IAA to 2,4-D, this percentage decreased from 48 to 26. It is suggested that one of the reasons that 2,4-D suppresses organogenesis in tobacco cultures (and possibly why it also functions as a herbicide) is that it reduces the stability of transcellular currents and disrupts the electrical patterns of cells so that they become less capable of organized polar growth.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid The authors are indebted to the Agricultural and Food Research Council of the UK for their financial support and to the Royal Society for the provision of the vibrating probe. We would also like to thank Dr. A. Lagoa for his help in culturing the cells.     

The origin and organization of cortical microtubules during the transition between M and G1 phases of the cell cycle as observed in highly synchronized cells of tobacco BY-2

The organization of microtubules (MTs) during the transition from the M phase to the G₁ phase of the cell cycle was followed in highly synchronized suspension-cultured cells ofNicotiana tabacum L. (tobacco BY-2) by sequential treatment of cells with aphidicolin and propyzamide. Short MTs were first formed in the perinuclear regions at the expense of phragmoplasts, but when these short MTs elongated to reach the cell cortex, they grew parallel to the long axis and towards the distal end of the cells. As soon as, or shortly before the tips of elongated MTs reached the distal end, transverse cortical MTs were formed in the region proximal to the division plane. Thereafter, almost all cells retained cortical MTs which were transversely orientated to the long axis of cells and could be observed in the G₁ phase. Thus, in the organization of cortical MTs, there are two steps that have been overlooked thus far. This novel observation provides a new scheme for the organization of cortical MTs, which could unify two contrasting hypotheses, i.e. organization in the perinuclear regions versus that in the cell cortex. These observations are discussed in relation to the microtubule-organizing center of plant cells.     

Kinetics of prolyl hydroxylation,intracellular transport and C-terminal processing of the tobacco vacuolar chitinase

The dynamics of intracellular transport and processing of one of the vacuolar chitinases of tobacco (Nic-otiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in conjunction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-binding domain and the catalytic domain, linked by a short peptide spacer containing several hydroxyprolines. It is synthetized as a preproprotein with a signal peptide for cotranslational transport into the endoplasmic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolytic cleavage. We investigated transformed N. sylvestris plants constitutively expressing CHN A or a mutant CHN A lacking the chitin-binding domain and spacer (CS CHN A), as well as N. plumbaginifolia protoplasts transiently expressing the same constructs. Processing and transport in the two systems was very similar. A shift in the apparent molecular weight of chitinase, indicative of prolyl hydroxylation, was detectable only 30 min after appearance of newly synthesized prochitinase, indicating that it might occur in a post-ER compartment. In total, labelled chitinase was detected in the microsomal fraction for up to 90–120 min as a prochitinase, bearing the VTP. Later, it appeared only in the soluble fraction (comprising the vacuolar sap) as the mature CHN A without the VTP. In both systems, intracellular transport and processing of CS CHN A was faster than that of the wildtype form, indicating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vacuole.Abbbreviations CBD chitin-binding domain - CHN A chitinase A - PBS phosphate-buffered saline - S proline-rich spacer - VTP vacuolar targeting peptide - CS deletion of CBD and S; - VTP deletion of VTP We thank M. Müller and T. Hohn, Friedrich Miescher-Institute, Basel, for the preparation of the protoplasts and F. Fischer, Friedrich Miescher-Institute, Basel, for the synthesis of the peptide. This work was supported by the Swiss National Science Foundation, Grants 31-26402.89 and 3100-037434.93.     

Modulation of the number of membrane-bound auxin-binding sites during the growth of batch-cultured tobacco cells

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.Abbreviations MES 4-morpholinoethanesulphonic acid - NAA 1-naphthaleneacetic acid     

Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10⁵ copies in the genome, can escape de-novo methylation.Abbreviation AzaC 5-azacytidine This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic. We thank Ms. Libue Jedliková, Ms. Hana Suchánková, and Ms. Emilie Koudelková for technical assistance.