Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain ofAgrobacterium tumefaciens

We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms ^–) A66 strain of Agrobacterium tumefaciens. Normally, tms ^– tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated from tms ^– tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology of tms ⁺ tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found in tms ⁺ tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 M) of -naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 M, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 M). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype of tms ⁺ cells while retaining the low auxin content and low auxin sensitivity of tms ^– cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - MACC N-malonyl ACC - NAA naphthaleneacetic acid - tms tumor morphology shooty, the auxin biosynthesis locus of Agrobacterium Ti plasmids The authors thank Dr. Andrew Binns (University of Pennsylvania, Philadelphia, USA) for providing cell lines TA6-5 and TA66C3-78, and Mr. James Dacey for preparation of the composite photograph used in Fig. 1. Support for this work by the National Science Foundation (DMB84-17087) and the U.S. Department of Agriculture (86-CRCR-1-2150) is gratefully acknowledged.     

Auxin synthesis in Agrobacterium tumefaciens and A. tumefaciens-transformed plant tissue

Problems of IAA biosynthesis, IAA precursors and IAA biosynthetic pathways in Agrobacterium tumefaciens-transformed plant tissue, especially in tobacco tissue culture are reviewed. The knowkedge about levels of IAA and the IAA biosynthetic pathways in Agrobacterium tumefaciens itself is also summarized.Dedical to the late Professor M. Kutáek, a pioneer in auxin biosynthesis research.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

Substituted nitroguanidines provide cytokinin activity during in vitro cultivation of plant tissues

Synthetic nitroguanidine derivatives can be used as alternatives to the traditional adenine-containing cytokinins used in plant tissue culture. First, nitroguanidine derivatives (NG) mimicked the typical activity of two standard cytokinins, 6-benzylaminopurine (BAP) and 2-isopentenyladenine (2iP) in the soybean callus (Glycine max) growth bioassay. NGs caused unanticipated responses as well, as demonstrated in three lines of tobacco (Nicotiana tabacum), when the auxin concentration was reduced from the standard concentration of 2 ug/ml NAA, to much lower concentrations of 0.01 ug/ml NAA or 0.02 ug/ml IAA. At the low auxin concentrations, kinetin lost the ability to promote either growth or differentiation, while the NG cytokinins were fully able to promote both. NGs promoted growth and differentiation in the presence of 0.01 ug/ml NAA in a newly initiated, totipotent line of Coker 319 tobacco. NGs plus 0.02 ug/ml IAA also promoted callus growth in a cytokinin-habituated tobacco line, Havana 425-CH. Lastly, NGs stimulated the outgrowth of healthy callus from aged callus that had been allowed to deteriorate through lack of subculture. Upon transfer of aged NTP callus to fresh media with NGs and 0.02 ug/ml IAA, healthy cell clusters were rapidly produced. In all three cases cited above, kinetin was ineffective at the low auxin concentrations. The NGs are therefore cytokinins, with the additional possibility of reducing the level of auxin required for their activity to be expressed.Abbreviations BAP 6-benzylaminopurine or N⁶- benzyladenine - IAA indoleacetic acid - 2iP N⁶-(²- isopentenyl)adenine - NAA -naphthaleneacetic acid - NG nitroguanidine derivative     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Ethylene production by shoot-forming and unorganized crown-gall tumor tissues of Nicotiana and Lycopersicon cultured in vitro

We have studied ethylene biosynthesis in cloned crown-gall cell lines of Nicotiana tabacum L., N. glutinosa L., and Lycopersicon esculentum (L.) Mill. transformed by the A6 strain of Agrobacterium tumefaciens (Smith and Townsend) Conn. or a tms (shooty) mutant strain, A66. Both the synthesis of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and the conversion of ACC to ethylene were affected by crown-gall transformation. All A6-transformed cell lines contained about 50 times more ACC than the A66-transformed cell lines, indicating that the tms genes stimulate ACC synthesis. On the other hand, A6-transformed N. tabacum and L. esculentum cell lines showed a very low capacity to convert ACC to ethylene when compared with A66-transformed cells of the same species. These differences in ACC-dependent ethylene formation were stable and could not be modified by supplying auxin to the culture medium. In contrast, both the A6- and A66-transformed N. glutinosa cell lines showed a low capacity for ACC-dependent ethylene production. Thus, the low-ethylene-forming phenotype did not seem to be under direct control of the tms genes and appeared to be part of the host response to crown-gall transformation. All cell lines exhibiting the low-ethylene-forming phenotype grew as unorganized tissues in culture, whereas cell lines showing a high capacity to convert ACC to ethylene formed shoots. Thus, ACC-dependent ethylene formation may be useful for studying host factors important in determining tumor phenotype.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA -naphthalencacetic acid     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Phototropic fluence-response relations for Avena coleoptiles on a clinostat

Phototropism of Avena sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14° is expressed within 180 min following the single pulse; a final curvature of about 62° in about 240 min following five pulses without pre-irradiation; and a curvature of over 125° in 360 min following five pulses after the RL pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 pmol·cm^-2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol·cm^-2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5-min RL co-irradiation increases with fluence to a maximum of about 60° at about 10 pmol·cm^-2, and then decreases to 0° at about 200 pmol·cm^-2. Curvature induced by five BL pulses following a 2-h RL pre-irradiation increased with fluence from a threshold of about 0.05 pmol·cm^-2 to a maximum of 90° at about 10 pmol·cm^-2, and then gradually decreased with fluence to 50° at 1 000 pmol·cm^-2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation downstream of the photoreceptor pigment for phototropism, and that the additivive effect of pulsed BL is distinct from the potentiating effect of RL on phototropism. Thus, any degree of curvature from 0° to over 90° may be induced by a fluence in the ascending arm of what is traditionally called the first positive phototropic response.Abbreviations BL blue light - RL red light     

Plants regenerated from auxin-auxotrophic variants are inviable

Summary Shoots regenerated from auxin-auxotrophic variants of Nicotiana plumbaginifolia were inviable when cultured in vitro in the absence of auxin. Variant shoots survived longer when grafted to wild-type stocks but eventually died after a characteristic pattern of degeneration. The auxin auxotrophs were isolated after mutagen treatment by a total isolation method as infrequent variants amongst haploid protoplast-derived cell colonies. The variants responded to several active auxins but, unlike the wild type, not to cytokinin. Plant regeneration from variant cultures ceased at early stages of shoot formation after complete withdrawal of auxin from the medium.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthaleneacetic acid     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

The Relationship of Indole-3-Acetic Acid Content and Growth of Crown-Gall Tumor Tissues of Tobacco in Culture

We have measured the content of the auxin, indole-3-acetic acid (IAA), in a cloned, crown-gall teratoma line of Nicotiana tabacum L. cv. 'Turkish' by a highly specific and sensitive radioimmunoassay. This tissue line, which does not require auxin for continuous growth in culture, exhibits two phases of growth. During the first phase, which lasts about two weeks after subculturing, growth is exponential on a fresh weight basis and the content of IAA is about 10⁻⁷–10⁻⁶ mol. kg⁻¹, but variable. During the second phase, growth rate declines gradually and the IAA content of the tissue drops dramatically; however, this drop does not result from a net loss of auxin by the tissue. The rate of growth during the exponential phase was not correlated with the IAA content of the tissue, but was strongly correlated with the IAA content of the inoculum. We found that rapidly growing leaves of Turkish tobacco have roughly the same IAA content as cultured teratoma tissues. Moreover, both tissues exhibited a similar relationship between auxin content expressed per leaf or per tissue explant and growth. These findings do not support the hypothesis that the autonomous growth of plant tumors results from an abnormally high content of auxin in the tissue.     

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K _m = 2.2 M;V _max = 23 nmol·(g FW)^–1·h^–1]; (ii) a low-affinity, high-capacity component [K _m = 159 M;V _max = 742 nmol·(g FW)^–1·h^–1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)^–1 h^–1];·mM^–1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K _m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K _i = 1–5 M; and a low affinity (K _i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.Abbreviations AEC S-2-aminoethyl-l-cysteine - K _i equilibrium constant - WT wild-type     

Initiation of auxin autonomy in Nicotiana glutinosa cells by the cytokinin-biosynthesis gene from Agrobacterium tumefaciens

Agrobacteria carrying mutations at the auxin-biosynthesizing loci (iaaH and iaaM of the Ti plasmid) induce shoot-forming tumors on many plant species. In some cases, e.g. Nicotiana glutinosa L., tumors induced by such mutant strains exhibit an unorganized and fully autonomous phenotype. These characteristics are stable in culture at both the tissue and cellular level. We demonstrate that the cytokinin-biosynthesis gene (ipt) of the Ti plasmid is responsble for the induction of both auxin and cytokinin autonomy in N. glutinosa. Cloned cell lines carrying an ipt gene but lacking iaaH and iaaM are capable of accumulating indole-3-acetic acid. Interestingly, non-transformed N. glutinosa tissues exhibit an auxin-requiring phenotype when they are grown on medium supplemented with an exogenous supply of cytokinin. These results strongly indicate that exogenously supplied cytokinin does not mimic all the effects of the expression of the ipt gene in causing the auxin-autonomous growth of N. glutinosa cells.Abbreviations FW fresh weight - IAA indole-3-acetic acid - I⁶ Ado isopentenyladenosine - kb kilobase - MS Murashige and Skoog (medium) - NAA -naphthaleneacetic acid - NAM -naphthaleneacetamide - T-DNA transferred DNA     

The appearance of nitrate reductase activity in nitrogen-starved cells of Ankistrodesmus braunii

Summary Crown-gall teratoma tissues of tobacco, when grown in culture, require exogeneous auxin (-naphthaleneacetic acid) or high concentrations of K⁺ in the medium to utilize ammonium glutamate as a nitrogen source. These factors are not required to utilize NO ₃ ^- or glutamine. The effects of K⁺ and auxin on glutamate utilization differ in that NH ₄ ⁺ is required for the action of K⁺, but not for the action of auxin. The tissues grew optimally when the ratio of NH ₄ ⁺ to glutamate was approximately one or greater. These results indicate that glutamate utilization involves at least two different mechanisms: one mechanism requires K⁺ and stoichiometric amounts of NH ₄ ⁺ , the other mechanism requires auxin. Experiments using explants of tobacco pith show that both mechanisms function in normal as well as crown-gall tissues of tobacco.