Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [¹⁴C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.     

Conjugation of aspartic acid with 4-bromophenylacetic acid,an auxin analogue of aspartic acid

Tobacco mesophyll protoplasts conjugate the auxins indoleacetic acid and naphthaleneacetic acid with aspartic acid very efficiently. This conjugation was found to be correlated with the toxicity of these molecules to protoplast-derived cells grown at low densities. Among a series of halogenated phenylacetic acids, 4-bromophenylacetic was toxic to cells grown at low densities although not able to stimulate proliferation at high cell densities, as opposed to indoleacetic acid and naphthaleneacetic acid. [¹⁴C-car☐yl]-4-bromophenylacetic acid was conjugated with aspartic acid by tobacco protoplasts. Although 4-bromophenylacetic acid is not an auxin, this molecule shares with auxins some of their properties.     

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.     

Effect of cycloheximide on IAA-induced proton excretion and IAA-induced growth in abraded Avena coleoptiles

IAA-induced proton excretion in peeled or abraded oat ( Avena saliva L. cv. Victory) coleoptiles is closely associated with IAA-induced growth. It was attempted to separate these two processes by using cycloheximide to inhibit them differentially. Growth of abraded coleoptile segments was measured by a shadow graphic method, and their IAA-induced acidification of the external solution was monitored with a pH meter. IAA stimulated proton excretion in abraded Avena coleoptile segments after a 13 min lag. IAA-induced proton excretion was inhibited within 5 min by cycloheximide at concentrations of 1.8 × 10⁻⁶, 3.6 × 10 or 3.6 × 10⁻⁵ M. Cycloheximide at these concentrations, added within 4 min of IAA, prevented IAA-induced acidification of the medium for at least 60 min. However, it did not prevent IAA-induced growth during this time. It is concluded that some of the initial IAA-induced growth seen in Avena coleoptiles is independent of detectable IAA-induced proton excretion.     

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     

Relationship between Auxin and Amino Acid Metabolism of Tobacco Protoplast-Derived Cells

Single amino acids were found to be highly toxic to protoplast-derived cells of tobacco (Nicotiana tabacum cv Xanthi) cultured at low density in a culture medium containing a low naphthaleneacetic acid concentration (0.05 micromolar). The cytotoxicities of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, proline, and valine were reduced when the naphthaleneacetic acid concentration of the culture medium was increased to 1 micromolar. This selective modification of amino acid toxicity by naphthaleneacetic acid could not be correlated with modifications of uptake rates or incorporation of these amino acids into protein or amino acid-auxin conjugates. A mutant clone resistant to high naphthaleneacetic acid concentrations and affected in root morphogenesis did not display, at the cellular level, the naphthaleneacetic acidmediated modification of amino acid cytotoxicity.     

Culture conditions of protoplast-derived cells of nicotiana sylvestris for mutant selection

Large numbers of protoplasts showing reproducible high plating efficiency can be isolated from in vitro propagated, haploid and diploid, plants of Nicotiana sylvestris. Their successful use in the selection of biochemical mutants depends on the establishment of suitable selection parameters: culture medium, cell density, age of cells at selection etc. Plating of protoplasts at low densities as well as simulation and reconstruction experiments of mutant selection were employed to optimize such selection parameters. The results show that some of the principles determined for tobacco protoplast cultures manipulated at low densities or in view of mutant selection are of more general value. However, requirements specific to N. sylvestris protoplast cultures have also been established; they play a decisive part in the successful isolation of resistant mutants in this species.Abbreviations AEC S-aminoethylcysteine - BA benzyl-adenine - NAA napthaleneacetic acid - p-cells or p-colonies protoplast-derived cells or colonies     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in Azuki bean epicotyls

Brassinolide, a plant hormone newly isolated from pollen, promotes growth of the stem of a number of plant species. Similar effects are induced by a brassinosteroid (BR), the synthetic 24-epibrassinolide. In this paper the effects of BR on acid secretion and transmembrane electrical potential difference in Azuki bean ( Vigna angularis , Ohwi and Ohashi cv. Takara) epicotyls were determined in short term experiments and compared with the effects on growth. At concentrations between 10^-7 to 10^-5 M , BR stimulates, similarly to indole-3-acetic acid (IAA), growth and H⁺ extrusion and hyperpolarizes the transmembrane electric potential (PD). These effects of BR, as well as those of IAA, are suppressed by inhibitors of RNA and protein synthesis. All these effects of BR and IAA appear roughly additive, even when both hormones are present at their optimal concentrations. The data are interpreted as showing that the action of BR on growth is at least in part mediated by its capability to activate electrogenic proton extrusion. The additivity of the effects of BR and IAA suggests that the primary mechanism of action of the two hormones is different.     

Changes in free IAA level in the leaves of short-and long-day tobacco during flowering and the effect of applied IAA on the transition to flowering

Changes in free IAA level were studied in the leaves of the central stem zone of short-day tobacco (Mcotianatabacum, cv. Maryland Mammoth) and long-day tobacco(Nicotiana silvestris) in inductive photoperiodic regime after 10, 20, 30 and 40 d, respectively. The leaves of SD tobacco Mammoth showed a high free IAA level in vegetative plants kept under long days but it significantly decreased (by ca. 50 %) after 10, 30 and 40 short days, respectively. After 20 short days the IAA level was as high as in the leaves of plants at the beginning of inductive treatment. The changes of freeIAA level in the leaves of LD tobacco N.silvestris were similar to those of SD Mammoth, but the IAA level in this species was significantly lower than that of Mammoth throughout the investigated period. Consequently, the changes observed in N. silvestris were much less pronounced. Plants of both tobacco species were fully induced to flowering by 30 inductive days and this was associated with differentiation of the flower organs. Application of 10 -4 M IAA during the last 10 d of the inductive treatment of 30 d significantly reduced flowering in SD tobacco Mammoth without changing the stem length and apex width. Apex length was slightly reduced. IAA application elicited almost no effect inN. silvestris. The results are discussed with respect to the possible role of IAA in flower induction in SD and LD plants.     

Valine-Resistance, a Potential Marker in Plant Cell Genetics. I. Distinction between Two Types of Valine-Resistant Tobacco Mutants Isolated from Protoplast-Derived Cells

In previous experiments, seven lines of valine-resistant plants were regenerated from protoplast-derived haploid tobacco mesophyll cells which had been UV mutagenized and submitted to selection by toxic concentrations of valine. In this study we described the transmission of valine-resistance to progeny and a preliminary phenotypical and biochemical characterization of the resistant plants.—Two types were thus distinguished among the seven mutant lines. Valine-resistance of the mutants of the first type (three lines) was transmitted as a single Mendelian dominant character (Vr1), whereas valine-resistance of the second type (four lines) was transmitted as a digenic recessive character (vr2 and vr3). Allelism tests revealed that the four recessive mutant lines yielded resistant progeny when intercrossed and, therefore, bear recessive mutant alleles at the same two unlinked loci.—When cultured at a density of 100 cell/ml, protoplast-derived cells of mutants of the first type had a low level of resistance to valine, whereas protoplast-derived cells of mutants of the second type displayed a high level of resistance to valine and to other amino acids.—According to the results of ¹⁴C-labelled amino acid uptake experiments, the amino acid resistance of mutants of the second type, but not valine-resistance of the first type, could be accounted for by reduced uptake of several amino acids. Possible uses of valine-resistance as a marker in plant cell genetics are discussed.     

Mesophyll cell protoplasts of potato: isolation, proliferation, and plant regeneration

Mesophyll cell protoplasts were isolated from potato (Solanum tuberosum L. cv. Russet Burbank) leaves and induced to proliferate in culture. Protoplast division was observed only among preparations isolated from plants previously conditioned under short periods of low intensity illumination. Sustained growth and development of protoplast-derived calli (p-calli) occurred when they were maintained on defined media at 24 C under 500 lux lighting. Shoot bud development within p-calli was controlled by a number of factors including light, temperature, basic medium composition, nature and source of phytohormones, the continued presence of an osmoticum, low concentrations of a utilizable carbohydrate, and the developmental stage of the p-callus.     

Evidence for Two Indoleacetic Acid-Induced Growth Responses in the Avena Straight-Growth Indoleacetic Acid Assay

Floating Avena sativa L. cv Victory coleoptile segments were used to determine whether the straight-growth indoleacetic acid (IAA) assay can be reconciled with the Avena curvature assay and the Cholodny-Went theory of photo- and gravitropism. Measurements of segment length after 5 h yield sigmoid-shaped IAA dose-response curves with the growth rate leveling off at 1 [mu]M. However, measurements made at 24 h generate bell-shaped curves with maximal growth being induced by 10 [mu]M IAA. The difference between short- and long-term IAA dose-response curves is not due to IAA degradation; instead, it is the result of two growth responses to IAA. The initial one is rapid, responds to low concentrations of IAA, and lasts for 12 h. The second response is less sensitive to IAA than the first one. It appears after 6 h but is not obvious until the last 12 h of a 24-h incubation. The profile of short-term IAA dose-response curves reflects the initial growth response, whereas that of the 24-h curve is the sum of both growth responses. Linear-linear plots of 5- and 24-h dose-response curves show that coleoptile segment growth rate is proportional to IAA concentration up to 0.3 [mu]M. When the efficiency of IAA action is taken into account, it is found that the most effective IAA concentration for short and long incubations is 0.4 [mu]M. It is concluded that the Avena straight-growth IAA assay is as sensitive as the Avena coleoptile curvature assay, and that it is consistent with the Cholodny-Went theory.     

Branch development in Lupinus angustifolius L.#II. Relationship with endogenous ABA, IAA and cytokinins in axillary and main stem buds

The concentrations of indole-3-acetic acid (IAA), cytokinins (CK) and abscisic acid (ABA) were measured in buds of different regions (main stem and lateral branches) of Lupinus angustifolius L. (cv. Merrit) and at different stages in the development of branches. In lupin, branching patterns are the result of discrete regions of axillary branches (upper, middle and basal) which elongate at much different rates. Early in development only the main shoot elongates, followed usually by basal branch growth and then rapid upper branch growth. Branches in the middle of the main stem grow only weakly or fail to develop. Levels of IAA were generally high in the apical buds of slowly growing branches and low in buds from strongly growing branches, whereas CK levels showed the opposite relationship. CK:IAA ratio showed a closer relationship with the rate of growth of a particular branch better than the levels of either CK or IAA alone. During early stages of growth ABA concentration did not follow the rate of branch growth. However, later in development, where growth did not closely match the ratio of CK:IAA, ABA level showed a strong negative relationship with growth. A significant decrease in ABA was associated with continued strong growth of the main stem apex following a decline in CK:IAA ratio. Overall, the best relationship between the level of growth factors in apical buds and branching pattern in lupin was the ratio of CK:IAA, implying that high CK:IAA at a given bud would promote growth. ABA level appeared to play a secondary role, as a growth inhibitor.     

Differential effect of short-term cold stress on growth, anatomy, and hormone levels in cold-sensitive versus -resistant cultivars of Digitaria eriantha

Two cultivars of Digitaria eriantha (cold-sensitive cv. Sudafricana and cold-resistant cv. Mejorada INTA) were exposed to cold stress (5?°C) for 0, 6, 24, or 72?h, and compared in terms of leaf and root growth, recovery period, shoot and leaf anatomy, and levels of chlorophyll, auxin (indole-3-acetic acid, IAA) and cytokinins (CKs). In Sudafricana, cold treatment caused reduced growth, slight changes in chlorophyll level, reduced levels of IAA and CK iso-pentenyladenine (iP), and reduced leaf dry weight (DW) and fresh weight (FW) during the recovery period. Anatomical damage was observed in chloroplasts, main stem, and axillary buds. Ultrastructural study showed reduced numbers of starch grains in chloroplasts of the bundle sheath and mesophyll. In Mejorada, cold treatment had no significant effect on growth or chlorophyll level. Leaf DW and FW quickly returned to normal levels during the recovery period. Anatomy of ground meristem was affected, but ultrastructure of bundle sheath and mesophyll chloroplasts was not. The cold tolerance of cv. Mejorada appears to be related to the stability of chlorophyll and CK levels, increase of IAA, and maintenance of normal shoot and leaf anatomy and ultrastructure.     

Synthesis and localization of pathogenesis-related proteins in tobacco.

The PR1 family of pathogenesis-related proteins from tobacco (Nicotiana tabacum L.) leaves is induced by a variety of pathogenic and chemical agents and is associated with resistance to tobacco mosaic virus. The majority of the PR1 proteins did not copurify with mesophyll protoplasts (the major cell type of the leaf) isolated from tobacco mosaic virus-infected N. tabacum cv. Xanthi-nc leaves. However, these isolated protoplasts were capable of synthesizing and selectively secreting the PR1 proteins. Using monoclonal antibodies for immunofluorescence microscopy, we localized these proteins to the extracellular spaces predominantly in regions adjacent to viral lesions as well as in xylem elements of infected leaves.     

Magnitude and Kinetics of Stem Elongation Induced by Exogenous Indole-3-Acetic Acid in Intact Light-Grown Pea Seedlings

Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tall (cv Alaska) peas (Pisum sativum L.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3 M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effectively promotes growth as it is transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (IGR) and prolonged growth response (PGR) induced by auxin. The IGR was inducible by at least 1 order of magnitude lower IAA concentrations than the PGR, suggesting that the process underlying the IGR is more sensitive to auxin induction. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tall seedlings. These results suggest that endogenous IAA is more growth limiting in dwarf plants than in tall plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of action as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tall plants, auxin cannot be the only limiting factor for stem growth in peas.     

Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts

The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.-Using cell densities of approximately 10( 4) cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Val(r)-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10(-6) per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10(-5) to 5 x 10(-4) resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F(1) double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were compared to those observed in wild-type cells. The results are considered in light of the amphidiploid structure of the tobacco genome.     

Development of Asparagus plumosus Shoot Tips Grown in vitro

Lateral shoot tips from young Asparagus setaceus (Kunth) Jessop (syn. A. plumosus Baker) shoots were grown on a modified Murashige and Skoog medium. Tips from 5 to 20 mm lateral shoots had significantly better growth and development than tips from lateral shoots (2 mm) still covered by leaf-scale. The optimum temperature for growth and development of the explants was 17 to 24°C. The initial growth was fast at 24°C but stopped after about 4 weeks. At 17°C the growth was slow but in return the cultures continued to grow. Kinetin was necessary for growth. Without any kinetin all cultures died. Optimum growth was found with 2 mg/l kinetin. There was no growth at all with IAA alone. A low IAA concentration had no effect, but at high concentrations IAA inhibited the kinetin induced growth.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

The Role of Auxins in Root-Knot Nematode-Induced Growth on Excised Tobacco Stem Segments

Root-knot nematodes, Meloidogyne incognita, induced lumps of callus tissue on the cambial surfaces of peeled tobacco stem segments cultured in vitro. Except for a layer 1 to 3 cells thick, callus was limited to the basal ends of control segments. Indole-3-acetic acid (IAA) applied in agar blocks to the centers of stem segments, when it had any effect on the cambial surface, induced streaks of callus extending from the blocks toward the basal ends of the segments. IAA in agar blocks also increased callus growth at the basal ends of the segments, increased the growth of pith on the undersides of the segments, promoted root initiation, but inhibited bud initiation. Nematodes produced none of these effects, nor did they change the type of organs induced by various concentrations of IAA in the medium. Callus tissue did grow on the cambial surface of stem segments surrounding agar blocks containing 2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport. Paraffin sections showed that the nematodes were confined to the callus tissue on the cambial surfaces of the segments. Except for occasional syncytia and areas of cell division, nematode-induced callus was composed of thin-walled, irregularly shaped cells arising from the cambium. Differences between the responses of tobacco stem segments to root-knot nematodes and IAA-agar blocks indicate that auxins were not freed from the plant tissue nor secreted by the nematodes. Instead, it is suggested that nematodes enabled the tissue to retain and use endogenous auxins that otherwise would have been transported to the basal ends of the segments.