T-DNA genes to study plant development: precocious tuberisation and enhanced cytokinins in A. tumefaciens transformed potato

Summary Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100–200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3–10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.     

Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines

An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg-1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg-1 FW compared to approximately 15 pg mg-1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.     

Control of tuberisation in potato by gibberellins and phytochrome B

Phytochrome B-deficient plants exhibit increased gibberellin (GA) levels or responsiveness, which may contribute to their elongated growth and reduced chlorophyll levels. We have investigated the effects of applications of gibberellic acid and an inhibitor of gibberellin biosynthesis, ancymidol, on wild-type and phytochrome B-antisense potato (Solanum tuberosum ssp. andigena) plants. The results showed that some phenotypes of the phytochrome B-antisense plants, i.e. increased stem length and reduced chlorophyll, can be mimicked by treating wild-type plants with gibberellic acid. However, another phenotype, i.e. tuberisation response in long days, is mimicked by application of a GA biosynthesis inhibitor ancymidol, thus appearing to be the result of a reduction in the gibberellin levels. A simple increase in gibberellin levels or sensitivity is, therefore, not sufficient to explain the phenotype of the antisense plants.     

Indole-3-acetic acid and indole-3-acetylaspartic acid isolated from seeds of Heracleum laciniatum Horn

Seeds from mature flowers of Heracleum laciniatum were collected locally (Tromsø, Norway). Seed coats were removed and the seeds were analyzed for their content of free, free plus ester-conjugate, and total indole-3-acetic acid (IAA) by quantitative gas chromatography-mass spectrometry. Seeds contained high levels of free and amide-linked IAA relative to other dicotyledonous seeds for which values have been published. The major amide conjugate in this material was identified as indole-3-acetylaspartate by gas chromatography-mass spectrometry of its bis-methyl ester.     

Channelling auxin action: modulation of ion transport by indole-3-acetic acid

The growth hormone auxin is a key regulator of plant cell division and elongation. Since plants lack muscles, processes involved in growth and movements rely on turgor formation, and thus on the transport of solutes and water. Modern electrophysiological techniques and molecular genetics have shed new light on the regulation of plant ion transporters in response to auxin. Guard cells, hypocotyls and coleoptiles have advanced to major model systems in studying auxin action. This review will therefore focus on the molecular mechanism by which auxin modulates ion transport and cell expansion in these model cell types.     

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Influence of indole-3-acetic acid,kinetin and abscisic acid on the estrogens content of beans

No influence of IAA on the endogenous estrogen content in bean plants was stated. At the same time kinetin was found to increase and abscisic acid to decrease the amounts of estrogens.     

Polyamines,indole-3-acetic acid and abscisic acid in rice phloem sap

Putrescine, spermidine, spermine and cadaverine have been identified and quantified in rice phloem sap and shoot extracts by HPLC. It is suggested that diamines, putrescine and cadaverine, easily migrate into the phloem, while movement of a triamine, spermidine, and a tetramine, spermine, tend to be restricted. Spermine especially seems to be the most immobile among polyamines. Thus it is indicated that movement of polyamines into phloem is decreased with increasing number of amino groups. Indole-3-acetic acid and abscisic acid in rice phloem sap were also analyzed by HPLC and it is suggested that indole-3-acetic acid is transported freely into phloem, while abscisic acid is much more actively exuded into phloem.     

GC-MS-SIM analysis of abscisic acid and indole-3-acetic acid in shoot bark of apple rootstocks

Concentrations of abscisic acid and indole-3-acetic acid were measured by GC-MS-SIM in the shoot bark of clonal apple rootstocks (M.27, M.9, MM.106 and MM.111) when the rootstocks were growing actively in the UK. These rootstocks are known to exhibit a wide range of control of tree size when grafted to a common scion. Shoot bark of the dwarfing rootstocks (M.27 and M.9) contained higher concentrations than the more vigorous rootstocks (MM.106 and MM.111) of ABA. Concentrations of ABA increased from May to July, followed by a decline in August. Only the month of sampling showed any significant influence on the concentration of IAA in shoot bark; however, there was a general increase, although not significant statistically, in IAA concentration with the increasing invigoration-capacity of the rootstock. At each sampling date the dwarfing rootstocks showed greater ratios of ABA:IAA than the invigorating rootstocks and generally the ratio for each rootstock increased from May to July, except for M.27 which showed the smallest ratio in June and the largest ratio in August. The results are discussed in relation to the generally accepted control exerted by the rootstocks on tree size and the possible influence of ABA on polar auxin transport.     

Uptake and decarboxylation of indole-3-acetic acid during auxin-induced growth in lupin hypocotyl segments

The elongation growth of etiolated hypocotyl segments of lupin (Lupinus albus L.) was stimulated by acid pH (4.6 versus 6.5) and by IAA for periods of up to 4 h. After this time, the segments were unable to grow further. In the presence of an optimal IAA concentration (10 μM), acid pH increased the growth rate but had no effect on final growth. With suboptimal IAA (0.1 μM), however, acid pH increased growth in a more than additive way, suggesting a synergistic action between the two factors. This synergism may be explained by the increased IAA uptake and decarboxylation seen at an acid pH. These results reinforce the view that the effects of low pH and IAA on growth are not independent. Vanadate inhibited growth and also IAA uptake and decarboxylation. This inhibitor, therefore, probably inhibits growth not only by decreasing ATPase-mediated acidification but also by decreasing H⁺-dependent IAA uptake from the apoplasm. This dependence of IAA uptake on ATPase may be mediated by apoplasmic acidification. The amount of IAA decarboxylated increased when the assay conditions favored the growth of segments, indicating that IAA could be destroyed by decarboxylation during the auxin-induced growth.     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -     

Deuterium-labelled indole-3-acetic acid neo-synthesis in plantlets and excised roots of maize

Synthesis of indole-3-acetic acid (IAA), using stable-isotope incorporation, was investigated in Zea mays L. Incorporation of ²H from ²H₂O into IAA molecules was shown to occur in intact plantlets and excised primary roots cultured in vitro. This demonstrates the de-novo formation of IAA, a process which is quantitatively well defined and is initiated early in germination.Abbreviations IAA indole-3-acetic acid     

Distribution of indole-3-acetic acid in relation to the growth of etiolated Lupinus albus hypocotyls

The free indole-3-acetic acid (IAA) in methanolic extracts of etiolated hypocotyls of lupin ( Lupinus albus L., from Bari, Italy) was determined by fluorimetry. The distribution of IAA along the hypocotyls was parallel to the growth, but when growth ceased oscillations occurred in the auxin level. These oscillations could be related to processes of differentiation mediated by IAA. The oscillations did not obey any impulses from the apex, since the application of [1-¹⁴C]-IAA to decapitated plants gives a distribution of radioactivity which also presents an undulatory pattern. Our results support the hypothesis that morphogenesis can be regulated by information transmitted by the translocation of waves of auxin.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

Effects of illumination source, culture ventilation and sucrose on potato (Solanum tuberosum) microtuber production under short days

The culture yield of a simple method of microtuber production of potato was increased by assessing the interactions of illumination source (Thorn Lighting (Philips) “Colour 84” lamps (TL‐84) or Grolux lamps (Sylvania) in a conventional growth room or natural light in a glasshouse cabinet), type of vessel closure (unventilated or ventilated) and sucrose concentration (1%, 2%, 4% or 8%). Microtuber initiation and growth in unventilated cultures was 100% at 8% sucrose falling to 40–50% at 4% sucrose and was absent at 1% or 2%. With ventilation, rapid tuberisation (90–100%) occurred at initial sucrose concentrations of 2–8%, but only when the medium was allowed to dry before transfer of cultures to short days. Water supplementation of cultures at day 28 prevented tuberisation at 1–4% sucrose up to day 56. The fresh weight and dry weight of microtubers per plant increased significantly with sucrose concentration, with ventilation of cultures and under natural light. In ventilated cultures, the mean number of usable microtubers (± 0.1 g weight) increased from between 1.0–1.4 per plant at 8% sucrose to between 1.6–2.6 per plant at 4% sucrose, with the highest numbers (1.8–2.6 per plant) produced under natural light for the cvs Desirée and King Edward. The mean % dry matter content of microtubers was reduced to 11.3% at 4% sucrose compared with 17.3% at 8% sucrose, but the survival rate of microtubers after 6 months storage was unaffected. Microtuber production under short days was improved at a higher intensity of natural light with culture ventilation in a partially‐shaded glasshouse cabinet, whilst using reduced inputs (lower sucrose supply and no lamps).     

In vitro PPFD and media composition affect both in and ex vitro performance of Alstroemeria Butterfly-hybrids

The objective was to reduce in vitro production costs while retaining or improving plant quality, in particular the suitability for pot plant production. Plants were grown at photosynthetic photon flux densities (PPFD) of 0–40 μmol m^-2 s^-1 and sucrose concentrations of 3–7% during the multiplication phase and the effects of sucrose, BA, and NAA during root formation were investigated. Ex vitro growth were tested in both experiments. A small reduction in the rhizome multiplication rate was found with increasing PPFD and sucrose concentration. Increasing sucrose concentration reduced the number of aerial shoots. Aerial shoots were etiolated when cultured in darkness and their number increased with increasing PPFD at 3% sucrose, whereas PPFD did not affect the number of aerial shoots at 5 or 7% sucrose. During the multiplication phase a synergistic promoting effect of PPFD and sucrose was observed on root formation. Root formation after transfer to rooting medium was affected by sucrose and PPFD during the multiplication phase. PPFD did not influence root formation after propagation on 7% sucrose, whereas on 3 or 5 % sucrose root formation was gradually inhibited when PPFD was decreased below 17 μmol m^-2 s^-1. The formation of thick roots was promoted by propagation in light, and not influenced by sucrose concentration. Ex vitro growth was not affected by in vitro conditions, except for 7% sucrose during the multiplication phase that reduced flowering. Root formation on rooting medium was reduced by BA and promoted both by NAA and high levels of sucrose. The root inhibiting effect of BA could not completely be overcome by simultaneous application of NAA and high sucrose concentrations. Thick roots were only produced in the presence of NAA, and not affected by sucrose treatment. Ex vitro flowering was negatively influenced by the presence of BA during root formation and by high levels of sucrose if BA was absent in the rooting medium. High sucrose levels and NAA could partially compensate for the negative effect of BA on flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid

Etiolated pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μ M . Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999