Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Ontogeny of intruding non-periplasmodial tapetum in the wild chicory,Cichorium intybus (Compositae)

The tapetal development ofCichorium intybus L. is investigated using LM and TEM and discussed in relation to the development in other species. During the second meiotic division the tapetal cells become binucleate and lose their cell walls. They intrude the loculus at the time of microspore release from the meiotic callose walls, which means that a locular cavity is never present in this species. During pollen development they tightly junct the exine, especially near the tips of the spines. During the two-celled pollen grain stage they degenerate and most of their content turns into pollenkitt. Until anther dehiscence they keep their individuality, which means that these intruding tapetal cells never fuse to form a periplasmodium. The ultrastructural cytoplasmatic changes during this development are discussed in relation to possible functions.     

Nitrate (15NO3) limitation affects nitrogen partitioning between metabolic and storage sinks and nitrogen reserve accumulation in chicory (Cichorium intybus L.)

In chicory, we examined how NO₃ ⁻ supply affected NO₃ ⁻ uptake, N partitioning between shoot and root and N accumulation in the tuberized root throughout the vegetative period. Plants were grown at two NO₃ ⁻ concentrations: 0.6 and 3 mM. We used ¹⁵N-labelling/chase experiments for the quantification of N fluxes between shoot and root and for determining whether N stored in the tuberized root originates from N remobilized from the shoot or from recently absorbed NO₃ ⁻. The rate of ¹⁵NO₃ ⁻ uptake was decreased by low NO₃ ⁻ availability at all stages of growth. In young plants (10–55 days after sowing; DAS), in both NO₃ ⁻ treatments the leaves were the strongest sink for ¹⁵N. In mature (tuberizing) plants, (55–115 DAS), the rate of ¹⁵NO₃ ⁻ uptake increased as well as the amount of exogenous N allocated to the root. In N-limited plants, N allocation to the tuberized root relied essentially on recent N absorption, while in N-replete plants, N remobilized from the shoot contributed more to N-reserve accumulation in the root. In senescing plants (115–170 DAS) the rate of ¹⁵NO₃ ⁻ uptake decreased mainly in N-replete plants whereas it remained almost unchanged in N-limited plants. In both NO₃ ⁻ treatments the tuberized root was the strongest sink for recently absorbed N. Remobilization of previously absorbed N from shoot to tuberized root increased greatly in N-limited plants, whereas it increased slightly in N-replete plants. As a consequence, accumulation of the N-storage compounds vegetative storage protein (VSP) and arginine was delayed until later in the vegetative period in N-limited plants. Our results show that although the dynamics of N storage was affected by NO₃ ⁻ supply, the final content of total N, VSP and arginine in roots was almost the same in N-limited and N-replete plants. This indicates that chicory is able to build up a store of available N-reserves, even when plants are grown on low N. We also suggest that in tuberized roots there is a maximal capacity for N accumulation, which was reached earlier (soon after 100 DAS) in N-replete plants. This hypothesis is supported by the fact that in N-replete plants despite NO₃ ⁻ availability, N accumulation ceased and significant amounts of N were lost due to N efflux. Received: 14 October 1996 / Accepted: 4 February 1997     

Putrescine influences growth and production of coumarins in transformed and untransformed root cultures of witloof chicory (Cichorium intybus L. cv. Lucknow local)

The effect of putrescine on growth and production of two coumarins, esculin, and esculetin in the transformed and untransformed roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of putrescine (Put) on growth and production of coumarins, polyamine inhibitors namely α-DL-difluromethylornithine (DFMO) and α-L-difluromethylarginine (DFMA) were used at 1 mM levels. Treatment with 1.5 mM of putrescine (Put) produced 1.96 - fold and 4.0 - fold increase in the growth of transformed and untransformed roots of chicory, respectively. The treatment with polyamine inhibitors showed much lower growth, as well as production compared with both 1.5 mM putrescine treatment and control in both transformed and untransformed chicory roots. The endogenous polyamines, both free and conjugated, were studied over the whole culture period, and it was seen that conjugated titers of all three polyamines viz., putrescine, spermidine and spermine were higher than level of free polyamines, throughout the culture period in both transformed and untransformed roots of chicory. Treatment in which polyamine inhibitors were used showed lower level of endogenous polyamines as compared with the 1.5 mM putrescine treated sample in both the systems. The treatment wherein putrescine was added at 1.5 mM level showed maximum accumulation of endogenous conjugated putrescine (2098.86±157.6 nmoles g⁻¹ FW; 896.8±67.2 nmoles·g⁻¹ FW), on the 14^th day in both transformed and untransformed roots respectively. The production of esculin and esculetin was strictly correlated with growth in every treatment in both systems. Putrescine at 1.5 mM resulted in greater length of primary root in transformed (18.3±1.4 cm) and untransformed (6.86±0.51 cm) as compared with their respective controls (11±0.9 cm; 2.9±0.1cm) and greater number of secondary and tertiary roots. This study suggests that putrescine influences plant root development and differentiation, and it also provides insight into the morphological changes that occur in roots in response to the external supply of polyamines.     

Genetic variability of nitrogen accumulation during vegetative development and remobilization during the forcing process in witloof chicory tuberized root (Cichorium intybus L.)

A better knowledge of genetic variability of traits related to nitrogen use efficiency (NUE) is a potential strategy to optimize N fertilization and to reduce environmental pollution without decreasing marketable yield and quality. To this aim, in this study, 13 cultivars of witloof chicory were compared with three reference cultivars known for their adaptation to low, intermediate and high N availability in the field during the vegetative phase of development. Pertinent criteria used for this study were determined by a thorough comparison of nitrogen reserve accumulation in tuberized roots during vegetative development and mobilization during the forcing process in the three reference cultivars. Cluster analysis allowed us to sort the cultivars into four main groups we named G1, G2, G3 and G4. Cultivars of group G4, better adapted to soils with high nitrogen contents (N-demanding cultivars), showed higher total N, nitrate, total amino acids (AA), glutamine contents and lower total N and AA mobilization for chicon growth than did cultivars of group G1, adapted to soils with low nitrogen content (N-sensitive cultivars). An intermediate behavior was exhibited by cultivars of groups G2 and G3, characterized as N tolerant. It is proposed that either chicory growers or breeders may take advantage of the genetic variability revealed in the present study to gain flexibility in choosing the right cultivar for the type of soil available (N-rich soil vs N-poor soil) or to adapt the level of N fertilization to the type of cultivar (N-demanding vs N-sensitive) in order to target the highest NUE for the best chicon yield and trade quality.     

An experimental study of 226Ra and 45Ca accumulation from the aquatic medium by freshwater turtles (fam. Chelidae) under varying Ca and Mg water concentrations

Snapping turtles Elseya dentata (Gray) from Magela Creek, Northern Territory, were exposed under laboratory conditions for up to 30 days to waters resembling the inorganic composition of Magela Creek water during the Wet season, with background and elevated Ca and Mg concentrations, that were labelled with ²²⁶Ra and ⁴⁵Ca. The resulting concentrations of ⁴⁵Ca in muscle, skin, gut, liver, shell bone and leg bone of E. dentata equilibrated or approached equilibrium by 12–18 days. Among the experiments, the concentrations of ⁴⁵Ca in all six tissues were inversely related to turtle mass. An increase in the Ca water concentration by a factor of 15 increased the ⁴⁵Ca concentration in all six tissues. The arithmetic factors of increase in the concentration in each tissue were proportional or more than proportional to the factor of increase in Ca water concentration; this factor was highest for muscle tissue (26.6). An increase in the Mg water concentration by a factor of 48 reduced the ⁴⁵Ca concentration in all tissues except skin where it increased. The concentration of ²²⁶Ra in each tissue (except the gut) was positively related to its ⁴⁵Ca concentration and inversely related to turtle mass in muscle, skin and liver. With the exception of the skin, the increased Ca water concentration did not reduce the ²²⁶Ra in the tissues but increased the ²²⁶Ra concentration in bone and muscle. The increased Mg water concentration had an inverse effect on the ²²⁶Ra concentrations in all tissues except shell. With the exception of the skin the effects of increased Ca and Mg water concentrations and turtle size on ²²⁶Ra concentrations in the tissues of E. dentata were similar to their effects on ⁴⁵Ca tissue concentrations, indicating the similar metabolic behaviour of ²²⁶Ra and ⁴⁵Ca in E. dentata.Exposures of the species Elseya latisternum (Gray), Emydura signata (Ahl) and Chelodina longicollis (Shaw), which are the same or closely related to species reported to occur in Magela Creek, to ⁴⁵Ca-labelled Sydney tap water for 7 days demonstrated their ability to also accumulate ⁴⁵Ca from their aquatic medium. The patterns of ⁴⁵Ca concentrations in the tissues of these species indicated that they were inversely related to turtle mass, as demonstrated in E. dentata. The concentrations of ⁴⁵Ca accumulated in the tissues were also comparable to those found in single specimens of E. dentata and E. victoriae (Gray) that were exposed for 7 days to simulated Magela Creek water. The data also indicated the larger long-necked C. longicollis accumulated less ⁴⁵Ca per gram of tissue than similar-sized, short-necked species E. signata and E. latisternum, suggesting that long-necked turtles from Magela Creek would accumulate less ²²⁶Ra from their aquatic medium than similar-sized short-necked species.The capacity of E. dentata to accumulate ²²⁶Ra from the aquatic medium is about two orders of magnitude less than that of the tissue of the freshwater mussel Velesunio angasi (Sowerby) exposed under similar experimental conditions.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures

The influx of K⁺(⁸⁶Rb⁺) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K⁺-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K⁺ influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K⁺ influx into the apical (0–2 cm) region of the seminal root which is associated with a compensatory increase in K⁺ influx into the more mature, basal regions of the root. Values of V _max and apparent K _m for K⁺ influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K _m at 8° C. The influx of K⁺ from solutions containing 0.6 mol·m^-3 K⁺ into both WG and DT plants was found to be linearly related to assay temperature over the range 2–27° C, and the temperature sensitivity of K⁺ influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K⁺ influx at 20° C:K⁺ influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K⁺ influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K⁺ influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K⁺]int). This result indicates some change in the regulation of K⁺ influx by [K⁺]int in plants exposed to low root temperatures. We suggest that K⁺ influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K⁺ influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.Abbreviations DT differential temperature pretreatment - [K⁺]int root potassium concentration - [K⁺]ext potassium concentration of nutrient medium - WG warm-grown pretreatment     

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

Summary The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l⁻¹ level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Copper uptake and translocation in chicory ( Cichorium intybus L. cv. Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv. Rondy) plants grown in NFT system. I. Copper uptake and distribution in plants

The uptake and distribution of copper was examined in chicory (Cichorium intybus L. cv. Grasslands Puna) and tomato (Lycopersicon esculentumMill. cv. Rondy) plants grown in a Nutrient Film Technique System (NFT) with addition of 0.05, 5, 10 and 20 mg Cu L^-1. Biomass production of shoots and roots of both chicory and tomato was strongly depressed by Cu concentrations higher than 5 mg Cu L^-1 in the rooting media. Although Cu concentrations in both shoots and roots of both species increased with increasing Cu concentrations in the rooting media, the increase in roots was very much greater than that in shoots, in which the range of concentrations was small. A large proportion of total Cu uptake was retained by roots except when plants were grown in solution Cu concentrations of 0.05 mg Cu L^-1. Copper retention by roots limited Cu translocation to xylem and shoots. Copper adsorption by the root appears to buffer against increases of Cu in the rooting media. A cupric-sensitive electrode used in conjunction with total Cu analysis by graphite furnace atomic absorption spectrophotometry (GFAAS) indicated that more than 99.6% of total Cu in xylem sap was in a complexed form. Large differences between measured and predicted Cu accumulation by shoots of tomato (0.134–0.243 mg Cu plant^-1, 0.660–4.274 mg Cu plant^-1, respectively) and chicory (0.095–0.203 mg Cu plant^-1, 0.626–1.620 mg Cu plant^-1, respectively) suggest that some xylem transported Cu is recirculated to roots via the phloem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots

During the first 4 d after the removal of SO ₄ ^2- from cultures of young barley plants, the net uptake of ¹⁵N-nitrate and the transport of labelled N to the shoot both decline. This occurred during a period in which there was no measurable change in plant growth rate and where the incorporation of [³H]leucine into membrane and soluble proteins was unaffected. Reduced N translocation was associated with six- to eightfold increases in the level of asparagine and two- to fourfold increases in glutamine in root tissue; during the first 4 d of SO ₄ ^2- deprivation there were no corresponding increases in amides in leaf tissue. The provision of 1 mol · m^–3 methionine halted, and to some extent reversed the decline in NO ₃ ^- uptake and N translocation which occurred during continued SO ₄ ^2- deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO ₄ ^2- deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO ₄ ^2- -deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO ₄ ^2- deprivation for 5 d was found to lower stomatal conductance, transpiration and photosynthesis, in the order given, to 33%, 37% and 18% of control values. The accumulation of amides in roots is probably explained by a failure to export either the products of root nitrate assimilation or phloem-delivered amino-N. This may be correlated with the lowered hydraulic conductivity. Enhanced glutamine and-or asparagine levels probably repressed net uptake of NO ₃ ^- and ¹³NO ₃ ^- influx reported earlier (Clarkson et al. 1989, J. Exp. Bot. 40, 953–963). Attention is drawn to the similar hydraulic signals occurring in the early stages of several different types of mineral-nutrient stresses.Abbreviations Asn asparagine - Gln glutamine - Lp hydraulic conductivity J.L.K. is extremely grateful to the British Council for supporting his working visit to Long Ashton. We thank John Radin for helpful discussion and encouragement.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

In-ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.)

A convenient and reliable method for culturing cotton embryos is needed to obtain interspecific hybrids of this genus. C.A. Beasley and I.P. Ting (Amer. J. Bot. 60, 130, 1973) developed a phytohormone-supplemented medium (BTP) upon which the growth of ovules was similar that of in situ ovules. This medium was examined for in-ovulo embryo culture. Although good ovule growth occurred on BTP no embryos developed to maturity. However, when the medium was supplemented with NH ₄ ⁺ , more than 50% of the ovules produced mature embryos, and many of these germinated precociously after 8–10 weeks of culture. After germination seedlings were established on a separate medium designed to give balanced root and shoot growth. Subsequently young plants could be transferred to pots for greenhouse culture.     

Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.)

Rates of CO₂ fixation during the light period and the rates of CO₂ release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 atom C · (mg Chl)^–1 · h^–1] are reduced in the dark period to 16 atom C · (mg Chl)^–1 · h^–1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Dieter Heineke for stimulating discussions and Mrs. Petra Hoferichter and Mrs. Marita Feldkämper for their technical assistance.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic acid