Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.)

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.     

Purification of a Single Major Form of Microsomal Cytochrome P-450 from Tulip Bulbs (Tulipa gesneriana L.)

Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68 nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52,500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs.     

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.     

Primary stress response induced by different elements is mediated through auxin signalling in barley root tip

Short-term exposure (15 min) of barley roots to different chemical elements revealed that Cd, Cu, Hg and Pb were the most toxic ones causing a marked root growth inhibition even at µM concentrations. Gd, La, Al, Cr, As, Zn, Ni and Se inhibited root growth to a similar extent only at mM concentrations. Despite the high 20 mM concentration, Co caused only a slight, while Mn, Mg or Ca did not evoke any root growth inhibition. Elements at concentrations inhibiting root growth caused a considerable accumulation of indole-3-acetic acid in the root apex. While Cr, As and Zn inhibited, Cd, Cu, Hg, Pb, Gd, La and Al markedly stimulated the generation of reactive oxygen species in the beginning of differentiation zone. Auxin signalling inhibitor alleviated or prevented root growth inhibition, reactive oxygen species generation and the stimulation of lipoxygenase and glutathione peroxidase activity by various elements, indicating a key role of auxin signalling in the stress response of barley root tip. On the other hand, it did not affect or even had an additive effect on dehydroascorbate reductase and ascorbic acid oxidase activity in combination with different elements. Our results indicate that the primary response of barley roots to the presence of various chemical elements during the short-term treatment is not a specific but rather a general adaptive stress response enabling the plant to survive adverse conditions.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

Resistance to broomrape (Orobanche spp.) in sunflower (Helianthus annuus L.) is temperature dependent

The effects of various temperature regimes in the range 29-17/21-9 degrees C day/night on each stage of the parasitism process of Orobanche cumana and O. aegyptiaca on sunflower were studied under controlled conditions in polyethylene bags. The response of the resistant sunflower variety 'Ambar' was expressed as the degeneration of the parasite tissues after its establishment in the plant roots, and this stage was found to be temperature dependent. The degeneration rate of Orobanche tubercles in the resistant sunflower variety was also found to be temperature dependent and was about five times as great as that in the sensitive variety in the highest temperature regime tested of 29/21 degrees C day/night. The ability to reject the parasite by causing its degeneration and death is the main factor that determines the resistance. As the temperature rises, more tubercles degenerate and die, that is the sunflower plant expresses higher levels of resistance.     

Reperti Teratologici in Tulipa Gesneriana L. Var. Spathulata (Bert.)

Summary

Here is described by the author a teratological case of Tulipa Gesneriana L. v. spathulata (Bert.) which is notable for the concomitance of several anomalies in a single individual:

a) irregular number of the leaves;

b) scape ending with three flowers which are anomalous;

c) two from the above mentioned flowers have tetracarpellary pistil;

d) the third flower has an androecium which is formed by three external stames and, besides, by three other stames which are smaller than the first-ones and are also inglobed in a bypertrophic esacarpellary pistill.     

Stimulation by auxin of phospholipase A in membrane vesicles from an auxin-sensitive tissue is mediated by an auxin receptor

Microsomal vesicles were prepared from zucchini (Cucurbita pepo L.) hypocotyls containing radioactive phosphatidylethanolamine or phosphatidylcholine, and these lipids were used as substrates by phospholipase A which is activated by auxins. Phospholipase D and phospholipase C hydrolysed the same substrates but were not influenced by auxin. Phospholipase A was activated by the auxins indolyl-3-acetic acid, 2,4-dichlorophenoxyacetic acid and, to a lesser extent, by -naphthaleneacetic acid whereas the weak auxins 2,3-dichlorophenoxyacetic acid and -naphthaleneacetic acid were almost inactive. This hormone specificity was also found in growth tests with etiolated zucchini hypocotyls. Phospholipase A activation by auxin was blocked by a polyclonal antibody against the maize auxin-binding protein. We propose that phospholipase A activation is a primary reaction in the signal transduction leading from hormone-binding to the growth response.Abbreviations IAA indolyl-3-acetic acid - 2,3-D, 2,4-D 2,3- and 2,4-dichlorophenoxyacetic acid - -NAA; -NAA - and -naphthaleneacetic acid This work was supported by the Deutsche Forchungsgemeinschaft. We thank D. Klämbt (Botanical Institute, University of Bonn, FRG) for a generous gift of polyclonal antibody (IgG fraction) against auxin-binding protein and U. Kutschera (Botanical Institute, University of Bonn, FRG) for advice with the growth tests.     

OsARF16, a transcription factor,is required for auxin and phosphate starvation response in rice (Oryza sativa L.)

Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (?Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and ?Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and ?Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and ?Pi treatments. In ?Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation‐induced genes (PSIs) were less induced by ?Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under ?Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and ?Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice.     

Intraspecific variation in the response of rice (Oryza sativa L.) to increased CO2 and temperature: growth and yield response of 17 cultivars

Seventeen rice (Oryza sativa L.) cultivars of contrasting ecosystemsand origins were exposed to two CO₂ concentrations (373 [ambient]and 664 µl l^–1 CO₂ [elevated]) at two differentday/night temperatures (29/21 C and 37/29 C) in glasshousesat the International Rice Research Institute phytotron duringthe dry seasons of 1994 and 1995. Growth at elevated CO₂ (asdetermined by total plant biomass at maturity) increased byan average of 70% and 22%, respectively, for all cultivars forgrowth temperatures of 29/21 C and 37/29 C relative to theambient CO₂ treatment. At the 29/21 C optimal growth temperature,grain yield increased on average c. 50% with enriched CO₂. Incontrast, at the higher growth temperature (37/29 C), grainyield was almost zero, presumably due, in part, to temperature-inducedinfertility (i.e. the percentage of filled spikelets was <1%).Among cultivars, lAC 165, a tropical japonica from Brazil, showedthe largest relative increase in both biomass and grain yield.While the range of responses to increased CO₂ and/or temperaturewere quite large (e.g. 10–250%) and may not be applicableto field conditions, data indicate that lines are availablewhich could maximize productivity as CO₂ concentration increases.Additional work, however, would be needed to identify cultivarswhich would maintain maximum yields in a high CO₂, high temperatureenvironment. Key words: Plant growth analysis, experimental design, computational methods, relative growth rate, net assimilation rate     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.     

Low temperature enhances photosynthetic down-regulation in French bean (Phaseolus vulgaris L.) plants

The mechanisms of photosynthetic adaptation to different combinations of temperature and irradiance during growth, and especially the consequences of exposure to high light (2000 micro mol m(-2) s(-1) PPFD) for 5 min, simulating natural sunflecks, was studied in bean plants (Phaseolus vulgaris L.). A protocol using only short (3 min) dark pre-treatment was introduced to maximize the amount of replication possible in studies of chlorophyll fluorescence. High light at low temperature (10 degrees C) significantly down-regulated photosynthetic electron transport capacity [as measured by the efficiency of photosystem II (PSII)], with the protective acclimation allowing the simulated sunflecks to be used more effectively for photosynthesis by plants grown in low light. The greater energy dissipation by thermal processes (lower F(v)'/F(m)' ratio) at low temperature was related to increased xanthophyll de-epoxidation and to the fact that photosynthetic carbon fixation was more limiting at low than at high temperatures. A key objective was to investigate the role of photorespiration in acclimation to irradiance and temperature by comparing the effect of normal (21 kPa) and low (1.5 kPa) O(2) concentrations. Low [O(2)] decreased F(v)/F(m) and the efficiency of PSII (Phi(PSII)), related to greater PSII down-regulation in cold pre-treated plants, but minimized further inhibition by the mild 'sunfleck' treatment used. Results support the hypothesis that photorespiration provides a 'safety-valve' for excess energy.     

In vitro micropropagation of Basilicum polystachyon (L.) Moench and identification of endogenous auxin through HPLC

An efficient plant regeneration protocol was developed for Basilicum polystachyon (L.) Moench using shoot tip from in vitro germinated plant. Both shoot multiplication and root induction were initiated from shoot tip explants in Murashige and Skoog’s (MS) basal medium supplemented with N⁶-benzylaminopurine (BAP) and 6-furfurylaminopurine (Kin) combination with 1-naphthaleneacetic acid (NAA) and without any plant growth regulator. Among the different concentrations and combinations of growth regulators, the highest number of shoots per explants was induced on 13.32 μM BAP with 0.53 μM NAA. It was also found that the multiplication of shoots along with roots induced in MS medium without any plant growth regulators. The in vitro grown plants were successfully hardened and acclimatized in the field with a 99% survival rate. The results obtained from HPLC analysis established the presence of a significant amount of endogenous auxin, viz. indole-3-acetic acid acid and indole-3-butyric acid in the shoot and root tips of B. polystachyon. This is the first report of a successful multiplication of B. polystachyon in absence of plant growth regulators and the presence of an abundant quantity of endogenous auxin in root and shoot tips using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) coupled with ultraviolet–visible (UV–Vis) detector.