The biosynthesis and translocation of 14C-IAA in Ricinus communis

The biosynthesis of ¹⁴C-IAA from ¹⁴C-tryptophan applied to abraded leaves of Ricinus communis and its subsequent export through the phloem were studied. Phloem sap was collected at intervals from incisions made in the stem below the IAA fed leaf. Any upward movement of label through the phloem or downward movement of phloem mobile compounds from leaves above the treated one were restricted by bark-ringing the plants.TLC and HPLC analyses of the collected sap indicate that some conversion of ¹⁴C-tryptophan to ¹⁴C-IAA had occurred. Subsequent GC-MS analysis of the HPLC purified samples of phloem sap revealed high levels of endogenous IAA transported from the fed leaf. The high ratio of unlabelled/labelled IAA in the phloem sap makes unequivocal confirmation by GC-MS of the predicted biosynthesis of ¹⁴C-IAA impossible. It is postulated that IAA is synthesised from tryptophan in mature leaves and exported to developing sink tissues with the flow of photoassimilates in the phloem.     

Effects of auxins and cytokinins on formation of Catharanthus roseus G. Don multiple shoots

The effects of different combinations of plant growth regulators and light intensity on the formation of multiple shoots of Catharanthus roseus (L.) were studied. By composing three dimension surfaces and their topo views from experimental data, it was clear that Murashige-Shoog (MS) medium supplemented with 7.0 mg l^-1 BA and 1.0 mg l^-1 NAA strongly stimulated the formation of shoots, whereas medium supplemented with 2,4-d suppressed the formation of shoots or caused shoot dedifferentiated. Light intensities of 550–700 Lux were found to be beneficial to the formation of shoots when MS medium was supplemented with 2 mg l^-1 6-BA and 0–1.0mg l^-1 NAA.Abbreviations BA-6 benzyladenine - NAA -naphthalenacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Loading and translocation of various cytokinins in phloem and xylem of the seedlings of Ricinus communis L.

The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated.     

Role of phloem in sucrose transport by Ricinus cotyledons

Sucrose is taken up and accumulated by cotyledons of Ricinus communis L. Autoradiographic studies reveal a predominant accumulation of sucrose in the phloem of the cotyledons. The export of sucrose from the cotyledons to hypocotyl and roots proceeds in the phloem by mass flow. These results, taken together with previous data, are experimental evidence for proton-sucrose symport as the mechanism of phloem loading.     

Phloem transport of sulfur in Ricinus

Mature leaves of Ricinus communis fed with ³⁵SO ₄ ^2- in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of ³⁵S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the ³⁵S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.Abbreviations CySH cysteine - GSH glutathione - GSSG glutathione disulfide - NEM N-ethylmaleimide - CyS-SCy cystine     

Phloem translocation of Fe,Cu, Mn,and Zn in Ricinus seedlings in relation to the concentrations of nicotianamine,an endogenous chelator of divalent metal ions,in different seedling parts

During the first 8 days of germination the Ricinus seedling is supplied with all nutrients by the endosperm via phloem transport. In 4- to 8-days-old seedlings the concentrations and contents of Fe, Cu, Mn and Zn, and nicotianamine (NA) in the endosperm, cotyledons, hypocotyl and roots were estimated. From the data obtained translocation rates and flow profiles for the metals were established. The main sink for Fe, Mn and Zn were the cotyledons whereas Cu was mainly imported into the hypocotyl. Maximum flow rates occurred between days 5 and 7, for Zn between days 6 and 8.The time kinetics of NA and divalent metal ion concentrations and contents are interpreted as co-transport. The role of NA as transport vehicle of micronutrients in the sieve tubes is discussed.     

Role of auxins and cytokinins in the development of lateral roots in wheat plants with several roots removed

The removal of four of five roots of 7–8-day-old wheat plants resulted in the activation of lateral root growth and the initiation of lateral root primordia on the remained root as compared to the main root of intact plants. The extent of this growth response depended on placing cut surface above or beneath the surface of the nutrient solution. The measurement of the IAA and cytokinin contents showed accumulation of these hormones in the root of experimental plants as compared to the main root of intact plants. IAA accumulation was correlated with the number of lateral roots and their primordia. The analysis of hormonal balance and their transport from the shoot to the root permits discussing the involvement of these hormones and their interaction in the control of root growth at the stages of both primordium initiation and development and lateral root elongation.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

Free tryptophan and indole-3-acetic acid levels in the leaves and vascular pathways of Ricinus communis L.

Levels of free tryptophan in the leaves, phloem and xylem saps of Ricinus communis L. were determined by colorimetric assay. Values of 0.38 g ml^-1 in root pressure sap and 96.0 g ml^-1 in phloem sap were recorded. Tryptophan levels were highest in mature and senescing leaves. Levels of indoleacetic acid (IAA) in the phloem sap and leaves were determined by gas chromatography—mass spectrometry using a deuterated internal standard. A mean value of 13.0 ng ml^-1 was recorded in phloem sap. The distribution in the leaves showed an inverse relationship to that of tryptophan, being highest in young leaves.Abbreviations IAA indoleacetic acid - GC-MS Gas chromatography-mass spectrometry - PFP-derivative pentafluoropropionyl-derivative - TLC thin layer chromatography     

Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants

Somatic embryos of cassava (Manihot esculenta Crantz) cultivar ‘Nanzhi 188’ were isolated and cut into fragments to be cultured on media with various cytokinins and auxins. Cytokinin induced of cassava shoot organogenesis, while auxin stimulated somatic embryogenesis. The effectiveness on organogenesis was different based on different cytokinins and in combination with auxins. Benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and N-isopentenyladenine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phloem transport,solute flux and the kinetics of sap exudation in Ricinus communis L.

The osmotic characteristics of phloem-sap exudation were examined in soil-grown and watercultured plants of Ricinus communis L. Prolonged exudation occurred from bark incisions in water-cultured plants. Fresh incisions caused large alterations in solute flux, but phloem-sap solute potential _s changed by less than ±8% over a period of 7 h. This was associated with a constancy in the levels of sucrose and K⁺, the principal solutes in the sap. Studies with foliar-applied tracers and leaf-excision experiments suggested that exudation was maintained by solute loading from mature leaves. A wide range of mass transfer values through the phloem was found, these being a function of exudation rate. We consider that the exudation process possesses essentially similar characteristics to phloem transport in the intact plant. The way in which bark incisions bring about large changes in solute flux is discussed in terms of the physical properties of the sieve-tube system.Abbreviations water potential - _s solute potential - _p pressure potential     

Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation

Decapitation or treatment with naphthylphthalamic acid (NPA) ndash; an inhibitor of IAA transport ndash; or a synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) caused a decrease in rooting of pea cuttings. However the mode of action of the above treatments was different. Decapitation resulted in a decrease of indole-3-acetic acid (IAA) and a simultaneous increase in cytokinin content at the base of the cuttings. NPA decreased IAA even more, but did not influence cytokinins. CPPU alone or in combination with amino-ethoxy-vinylglycine (AVG) ndash; an ethylene biosynthesis inhibitor ndash; increased IAA at the rooting zone, but CPPU was transported from apex to the base of the cuttings where it inhibited rooting. NAA, applied alone after decapitation, stimulated rooting, probably partly by being an auxin, and partly by inhibiting the accumulation of cytokinins at the base of the cuttings. Treatment with AVG neither influenced rooting nor auxin or cytokinin content. Therefore, ethylene production does not seem to be one of the main factors involved in the reduced rooting after the various treatments.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Determinations of indole-3-acetic acid in xylem sap of Ricinus communis L. using mass fragmentography

Mass fragmentography employing a deuterated internal standard was used to make quantitative analyses of indole-3-acetic acid in xylem sap collected from Ricinus communis L. When contamination of the sap by microorganisms was reduced by frequent collection, levels of IAA were found to be less than 0.5 ng ml^-1. It is therefore proposed that the transpiration stream does not play a significant role in the transport of IAA within the plant.Abbreviations IAA indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - BSTFA bis-Trimethylsilytrifluoroacetamide - TMCS trimethylchlorosilane - BSA bis-Trimethylsilylacetamide - TMS₂-IAA bis-trimethylsilyl derivative of IAA     

Effects of auxins and cytokinins on tissue culture of Grateloupia dichotoma (Gigartinales,Rhodophyta)

The role played by plant growth regulators in algae is poorly known. In order to increase the knowledge about the function of auxins and cytokinins in seaweeds, explants such as apical and intercalary segments and callus-like structures (CLS) of Grateloupia dichotoma were cultured in semi-solid or liquid artificial media ASP 12-NTA. Two auxins, indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and one cytokinin, 6-benzylaminopurine (BA), at concentrations of 0.5 and 5.0 mg l^–1 were tested. Moreover, IAA and BA were tested together at concentrations of 1:5 and 5:1 mg l^–1. All treatments promoted the growth of CLS in intercalary segments; CLS from apical segments were significantly higher in treatments with 2,4-D or IAA:BA (1:5 mg 1^–1). The morphogenetic responses for auxins and BA were opposite, auxins inhibited while BA promoted the formation of lateral branches; however, auxins promoted the elongation of such branches. The process of plant regeneration observed on CLS was stimulated significantly by treatment with high concentration of BA or IAA:BA (1:5 mg 1^–1) in semi-solid and liquid media. The growth of upright axes was stimulated significantly by treatment with 2,4-D in semi-solid medium, and IAA:BA (1:5 mg l^–1) in liquid medium. These results show the importance that plant growth regulators could have in the control of growth, morphogenetic processes and micropropagation in red algae.This paper is part of the PhD thesis of NSY.     

Mineral uptake and transport in xylem and phloem of the proteaceous tree,Banksia prionotes

Xylem sap of sinker (tap) root, cluster feeding roots, lateral roots and from an age series of main stem extensions of 6-year trees of Banksia prionotes was collected and analyzed for principal organic and inorganic solutes. During the phase of root uptake activity in winter and spring, cluster roots were principal xylem donors of malate, phosphate, chloride, sodium, potassium and amino acid N whereas other parts of the root served as major sources to the shoot of other cations, nitrate and sulphate. Sinker root xylem sap was at all times less concentrated in solutes than that of lateral roots into which cluster roots were voiding exported solutes. Phosphate was abstracted from xylem by stem tissue during winter and it and a range of other solutes released back to xylem immediately prior to extension growth of the shoot in summer. Phloem sap collected from mid regions of stems was unusually low in potassium and phosphate relative to chloride and sulphate in comparison with phloem sap of other species, and its low potassium: sodium ratio relative to xylem indicated poor discrimination against sodium during phloem loading. Data are discussed in relation to the asynchronous seasonal cycles of nutrient uptake and shoot growth.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

Transport of magnesium ions in the phloem of Ricinus communis L. seedlings

During growth of Ricinus communis seedlings, magnesium ions are mobilized in the endosperm, taken up by and accumulated to very high levels (150 μmol·g FW^?1) in the cotyledons, and translocated to hypocotyl and roots. The magnesium gain from days 6 to 7 in the cotyledons and the seedling axis necessitates a total up-take rate of 600 nmol·h^?1-seedling^?1 and the phloem translocation rate must amount to 200 nmol·h^?1. seedling^?1. The phloem loading of magnesium and the regulatory properties of this process were investigated, making specific use of the ability to collect pure phloem sap from the cut hypocotyl of 6-d-old Ricinus seedlings. The concentration of magnesium in sieve-tube sap (5 mM) was fairly constant under many incubation conditions, e.g. incubation in magnesium-free buffer, incubation with different cations (K⁺, Na⁺, NH ₄ ⁺ ) or anions (Cl^?, NO ₄ ^- , SO ₄ ^2- ), or incubation with sucrose and amino acids. Even addition of magnesium chloride to the cotyledons did not enhance phloem loading of magnesium ions. Therefore the high magnesium content of the cotyledons was sufficient for continuous phloem loading of magnesium, irrespective of external ionic conditions. Also, the flow rate of sieve-tube sap did not influence the magnesium concentration in the sap. Only the incubation with sulfate and phosphate ions increased the magnesium-ion concentration in the phloem. Magnesium sulfate offered to the cotyledons caused a threefold increase of magnesium ions in the sieve-tube sap, which was inhibited by Na⁺, NH ₄ ⁺ and Ca²⁺ in rising order, but not by K⁺. Incubation with phosphate for a prolonged period (8 h) led to an increased mobilization of intra-cotyle-donary magnesium and an enhanced phloem loading of mobilized magnesium. It is concluded that phosphate availability is a decisive factor for mobilization and translocation of magnesium ions within the plant.