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     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

Glycosylation of exogenous protein by endoplasmic-reticulum membranes from castor-bean (Ricinus communis) endosperm.

Endoplasmic-reticulum membranes isolated from the endosperm tissue of 3-day-old castor-bean (Ricinus communis) seedlings catalysed the enzymic transfer of the sugar moiety from an oligosaccharide--lipid to a chemically unfolded form of ribonuclease A.     

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     

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.     

The concentration of xylem sap constituents in root exudate, and in sap from intact, transpiring castor bean plants (Ricinus communis L.)

Root exudates were sampled from detopped root systems of castor bean (Ricinus communis). Different volume flux rates were imposed by changing the pneumatic pressure around the root system using a Passioura-type pressure chamber. The concentrations of cations, anions, amino acids, organic acids and abscisic acid decreased hyperbolically when flux rates increased from pure root exudation up to values typical for transpiring plants. Concentrations at low and high fluxes differed by up to 40 times (phosphate) and the ratio of substances changed by factors of up to 10. During the subsequent reduction of flux produced by lowering the pneumatic pressure in the root pressure chamber, the concentrations and ratios of substances deviated (at a given flux rate) from those found when flux was increased. The flux dependence of exudate composition cannot therefore be explained by a simple dilution mechanism. Xylem sap samples from intact, transpiring plants were collected using a Passioura-type root pressure chamber. The concentrations of the xylem sap changed diurnally. Substances could be separated into three groups: (1) calcium, magnesium and amino acid concentrations correlated well with the values expected from their concentration-flux relationships, whereas (2) the concentrations of sulphate and phosphate deviated from the expected relationships during the light phase, and (3) nitrate and potassium concentrations in intact plants varied in completely the opposite manner from those in isolated root systems. Abscisic acid concentrations in the root exudate were dependent on the extent of water use and showed strong diurnal variations in the xylem sap of intact plants even in droughtstressed plants. Calculations using root exudates overestimated export from the root system in intact plants, with the largest deviation found for proton flux (a factor of 10). We conclude that root exudate studies cannot be used as the sole basis for estimating fluxes of substances in the xylem of intact plants. Consequences for studying and modelling xylem transport in whole plants are discussed.     

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     

Enhanced accumulation of Cd in castor (Ricinus communis L) by soil-applied chelators

Phytoextraction has been identified as one of the most propitious methods of phytoremediation. This pot experiment were treated with varying amounts of (ethylenediamine triacetic acid) EDTA 3–15, (Nitriloacetic acid) NTA 3–10, (Ammonium citrate) NH₄ citrate 10 – 25 mmol and one mg kg^–1Cd, filled with 5 kg soil. The addition of chelators significantly increased Cd concentration in soil and plant. The results showed that maximum Cd uptake was noted under root, shoot and leaf of castor plant tissue (2.26, 1.54, and 0.72 mg kg^–1) under EDTA 15, NTA 10, and NH₄ citrate 25 mmol treatments respectively, and in soil 1.08, 1.06 and 0.52 mg kg^–1 pot^–1 under NH₄ citrate 25, NTA 10 and EDTA 15 mmol treatments respectively, as against to control (p < 0.05). Additions of chelators reduction biomass under the EDTA 15 mmol as compared to other treatments, However, Bioconcentration factor (BCF), translocation factor (TF) and remediation factor (RF) were significantly increased under EDTA 15 and NH₄ citrate 25 mmol as against control. Our results demonstrated that castor plant proved satisfactory for phytoextraction on contaminated soil, and EDTA 15 and NH₄ citrate 25 mmol had the affirmative effect on the Cd uptake in the artificial Cd-contaminated soil.     

Fusarium pallidoroseum L. on castor (Ricinus communis) in Samaru,Nigeria

Diseased castor leaves were collected from the Institute for Agricultural Research (IAR) fields and taken to the laboratory for isolation. Leaves were grown on Potato Dextrose Agar with Streptomycin and incubated for seven days. Grown cultures were observed under microscope and Fusarium pallidoroseum was isolated as confirmed by IMI. Inoculated leaves showed symptoms of wilts and blight.     

Cloning and characterization of the calreticulin gene from Ricinus communis L.

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase.Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5-untranslated genomic sequence fused to the -glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.     

Delivery rates of abscisic acid in xylem sap of Ricinus communis L. plants subjected to part-drying of the soil

Abscisic acid (ABA) moving from roots to shoots in the transpirationstream is a potential hormonal message integrating perceptionof a root stress with adaptive changes in the shoot. A twinroot system was used to study ways of estimating effects ofdroughting the upper roots of Ricinus communis L. on abscisicacid (ABA) transport to the shoot in the transpiration stream.Droughted plants transpired more slowly than controls. Droughtingalso increased concentrations of ABA up to I I-fold in sap inducedto flow from the roots of freshly decapitated plants at ratesof whole plant transpiration. However, because of dilution effectsarising from the different sap flows in control and droughtedplants, these changes in ABA concentration in the xylem sapdid not accurately reflect amounts of ABA transported. To overcomethis problem, delivery rates were calculated by multiplyingconcentration with sap flow rate to generate ABA delivery interms of µmol s–1 per plant. Droughting for 24 hor more increased ABA delivery from roots to shoots by 5-fold.Since droughting can alter the relative sizes of the roots andshoots and also the root:shoot ratio these delivery rates wererefined in several ways to reflect both the amount of root generatingthe ABA message and the size of the recipient shoot system. Key words: Abscisic acid, Ricinus communis L., soil drying, xylem sap     

Transport of hexoses by the phloem of Ricinus communis L. seedlings

The sieve-tube sap of Ricinus communis L. seedlings has been analysed to determine whether or not hexoses can be taken up by the phloem. Under natural conditions, i.e. with the endosperm attached to the cotyledons, glucose and fructose occurred only in trace amounts in the sieve-tube sap. Incubation of the cotyledons with hexoses in the concentration range 25–200 mM caused a rapid and substantial uptake of hexoses into the phleom, where they appeared eventually in the sieve-tube sap at the same concentration as in the incubation medium. Phloem loading of glucose, 3-O-methyl-glucose and sorbitol occurred easily, whereas fructose was less well loaded. glucose and to a larger extent fructose were also transformed to sucrose, which was loaded into the phloem. The loading of hexoses into the sieve tubes as observed in the experimental exudation system also occurred in the intact seedling, but transloction in the latter soon came to a standstill, probably because of lack of consumption by the sink tissues. These results indicate that the virtual absence of hexoses in the sievetube sap under in-vivo conditions is not because of the inability of the phloem-loading system to transport the monosaccharides but because of the absence of sufficiently high concentrations in the apoplast.     

Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L. and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes

Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10–30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individual STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3′ untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (K _m) for reduced glutathione and cysteine were 2 mM and 50 μM, respectively. Besides l-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins. Received: 13 December 1996 / Accepted: 28 December 1996     

Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties

The activity of nitrate reductase (+Mg(2+), NR(act)) in illuminated leaves from spinach, barley and pea was 50-80% of the maximum activity (+EDTA, NR(max)). However, NR from leaves of Ricinus communis L. had a 10-fold lower NR(act), while NR(max) was similar to that in spinach leaves. The low NR(act) of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinus extracts inhibiting NR were not found. NR(act) from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. NR(max) had a broad pH response profile that was similar for the spinach and the Ricinus enzyme. Accordingly, the Mg(2+)-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg(2+)concentration. Following a light-dark transition, NR(act) from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NR(max). In contrast to the spinach enzyme, Ricinus-NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR contains the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg(2+)-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.     

农杆菌介导的蓖麻转化体系优化

针对影响农杆菌侵染效率的主要因素进行条件优化,确立了以OD600为0.8的菌液侵染、预培养5 d后的外植体为最佳侵染条件;侵染外植体经过5 d的共培养并除菌后,转到含有250 mg/L Kan的培养基中进行再生诱导,获得了多株具有Kan抗性的再生植株;通过在共培培养基中添加20 mg/L AS,124 mg/L Na2S2O3和152.4 mg/L DTT,有效地抑制了侵染后外植体的褐化现象,提高了筛选阶段抗性芽的成活率;对具有Kan抗性的再生植株进行PCR和Southern blot验证,共获得18株包含外源基因的阳性转化株。     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Phloem transport of abscisic acid in Ricinus communis L. seedlings

Sieve tube sap exuded from the cut hypocotyl of castor bean seedlings (Ricinus communis L.) was found to contain 0.2–0.5 mmol m^?3abscisic acid (ABA). The ABA concentration in the sieve tube sap always exceeded that in root pressure exudate under a wide range of water supply. Exudation of sieve tube sap from the cut hypocotyls caused water loss, and this induced ‘water shortage’ in the cotyledons which resulted in the ABA concentration in the cotyledons increasing by 3-fold and that in the sieve tube sap increasing by up to 50-fold within 7h. The wounded surface of the cut hypocotyl was not responsible for the ABA increase. Incubation of the cotyledons of endosperm-free seedlings in various ABA concentrations (up to 100 mmol m^?3) increased the ABA concentration in sieve tube sap. The concomitant increase in ABA, both in cotyledons and in sieve tube sap, had no effect on the phloem loading of sucrose, K⁺ and Mg²⁺ within the experimental period, i.e. up to 10h. It can be concluded that (i) the phloem is an important transport path for ABA, (ii) water stress at the phloem loading sites elevates phloem-mobile ABA, which may then serve as a water stress signal for sinks, for example stem and roots (not only for stomata), and (iii) the ABA concentration of cells next to or in the phloem is more important than the average ABA content in the whole cotyledon for determining the ABA concentration in sieve tube sap.     

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.