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.     

Foliar application of nitrate or ammonium as sole nitrogen supply in Ricinus communis. II. The flows of cations, chloride and abscisic acid

Following a precultivation with pedospheric nitrogen nutrition, Ricinus plants were supplied with nitrogen solely by spraying nitrate or ammonium solution onto the leaves during the experimental period. The chemical composition of tissues, xylem and phloem exudates was determined and on the basis of the previously determined nitrogen flows (Peuke et al., New Phytologist (1998), 138 , 657–687) the flows of potassium, sodium, magnesium, calcium, chloride and ABA were modelled. These data, which permit quantification of net-uptake, transport in xylem and phloem, and utilization in shoot and root, were compared with results obtained in plants with pedospherically-supplied nitrate or ammonium and data in the literature. Although the overall effects on the chemical composition of supplying ammonium to the leaves were not as pronounced as in pedospherically supplied plants, there were some typical responses of plants fed with ammonium (ammonium syndrome). In particular, in ammonium-sprayed plants uptake and transport of magnesium decreased and chloride uptake was increased compared with nitrate-sprayed plants. Furthermore, acropetal ABA transport in the xylem in ammonium-sprayed Ricinus was threefold higher than in nitrate-sprayed plants. Additionally, concentrations of anions were more or less increased in tissues, particularly in the roots, and transport fluids. The overall signal from ammonium-sprayed leaves without a direct effect of ammonium ions on uptake and transport systems in the root is discussed.     

Vascular transport of auxins and cytokinins in Ricinus

Analyses of vascular saps supplying source and sink organs havedemonstrated the presence of major endogenous hormones and/or theirprecursors. Indol-3yl-acetic acid, a number of gibberellins, cytokininsand abscisic acid, as well as the precursor for ethylene production havebeen found in these vascular saps, allowing the sites of hormonalsynthesis and putative target tissues to be deduced. Exogenously appliedhormones are also readily loaded into these vascular pathways and may betranslocated over considerable distances from a point of application.Observations such as these indicate a possible co-ordination systembetween source and sink regulated by the synthesis and transport ofendogenous hormones. It is widely accepted that the partitioning ofassimilates between photosynthetic source organs and utilising sinkorgans is regulated by endogenous plant hormones. The key intermediatesteps involved in assimilate transport, such as phloem loading andunloading, have been shown to be responsive to applied hormones,although the role of endogenous hormones in these processes remainsessentially unresolved. Results of the analyses of vascular saps fromRicinus communis, which have been obtained using a range ofphysicochemical methods, are compared and contrasted with those obtainedby the application of exogenous hormones or their precursors. Theseresults are evaluated critically and interpreted in the light of currentmodels of source:sink regulatory processes and the long-distancetransport of auxins and cytokinins in higher plants.     

Response of sugar metabolism in the cotyledons and roots of Ricinus communis subjected to salt stress

• Ricinus communis is an important oilseed crop worldwide and is also considered one of the best potential plants for salt-affected soil improvement in northeast China. However, little is known about photosynthesis and carbohydrate metabolism in this plant, nor the distribution of carbohydrates in cotyledons and roots under salinity stress.
• In the present study, seedling growth, gas exchange parameters (P_N, E, g_s and C_i), carbohydrate (fructose, sucrose, glucose, soluble sugar and starch) metabolism and related enzymes and genes were measured in Ricinus plants.
• Under salt stress, P_N of cotyledons decreased significantly (P < 0.05), resulting in weak photosynthetic capacity. Furthermore, salt stress increased sucrose and glucose content in cotyledons, but decreased soluble sugar and starch content. However, sucrose increased and starch decreased in roots. This may be correlated with the increasing sugar metabolism under salinity, including notable changes in sugar-related enzyme activities (SPS, SuSy, α-amylase and β-amylase) and gene expression of RcINV, RcSUS, RcAmY, RcBAM and RcGBE1.
• The results suggest that salinity reduces photosynthesis of cotyledons, alters carbohydrate allocation between cotyledons and roots and also promotes starch utilization in cotyledons and starch biosynthesis in roots, leading to a functional imbalance between cotyledons and roots. Together, these findings provide insights into the crucial role of sugar metabolism in improving salt-tolerance of Ricinus during the early seedling growth stage.

     

Effects of drought on nutrient and ABA transport in Ricinus communis

We studied the effects of variations of water flux through the plant, of diurnal variation of water flux, and of variation of vapour pressure deficit at the leaf on compensation pressure in the Passioura-type pressure chamber, the composition of the xylem sap and leaf conductance in Ricinus communis. The diurnal pattern of compensation pressure showed stress relaxation during the night hours, while stress increased during the day, when water limitation increased. Thus compensation pressure was a good measure of the momentary water status of the root throughout the day and during drought. The bulk soil water content at which predawn compensation pressure and abscisic acid concentration in the xylem sap increased and leaf conductance decreased, was high when the water usage of the plant was high. For all xylem sap constituents analysed, variations in concentrations during the day were larger than changes in mean concentrations with drought. Mean concentrations of phosphate and the pH of the xylem sap declined with drought, while nitrate concentration remained constant. When the measurement leaf was exposed to a different VPD from the rest of the plant, leaf conductance declined by 400mmol m^?2 s^?1 when compensation pressure increased by 1 MPa in all treatments. The compensation pressure needed to keep the shoot turgid, leaf conductance and the abscisic acid concentration in the xylem were linearly related. This was also the case when the highly dynamic development of stress was taken into account.     

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     

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     

Simultaneous measurement of water flow velocity and solute transport in xylem and phloem of adult plants of Ricinus communis over a daily time course by nuclear magnetic resonance spectrometry

A new method for simultaneously quantifying rates of flow in xylem and phloem using the FLASH imaging capabilities of nuclear magnetic resonance (NMR) spectrometry was applied in this study. The method has a time resolution of up to 4 min (for the xylem) and was used to measure the velocity of flows in phloem and xylem for periods of several hours to days. For the first time, diurnal time course measurements of flow velocities and apparent volume flows in phloem and xylem in the hypocotyl of 40‐d‐old Ricinus communis L were obtained. Additional data on gas exchange and the chemical composition of leaves, xylem and phloem sap were used to assess the role of leaves as sinks for xylem sap and sources for phloem. The velocity in the phloem (0·250 ± 0·004 mm s^?1) was constant over a full day and not notably affected by the light/dark cycle. Sucrose was loaded into the phloem and transported at night, owing to degradation of starch accumulated during the day. Concentrations of solutes in the phloem were generally less during the night than during the day but varied little within either the day or night. In contrast to the phloem, flow velocities in the xylem were about 1·6‐fold higher in the light (0·401 ± 0·004 mm s^?1) than in the dark (0·255 ± 0·003 mm s^?1) and volume flow varied commensurately. Larger delays were observed in changes to xylem flow velocity with variation in light than in gas exchange. The relative rates of solute transport during day and night were estimated on the basis of relative flow and solute concentrations in xylem and phloem. In general, changes in relative flow rates were compensated for by changes in solute concentration during the daily light/dark cycle. However, the major solutes (K⁺, NO₃^?) varied appreciably in relative concentrations. Hence the regulation of loading into transport systems seems to be more important to the overall process of solute transport than do changes in mass flow. Due to transport behaviour, the chemical composition of leaves varied during the day only with regard to starch and soluble carbohydrates.     

Oxygen isotope enrichment of organic matter in Ricinus communis during the diel course and as affected by assimilate transport

The oxygen isotope composition in leaf water and organic compounds in different plant tissues is useful for assessing the physiological performance of plants in their environment, but more information is needed on Delta(18)O variation during a diel course. Here, we assessed Delta(18)O of the organic matter in leaves, phloem and xylem in stem segments, and fine roots of Ricinus communis during a full diel cycle. Enrichment of newly assimilated organic matter in equilibrium with leaf water was calculated by applying a nonsteady-state evaporative enrichment model. During the light period, Delta(18)O of the water soluble organic matter pool in leaves and phloem could be explained by a 27 per thousand enrichment compared with leaf water enrichment. Leaf water enrichment influenced Delta(18)O of phloem organic matter during the night via daytime starch synthesis and night-time starch remobilization. Phloem transport did not affect Delta(18)O of phloem organic matter. Diel variation in Delta(18)O in organic matter pools can be modeled, and oxygen isotopic information is not biased during transport through the plant. These findings will aid field studies that characterize environmental influences on plant water balance using Delta(18)O in phloem organic matter or tree rings.     

Sucrose unloading in the hypocotyl of the Ricinus communis L. seedling measured by 13C-nuclear magnetic resonance spectroscopy in vivo

The unloading of sucrose in the apical part of the hypocotyl of Ricinus communis L. seedlings was measured by ¹³C-nuclear magnetic resonance (NMR) spectroscopy. The cotyledons of the seedling were immersed in 5 mM Mes buffer containing 100 mM ¹³C-labeled sucrose. At intervals of 70–90 min, ¹³C-NMR spectra with broadband decoupling and nuclear Overhauser enhancement were acquired in vivo. The spectra showed growing ¹³C-resonances of the labeled positions in the sucrose molecule reaching steady-state labeling within 7–8 h. The specific ¹³C labeling of sucrose in the G₁-position changed from 0.38 in the supplied sucrose solution to 0.16 in the sucrose extracted from the hypocotyl piece at the end of the experiment (13 h). Labeling of starch (and other insolubles) in the hypocotyl piece was ca. 0.10. It is proposed that the decreased specific labeling of unloaded sucrose is mostly due to the separate local pools of sucrose in the cortex and pith parenchyma, respectively, and less to continuous starch degradation and conversion to sucrose. The report gives an example of the application of ¹³C-NMR spectroscopy in assimilate allocation studies. Received: 10 October 1998 / Accepted: 31 December 1998     

Assimilate export by leaves of Ricinus communis L. growing under normal and elevated carbon dioxide concentrations: the same rate during the day, a different rate at night

Castor bean (Ricinus communis L.) plants were grown for 5–7 weeks in a controlled environment at 350 μl l⁻¹ or 700 μl l⁻¹ CO₂. Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO₂ concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 μl l⁻¹ than at 350 μl l⁻¹. Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO₂ treatment. Respiration was 25% higher in high-CO₂ plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 μl l⁻¹ and 700 μl l⁻¹ CO₂. During the night the low-CO₂ plants had only 50% of the daytime export rate, in contrast to the high-CO₂ plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO₂ regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO₂ concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This “assimilation efficiency” was the same for both CO₂ regimes. It is speculated that under 350 μl l⁻¹ CO₂ the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night. Received: 2 February 1999 / Accepted: 19 April 1999     

Long-distance translocation of polyamines in phloem and xylem of Ricinus communis L. plants

Polyamine content and enzyme activities in the biosynthetic and degradative pathways of polyamine metabolism were investigated in sieve-tube sap, xylem sap and tissues of seedlings and adult plants of Ricinus communis L. Polyamines were present in tissues and translocation fluids of both seedlings and adult plants in relatively high amounts. Only free polyamines were translocated through the plant, as indicated by the finding that only the free form was detected in the phloem and the xylem sap. Removal of the endosperm increased the polyamine content in the sieve-tube exudate of seedlings. The level and pattern of polyamines in tissue of adult leaves changed during leaf age, but not, however, in the sieve-tube sap. Xylem sap was relatively poor in polyamines. Polyamine loading in the phloem was demonstrated by incubating cotyledons with [¹⁴O]putrescine and several unlabelled polyamines. Feeding cotyledons with cadaverine and spermidine led to a decrease in the level of putrescine in sieve-tube sap, indicating a competitive effect. Comparison of polyamine content in the tissue and export rate showed that the export would deplete the leaves of polyamines within 1–3 d, if they were not replenished by biosynthesis. Polyamine biosynthesis in Ricinus proceeds mostly via arginine decarboxylase, which in vitro is 100-fold more active than ornithine decarboxylase. The highest arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities were detected in cotyledons, while in sieve-tube sap only a slight arginine decarboxylase activity was found. Received: 18 March 1997 / Accepted: 20 August 1997     

Phloem turgor and the regulation of sucrose loading in Ricinus communis L.

The influence of plant water relations on phloem loading was studied in Ricinus communis L. Phloem transport was maintained in response to bark incisions even at severe water deficits. Water stress was associated with a net increase in the solute content of the sieve tubes, which resulted in maintenance of a positive phloem turgor pressure _p. There was a significant increase in solute flux through the phloem with decreasing xylem water potential (). In addition, sugar uptake by leaf discs was examined in media adjusted to different water potentials with either sorbitol (a relatively impermeant solute) or ethylene glycol (a relatively permeant solute). The limitations in this experimental system are discussed. The results nevertheless indicated that sucrose uptake can be stimulated by a reduction in cell _p, but that it is little affected by cell or solute potential _s. On the basis of these data we suggest that sucrose loading is turgor-pressure dependent. This may provide the mechanism by which transport responds to changes in sink demand in the whole plant.Abbreviations water potential - _s solute potential - _p pressure potential     

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H⁺-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H⁺-ATPase antibody clones, raised against the PM H⁺-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H⁺-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K⁺ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes. Received: 3 July 2000 / Accepted: 12 October 2000     

Radial transport of salt and water in roots of the common reed (Phragmites australis Trin. ex Steudel)

To understand the root function in salt tolerance, radial salt and water transport were studied using reed plants growing in brackish habitat water with an osmotic pressure (π_M) of 0.63 MPa. Roots bathed in this medium exuded a xylem sap with NaCl as the major osmolyte and did so even at higher salt concentration (π_M up to 1.3 MPa). Exudation was stopped after a small increase of π_M (0.26 MPa) using polyethylene glycol 600 as osmolyte. The endodermis of fine lateral roots was found to be the main barrier to radial solute diffusion on an apoplastic path. Apoplastic salt transfer was proven by rapid replacement of stelar Na⁺ by Li⁺ in an isomolar LiCl medium. Water fluxes did not exert a true solvent drag on NaCl. Xylem sap concentrations of NaCl in basal internodes of transpiring culms were more than five times higher than in medial and upper ones. It was concluded that the radial NaCl flux was mainly diffusion through the apoplast, and radial water transport, because of the resistance of the cell wall matrix to convective mass flow, was confined to the symplast. Radial salt permeation in roots reduced the water stress exerted by the brackish medium.