Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare

Using the facultative root hemiparasite Rhinanthus minor and its host Hordeum vulgare several aspects of water relations have been measured in this parasitic association. Extraction of xylem sap by the parasite from the host's roots is facilitated by con siderably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another root hemiparasite, Melampyrum arvense, parasitizing various grasses in the field, showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values and that of Rhinanthus seminal roots showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition, and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host's roots. This water uptake by the parasitized host, however, due to a parasite-induced reduction in the host's growth, was decreased by 22% as compared to non-parasitized barley. The overall changes in growth-related water deposition in the host and parasite pointed to decreased shoot growth and relatively favoured root growth in the host and to strongly favoured shoot growth in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitizing one barley plant.     

Contents and flows of assimilates (mannitol and sucrose) in the hemiparasiticRhinanthus minor/Hordeum vulgare association

Using the facultative root hemiparasiteRhinanthus minor andHordeum vulgare as a host, the flows and partitioning of mannitol in the parasite, and of sucrose in the host have been studied during the period of 41 to 54 days after planting, i.e, about 30 to 43 days after successful attachment of the parasite to the host. The biosynthesis of mannitol inRhinanthus shoots increased 16-fold by parasitism, resulting in a 15-fold higher mannitol flow in the phloem and a 10-fold higher deposition in the shoot. Under reduced nitrogen supply and with ammonium as the only N-form the concentrations of mannitol tended to be increased by approximately 2-fold. Xylem flows of mannitol were increased 10-fold after attachment. No mannitol was found in barley roots even in the direct vicinity of the haustoria. Compared to unparasitized barley, the net biosynthesis and deposition in the shoot and the phloem flow was decreased substantially. No sucrose has been detected in barley xylem sap and consequently there was no indication of a sucrose transfer from the host to the parasite. A possible involvement of mannitol in the abscisic acid relations of the parasite is discussed.     

Root-derived cytokinins as long-distance signals for NO3--induced stimulation of leaf growth

Leaf growth of many plant species shows rapid changes in response to alterations of the form and the level of N supply. In hydroponically-grown tomato (Lycopersicon esculentum L.), leaf growth was rapidly stimulated by NO(3)(-) application to NH(4)(+) precultured plants, while NH(4)(+) supply or complete N deprivation to NO(3)(-) precultured plants resulted in a rapid inhibition of leaf growth. Just 10 microM NO(3)(-) supply was sufficient to stimulate leaf growth to the same extent as 2 mM. Furthermore, continuous NO(3)(-) supply induced an oscillation of leaf growth rate with a 48 h interval. Since changes in NO(3)(-) levels in the xylem exudate and leaves did not correlate with NO(3)(-)-induced alterations of leaf growth rate, additional signals such as phytohormones may be involved. Levels of a known inhibitor of leaf growth, abscisic acid (ABA), did not consistently correspond to leaf growth rates in wild-type plants. Moreover, leaf growth of the ABA-deficient tomato mutant flacca was inhibited by NH(4)(+) without an increase in ABA concentration and was stimulated by NO(3)(-) despite its excessive ethylene production. These findings suggest that neither ABA nor ethylene are directly involved in the effects of N form on leaf growth. However, under all experimental conditions, stimulation of leaf growth by NO(3)(-) was consistently associated with increased concentration of the physiologically active forms of cytokinins, zeatin and zeatin riboside, in the xylem exudate. This indicates a major role for cytokinins as long-distance signals mediating the shoot response to NO(3)(-) perception in roots.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions.

Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed.     

Water Relations of the Hemiparasite Rhinanthus serotinus before and after Attachment

Growth of the hemiparasite Rhinanthus serotinus (Schönh.) Oborny was greatly stimulated after attachment of the parasite to the roots of the host plant, Hordeum vulgare L. Before attachment the hydrostatic pressure in the xylem, determined by the pressure bomb technique, was found to be lower in Rhinanthus than in the host. It increased after the formation of haustoria between host and parasite. Apparently, the water transport to Rhinanthus was facilitated. The hydrostatic pressure remained lower than that of the host, accounting for the flow of water and solutes in the direction of the parasite and indicating that there exists a resistance to water transport in the haustoria. Water and solutes were absorbed by the cells, which increased in size. The turgor pressure of the parasite rose steeply, but the osmotic potential was hardly affected.     

Mobility of boron-polyol complexes in the hemiparasitic association between Rhinanthus minor and Hordeum vulgare: the effects of nitrogen nutrition

Boron (B) is an essential nutrient required for plant growth and physiological processes. Long-distance B transport is facilitated by the formation of B–polyol complexes. We investigated B uptake and distribution in response to differing levels of exogenous nitrogen supply in the hemiparasitic association between Rhinanthus minor and Hordeum vulgare (barley) and in unparasitised barley and single Rhinanthus plants. In this system, the polyol mannitol is the major assimilate in Rhinanthus , whereas polyols are not detectable in barley. Furthermore, previous studies have shown that the accumulation of polyols within Rhinanthus is negatively affected by the application of exogenous nitrogen. Within the association, the strongest accumulation of B was detected in lateral buds and inflorescences of Rhinanthus , consistent with the greatest B demand in strong sink organs supplied through the phloem that contain high concentrations of mannitol. In the host, the strongest B accumulation was found in xylem-supported leaf lamellae. Roots and sheaths did not accumulate substantial amounts of B, while re-circulation of B through the phloem vessels accounted for only 10% (unparasitised) and 8% (parasitised) of the xylem sap-imported B in the mannitol-free barley hosts. In contrast, 53% (attached) and 39% (in the absence of a host) of the xylem sap-imported B was re-circulated in the phloem in the mannitol-rich Rhinanthus . We therefore present the first quantitative uptake and flow models of long-distance B transport in polyol-rich and polyol-free plants. Our findings are consistent with a close relationship between B re-translocation and mannitol concentrations in phloem vessels.     

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

Rapid and tissue-specific changes in ABA and in growth rate in response to salinity in barley leaves

The addition of 100 mM NaCl to the root medium of barley plantscaused the rapid cessation of elongation of the growing leafthree, followed by a sudden resumption of growth during thefollowing hour. The idea that resumption of growth is precededand mediated by rapid and tissue-specific changes in ABA concentrationand by changes in transpiration was tested. Leaf elongationvelocity was recorded continuously using linear variable displacementtransducers (LVDT), ABA was determined by immunoassay, and transpirationand stomatal conductivity were measured gravimetrically andby porometry, respectively. Within 10 min following additionof salt, ABA increased 6-fold in the distal portion of the leafelongation zone; in the proximal portion, ABA accumulated witha delay. In the portion of the growing blade that had emergedABA increased 3-fold and remained elevated during the following20 min. This preceded a decrease in transpiration and stomatalconductivity, which, in turn, coincided with growth resumption.Twenty hours following the addition of salt, the ABA concentrationshad returned to the level before stress. Leaf elongation velocitywas still reduced. It is concluded that NaCl causes a rapidincrease in ABA in the transpiring portion of the growing leaf.This leads to a decrease in transpiration. As a result, xylemwater potential is expected to rise. The moment that the waterpotential gradient between the xylem and the peripheral cellsin the growth zone favours water uptake again into the latter,leaf elongation resumes. The results suggest that ABA causesdifferent responses in different leaf regions, all aimed atpromoting the resumption of leaf growth. Key words: Abscisic acid, cell elongation, Hordeum vulgare, leaf growth, salinity, water relations.     

Physiological Changes in the Hemiparasite Rhinanthus serotinus before and after Attachment

Growth of the hemiparasite Rhinanthus serotinus (Schönh.) Oborny was greatly stimulated after attachment of the parasite to the roots of the host plant, Hordeum vulgare L. In order to find the limiting factors for the growth of Rhinanthus without a host, unattached and attached Rhinanthus plants were compared. Within I day after attachment the contents of nitrogen, phosphorus, potassium, magnesium, and sodium increased considerably. Organic nitrogen and phosphorus compounds were rapidly synthesized in attached Rhinanthus. The accumulation of sugars in unattached Rhinanthus and the decrease in sugar content after attachment suggested that the main requirement from the host was not for carbohydrates.     

Root-to-shoot signalling when soil moisture is heterogeneous: increasing the proportion of root biomass in drying soil inhibits leaf growth and increases leaf abscisic acid concentration

To determine whether root-to-shoot signalling of soil moisture heterogeneity depended on root distribution, wild-type (WT) and abscisic acid (ABA)-deficient (Az34) barley (Hordeum vulgare) plants were grown in split pots into which different numbers of seminal roots were inserted. After establishment, all plants received the same irrigation volumes, with one pot watered (w) and the other allowed to dry the soil (d), imposing three treatments (1 d: 3 w, 2 d: 2 w, 3 d: 1 w) that differed in the number of seminal roots exposed to drying soil. Root distribution did not affect leaf water relations and had no sustained effect on plant evapotranspiration (ET). In both genotypes, leaf elongation was less and leaf ABA concentrations were higher in plants with more roots in drying soil, with leaf ABA concentrations and water potentials 30% and 0.2 MPa higher, respectively, in WT plants. Whole-pot soil drying increased xylem ABA concentrations, but maximum values obtained when leaf growth had virtually ceased (100 nm in Az34, 330 nm in WT) had minimal effects (<40% leaf growth inhibition) when xylem supplied to detached shoots. Although ABA may not regulate leaf growth in vivo, genetic variation in foliar ABA concentration in the field may indicate different root distributions between upper (drier) and lower (wetter) soil layers.     

Abscisic acid in the xylem: where does it come from, where does it go to?

Abscisic acid is a hormonal stress signal that moves in the xylem from the root to the different parts of the shoot where it regulates transpirational water loss and leaf growth. The factors that modify the intensity of the ABA signal in the xylem are of particular interest because target cells recognize concentrations. ABA(xyl), will be decreased as radial water flow through the roots is increased, assuming that radial ABA transport occurs in the symplast only. Such dilutions of the plant hormone concentration can be compensated in different ways, which help to keep the ABA-concentrations in the xylem constant: (i) apoplastic bypass flows of ABA, (ii) ABA flows between the stem parenchyma and the xylem during transport and (iii) the action of beta-D-glucosidases that release free ABA from its conjugates to the root cortex and the leaf apoplast. The significance of reflection coefficients (sigma(ABA)), permeability coefficients of membranes (P(S)(ABA)) and apoplastic barriers for ABA is discussed.     

Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC,the main anion conductance in xylem-parenchyma cells of barley roots

We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.     

Flows of elements, ions and abscisic acid in Ricinus communis and site of nitrate reduction under potassium limitation.

In a pot experiment Ricinus communis plants were cultivated in quartz sand and supplied daily with a nutrient solution which contained 4 mol m(-3) nitrate as the nitrogen source and either full strength potassium (1.3 mol m(-3), control) or 8% potassium (0.1 mol m(-3), K(+)-limitation). Although the final fresh weight of the whole plant was not affected by K(+)-limitation, the root-shoot ratio was increased due to a relatively increased root growth and inhibited development of younger shoot parts. Owing to K(+)-limitation, photosynthesis was slightly decreased, while dark respiration of the shoot markedly decreased and root respiration was nearly doubled. The transport of carbon in the phloem, and to some extent in the xylem, was greater and the root was favoured in the partitioning of carbon. This was also true for nitrogen and potassium which were both taken up at lower rates, particularly potassium. In these two cases a high remobilization and recycling from the old part of the shoot was observed. By contrast, uptake of sodium was 2.4-fold higher under K(+)-limitation and this resulted in increased flows in the plants, which was discussed generally as a means for charge balance (in combination with a slight increase in uptake of magnesium and calcium). Nitrate reduction took place in the same portion in the root and shoot. This was a shift to the root compared to the control and points to an inhibition of xylem transport caused by limitation of K(+) as an easily permeating countercation. Low K(+) supply also resulted in an increased biosynthesis of ABA in the roots (265%). This caused a slightly increased deposition of ABA in the roots (193%) and a 4.6-fold higher root-to-shoot and a doubled shoot-to-root ABA signal in the xylem or phloem, respectively. The high degradation of ABA in the shoots prevented ABA accumulation there.     

The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. III. Long-distance transport of abscisic acid depending on nitrogen nutrition and salt stress

Seedlings of Ricinus communis L. were cultivated in quartz sandand supplied with media which contained either different concentrationsof nitrate or ammonium nitrogen and were treated with a lowsalt stress. The concentration of ABA was determined in tissuesand in xylem and phloem saps. Between 41 and 51 day after sowing,abscisic acid (ABA) flows between roots and shoots were modelled.Long-distance transport of ABA was not stimulated under conditionsof nitrate deficiency (0.2 mol m^–3). However, when ammoniumwas given as the only N source (1.0 mol m^–3), ABA transportin both xylem and phloem was increased significantly. Mild saltstress (40 mol m^–3 NaCl) increased ABA transport in nitrate-fedplants, but not in ammonium-fed plants. The leaf conductancewas lowered by salt treatment with both nitrogen sources, butit was always lower in ammonium-fed compared to nitrate-fedplants. A negative correlation of leaf conductance to ABA levelsin leaves or flow in xylem was found only in comparison of ammonium-fedto nitrate-fed plants. Key words: Abscisic acid, ammonium, Ricinus communis, phloem, xylem, transport, nitrate, nitrogen nutrition     

Functional anatomy of haustoria formed by Rhinanthus minor: linking evidence from histology and isotope tracing

The root parasite Rhinanthus minor feeds on the xylem of a diverse range of species. Grasses and legumes are the best hosts, while on forbs R. minor typically shows poorer growth. It has been hypothesized that host quality is linked to the expression of defences against the parasite seen in forb roots, but never in grasses. The efficacy of these defence mechanisms in preventing resource loss has not, however, been measured directly. Here we combine histological characterization of haustoria formed on Cynosurus cristatus (a grass), Leucanthemum vulgare and Plantago lanceolata (forbs) with (15)N tracers supplied to the host to quantify the efficacy of these defence responses. Rhinanthus minor penetrated only the xylem of C. cristatus, abstracting an average of 17% of the (15)N tracer taken up, but only 2.5 and 0.2%, respectively, when attached to L. vulgare and P. lanceolata. For the first time, this study has established that the resistance mechanisms of the forbs are effective in preventing the parasite from directly accessing their xylem solutes.     

Does legume nitrogen fixation underpin host quality for the hemiparasitic plant Rhinanthus minor?

The high quality of leguminous hosts for the parasitic plantRhinanthus minor (in terms of growth and fecundity), comparedwith forbs (non-leguminous dicots) has long been assumed tobe a function of the legume's ability to fix atmospheric nitrogen(N) from the air and the potential for direct transfer of compatibleamino compounds to the parasite. Using associations betweenRhinanthus minor and Vicia faba (Fabaceae) that receive N eitherexclusively via symbiotic associations with rhizobia supplyingorganic N fixed from N₂ or exclusively through the supply ofinorganic nitrate to the substrate, the underlying reasons forthe quality of legumes as hosts for this parasite are unravelled.It is shown that sole dependence of the host, V. faba, on Nfixation results in lower growth of the attached parasite thanwhen the host is grown in a substrate supplied exclusively withinorganic N. In contrast, the host plants themselves achieveda similar biomass irrespective of their N source. The physiologicalbasis for this is investigated in terms of N and abscisic acid(ABA) partitioning, haustorial penetration, and xylem sap aminoacid profiles. It is concluded that legume N fixation does notunderpin the quality of legumes as hosts for Rhinanthus butrather the well-developed haustorium formed by the parasite,coupled with the lack of defensive response of the host tissuesto the invading haustorium and the presence of sufficient nitrogenouscompounds in the xylem sap accessible to the parasite haustoria,would appear to be the primary factors influencing host qualityof the legumes. Key words: ABA, haustorium, legume, nitrogen fixation, nodules, parasitic plant Received 14 November 2007; Revised 7 January 2008 Accepted 8 January 2008     

Long Distance Transport of Abscisic Acid in NaCI-Treated Intact Plants of Lupinus albus

Plants of Lupinus albus were grown for 51 d under control (1.1mol m^–3 NaCl) and saline (40 mol m^–3 NaCl) conditions.Plants were harvested and changes of carbon, nitrogen and abscisicacid (ABA) contents of individual organs were determined 41d and 51 d after germination. In the period between the twoharvests xylem and phloem saps were collected and respirationand photosynthesis of individual organs were measured. Usingflows of carbon, C/ABA ratios and increments of ABA flows ofABA in phloem and xylem and rates of biosynthesis and degradationof ABA were calculated. Both under control and saline conditionsnet biosynthesis occurred in the root, the basal strata of leavesand in the inflorescence. Metabolic degradation of ABA tookplace in the stem internodes and apical leaf strata. Salt stress increased xylem transport of ABA up to 10-fold andphloem transport to the root up to 5-fold relative to that ofthe controls. A considerable amount of ABA in the xylem saporiginated from biosynthesis in the roots, i.e. 55% in salt-treatedand smaller than 28% in control plants. The remaining part ofABA in the xylem sap originated from the shoot: it was translocatedin the phloem from fully differentiated leaves towards the rootand from there it was recirculated back to the aerial partsof the plant. The data suggest that ABA may serve as a hormonalstress signal from the root system. Key words: Lupinus albus, salt stress, abscisic acid, long distance transport     

Extracellular beta-glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves

Abscisic acid conjugate concentrations increased in barley xylem sap under salinity, whereas it remained at a low level in the intercellular washing fluid (IWF) of barley primary leaves (Hordeum vulgare cv. Gerbel). Here it is shown that IWF contains beta-glucosidase activity which releases abscisic acid (ABA) from the physiologically inactive ABA-glucose conjugate pool in the leaf apoplast. The following data support this conclusion and give the first biochemical and physiological characterization of the extracellular glucosidase activity in barley. Free ABA was released by the incubation of ABA glucose ester with IWF. The product exhibited the retention time of authentic ABA upon separation by thin layer chromatography and was identified by ABA-ELISA. p-Nitrophenol-beta-D-glucopyranoside (pNPG) was used as the substrate for beta-glucosidases. The K(M)(pNPG) was 1.8 mmol l(-1). The activity was affected by ABA glucopyranoside in a competitive type of inhibition with a K(I) of 400 micromol l(-1). Various hormone conjugates were compared with respect to their inhibitory effect on beta-glucosidase activity. Inhibition was highest for the ABA glucopyranoside and the zeatin riboside, but insignificant for ABA methyl ester and zeatin-9-beta-D-glucoside. The specific activity of the beta-glucosidase was 16-fold greater in IWF as compared to crude leaf extracts confirming its extracellular compartmentation. The activity of beta-glucosidase was strongly increased after growth in hydroponic medium supplemented with NaCl. The data support the hypothesis that the glucose conjugate is a long-distance transport form of ABA.