Frank Dannel

Isolation of apoplasmic fluid from sunflower leaves and its use for studies on influence of nitrogen supply on apoplasmic pH

Summary A centrifugation method for collection of apoplasmic fluid from isolated sunflower leaves has been developed. As indicated by the marker enzymes hexose phosphate isomerase EC 5.3.1.9, glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.13 and malate dehydrogenase EC 1.1.1.37, the symplasmic contamination of the obtained fluid is smaller than 1.6 %. Experiments with the apoplasmic tracer Sulphorhodamine G showed that no dilution with membrane filtered cell sap occurred. The described method allows quick and easy collection of apoplasmic fluid that is representative of its composition in intact leaves. The amounts of fluid obtained enable most analyses to be carried out. The method has been used to study the influence of ammonium or nitrate supply to roots on the apeplasmic pH in sunflower leaves. In plants supplied with either 2 mol m -3 nitrate or ammonium the pH of the xylem exudate at the stem base was 5.80 and 5.79, respectively, and apoplasmic pH was 6.77 and 6.87, respectively. In plants supplied with 4 mol m -3 nitrate, pH of the xylem exudate was 5.87 and apoplasmic pH 7.42. The results suggest that high apoplasmic pH in leaves is caused by high proton consumption during proton-anion cotransport across the plasma membrane.

Compartmentation of boron in roots and leaves of sunflower as affected by boron supply

Summary A method for studying the compartmentation of B in different plant organs has been developed. The basic steps of this method are the preparation of cell sap by squeezing the plant material after a freezethaw-cycle and the subsequent preparation of the water insoluble residue (WIR) by washing the remaining residue after homogenization. These two fractions represent B pools of different solubility and physiological behaviour. The WIR B seems to be closely bound to cell wall polymers, whereas the cell sap B mainly represents the soluble B in the symplasm which seems to be directly available for possible physiological functions in the cell. Using the described method, the compartmentation of B in roots and leaves of sunflower plants, cultivated with a B supply of 0.1 to 1600 μmol · L −1 , was studied as well as the B concentrations on the pathway from the nutrient solution to the symplasm of the leaf cells which was characterized by analyzing a sequence of liquid compartments (root cell sap, xylem exudate and leaf cell sap). Although the external supply increased by a factor of 16,000, B concentration in root and leaf cell walls increased only by a factor of 2.8 and 22, respectively. This indicates that the cell wall does not contribute effectively to the detoxification of excess B taken up by the plant by providing additional binding sites in response to B toxicity. B concentrations in root symplasm and xylem exudate suggest that two mechanisms of B uptake are present in sunflower. At low supply there seems to be a concentration mechanism, which is downregulated in activity when supply increases from 0.1 to 10 μmol · L −1 . At a supply above 100 μmol · L −1 passive diffusion of boric acid from the external solution across the plasma membranes into the cells of the root cortex seems to be responsible for B uptake. At the passage of the casparian strip into the stele and/or at xylem loading itself a considerable retention of B was observed.

Characterization of root boron pools, boron uptake and boron translocation in sunflower using the stable isotopes 10 B and 11 B

The B pools in the roots and the characteristics of B uptake and its loading into the xylem were investi-gated in sunflower (Helianthus annuus L.) plants precultured with high (100 M) or low (1 M) 11 B supply. In order to study B fluxes and their dependence on root metabolic activity, short-term treatments with differential 10 B supply in combination with metabolic inhibition treatments (50 M 2,4-dinitrophenol; root zone temperature of 7˚C) or with no further treatment (control) were carried out. Subsequently, xylem exudate was collected, and roots were harvested and fractionated into two B pools that differed in their water-solubility as well as in their exchangeability. The exchange or release of 11 B initially present during the 3 h treatment was maximal at 18% in the cell wall pool, whilst it was up to 94% in the symplasmic pool. All observed alterations in the cell wall-bound B can be explained by passive processes. Control plants precultured with high B supply showed a linear response of the 10 B concentrations in the root cell sap and in the xylem exudate to the differential short-term 10B supply, and this was not affected by the metabolic inhibition treatments. In the control plants precultured with low B supply, the response of the 10 B concentrations in the root cell sap and xylem exudate to the differential short-term 10 B supply appeared to be a com-bination of a saturable and a linear component. The metabolic inhibition treatments turned off the saturable compo-nent and the response became linear. In summary, the results suggest that B uptake into the root symplasm, as well as xylem loading, are performed by two transport mechanisms, with the linear components representing B transport by passive diffusion. The saturable components may represent unknown carrier- or channel-mediated transport of B, which is dependent on metabolic energy.

Defect in Root-Shoot Translocation of Boron in Arabidopsis thaliana Mutant bor 1-1

Summary Compartmental analysis of B in wild type and bor 1-1 mutant plants of Arabidopsis thaliana were carried out to elucidate reports that the mutant bor 1-1 has a defect(s) in absorption andlor translocation of B, which results in reduced B contents in its shoot (Noguchi et al., 1997b). The plants were grown hydroponically and B contents in water soluble fractions (cell sap) and in water insoluble residues (WIR) were determined. At low B supply (3 μmol · L -1 ), in shoots of bor 1-1 mutant plants, concentrations of water soluble B (B in cell sap) were more than 20-fold lower than in wild type plants. The difference in B contents was less distinct in WIRs. At higher B supply (30 and 100 μmol · L -1 ), there were only marginal or no differences in B contents of both fractions between the mutant and wild type plants. Consequently, the reduced B contents in shoots of the mutant plants at low B supply were due mainly to reduced B contents in the water soluble fractions (cell sap). The results suggest that the bor 1-1 mutation has little or no effect on the binding of B in the cell wall, since B in WIRs mainly represents cell wall bound B. In wild type plants, concentrations of B in xylem exudates were about 3.4-fold higher than those in root cell saps at low B supply (3 μmol · L -1 ), whereas B concentrations were similar in both fractions when B was sufficiently supplied (30 μmol · L -1 ), suggesting that A. thaliana plants possess mechanisms to concentrate B in the process of root-to-shoot transport only at low B supply. In bor 1-1 mutant plants, concentrations of B in shoots, xylem exudates and root cell saps were about 25, 30 and 65%, respectively, of those in wild type plants at low B supply, whereas B concentrations were similar in both genotypes under B sufficient conditions. These data suggest that the major defect in the bor 1-1 mutant is at the B concentrating mechanism in the root-to-shoot transport of B that functions only at low B supply.

Are there two Mechanisms for Boron Uptake in Sunflower

Summary Uptake mechanisms for B have been characterized in young sunflower plants ( Helianthus annuus L. cv. Frankasol), precultured with either low B supply (1 μmol L -1 ) or high B supply (100 gmol L -1 ), by investigating the distribution of B between cell sap and water insoluble residue in roots and the fluxes of B to the shoots in response to short term changes in B supply. Complementary studies were carried out on soluble B complexes in roots and xylem sap and on the effect of inhibition of metabolic processes in the roots on B uptake. Two mechanisms seem to bring about B uptake into the roots, dependent on B supply during plant culture. When B supply is high, passive diffusion is responsible. With low B supply, a concentration mechanism for B is established, which can be turned off by increasing B supply as well as by the inhibition of metabolic processes by low root zone temperature or 2,4-Dinitrophenol. For building up and maintaining the concentration gradient between the root and the external solution, the formation of B complexes within the cell seems at least partially to be involved. The uptake of B from the external medium into the root is likely to be the key step for translocation of B to the shoot, whereas the mechanism of xylem loading is still unclear.

Effect of pH and boron concentration in the nutrient solution on translocation of boron in the xylem of sunflower

The influence of the pH in the nutrient solution (pH 3 to 10) on uptake and translocation of boron (B) in the xylem of young sunflower plants was investigated over a three hour treatment. Two B concentrations were supplied during the whole growth period of seven days: a marginal (1 μM) and a sufficient supply (100 μM). Boron concentrations in the xylem exudate responded both to B and pH treatments. When B supply was sufficient, B concentration in the xylem exudate was the same or lower than that in the nutrient solution and inversely related to the exudation rate. In contrast at marginal supply, B concentration in the xylem exudate was up to 25-fold higher than in the nutrient solution and decreased in direct proportion to the pH dependent concentration of undissociated boric acid. In conclusion, the results indicate that there might be two different mechanisms for uptake and translocation of B in sunflower. At sufficient supply, it seems that a passive diffusion process of boric acid, partly restricted by permeability of membranes, is responsible for uptake. At marginal supply, a concentration mechanism for boric acid builds up a gradient against the nutrient solution, obviously to satisfy the B demand of the plant.

Compartmentation of boron in roots and its translocation to the shoot of sunflower as affected by short term changes in boron supply

The effect of short term changes in boron (B) supply on B uptake and compartmentation in the roots and its translocation to the shoot was studied using sunflower plants, precultured for seven days with a sufficient (100 µM) and a marginal (1 µM) B supply. Boron translocation to the shoot was determined by analysis of xylem exudate. For compartmental analysis of the roots, B was determined in preparations of cell sap (soluble B in the symplasm) and water insoluble residue (cell wall bound B). Boron supply during preculture and short term treatments influenced both the B concentration in the water insoluble residue and in the cell sap, the latter to a larger extent. Boron bound in the cell wall of the roots during the preculture with sufficient B supply could not be removed or mobilized after short term change to marginal B supply. From this it can be concluded, that this bound B plays a structural role in the cell wall and does not function as a storage pool for further use in the plant. The response of the B concentrations in root cell sap and xylem exudate to the short term treatments suggests, that B uptake at sufficient supply is achieved by passive diffusion, whereas at low supply there is a mechanism to concentrate B against the nutrient solution, which helps to satisfy the B demand of the plants. Additionally, it can be concluded that the key step of the concentration mechanism is the uptake of B from the nutrient solution into the symplasm of the root cells, followed by a passive loading of the xylem. The following three concentration mechanisms are discussed: anion trapping, active energy dependent uptake, and concentration in the symplasm by formation of B complexes.

Isolation of Soluble Boron Complexes and their Determination Together with Free Boric Acid in Higher Plants

Summary Isolation of soluble boron complexes and their quantitative determination together with free boric acid in higher plants was carried out by gel permeation chromatography in the liquid compartment of plant leaves and roots. The described method,is suitable not only for the isolation of B complexes from plant cell sap, but also allows for their quantitative determination together with free boric acid, which is possible for samples with a B concentration of at least 157#x03BC;molL −1 (Table 2). This gel permeation chromatography method therefore complements methods that allow characterization, but not quantitative determination of B complexes in plant samples. Soluble B complexes have been isolated from sunflower ( Helianthus annuus L.) grown with a marginal to toxic B supply and curly kale ( Brassica oleracea L.). In sunflower root symplasm there was only a small concentration of B complexes that was not dependent on variations in B supply from the marginal to the toxic level. The resulting large variations in total symplasmic B were therefore only reflected in the concentrations of free boric acid. In contrast, the concentration of B complexes in sunflower leaf symplasm was about 50-fold larger than in the root symplasm and made up 31 % of the total symplasmic B. In curly kale leaves even 59 % of the total symplasmic B was present as B complexes. Further experiments are necessary to characterize the B complexes in sunflower and curly kale to investigate their physiological role in plant metabolism.

Characterization of Boron Uptake in Higher Plants

The only clearly defined function of boron (B) in higher plants is linked with the stabilisation of cell walls where B preferentially binds and thus stabilises the cell wall Matoh (1997). There is growing evidence however for a role of B in maintaining the integrity of membranes (Cakmak and Romheld, 1997), and Pfeffer et al., (2001) have used evidence of cytoplasmic homeostasis to suggest that B may function as in the cytoplasm. In organisms without cell walls such as zebrafish, it has recently been shown that inadequate B supply results in severe growth inhibition (Rowe and Eckhert, 1999).

Characteristics of boron uptake in roots of sunflower by a putative boron transporter

The mechanisms of boron (B) uptake in roots of sunflower (Helianthus annuus L.) plants were studied. To characterize the high affinity B uptake system, which is induced by low B supply, plants were pre-cultured with 100 µmol1−1 11B (high B) or 1 µmol 1−1 11B (low B) supply. Subsequently, short term (5, 10, 15 min) B uptake experiments with varying 10B supply were carried out and the B pools in roots were analysed. The saturable component of B uptake (i. e. the high affinity B uptake system) followed Michaelis-Menten kinetics with an apparent Km of 15 µmol 1−1 and a Vmax of 30nmol groot FW −1 h−1, which corresponds fairly well with data for high affinity uptake systems for other mineral nutrients.