Regulation of phosphate deficiency-induced carboxylate exudation in cluster roots of white lupin (Lupinus albus L.)

................................................................................................................ 156-160 Abstract (German) ................................................................................................. 161-165 References ............................................................................................................. 166-187 List of figures shown I ———————————————————————————————————— List of figures shown Figure No. Title page 1 Models for morphological and root-induced chemical phosphate mobilization in the rhizosphere 3 2 Schematic representation of P-deficiency-induced metabolic processes, as indicated by heavy arrows, that may circumvent P-dependent basic reactions of carboxylate metabolism, resulting in carboxylate production 16 3 Schematic representation suggesting metabolic processes being inhibited under P deficiency, as indicated by „ = “, promoting a reduced turnover of citrate 18 4 Schematic representations of P-deficiency-induced modifications of respiration with potential impact on citrate accumulation (a) reduced respiration leading to a feedback inhibition of the TCA cycle by overreduction of the reduction equivalents or (b) increased production of H2O2, caused by an impaired respiration, leading to inhibition of the aconitase enzyme 20 5 Incubation of part of the root system with metabolic inhibitors in a separate jar 34 6 Immunoblot analysis of PEP-C in different white lupin root segments 36 7 Malic enzyme (ME) activity in different white lupin root segments 37 8 Pyruvate tissue concentrations in different white lupin root segments 38 9 aconitase and NADP-dependent ICDH activity in different white lupin root segments 40 10 Peroxide concentrations in different white lupin root segments 41 11 Malondialdehyde (MDA) concentrations in different white lupin root segments 42 12 Malate and citrate concentrations and citrate/malate tissue concentration ratios in young cluster root segments after incubation with the aconitase inhibitor H2O2, applied at the concentrations of 10 mM and 5 mM for 1.5 h, 3 h and 5 h. 43 13 Malate and citrate tissue concentrations and citrate/malate tissue concentration ratios in young and mature cluster root segments after incubation with 10 mM the aconitase inhibitor monofluoroacetate (MFA) for 8 h. 44 14 Malate (left) and citrate (middle) exudation rates and citrate/malate exudation ratios (right) in young and mature cluster root segments after incubation with 10 mM the aconitase inhibitor monofluoroacetate (MFA) for 8 h 45 15 Organic acid exudation rates in seedling root tips after incubation with 10 mM the aconitase inhibitor monofluoroacetate (MFA), 20 μM Al, and a combination of both (Al+MFA) for 12 h, followed by a localized root exudate collection for 2 h 47 16 aconitase and NADP-dependent ICDH activity in young and mature white lupin root segments after incubation with 10 mM MFA for 8 h 48 17 Histological formazan staining of dehydrogenase activities in different root zones of P-deficient white lupin with citrate and succinate as substrates 50 18 Respiration, measured as O2 depletion, in different white lupin root segments 52 19 Western blot analysis of alternative oxidase protein in different white lupin root segments 53 List of figures shown II ———————————————————————————————————— 20 The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) with the concentrations of 0; 0.2; 1; 10, and 20 μM on respiration of different white lupin root segments 54 21 The effect of partial root incubation with the respiration inhibitors azide (1 mM) and SHAM (7.5 mM) for 4 h and 8 h on malate and citrate concentrations in young (-P y) and mature (-P m) cluster roots of white lupin 56 22 Malate (left) and citrate (middle) concentrations and citrate/malate tissue concentration ratios (right) in young cluster root segments after incubation with the nitrate reductase inhibitor Na2WO4, applied at the concentrations of 300 μM, 600 μM and 1000 μM for 16 h 57 23 reaction scheme of the enzyme ATP-citrate lyase 58 24 A: malate and citrate root segment tissue concentrations. B: in vitro activities of ATP-citrate lyase in different white lupin root segments; C: transcript levels of ACL 58 25 Malate and citrate concentrations and citrate/malate tissue concentration ratios in young cluster root segments after incubation with the citrate lyase inhibitor hydroxycitrate (HC), applied at the concentrations of 5 mM for 12 h and of 100 mM for 8 h 59 26 Spatial variation of pH, plasma membrane H-ATPase activity, and exudation of citrate along cluster roots of P-deficient white lupin 98 27 Development of citrate exudation rate per plant and pH in the nutrient solution in +P control plants and P-deficient plants during plant growth 98 28 Vanadate sensitive PM H-ATPase hydrolytic activity measured as phosphate release by ATP cleavage in PM vesicles derived from different segments of cluster roots of P deficient plants. A: H-ATPase activity per cluster; B: per cluster fresh weight; C: per protein. 99 29 PM H-ATPase hydrolytic activity measured as phosphate release by ATP cleavage in PM vesicles derived from roots of two to five weeks old P sufficient (+P) or P deficient (-P) plants 100 30 PM H-ATPase hydrolytic activity measured as phosphate release by ATP cleavage in PM vesicles derived from roots of five weeks old P sufficient (+P) or P deficient (-P) plants at different pH values in the assay solution. Maximum activity for +P at pH 6.50; for -P at pH 6.35 100 31 PM H-ATPasehydrolytic activity measured as phosphate release by ATP cleavage in PM vesicles derived from -P and +P control plant roots dependent on citrate (A) and malate (B) concentrations in the assay solution. 101 32 Effect of NO3 (100 mM), vanadate (0.1 mM), malate (7.5 mM) and citrate (5 mM) on the pH gradient formation in PM vesicles, determined by the absorbance change of acridine orange at λ = 492 nm. Vesicles were isolated from P deficient roots. The acidification was stopped by the addition of 10 mM EDTA-BTP 102 33 Immunodetection of the H-ATPase protein by Western Blotting of PM vesicles isolated from roots of +P control plants or of P-deficient (-P) clustercontaining plants 103 34 Citrate and malate concentrations in different root segments of white lupin as affected by buffering the external pH and application of propionate 105 35 Protoplasts isolated from A+B: mature cluster root laterals. Red colouration inside the protoplasts means low viability. C: from cotyledons. D-G: root hair protoplasts isolated according to the methods of Gassmann and Schroeder (1994) and Cocking (1985) 108 List of figures shown III ———————————————————————————————————— 36 Shoot and root fresh weight of P deficient white lupin plants under ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 128 37 P deficient white lupin plants cultivated at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations after 35 days of growth. below: shoots and roots of P-deficient white lupin plants cultivated at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations 29 days after sowing 129 38 Root/shoot mass ratio of P deficient white lupin plants under ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 130 39 Total number of cluster roots per plant of P-deficient white lupin plants at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 130 40 Total number of cluster roots per root fresh biomass of P deficient white lupin plants at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 131 41 Distribution of cluster roots of different developmental stages depending on plant age and atmospheric CO2 concentrations 131 42 Malate and citrate exudation rates in different white lupin cluster root segments at six different days after sowing at ambient (400 μmol mol) and elevated (800 μmol mol) CO2 concentrations 132 43 Pi concentrations per root biomass in different cluster root segments at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 133 44 Pi concentrations per shoot biomass in the shoot tip at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations at six different days of harvest (days after sowing) 134 45 Shoot and root dry weight and total plant dry weight of white lupin plants grown in rhizoboxes at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations with sufficient (+P) and without (-P) external P supply, 35 days after sowing 136 46 Distribution of cluster roots in different developmental stages (four harvest dates) of white lupin plants grown in rhizoboxes, at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations grown with sufficient (+P) and without (-P) external P supply (data from J. Wasaki and A. Rothe) 137 47 Citrate exudation from root segments of +P and P-deficient plants 35 days after sowing at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations (data from G. Neumann) 138 48 Rhizosphere acid and alkaline phosphatase activity [nmol substrate turnover h g rhizosphere soil] of white lupin plants grown in rhizoboxes at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations grown with sufficient (+P) and without (-P) external P supply 35 days after sowing (data from J. Wasaki and A. Rothe) 139 49 Shoot and root total P concentration per dry weight and P root and shoot contents of white lupin plants grown in rhizoboxes at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations grown with sufficient (+P) and without (-P) external P supply 35 days after sowing. (data from J. Wasaki and A. Rothe) 140 List of tables shown I ———————————————————————————————————— List of tables shown Table No. Title page 1 Characteristics of different developmental stages of cluster roots I 35 2 Characteristics of different developmental stages of cluster roots II 36 3 The effect of partial root incubation with the respiration inhibitors azide (1 mM) and SHAM (7.5 mM) for 4 h and 8 h on citrate/malate tissue concentration ratios in young (-P y) cluster roots of white lupin 56 4 Homogenization stock solution (MO) for vesicle isolation 87 5 GS stock solution 87 6 MR stock solution (resuspension medium) 87 7 Homogenization solution 87 8 Gradient Solution (GS) 88 9 Different inhibitor treatments to determine the activities of the different ATPases in the vesicle suspension derived from different subcellular membrane fractions 89 10 Solution 1 to stop the ATPase reaction 90 11 Solution 2 for the colour reaction with Pi 90 12 Vanadate-sensitive and –insensitive plasma membrane ATPase reaction medium 90 13 pH-dependent vanadate-sensitive and –insensitive plasma membrane ATPase reaction medium 91 14 Incubation solution for C-citrate transport determination 92 15 Washing solution 92 16 Solutions used for protoplast isolation 94 17 Solutions used protoplast isolation from root hairs 95 18 Purity of vesicle preparations characterized by inhibition of marker enzymes (ATPases). 97 19 Uptake of C labelled citrate and H pumping activity by addition of citrate alone or citrate + Mg-ATP for energetization in PM vesicles derived from Pdeficient (-P) and +P control plants. 104 20 Preparation of the mixed reagent for Pi determination 125 21 Preparation of acids and molybdate-vanadate solution to determine Phosphate-P 126 22 Number of cluster roots of white lupin plants removed for further investigations at different harvest times. Plants were grown in rhizoboxes at ambient (400 μmol mol) and elevated (800 μmol mol) atmospheric CO2 concentrations grown with sufficient (+P) and without (-P) external P supply (data from Wasaki and Rothe) 137