Water, mineral nutrient and hormone flows and exchanges in the hemiparasitic association between root hemiparasite Rhinanthus minor and the host Hordeum vulgare Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Fan Jiang aus China Würzburg 2004 Eingereicht am: Mitglieder der Prüfungskommission: Vorsitzender: Prof. Dr. U. Scheer Gutachter: Prof. Dr. W. Hartung Gutachter: Prof. Dr. M. Riederer Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: Contents 1. Introduction..............................................................................................................4 1.1 Parasitic plants: a general introduction..............................................................................4 1.2 Haustoria ...............................................................................................................................5 1.3 Water relations ......................................................................................................................7 1.4 Nutritional relationships of parasites and their host..........................................................9 1.4.1 Nitrogen.........................................................................................................................10 1.4.2 Ion accumulation..........................................................................................................11 1.4.3 Carbon assimilation .....................................................................................................12 1.5 Phytohormones in hemiparasitic associations ..................................................................14 1.6 Project aims .........................................................................................................................15 2. Materials and methods ..........................................................................................16 2.1 Plant culture ........................................................................................................................16 2.2 Harvests of plant tissue.......................................................................................................18 2.3 Collections of xylem sap and of phloem exudate..............................................................18 2.4 Modelling of water flows ....................................................................................................19 2.5 Analysis of plant tissue and transport fluids for mineral elements, sugar, anions and amino acids ................................................................................................................................20 2.6 Estimation of net flows of mineral nutrients (N, P, K+, Mg2+) through xylem and phloem in unparasitised barley and unattached Rhinanthus and within the association of barley and Rhinanthus..........................................................................................................................21 2.7 ABA analysis........................................................................................................................22 2.7.1 Extraction of tissues.....................................................................................................22 2.7.2 Principle of the analysis...............................................................................................22 2.7.3 Procedures of ELISA ...................................................................................................23 2.8 Cytokinins analysis .............................................................................................................24 2.8.1 Extraction of tissue.......................................................................................................24 2.8.2 Procedures of ELISA ...................................................................................................25 2.9 Modelling of ABA flows ......................................................................................................26 2.10 Rough estimates of the movements of sucrose in barley................................................26 2.11 Precision of models............................................................................................................27 2.12 Leaf conductance...............................................................................................................27 2.13 Scanning electron microscopy..........................................................................................28 2.14 Hydraulic conductivity of roots .......................................................................................28 2.15 Collection of root exudates from barley and Rhinanthus seedlings..............................29 2.16 Stain for haustoria sections ..............................................................................................30 2.17 Immunolocalisation of ABA .............................................................................................30 2.17.1 Tissue preparation......................................................................................................30 2.17.2 Immunocytochemistry ...............................................................................................31 3. Results .....................................................................................................................32 3.1 Growth of single and parasitising Rhinanthus..................................................................32 3.2 Haustorium..........................................................................................................................34 3.3 Water flows in the parasitic associaton Rhinanthus minor/Hordeum vulgare................36 1 Contents 3.3.1 The stomatal movements of Rhinanthus and Melampyrum arvense ........................36 3.3.2 Hydraulic conductivity of roots of Rhinanthus and Hordeum vulgare.....................39 3.3.3 Water flows in the parasitic associaton Rhinanthus minor/Hordeum vulgare - supplied with 5 mM NO3 .....................................................................................................42 3.3.4 Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare - + supplied with 1 mM NO3 or 1 mM NH4 ............................................................................43 3.3.5 Comparision of water flows in the parasitic association Rhinanthus minor/Hordeum - - vulgare supplied with 5 mM NO3 or 1 mM NO3 ...............................................................47 3.4 Nutritional flows from Hordeum vulgare to the hemiparasite Rhinanthus minor and the influence of infection on host and parasite nutrient relations...............................................49 - 3.4.1 Plants supplied with 5 mM NO3 .................................................................................49 3.4.1.1 Biomass accumulation ..........................................................................................49 3.4.1.2 Mineral nutrients partitioning in control barley, in solitary Rhinanthus and in the association between barley and Rhinanthus .............................................................49 3.4.1.3 Concentrations of mineral nutrients, amides and amino acid-N in xylem sap 50 3.4.1.4 Net flows of mineral nutrients within uninfected barley, unattached Rhinanthus and in the association between barley and Rhinanthus .................................................53 - + 3.4.2 Plants supplied with 1 mM NO3 and 1 mM NH4 .....................................................58 3.4.2.1 Biomass accumulation ..........................................................................................58 3.4.2.2 Mineral nutrients partitioning in control barley, in solitary Rhinanthus and in the association between barley and Rhinanthus .............................................................59 3.4.2.3 Concentrations of mineral nutrients, amides and amino acid-N in xylem sap 65 3.4.2.4 Net flows of mineral nutrients within uninfected barley, unattached Rhinanthus and in the association between barley and Rhinanthus .................................................67 3.4.2.5 Comparision of mineral relations in the parasitic association Rhinanthus - minor/Hordeum vulgare supplied with 5 or 1 mM NO3 .................................................75 3.5 Contents and flows of assimilates (mannitol and sucrose) in the hemiparasitic Rhinanthus minor/Hordeum vulgare association ....................................................................78 3.5.1 Contents and flows of mannitol and sucrose..............................................................78 3.5.2 Mannitol in xylem sap and mannitol/K in phloem sap .............................................80 3.5.3 Flows of mannitol and sucrose ....................................................................................80 3.6 Abscisic acid (ABA) flows from Hordeum vulgare to the hemiparasite Rhinanthus minor and the influence of infection on host and parasite abscisic acid relations..........................88 3.6.1 Abscisic acid content in single and attached barley and Rhinanthus minor............88 3.6.2 ABA in the xylem sap ...................................................................................................88 3.6.3 ABA flows......................................................................................................................91 3.6.4 Comparision of ABA in Rhinanthus/Hordeum associations