Regulation of phosphate deficiency induced carboxylate exudation in cluster roots of white lupin (Lupinus albus L.) [Elektronische Ressource] / presented by Angelika Kania

Institute of Plant Nutrition
University of Hohenheim
Field: Rhizosphere and Fertilization Prof. Dr. Volker Römheld
(Supervisor)



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


Dissertation

Submitted in fulfilment of the requirements for the degree "Doktor der Agrarwissenschaften"
(Dr. sc.agr. /Ph.D. in Agricultural Sciences)

to the

Faculty Agricultural Sciences

presented by





Angelika Kania
from Böblingen


2005

















This thesis was accepted as a doctoral dissertation in fulfilment of the requirements for the
degree "Doktor der Agrarwissenschaften" by the Faculty Agricultural Sciences at the
University of Hohenheim on March 22, 2005.



Date of oral examination: May 13, 2005



Examination Commitee

Supervisor and Review Prof. Dr. V. Römheld

1. Co-Reviewer Prof. Dr. R. Böcker

2. Co-Reviewer Prof. Dr. E. Kandeler

Vice-Dean and Head of the Committee Prof. Dr. K. Stahr





























Table of contents I
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯


Table of contents


Page

General introduction .............................................................................................. 1-10
Phosphorus availability in soils ........................................................................ 1
Mechanisms of P acquisition in higher plants .................................................. 1
Perspectives to improve P nutrition in crop plants ........................................... 6
Fertilization management and cropping systems ........................................ 6
Selection and breeding of P-efficient plants ................................................ 7
Lupinus albus – a model plant to study chemical P acquisition in plants ........ 9

General Methods ................................................................................................... 11-13
Plant cultivation and harvest ............................................................................ 11
Determination of organic acids by HPLC ........................................................ 12
Root tissue concentrations ........................................................................... 12
Root exudate collection ............................................................................... 12
Conditions for HPLC ................................................................................... 12
Statistics ....................................................................................................... 13

Chapter 1: Phosphorus deficiency-induced alterations of organic acid
metabolism during cluster root development

Introduction ........................................................................................................... 14-21
Phosphorus deficiency-induced biosynthesis of carboxylates ......................... 14
Phosphorus deficiency-induced inhibition of citrate turnover ......................... 17
Inhibition of aconitase ................................................................................. 17
Reduced assimilation of nitrate with 2-oxoglutarate as N acceptor ............ 17
ATP-citrate lyase ......................................................................................... 18
Reduced respiration ..................................................................................... 19

Materials and methods .......................................................................................... 22-34
Enzyme assays .................................................................................................. 22
PEP-C immunodetection .................................................................................. 22
Pyruvate determination ..................................................................................... 22
Malic enzyme (ME) activity determination ..................................................... 24
+ Aconitase and NADP -Isocitrate-Dehydrogenase (NADP-ICDH) activity
assay ................................................................................................................. 25
Hydrogen-peroxide (H O ) determination ....................................................... 262 2
Malondialdehyde determination ....................................................................... 27
Histology of dehydrogenase activities ............................................................. 28
Respiration ........................................................................................................ 28
Root O uptake ............................................................................................ 282
AOX Western Blot analysis ........................................................................ 29
ATP-citrate lyase assay .................................................................................... 32
Inhibitor treatments .......................................................................................... 33





Table of contents II
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯

Results.................................................................................................................... 35-59
Phosphorus deficiency-induced biosynthesis of carboxylates ........................ 35
Carboxylate accumulation and metabolic activity status in different
developmental stages of cluster roots .......................................................... 35
Malic enzyme activity ................................................................................ 37
Pyruvate concentrations .............................................................................. 38nduced inhibition of citrate turnover ........................ 39
Inhibition of Aconitase and NADP-dependent isocitrate-dehydrogenase
(NADP-ICDH) ............................................................................................ 39
H O – a natural inhibitor of aconitase in P-deficient white lupin ? ................ 402 2
Peroxide (H O ) concentrations .................................................................. 402 2
Malondialdehyde (MDA) concentrations .................................................... 41
Effects of the external application of aconitase inhibitors on accumulation
and root exudation of citrate ............................................................................. 42
Inhibition of the enzyme aconitase by hydrogen peroxide (H O ) ............. 422 2
Inhibition of the enzyme aconitase by monofluoroacetate (MFA) and its
influence on carboxylate accumulation in young cluster roots ................... 44me
influence on carboxylate exudation from young cluster roots .................... 45me
influence on carboxylate exudation from P-sufficient seedling root tips .... 46
Inhibition of the enzyme
influence on aconitase and NADP-ICDH activities .................................... 47
In situ activity of dehydrogenases involved in citrate turnover ................... 49
Reduced respiration .......................................................................................... 51
Respiration rates and cytochrome and alternative oxidase (AOX)
capacities .................................................................................................... 51
Western Blot analysis ............................................................................. 52
Uncoupling of respiration ....................................................................... 53
Oxidative damage of the respiratory chain ? ............................................... 54
Partial root incubation with respirat