RT Dissertation/Thesis
T1 Regulation of phosphate deficiency-induced carboxylate exudation in cluster roots of white lupin (Lupinus albus L.)
A1 Kania,Angelika
WP 2005/08/17
AB In many tropical and subtropical areas crop production is severely limited by a deficiency of plant-available phosphorus (P) in the soils. Therefore plant mechanisms to mobilize the sparingly soluble P fraction are of high interest. One such mechanism of P-deficient plants is the exudation of carboxylates and protons from roots. White lupin (Lupinus albus L.) was chosen as a model system to investigate plant metabolism under P deficiency which enable the plant to release ex-traordinarily high amounts of citrate and protons from its cluster roots (bottlebrush-like clusters of short rootlets of determinate growth which form along secondary lateral roots). The aim of this work was to determine the reasons for the high citrate acccumulation observed in mature cluster roots of P-deficient white lupin and to characterize the regulation of citrate release. 
A threshold citrate concentration is seen as a prerequisite for the transient pulse of intense citrate exudation associated with rhizosphere acidification which occurs over a time period of 2-3 days. 
Biochemical changes on the anabolic side of citrate metabolism such as increased activities of phosphoenolpyruvate carboxylase (PEP-C) or malate dehydrogenase (MDH) cannot solely ex-plain the very high citrate accumulation observed during cluster root development, although these reactions supply the cluster roots with citrate precursors. In addition, pyruvate concentra-tions decrease in developing cluster roots, probably in relation to the decreasing malic enzyme activities in the respective clusters. 
Citrate accumulation might also be caused by an impaired citrate turnover. Aconitase, the en-zyme catalyzing the turnover of citrate via cis-aconitate to isocitrate, showed decreasing activi-ties during cluster root development. NADP-isocitrate dehydroganase (NADP-ICDH) activities, as the next metabolic reaction which oxidizes isocitrate to 2-oxoglutarate, paralleled aconitase activities in all the different root segments investigated, although on a two- to threefold higher level. For this, aconitase rather than NADP-ICDH activities seem to limit citrate turnover. Spe-cific activities of aconitase and NADP-ICDH were the same in all the root segments investi-gated. 
Aconitase is rapidly inactivated by H2O2, which can be produced at increased rates under P limitation. However, neither H2O2 concentrations nor malondialdehyde concentrations as a marker for lipid peroxidation under oxidative stress were increased in clusters with low aconitase activities. Artifical inhibition of aconitase by incubating young cluster roots with high amounts of externally applied H2O2 did not change citrate and malate concentrations in these root seg-ments. However, a strong increase in citrate concentrations and a strong decrease in malate con-centrations in young cluster roots, together with high citrate exudation rates, could be observed when monofluoroacetate (MFA) as another aconitase inhibitor was applied. Inhibition of the aconitase enzyme therefore forced still young clusters to react like mature ones. This hints to aconitase as a key metabolic step in citrate turnover. High rates of carboxylate exudation were measured even from seedling root tips when incubated with MFA. 
Decreasing dehydrogenase activities as found during cluster root development by in situ staining with formazan were independent of the substrate supplied (citrate, aconitate, isocitrate, succinate, malate). This is in accordance with the decreasing enzyme activities measured in the different root segments such as aconitase, NADP-ICDH or malic enzyme in vitro. A reduced nitrate re-ductase (NR) activity under P deficiency, resulting in a lower drainoff of 2-oxoglutarate for N assimilation, seems not to play an important role for citrate accumulation, since an artificial NR inhibition with tungstate did not significantly increase citrate concentrations in young cluster roots. 
The change from malate to citrate accumulation during cluster root development is paralleled by a reduction in ATP-citrate lyase (ACL) activity, an enzyme cleaving citrate to oxaloacetate and acetyl-CoA. The good correlation between the citrate/malate ratio in root exudates and ACL ac-tivities indicates that ACL plays a key role as a metabolic switch between malate and citrate ac-cumulation during cluster root development under P deficiency. The enzyme might prevent high citrate concentrations under less severe P deficiency, when ACL activity is not limited by ATP availability. The attempt to inhibit the ACL enzyme by application of hydroxycitrate (HC) did not show any effect on citrate or malate concentrations in the young cluster roots. However, HC was probably not taken up into the root cells and could therefore not exert any inhibitory effects. 
Decreasing total respiration rates as found for developing cluster roots might affect citrate accu-mulation directly by reduced consumption of citrate in the TCA cycle or indirectly by H2O2-induced inhibition of aconitase activity. However, a reduced respiration rate did not result in higher H2O2 concentrations in white lupin. Cytochrome pathway capacity decreased parallel to total respiration, suggesting that the cytochrome pathway determines total respiration. An in-crease in alternative oxidase (AOX) capacity did take place in cluster roots, but was not high enough to compensate for the decreased cytochrome capacity. The AOX enzyme often occurs under P deficiency or under oxidative stress, probably to bypass a limiting Pi-and ADP-dependent cytochrome pathway. The amount of the AOX protein, determined by immunodetec-tion, paralleled AOX capacity. However, the availability of Pi and adenylates was not limiting  for total respiration, since uncoupling oxidative phosphorylation with CCCP did not increase the respiration rate. The citrate/malate ratio in young clusters with high rates of respiration and low inherent levels of citrate accumulation was only slightly increased by short-term application      (4 -8 h) of azide and SHAM as respiration inhibitors. 

The concomitant release of citrate and protons from mature cluster roots of P-deficient white lupin plants hints to a common regulation of citrate exudation and H+-ATPase activity in this specific root zone. Highly purified inside-out plasma membrane (PM) vesicles were isolated in a membrane-physiological approach to determine H+-ATPase characteristics involved in citrate exudation under P deficiency. 
Increased hydrolytic activity of the PM H+-ATPase derived from P-deficient plants parallels an increase in rhizosphere acidification and citrate exudation and hints to a causal relationship. Western blot analysis revealed a higher H+-ATPase protein amount under P deficiency. The op-timum pH of the H+-ATPase was shifted towards more acidic conditions under P-deficiency, which might be an adaptation to the supposedly decreased cytosolic pH brought about by the pH stat mechanism when carboxylates accumulate. Lower citrate concentrations (2 mM) stimulated PM vesicle acidification even in the absence of ATP, which was further enhanced by the addi-tion of Mg-ATP, and particularly expressed in PM vesicles isolated from roots of P-deficient plants. Accordingly, 14C-citrate was taken up at higher rates into vesicles derived from P-deficient white lupin compared with vesicles of P-sufficient control plants. Therefore citrate transport predominantly occurs in roots of P-deficient plants, and is linked with the activity of the PM H+-ATPase to maintain the electrochemical potential gradient which is reduced by citrate export out of the cell. Citrate exudation combined with an increase in H+-ATPase activity seems to prevent citrate accumulation up to concentrations which might exert inhibitory effects on the PM H+-ATPase. Such an inhibition was seen by diminished intravesicular proton accumulation, detected with the pH probe acridine orange, when 5 mM citrate were applied to the vesicle preparation. No such inhibitory effects were observed by malate application, which hints to a citrate-specific reaction. 
Lowering the cytosolic pH by external application of propionate stimulated citrate and malate exudation in non-cluster laterals and in young clusters. Therefore a causal relationship might exist between citrate accumulation and exudation by acidification of the cytosol. The threshold citrate concentration at which citrate exudation is triggered perhaps is reached when citrate ac-cumulation leads to acidification of the cytosol. Carboxylate exudation in young cluster roots and seedling root tips hints to a putative anion channel which already exists in young tissue and might be regulated in relation with H+-ATPase activity and cytosolic pH. 
Protoplasts, isolated from mature cluster roots, did only give very low yield and were not viable for seals high enough for patch-clamp studies. This might be due to the fast senescence in the developing clusters which also seems to change membrane integrity. High yields could only be gained from seedling root tips, or cotyledons. Similarly, protoplast isolation from root hairs also was only possible from seedling root tips or non-cluster lateral root tips, but even not from just emerging root hairs of young cluster roots. 

To determine the influence of a second growth factor in addition to P deficiency on citrate me-tabolism, white lupin was cultivated in nutrient solution and in rhizoboxes at ambient (400 μmol mol-1) and at elevated (800 μmol mol-1) atmospheric CO2 concentrations.
Plant development was accelerated at elevated CO2 concentrations, and P deficiency and senes-cence symptoms such as yellowing, wilting, and abscission of leaves could be seen much earlier.  When cultivated in nutrient solution, shoot growth was rather unaffected by the CO2 concentra-tion, whereas root growth was much faster at elevated CO2. Quite contrary, shoot growth was slightly higher at elevated CO2 concentrations in plants in rhizobox culture, but root growth was unchanged. However, the harvest of a higher amount of cluster roots from plants at elevated CO2 or the calcareous soil might have reduced root growth of the plants grown in rhizoboxes.
Higher root/shoot ratios under P deficiency were further increased at elevated CO2 concentra-tions. The amount of clusters was higher in plants grown in nutrient solution at 800 μmol mol-1 CO2 from day 21 to day 33 after sowing, but thereafter the differences disappeared. No signifi-cant differences between CO2 treatments were observed for the proportion of cluster roots rela-tive to the whole root system. Independent of the cultivation method, root exudation per cluster or per cluster root weight was unchanged by the elevated CO2 concentration. The distribution of citrate and malate exudation in different cluster root segments with decreasing malate exudation and a peak of citrate exudation in mature clusters was also confirmed at 800 μmol mol-1 CO2. The increased carbon distribution into the root at 800 μmol mol-1 CO2, seen in a higher root/shoot ratio, was not transformed into higher exudation rates from the single cluster. Acid and alkaline phosphatase activities in the rhizosphere of L. albus continually increased during cluster root development independent of the CO2 supply.
Phosphatase activities and carboxylate accumulation and exudation rates were essentially un-changed by different atmospheric CO2 concentrations. This might be due to also unaltered Pi concentrations in the respective root segments, because internal P concentrations seem to deter-mine these parameters. Since citrate accumulation and exudation probably depends on citrate degradation, which is not influenced by the amount of carbon supplied for anabolic processes, elevated CO2 concentrations do rather not change citrate concentration and exudation. Accord-ingly, no significant effects of different CO2 concentrations were seen on microbial diversity in the rhizosphere of white lupin.  
So far, no single cause or mechanism was found to be responsible for the high citrate concentra-tions measured in mature cluster roots, although citrate degradation seems to be important and aconitase probably plays a key role. A general impairment of metabolism due to decreasing con-centrations of Pi, adenylates, RNA, and proteins rather seems to bring about decreasing enzyme activities and reduced respiration. Various regulatory mechanisms via phosphorylation/ dephos-phorylation, phytohormones, nitric oxide, or others also have to be considered. 
K1 Citratstoffwechsel
K1 Organische Säuren
K1 Phosphormangel
K1 Exsudation <Botanik>
K1 Wurzel
PP Hohenheim
PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
UL http://opus.uni-hohenheim.de/volltexte/2005/104