Abstract
(Englisch)
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The aim of our part of the project was i) to describe in detail the vacuolar phosphate transporter and ii) to perform experiments in order to elucidate the mechanisms involved in organic acid excretion in white lupin.
i) Vacuolar phosphate transport
Vacuoles isolated from plants usually still contain a phosphate pool and showed negligible uptake of phosphate. Therefore it was not possible to analyze the vacuolar phosphate uptake system using vacuoles isolated from leaves. However, in collaboration with Prof. Mimura we succeeded to establish a cell culture system from Catharanthus roseus, which can easily be deprived in phosphate. We established a protocol for vacuole isolation and transport studies. Using isolated vacuoles we characterized the phosphate transport system. The vacuolar phosphate transporter has a low affinity for phosphate (Km 5 -8 mM). Phosphate uptake is strongly increased when the vacuole is energized by the addition of ATP. Since the ATP-dependent part of Pi uptake can be inhibited by bafilomycin and increased uptake can also be observed in the presence of PPi, the substrate for the vacuolar PPi-dependent proton pump, it must be concluded that the vacuolar proton pumps are implicated in the energization of Pi uptake. The strong inhibition of Pi uptake observed in the presence of SCN- but not NH4+, indicates that the driving force is the vacuolar inside positive membrane potential.
In order to study the uptake of phosphate in transgenic potato plants we have established a method to isolate vacuoles from young potato leaves. To localize the chimeric phosphate-a-TIP transporter we tested two different antibodies directed against the entire or a peptide from a-TIP. However, in both cases the reaction was not specific enough to identify the protein.
ii) Proteoid roots of white lupin
In order to elucidate how some specialized plants can survive in soils containing very low concentrations of phosphate or strongly bound phosphate we have analyzed the proteoid roots of white lupin. These root clusters are developed under phosphate stress and enable lupins to locally strongly acidify the rhizosphere. Furthermore, proteoid roots excrete also large amounts of organic acids, which can be exchanged in the soil with strongly bound Pi. To get more insights on the metabolism and development of proteoid roots, physiological, biochemical and molecular studies were performed.
As also shown for other plants grown under Pi stress, proteoid roots express a high affinity phosphate transporter if phosphate is limited. Based on kinetic data, a similar phosphate transporter is also induced in other parts of the root. However, based on fresh weight the vmax of proteoid roots is higher by a factor of about 1.5 than that measured in apical roots.
We localized in more details the excretion of organic acids. In the apex, as well as in juvenile proteoid roots, malate is excreted, while the amounts of excreted citrate is very low. On the base of mature proteoid roots excretion of malate is still comparable to that of the apical roots, however, citrate excretion is strongly increased when compared to juvenile proteoid roots. The situation is reversed in mature proteoid roots, where low amounts of malate but very high of citrate are excreted. Total citrate excretion can make up to 20% of the carbon fixed during photosynthesis.
Biochemical studies showed that PEP carboxylase is increased by a factor of about 1.5 in comparison to normal -P roots and about threefold when compared to roots grown in the presence of phosphate. Citrate synthase was not affected significantly, however, a slight but significant decrease of aconitase activity could be observed. Respiration increases slightly in juvenile proteoid roots compared to normal + and - P roots. However, in mature proteoid roots respiration is strongly inhibited.
Using the AFLP method we identified several upregulated genes coding for enzymes implicated in carbohydrate catabolism in mature proteoid roots. Such an upregulation can be explained by the high demand of energy for the synthesis of citrate. We have analyzed in detail the expression and activities of sucrose synthase, fructokinase and phosphoglucomutase. Fructokinase and phosphoglucomutase activities were increased in response to phosphorus starvation. In -P plants, the juvenile and mature proteoid roots had the highest activity. The activity of sucrose synthase, unlike fructokinase and phosphoglucomutase activities, was not enhanced by phosphorus starvation in normal roots, but strongly increased in juvenile and mature proteoid roots.
Low respiration and production of large amounts of organic acids indicate that the plant accumulate large amounts of reduction equivalents. A pathway to cope with too much NADH consists in the reduction of acetaldehyde originating from pyruvate decarboxylation and formation of ethanol. Indeed, alcohol dehydrogenase activity is strongly increased in citric acid excreting proteoid roots.
The results of the AFLP indicated that the activity of ATP-citrate lyase could be responsible for the switch between malate and citrate excretion. Indeed, we could show that in regions excreting malate a much higher activity of ATP-citrate lyase could be detected than in regions excreting no organic acids or citrate. Interestingly, in plants ATP-citrate lyase is a hetereodimer, while in animals the same enzyme is constituted by one sole polypeptide. Actually we are trying to elucidate in more detail the molecular nature and physiological function of ATP-dependent citrate lyase in plants.
One putative membrane bound protein kinase identified with the AFLP technique has a close homologue in Arabidopsis. Preliminary expression studies in Arabidopsis showed that this kinase is expressed in the whole plant in young seedlings, but root specific in adult plants. In these plants expression is strongly increased under phosphate limitation. Work to elucidate the role of this protein kinase in on the way.
Using the AFLP method more than hundred clones for enzymes and transcription factors putatively implicated in the regulation of proteoid development and function have been identified. In order to get more specific sequences and to take advantage of our increased knowledge of the different developmental stages of proteoid roots, we recently performed a substractive approach. The number of clones putatively involved in proteoid root formation was strongly decreased. Among the most interestingly a teosinte-like gene was identified. This gene and its product will be analyzed in more detail in future.
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