The gene for mitochondrial citrate synthase (CS) was isolated from Daucus carota (DcCS) and introduced into Arabidopsis thaliana (strain WS) using Agrobacterium tumefaciens-mediated transformation. Characteristics of citrate excretion were compared between T3 transgenic plants, which were derived from the initial transgenic plants by self-fertilization and homozygous for DcCS, and the control plants that had no DcCS. The highest CS activity 0.78 micromol protein min(-1) exhibited by the transgenic plants was about threefold greater than that found in the control plants (0.23-0.28 micromol protein min(-1)). Western analysis of the transgenic plants showed two CS signals corresponding to signals obtained from both D. carota and A. thaliana. Thus, it appears that the CS polypeptides by ectopic expression of DcCS were processed into the mature form and localized in the mitochondria of A. thaliana. The signal corresponding to the mature form of DcCS were greater in the transgenic plants having higher levels of CS activity. When the transgenic plants were grown in Al-phosphate media, a correlation between the levels of CS activity and the amounts of citrate excreted into the medium. The highest value (5.1 nmol per plant) was about 2.5-fold greater than that from control plants (1.9 nmol per plant). Both growth and P accumulation were greater in transgenic plants with high CS activity than that in control plants when they were grown on an acid soil where the availability of phosphate was low due to the formation of Al-phosphate. It appears that the overexpression of CS in A. thaliana improves the growth in phosphorous limited soil as a result of enhanced citrate excretion from the roots.

Organic acid excretion plays a key role in the superior P(i)-acquisition of barely soluble inorganic P sources from soils. Seedlings of white lupin (Lupinus albus L.) grown for 37 d in -P nutrient solution showed typical -P symptoms, such as low P content, increased root/shoot ratio and the development of cluster roots which released large amounts of citrate. Citrate concentration in the cluster roots was 21.5 micro mol (g FW)(-1), which corresponded to a 4.3- and 2.6-fold increase of +P and -P root apexes, respectively. Cluster roots possessed higher phosphoenolpyruvate carboxylase and phosphoenolpyruvate phosphatase activity than those in +P root apexes, which could result in increasing the supply of substrate for citrate synthase. On the other hand, the cytosolic pathway which converts citrate to 2-oxoglutarate consists of aconitase and NADP-specific isocitrate dehydrogenase activity that was lower in the cluster roots than in +P root apexes, and may contribute to citrate accumulation. Thus, metabolic balance with these alterations would play an important role in increasing citrate concentration in the cluster roots. The molecular characterization of NADP-specific isocitrate dehydrogenase indicated that the cytosolic isoenzyme functions as a hetero-dimer, and that the activity would be regulated by the transcript levels for both isoforms.

Wheat (Triticum aestivum cv. Kalyansona) seedlings were exposed to 0, 10, 50, and 100 μM Al in a 250 μM CaCl2 solution for 24 h at pH 4.5. Root elongation decreased gradually, while the Al content markedly increased with increasing supply of Al in the solution. The generation of H2O2 during Al stress was investigated biochemically and histochemically, as well as other events related to the decomposition of H2O2. After 24 h treatment, root elongation inhibition was detected at all the concentrations of Al. The level of lipid peroxidation at 10 μM Al after 24 h treatment was similar to that of the control, while a significant increase in lipid peroxidation was detected at 50 and 100 μM Al. H2O2 generation was higher at 50 and 100 μM Al than that at 10 μM Al. These results suggest that Al‐inducible lipid peroxidation may require excessive yield of H2O2. Time course experiment with 10 μM Al indicated that there is a strongly positive correlation between root elongation inhibition and cellular H2O2 yield. There were no significant changes in the activities of catalase (CAT) and ascorbate peroxidase (APX) in roots at 10 μM Al after 24 h treatment. However, a significant decrease in the activities of CAT and APX and a large increase in the activities of oxalate oxidase (OXO), guaiacol peroxidase (GPX), and coniferyl alcohol peroxidase (CA‐POX) as well as lignin deposition were observed at 100 μM Al. These results suggest the possible involvement of OXO in the production of a large amount of H2O2 under severe Al stress, whereas GPX and CA‐POX may be involved in the degradation of H2O2, leading to lignin deposition.

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Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 2461750 g/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.