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Previously, we have characterized two tonoplast intrinsic proteins ( TIPs) and four plasma membrane intrinsic proteins ( PIPs) from the 2-day-old petals of tulip ( Tulipa gesneriana). In this study, we analyzed the development of tulip petals and stems, temperature-dependent petal movement, the amount of 3H2O transported into petals and stems during petal movement, and the transcript levels of two TIP ( TgTIP1;1 and TgTIP1;2) and four TgPIP genes in petals and stems, from the first day of petal opening to day 12. The development of the petals and stems was completed by days 6 and 9, respectively, after the first day of petal opening. Temperature-dependent petal movement and the amount of 3H2O that was transported into petals could be detected at significant levels up to day 6 with petal movement reaching a peak at day 3. Real-time reverse transcription-polymerase chain reaction analysis revealed that TgTIP1;1 and TgTIP1;2 were expressed ubiquitously in petals, stems, leaves, bulbs and roots. However, the expression level of TgTIP1;2 was very low in bulbs. The expression of both TgTIP1 genes was upregulated in close association with the development of petals but not with that of the stem. The four TgPIP genes were expressed at almost the same level during the development of the petals and the stem. However, the levels of the TgTIP1 and TgPIP transcripts in petals decreased during the course of petal wilting from day 9 onwards. These results suggest that TgTIP1;1 and TgTIP1;2 may contribute to petal development. [ABSTRACT FROM AUTHOR]

l-Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP-d-mannose vial-galactose (d-Man/l-Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo-inositol oxygenase,d-galacturonate reductase andl-gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing variousd-Man/l-Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate,l-threonate andl-tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified. [ABSTRACT FROM AUTHOR]

In higher plants, ascorbate peroxidase (APX; EC 1.11.1.11), the major H2 O2 -scavenging enzyme, occurs in several distinct isoenzymes that are localized in cytosol and various cell organelles. Here, we have purified and characterized an APX from the soluble fraction of plastids of non-photosynthetic tobacco BY-2 cells. The plastidic APX was a monomer with a molecular weight of 34 000. The enzymatic properties of the plastidic APX, including the rapid inactivation by H2 O2 in ascorbate-depleted medium, were highly comparable with those of the chloroplastic stromal APX of spinach and tea leaves. However, the other chloroplastic APX isoenzyme, the thylakoid-membrane bound APX, was not detected in the plastids of the BY-2 cells. The N-terminal amino acid sequence of the plastidic APX was completely identical with the deduced amino acid sequence of a previously identified cDNA sequence of tobacco chloroplastic APX. When a green fluorescence protein gene tagged with the chloroplast-targeting signal sequence of APX was expressed in the BY-2 cells, the fluorescence protein exclusively localized into plastids, and not into mitochondria. We conclude that plastidic APX in non-photosynthetic tissues is the same as the chloroplastic APX that occurs in leaves. [ABSTRACT FROM AUTHOR]

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H2O2) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H2O2 by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP, were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H2O2. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbatedepleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea. an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamydomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena. The enzyme had a molecular mass of 34 kDa. The Km values of the enzyme for ascorbate and H2O2 were 5.2±0.3 and 25 ±3.4 μM, respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg-1 chlorophyll h-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite. and it diffused from the organelles into the medium. [ABSTRACT FROM AUTHOR]