Ascorbate Oxidase Gene Research Articles

Expression analysis of a set of genes related to the ripening of bananas and mangoes

During ripening many important physic-chemical changes contribute to fruit quality, and they are precisely determined by gene expression. Specific genes are essential to normal ripening; however, information on gene expression about the majority of tropical fruit, such as bananas and mangoes is limited. In this way, the present study was undertaken with the objective to provide preliminary access to the changes in expression of some genes potentially relevant to banana and mango ripening. To simultaneously evaluate the changes in gene expression, a small collection of genes related to ethylene biosynthesis, starch mobilization, cell wall disassembly, pigment synthesis and ascorbate metabolism was assembled in nylon membranes and probed with cDNA from unripe and ripe fruit. Some interesting differences were observed between gene expression in bananas and mangoes. In relation to starch metabolism, banana a-amylase was induced during ripening while phosphorylase was more induced in mangoes. Similarly, expression of cell wall-related genes for polygalacturonase and expansin were also different in those fruits. Fructanfructosyltransferase, chalcone synthase, and ascorbate oxidase genes were also induced in ripening mangoes, but not in bananas. Although the number of sequences involved was relatively small, this simple and feasible approach provided interesting preliminary data that can be starting points for more in depth studies.

Cloning and mapping of genes involved in tomato ascorbic acid biosynthesis and metabolism

Ascorbic acid is a key component of plant antioxidant system and it has a multitude of functions in organisms. The cDNA clones encoding homologues of dehydroascorbate reductase (DHAR) and ascorbate oxidase (AO) genes, which are involved in ascorbic acid metabolism, were isolated from tomato plants. Deduced amino acid sequences exhibited 50–88% identities with the corresponding sequences from other plant species. In order to screen for candidate genes linked to tomato ascorbic acid biosynthesis and metabolism, the ascorbic acid-associated genes were mapped. These genes were located on the tomato map using a population of introgression lines (ILs) having one segment of Lycopersicon pennellii (LA716) in a L. esculentum (M82) background. The 75 ILs allowed the genome to be segmented into 107 bins. Fourteen genes involved in ascorbic acid biosynthesis and metabolism were mapped to 15 loci and most of the probes revealed a single copy signal. Furthermore, the digital expression profiles of the genes associated with ascorbic acid biosynthesis and metabolism were described.

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.

Inhibition of Phosphoinositide‐Specific Phospholipase C Results in the Induction of Pathogenesis‐Related Genes in Soybean

The inositol 1,4,5-trisphosphate (IP3) content is decreased in soybean cells following infection with Pseudomonas syringae pv. glycinea (Psg). In this investigation, a differential display approach was applied to isolate soybean genes that are transcriptionally up-regulated by the inhibition of phosphoinositide-specific phospholipase C (PI-PLC) activity and to study if the transcription of those genes is altered following Psg infection. Four genes, transcriptionally activated following treatment with the PI-PLC-specific inhibitor U-73122, were cloned. Three of the four genes were induced following infection with Psg. The transcripts of a hydrolase homologue (GmHy) were induced in the incompatible but not compatible soybean-Psg interaction. The transcripts of a putative ascorbate oxidase gene (GmAO) were induced in both compatible and incompatible interactions. GmHy and GmAO may represent new classes of pathogenesis-related genes. In addition to these two novel genes, homologues of PR-10 and polygalacturonase inhibitor protein (GmPR10 and GmPGIP, respectively) were identified. These two genes have previously been reported as pathogenesis-related. Transcripts of GmPR-10, but not GmPGIP, were induced in both compatible and incompatible soybean-Psg interactions. Induction of these genes, except for GmPGIP, following inhibition of PI-PLC by either the U-73122 treatment or bacterial infection suggests that PI-PLC may negatively regulate the expression of defence genes.

The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain.

The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.

The function of ascorbate oxidase in tobacco.

The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-gamma-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA content.

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non‐synchronous, synchronous, and elongation cultures of tobacco BY‐2 cells. In non‐synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61 200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala 465Leu and Phe 463Trp/Ala 465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

Characterization and expression of a new class of zinc finger protein that binds to silencer region of ascorbate oxidase gene.

A unique A/T-rich sequence (5'-AAAAAGTAAAAA-GTAAAAAAGTAAAAAG-3), referred to as the AGTA repeat, is found in the silencer region of the pumpkin ascorbate oxidase gene. A cDNA for protein (AOBP) that binds to the AGTA repeat was isolated from pumpkin by the southwestern method. The AOBP protein has a new class of zinc/DNA-binding domain named Dof/MOA domain that is highly conserved in many plant proteins and is significantly related to those of steroid hormone receptors and GATA1. Gel retardation analysis indicated that AOBP bound to the AGTA repeat through the Dof/MOA domain. Metal chelators, 1,10-phenanthroline and EDTA, specifically inhibited the DNA binding of AOBP, indicating that metal coordination plays an important role in DNA binding of AOBP. Thus, the Dof/MOA domain acts as a zinc/DNA-binding domain in AOBP. Gel retardation analysis with mutated oligonucleotides suggested that the Dof/MOA domain recognized the AGTA core sequence. AOBP mRNA was expressed in mature tissues of pumpkin, but was expressed only in small amounts or was not expressed in growing tissues. Furthermore, the expression was auxin-independent. The expression pattern of AOBP and that of ascorbate oxidase did not show a positive correlation.

Cloning of the pumpkin ascorbate oxidase gene and analysis of a cis-acting region involved in induction by auxin.

A genomic clone encoding ascorbate oxidase was isolated from pumpkin (Cucurbita sp.). This gene is consisted of four exons and three introns. Analyses of the promoter fusion to beta-glucuronidase reporter gene by transient expression assay in pumpkin fruit tissues suggested the existence of a cis-acting region responsible for auxin regulation.

Melon ascorbate oxidase: cloning of a multigene family, induction during fruit development and repression by wounding.

A small family of at least four genes encoding melon ascorbate oxidase (AO) has been identified and three members of it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymes homologous to ascorbate oxidases from other plants and similar to other multicopper oxidases. We describe detailed molecular studies addressing melon AO expression during organ specific differentiation, fruit development and ripening, and in response to wounding. In particular, AO transcript accumulation was induced in ovaries and the outer mesocarp of mature preclimacteric melon fruits, before the expression of genes encoding the necessary enzymatic activities for ethylene biosynthesis. On the other hand, AO was not expressed in late stages of fruit ripening and was repressed in wounded fruits. The role of ethylene in transcriptional regulation of AO is discussed.

Sequence requirement of 5'-upstream region of the ascorbate oxidase gene for organ-specific expression in cucumber.

Sequence requirement of 5'-upstream region of the ascorbate oxidase gene for organ-specific expression in cucumber.

Regulation of Ascorbate Oxidase Gene Expression in Higher Plants

Regulation of Ascorbate Oxidase Gene Expression in Higher Plants