No abstract

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-␥-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.Ascorbate (AA) plays a key role in defense against oxidative stress and is particularly abundant in photosynthetic tissues (Foyer et al., 1983; Smirnoff, 2000). Most (over 90%) of the AA is localized in the cytoplasm, but unlike other soluble antioxidants, a substantial proportion is exported to the apoplast, where it is present at millimolar concentrations. Apoplastic AA is believed to represent the first line of defense against potentially damaging external oxidants such as ozone, SO 2 , and NO 2 (Plö chl et al., 2000; Barnes et al., 2002). In the apoplast, AA is oxidized to monodehydroascorbate (MDHA) by the enzyme ascorbate oxidase (AO). MDHA is an unstable radical and rapidly disproportionates to yield DHA and AA. DHA is then transported into the cytosol through the plasma membrane by a specific carrier that preferentially translocates the oxidized form in exchange for the reduced form, ensuring a continuous flux of reducing power to the cell wall (Horemans et al., 2000). Perhaps the most intriguing and poorly understood of the enzymes involved in AA metabolism in plants is the apoplastic AO. No clear biological functions for AO have been described to date. However, it is widely believed that AO plays a role in cell elongation because of its extracellular localization and its high activity in rapidly expanding tissues (Esaka et al., 1992; Moser and Kanellis, 1994; Ohkawa et al., 1994; Kato and Esaka, 1999). Recent work has shown that tobacco (Nicotiana taba...

The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca 21 channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.