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...

Previous studies with 95 bread wheat doubled haploid lines (DHLs) from the cross Chinese Spring (CS)xSQ1 trialled over 24 yearxtreatmentxlocations identified major yield quantitative trait loci (QTLs) in homoeologous locations on 7AL and 7BL, expressed mainly under stressed and non-stressed conditions, respectively. SQ1 and CS contributed alleles increasing yield on 7AL and 7BL, respectively. The yield component most strongly associated with these QTLs was grains per ear. Additional results which focus on the 7AL yield QTL are presented here. Trials monitoring agronomic, morphological, physiological, and anatomical traits revealed that the 7AL yield QTL was not associated with differences in flowering time or plant height, but with significant differences in biomass at maturity and anthesis, biomass per tiller, and biomass during tillering. In some trials, flag leaf chlorophyll content and leaf width at tillering were also associated with the QTL. Thus, it is likely that the yield gene(s) on 7AL affects plant productivity. Near-isogenic lines (NILs) for the 7AL yield QTL with CS or SQ1 alleles in an SQ1 background showed the SQ1 allele to be associated with >20% higher yield per ear, significantly higher flag leaf chlorophyll content, and wider flag leaves. Epidermal cell width and distance between leaf vascular bundles did not differ significantly between NILs, so the yield-associated gene may influence the number of cell files across the leaf through effects on cell division. Interestingly, comparative mapping with rice identified AINTEGUMENTA and G-protein subunit genes affecting lateral cell division at locations homologous to the wheat 7AL yield QTL.

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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.

Chlorine in the Earth is highly depleted relative to carbonaceous chondrites and solar abundances. Knowledge of the Cl concentrations and distribution on Earth is essential for understanding the origin of these depletions. Large differences in the stable chlorine isotope ratios of meteoritic, mantle and crustal materials have been used as evidence for distinct reservoirs in the solar nebula and to calculate the relative proportions of Cl in the mantle and crust. Here we report that large isotopic differences do not exist, and that carbonaceous chondrites, mantle and crust all have the same 37Cl/35Cl ratios. We have further analysed crustal sediments from the early Archaean era to the Recent epoch and find no systematic isotopic variations with age, demonstrating that the mantle and crust have always had the same delta37Cl value. The similarity of mantle, crust and carbonaceous chondrites establishes that there were no nebular reservoirs with distinct isotopic compositions, no isotopic fractionation during differentiation of the Earth and no late (post-core formation) Cl-bearing volatile additions to the crustal veneer with a unique isotopic composition.

Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O 3 ). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Overexpression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO overexpression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO overexpressing plants exposed to 100 nmol mol -1 ozone for 7 h day -1 exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO 2 assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO 2 assimilation rate when exposed to a brief ozone episode (300 nmol mol -1 for 8 h). Stomatal conductance, hence O 3 uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.Abbreviations AO: ascorbate oxidase AE A sat : CO 2 assimilation rate under light-saturated conditions AE AA: L-ascorbic acid AE AWF: apoplast washing fluid AE c a : atmospheric CO 2 concentration AE c i : intercellular CO 2 concentration AE CFA: charcoal/Purafil-filtered air AE DAT: days after transfer to the respective treatments AE DHA: dehydroascorbate AE GSH: reduced glutathione AE GSSG: oxidised glutathione AE g H2O : stomatal conductance to water vapour AE PPFD: photosynthetic photon flux density AE ROS: reactive oxygen species AE V cmax : maximum in vivo rate of Rubisco carboxylation Planta (2003) 216: 918-928

Although there is a great deal of concern about the effects of human activities on biodiversity, until recently there has been very little interest in the effects of ozone on the species that constitute the major part of the flora, the diverse herbaceous and shrubby species of natural and semi-natural communities. However, many wild species have been shown to be at least as sensitive to ozone as crops that show significant yield losses, so there is a pressing need for an evaluation of the risk to wild species posed by ozone. This review attempts to assess progress and highlight problems. It begins with a comment on semantics, discusses the difficulties involved in measuring relative ozone resistance and then proceeds to consider the effects of ozone on growth and resource allocation. The evidence for evolution of resistance is appraised and then the potential effects of several interactions (cutting\grazing, competition, soil water deficit and nutrition) are considered. The review ends with some remarks on observation of oxidant-induced changes in ecosystems.

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