| Summary: | Ascorbate peroxidase (EC: 1.11.1.11; APX) scavenges hydrogen peroxide. Hydrogen peroxide is a reactive oxygen intermediate (ROI) that is generated in numerous cellular reactions and is linked to signalling pathways associated with a diversity of environmental stimuli. If the production of hydrogen peroxide exceeds the capacity of scavenging systems to remove it, this ROI will accumulate and photooxidative stress may result. There are five known members of the APX gene family in Arabidopsis. This project focused on the role and regulation of the APX2 gene. In the Arabidopsis ecotype Columbia, expression of this gene is not detectable in the absence of stress. However, when leaves are exposed to high light or wounding, APX2 gene expression is rapidly, but transiently induced. Karpinski et al. (1999) proposed that APX2 gene expression is associated with acclimating Arabidopsis plants to photooxidative stress. The aim of this project was to isolate mutants with deregulated APX2 gene expression to provide an insight into how this gene is regulated and what effect altering its expression has on the response to photooxidative stress. A mutagenesiss creenw as developedt o isolateA rabidopsism utantst hat expressedth eA PX2 genew ithout high light or wound treatments.T o facilitate this screen,a transgenicl ine with a luciferaser eporter genew as mutagenisedT. hirty four thousandM 2 plantsw ere screenedfo r luciferasea ctivity undern on-stressc onditions.T wo allelic, recessivem utantsw ere identified that were alteredi n both luciferasea ctivity ande ndogenousA PX2 genee xpression;t hesew ere called regulators of ascorbatep eroxidase (rap]). The abundanceo f APX2 RNA in non-stressedm utant rosettes was not as high as that in stress treated leaves. Imaging of luciferase activity indicated that this expression varied between leaves. As in wildtype plants, APX2 gene expression was associated with the vascular system. rap] rosettesh ave alterede xpressiono f genest hat encodeo there nzymesa ssociatedw ith antioxidant defence, besides APX2. These genes encode isoforms that scavenge superoxide radicals (superoxided ismutase)a ndr educeo xidised forms of ascorbate( monodehydroascorbate and dehydroascorbatree ductases)T. he mutantsh ave approximatelyo ne third of the glutathione contento f wildtype rosettesT. his is consistentw ith the finding that APX2 genee xpressioni s impaired in Arabidopsis leaves treated with this thiol (Karpinski et al. 1997). Glutathione is an antioxidant and plants in which antioxidant defences are compromised might be expected to accumulater eactiveo xygen speciess ucha s hydrogenp eroxide.I ncreasedh ydrogen peroxide content inducesa low level of APX2 genee xpression( Karpinski et al. 1997),c omparablet o that detectedi n rap] rosettesH. owever, total foliar hydrogenp eroxidec oncentrationw as not detectably altered in the mutants. In response to high light and wounding, rapl rosettes accumulated higher concentrations of APX2 transcripts than wildtype plants. However, rapid fluorescence measurements indicated that there was no difference in the sensitivity of mutant rosettes to a high light stress. A numbero f Arabidopsise cotypesw ere identified that expressedth e APX2 geneu nder conditions that did not induce expression in Columbia plants.
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