Identification of Arabidopsis mutants with altered Ascorbate Peroxidase II gene expression.
Ascorbate peroxidase (EC: 188.8.131.52; 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
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.