|dc.description.abstract||Reactive oxygen species (ROS) are produced continuously in plants and act as important
signalling molecules in many cellular processes including stress and defence responses. ROS can
arise from external sources as well as being generated by the plant. Pollutants, such as ozone,
enter the leaf via stomata and dissolve in the apoplast. ROS can arise both intracellularly and
apoplastically: superoxide is produced during photosynthesis as well as by the plasma membrane
bound NADPH oxidase during the oxidative burst. Under abiotic stresses such as drought or
high light-intensity, superoxide production from photosynthetic electron flow is increased.
Ascorbate plays a crucial role in symplastic and apoplastic ROS metabolism. Intracellular
ascorbate metabolism is highly regulated; it is coupled to glutathione oxidation and reduction
and is under tight enzymic control. Export of ascorbate into the apoplast increases during ozoneinduced
stress. The apoplast redox state is considered to be more variable than the symplasm.
Ascorbate is thought to be taken up from the apoplast in its oxidised form, DHA, via specific
carriers, implying tight regulation of apoplastic/symplastic ascorbate transport.
An apoplastic ascorbate breakdown pathway has recently been described by Green and
Fry (2005). Ascorbate is oxidised and hydrolysed to yield oxalate via two novel intermediates,
cyclic oxalyl L-threonate (cyc.ox.thr.) and 4-O-oxalyl-L-threonate (ox.thr.) A novel esterase is
thought to catalyse the hydrolysis of ox.thr. to oxalate. Dehydro-L-ascorbate DHA was also
hydrolysed to L-2,3-diketogulonate (DKG) which broke down to two unidentified compounds, C
and E. It was not known whether this pathway operated intracellularly and how increased ROS
production might affect flux through this pathway. The pathway, described, in the culture
medium of 5-day-old rose cell suspension cultures but had not been investigated in planta.
Intracellular and extracellular metabolism of [14C]ascorbate in [14C]ascorbate-loaded
cells was investigated in response to oxidative stress induced by 0.1 and 1 mM H2O2 and 1 and
10 μM methyl viologen (MV2+). The symplasm became more oxidised in response to 0.1 mM
H2O2; DHA levels increased and ascorbate decreased, but ox.thr. and oxalate, products of
irreversible ascorbate breakdown, did not accumulate. Symplastic ox.thr. and oxalate
accumulated in response to MV2+ and 1 mM H2O2. Ox.thr. and oxalate were observed in-planta.
Flux through the pathway was increased in transgenic tobacco plants which overexpressed the
cell wall-located enzyme ascorbate oxidase, suggesting that the redox state of the apoplast could
increase apoplastic ascorbate breakdown via ox.thr. The rate of production of oxalate in vivo
compared to in vitro studies suggested that the esterase was located to the symplasm as well as
the apoplast. Oxalate did not appear to be metabolised further. Compounds C and E were neither
observed in planta nor in 10-day old rose cell cultures. DKG and cyc.ox.thr. were present only in
Export of 14C in [14C]ascorbate loaded cells increased in response to 1 and 5 mM H2O2.
Increased export was characterised by a rapid response during the first 2 min of H2O2 exposure.
In Arabidopsis and rose cell suspension cultures, export was often observed to occur in series of
pulses. The amplitude of pulses increased within the first 2 min of H2O2 exposure. This was not
thought to be a result of membrane disruption. 14C appeared to be exported as [14C]ascorbate and
taken up as [14C]DHA, with minimal oxidation in the culture medium.
These results provide more insight into intracellular ascorbate breakdown via ox.thr. and
suggest that oxalate could accumulate in response to oxidative stress in plants. The export of
ascorbate/DHA in pulses in response to H2O2 hints at novel mechanisms of regulation of
ascorbate/DHA transport across the plasma membrane.||en