Transport and metabolism of symplastic and apoplastic ascorbate during oxidative stress

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 low levels. 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.