Investigating the role of apparent free-space apoplasm in photoassimiliate translocation using H+-selective voltammetry
This thesis details the development of double-barreled proton (H +)-selective microelectrodes using an efficient new vapour-silaniztion protocol (patent pending), and their use in detecting [H+] in the intercellular apparent free-space (AFS) apoplasm in detached ' Pisum sativum' and 'Helianthus annuus' leaves. The electrophysiological technique was compared with established centrifugation/extraction protocols, and proved more accurate, and less invasive. Using microelectrodes, the [H+]AFS and AFS electrical potential (E AFS) were monitored 'in situ' while leaves were subjected to transitions between light and darkness, either in rapid intervals, or over a 16 h light:8 h dark cycle. To test the effects of photoperiod, host plants were exposed to either a 16 h light:8 h dark cycle (control plants) or a 24 h dark period (reduced-energy state plants) prior to leaf detachment. Generally, a rapid transition from dark to light caused a net increase in [H+ ]AFS and a concurrent hyperpolarization ('i.e'. increase) of the EAFS. A rapid light to dark transition caused a net decrease in [H+]AFS and a concurrent depolarization ('i.e'. decrease) of the EAFS in both control and reduced energy state leaves. The magnitude of the [Delta][H+] AFS was comparatively subdued in reduced energy state leaves, and both [Delta][H +]AFS and [Delta]EAFS were largely complete within the notably short interval of 180 s following stimulus. A hitherto unreported correlation between [H+]AFS and time of day was observed over the 16 h light:8 h dark cycle, where H+ AFS began to decrease in anticipation of the 8 h dark period. This circadian rhythm may explain certain discrepancies in both absolute [H+] AFS and [Delta][H+]AFS values reported throughout the literature. To correlate changes in [H+]AFS with net carbon exchange rate (NCER) and photoassimilate export rate (PER), ' Pisum sativum' leaves were exposed to either short-term atmospheric [CO2] (AC) (370.0 [mu]l l-1) or elevated [CO 2] (EC) (1000.0 [mu]l l-1). Short term exposure to EC caused an overall increase in NCER and PER in attached 'Pisum sativum' leaves, as well as a transient increase in [H+] AFS of ~40 fold in detached leaves. This novel transient increase was interpreted as the physicochemical result of high concentrations of dissolved inorganic carbon and the low capacity of apoplasmic H+ buffering. The transient nature of the response implies adaptive biological regulation of the AFS by adjacent cells. Concomitant with the transient increase in H +AFS was a transient increase in stomatal conductance and a notable, if minor, deflection in PER. When taken collectively, the results indicate that photosynthetic processes in apoplastic loaders affect the [H +]AFS in a feed-forward manner, which may in turn affect sucrose loading and export, assuming detached and attached leaves behave similarly.