<p>Soil salinity is a major threat to future food stability. Almost 20% of irrigated land is currently too saline to grow traditional crops. Moreover, rising sea levels, scarcity of fresh water, and more intense and prolonged periods of drought are exacerbating the problem. Saline soils severely reduce yields of most crop plants. By contrast, halophytes, which naturally thrive on saline substrates, have a variety of mechanisms to tolerate both the osmotic and cytotoxic components of salt stress. There has been concerted scientific effort worldwide to understand these mechanisms, and to introduce genes that may increase salinity tolerance in crop plants. Many halophytes in the Caryophyllales are pigmented red owing to a tyrosine-derived alkaloid called betacyanin. Recent studies using Disphyma australe, a succulent halophyte common on coastal dunes and rocky outcrops throughout New Zealand, have indicated a role for betacyanins in salinity tolerance. This thesis focuses on how the mechanism through which betacyanins might affect salt tolerance mechanisms in D. australe and whether the putative benefits of betacyanins on salt tolerance might be transferred to naturally non-betacyanic plants. Effects of betacyanin on Na+ distribution in salt-stressed leaves of red and green morphs of D. australe were studied using fluorescence microscopy, cryo-scanning electron microscopy with energy dispersive X-ray analysis, and atomic absorption spectrometry (AAS). In betacyanic leaves Na+ accumulated in the epidermis, while in green leaves Na+ was distributed more evenly across the epidermis and mesophyll. Both leaf types had similar numbers of salt glands, but salt secretion rates were higher in red than in green leaves. Betacyanic leaves under salt stress were able to maintain relatively high K+/ Na+ ratios, essential for many metabolic processes, while the leaves of green plants were not. Leaf sections stained with fluorescein diacetate and propidium iodide showed that mesophyll viability decreased significantly in green leaves under salt stress, while there was almost no decrease in mesophyll viability in the presence of betacyanins. Thus, betacyanic leaves might protect the photosynthetically active mesophyll from cytotoxic effects of Na+ by accumulating Na+ in the epidermis instead of the mesophyll. This in turn leads to more efficient salt secretion and higher K+/ Na+ ratios in the mesophyll, resulting in increased mesophyll viability under salt stress. Effects of high apoplastic sodium concentrations on ion flux kinetics in mesophyll tissue was studied using the non-invasive microelectrode ion flux estimation technique. Mesophyll cells of both betacyanic and green leaves showed a highly unusual K+ flux response; most crop plants leak K+ out of cells upon salt stress, but D. australe and the native Australian Disphyma crassifolium both showed K+ influx upon salt stress. Actively taking up K+ from the apoplast to maintain a high cytosolic K+/ Na+ ratio during salt stress might be an entirely new mechanism to combat the cytotoxic stress component of salinity stress in these halophytes. The salt induced K+ uptake was dependent on the presence of Cl- and Cl- was also taken up into mesophyll cells upon salt stress. Taking up both cations and anions at the same time could avoid membrane depolarisation. Voltage-gated channels, which are involved in the salt induced K+ efflux in glycophytes, would not be activated and this could be a new mechanism to avoid a K+ leak during salt stress. To test whether the beneficial effect of betacyanin production on salt tolerance could be transferred to naturally non-betacyanic plants, transgenic betacyanin-over-expression (BtOE) mutants of Nicotiana tabacum were generated by our colleagues at Plant & Food Research Ltd. Betacyanins in leaf discs of N. tabacum were associated with decreased chlorophyll degradation upon high light and high salt stress. Additionally, the decline in maximum quantum efficiency of PSII after high light and salt treatment was significantly greater in green than in betacyanic leaves. Placing a polycarbonate filter with a similar absorption spectrum to betacyanin over green N. tabacum leaf discs had a similar effect to the presence of betacyanin. Thus, betacyanins probably have a photoprotective effect in N. tabacum, which is essential as both high light and salinity can impair photosynthesis. To assess if the salt tolerance enhancing effect of betacyanin production observed in the leaf discs also occurs in whole N. tabacum plants, the ability to recover from exposure to saturating light was assessed. Betacyanic plants were able to fully recover quicker after exposure to saturation light than green leaves. This research shows that the presence of betacyanins during salt stress correlates with an altered Na+ distribution in leaf tissues and a higher salt secretion rate, which contributed to higher mesophyll viability. Moreover, a completely new ion flux response to salt stress was observed in D. australe and D. crassifolium. The observed salt induced K+ uptake into the mesophyll cells during salt stress might be an entirely new mechanism, to maintain a high K+/ Na+ ratio in the cytosol and avoid the cytotoxic effects of Na+ in photosynthetically active tissue. The beneficial effects of betacyanins could also be transferred to non-betacyanic species, by introducing betacyanin production. These results strongly suggest that betacyanins play a role in salt tolerance in halophytes and might be a valuable resource in increasing the salt tolerance of naturally non-betacyanic crop plants.</p>
Ion Transport through C-butyl-pyrogallol[4]arene-loaded Poly(Vinyl Chloride) Membranes