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It is now generally accepted that phenolic compounds such as flavonoids and tannins are produced by plants in response to and as part of a strategy to minimise cellular damage from oxidative stress (Agati and Tattini, 2010, Pollastri and Tattini, 2011). Of critical importance to plants is the need to protect their photosynthetic machinery as this is both the site where light is converted to chemical energy (powering growth and metabolic activities) and a major site of ROS production in plant.

The importance of photosynthetically active tissues and their involvement in ROS production is illustrated when one compares the difference in flavonoid and total antioxidant (gallic acid equivalents) production between the spongy parenchyma and the photosynthetically active palisade of the various salt-treated plants. The preferential concentration of flavonoids in the photosynthetic tissue as seen during this experiment has also been documented by several other researchers (Agati et al., 2007, Melgar et al., 2009). Agati et al (2007) were able to show flavonoid co-location with chloroplasts and proposed that their role was to directly scavenge singlet oxygen due to its short diffusion time and high reactivity. Other researchers have highlighted their ability to scavenge hydrogen peroxide, superoxide and other reactive species (Rafat Husain et al., 1987, Tattini et al., 2004).

Our hypothesis that non-optimal environmental conditions cause elevated ROS levels, which the plant needs to counteract through increased antioxidant (flavonoid, tannin) production, is supported by both our observations in the field and under controlled conditions. Higher producing plants were more exposed to sunlight (e.g. lower shelter index) than lower producing plants, under natural conditions. Given the geographical location of field sites, namely coastal foredunes, and the high levels of UV radiation in Australia, this higher exposure may lead to increased ROS production. Two mechanisms may contribute to this process. First, increased irradiance and photon interception may exceed the photochemical competence of PSII, raise superoxide production and lead to photoinhibition (Foyer and Noctor, 2000). Evidence of this is the decreased Fm (the number of open reaction centres of PSII) at higher producing cluster 2 sites. Secondly, UV-B component per se can destroy chlorophyll as well as carotenoids, in addition to degrading photosystem II and rubisco function (Zhang and Zhao, 2008). Other factors shown to be significantly different between

the high and low producing clusters (soil electrical conductivity, nutrient levels and stomatal density) can also be explained in the context of higher ROS and hence higher antioxidant (flavonoid, tannin) production.

Tannin and flavonoid production was positively correlated with higher stomatal density and daily evaporation (Appendix Chapter 2, Appendix Table 2.4), suggesting that ROS production (and, hence a need for antioxidant protection) was causally related to water stress. Plants respond to a lack of moisture by closing their stomata, which impairs the ability of photosynthetic machinery to fully utilise light that was absorbed by photosynthetic pigments and leads to increased ROS (superoxide radical) production in chloroplasts (Flexas and Medrano, 2002b, Flexas and Medrano, 2002a, Miller et al., 2010a). In addition, the lack of moisture leads to an increased rate of photorespiration. All these processes exacerbate the effects of increased exposure to irradiance (including UV) and photosystem impairment. The water stress experienced by the higher-producing cluster is a function of both being exposed to higher evaporative conditions (a lower shelter index) and having increased moisture loss via uncontrolled stomatal conductance i.e. a higher stomatal count providing more points to lose water from. The role individual plant physiology plays in ROS and hence subsequent antioxidant (flavonoid, tannin) production is further highlighted by the frequency analysis which showed 100% of tannin and flavonoid regression models included this factor.

In addition to the influence of climatic variables on ROS production, soil nutrient availability also plays an important role. This is evidenced by both the frequency with which soil K+ appears in the regression models and the differences in soil K+ and Na+ levels between the high (cluster 2) and low (cluster 1) producing clusters. Potassium is an essential nutrient for cellular metabolism since it is involved in the activation of over 50 enzymes (Marschner, 2012) and as suchK+deficiencies are likely to perturb metabolism, resulting in the increased production of ROS (Cakmak, 2005). The increased ROS concentration has been shown to induce K+ loss via outward rectifying potassium channels (KOR) (Cuin and Shabala, 2007) and if not addressed results in programmed cell death (Shabala, 2009). Therefore it is not surprising to see the plant produce antioxidants in an attempt to scavenge excessive ROS produced under K+ deficient conditions.

metabolically active palisade mesophyll tissue with the heavily vacuolated (storage organ) spongy parenchyma under controlled conditions. In the palisade cells, a clear reduction in potassium concentration occurs in response to increased salinity. High NaCl levels in the leaf apoplast have been reported to cause a massive K+ leak from photosynthetically active mesophyll tissues (Shabala et al., 2007, Shabala et al., 2005, Shabala, 2000). This process is mediated by depolarization-activated outward rectifying K+ channels (Shabala et al., 2006) and results in severe depletion of the cytosolic K+ pool. In addition, K+ leak from the cytosol may occur via ROS-activated K+ permeable channels, both selective (Cuin and Shabala, 2007) and non-selective (Demidchik and Maathuis, 2007, Demidchik et al., 2003). Thus, an increased ability to scavenge the excess ROS produced under saline conditions may be essential to control cytosolic K+ homeostasis and prevent PCD resulting from activation of caspase-like endonucleases and proteases (Reape and McCabe, 2010, Vartapetian et al., 2011) under low K+ conditions (Demidchik et al., 2010).

Of special interest was the negative correlation between amount of soil Na+ and flavonoid and tannin production in plants under field conditions. Normally, one would expect higher soil salinity to increase ROS production through perturbed metabolism and membrane depolarisation. However, CR is an obligate halophyte and as such has a physiological need for a minimum soil sodium concentration. Many halophytes use inorganic ions (such as Na+ and Cl-) stored in vacuoles to generate lower osmotic potential to draw water out of the soil and into the plant for growth processes (Flowers and Colmer, 2008). Because of this, the absence of Na+ in the growth media can be considered as a stress for CR species. This explains the inverse relationship between soil Na+ content and antioxidant activity under field conditions (Figure 2.4c) where soil salinity levels were not high enough (e.g. <50 mM NaCl) to provide optimal plant growth at any site and the lower Na+ concentration at cluster two sites simply increased stress levels. Whether the increased stress is due to reduced carbon fixations, a fall in turgor, changes in cell wall elasticity or futile cycling of ions remains unclear (Flowers and Colmer, 2008). However, the corresponding increase in antioxidant activity at suboptimal salinities indicate that it is not merely a physical (turgor) effect, but mediated by ROS signalling interplay and metabolic perturbation is clearly occurring.

including membrane depolarisation, potassium efflux, and interference with cellular enzymatic processes which can potentially lead to programmed cell death (Shabala, 2009, Shabala and Cuin, 2007). So, although insufficient and excess salt are physiologically different problems, both result in the plant experiencing stress, producing ROS and upregulating antioxidant (including flavonoid) production.

Highlighting the complex role ROS play in the plant signalling pathway is the fact that it was not a single factor alone that best predicted flavonoid/tannin production, but rather a complex interplay between several stress-causing variables. This interplay has been documented on many occasions where it has been shown that exposure to one stress agent affords protection against a second e.g.salinity to UV-B stress (Cakirlar et al., 2008, Melgar et al., 2009) and is referred as cross-tolerance (Bowler and Fluhr, 2000) due to the commonality of downstream targets in the signalling process. As such, when there are multiple stress agents present, a complex nonlinear effect is to be expected.

In addition to their ROS scavenging activity, flavonoids inhibit the transportation of auxins through tissue. Whether the flavonoid inhibition of auxin transport (Brown et al., 2001) or ROS induced ion fluxes (Demidchik and Maathuis, 2007) were responsible for the outcome was not determined, however the role of flavonoids as signalling molecules is widely known (recently reviewed in Pollastri and Tattini (2011)) and the effect on biomass production is most likely the result of a complex interplay between all of these factors.