Tree ring and stem cellulose oxygen isotope ratios can potentially indicate past climate change, such as shifts in relative humidity (RH) and temperature, as well as past changes in ecohydrological processes, such as shifts in the source of water used by plants. However, our understanding of how physiological and biochemical processes modulate the plant stem cellulose oxygen isotope ratio is still in its infancy. The greater our understanding of these processes, the greater will be our ability to interpret climatic and ecohydrological changes from the oxygen isotope ratio of stem cellulose. Currently, it is known that the bulk of the leaf and stem water oxygen isotopic signal is incorporated into stem cellulose by the exchange between oxygen from cellulose precursors, such as hexoses and trioses (Sternberg, 2009) and leaf and stem water. Tree ring cellulose records both the signature of the leaf water oxygen isotope ratio (δ18OLW), which is sensitive to RH among other factors, and the oxygen isotope signal of stem water (δ18OSW; (Eqn 1)), which is related to the isotopic composition of precipitation. The incorporation of the stem water oxygen isotope ratio in the stem cellulose molecule initiates as sucrose, carrying the isotopic signature of leaf water, is translocated from the leaf to the stem and cleaves into glucose and fructose. These molecules (glucose and fructose) are isomeric and interchange from one form to the other rapidly (Hill et al., 1995). Recent findings have indicated that this exchange can occur during phloem loading at the leaf and along the pathway from the leaf to the final destination, where sucrose is finally converted to cellulose (Gessler et al., 2013).
The participation of fructose in a futile cycle (Fig. 1) generates several carbonyl groups that can undergo hydration (oxygen from carbon 2, 3, 4 and 5) and exchange with cell water (Hill et al., 1995). Hydration of carbonyl groups involves the addition of oxygen from the surrounding water molecules (Fig. 2). When the carbonyl group dehydrates, the double bond from the carbonyl group is restored, but the remaining oxygen can be from either the original molecule or the water surrounding it (Sternberg & DeNiro, 1983) (Fig. 2). The carbonyl groups from trioses exchange oxygen with stem water rapidly, especially those of dihydroxyacetone which reach equilibrium in < 20 s (Model et al., 1968; Reynolds et al., 1971). When this exchange reaches equilibrium, the δ18O value of a carbonyl group is c. 27‰ higher than that of the water surrounding it (Sternberg & DeNiro, 1983; Sternberg, 1989; Yakir & DeNiro, 1990). The isotope enrichment of the carbohydrate oxygen above that of water is known as the biochemical fractionation (εbio).
The oxygen isotope ratio of stem water, by the above exchange, is passed on to fructose and glucose, the cellulose precursors. However, not all of the fructose molecules undergo the above futile cycle and not all of the oxygen attached to the hexose carbon exchanges with water during cellulose synthesis. Consequently, the labeling of oxygen by stem water during cellulose synthesis is incomplete and corresponds to c. 42% of all the oxygen found in the cellulose molecule (Sternberg et al., 1986; Roden et al., 2000; Sternberg & Ellsworth, 2011), the rest of which holds the isotopic signature of leaf water. Roden et al. (2000) represented the proportion of stem and leaf water oxygen isotopic contribution to δ18OCELL, assuming that εbio is a constant of 27‰, with the following equation:
where δ18OCELL represents the oxygen isotope ratio of stem cellulose.
However, recent observations have indicated that the proportion of oxygen exchange between stem water and carbohydrates (abbreviated as pex) and the biochemical fractionation factor (εbio) during cellulose synthesis may vary. For example, temperature can affect εbio (Sternberg & Ellsworth, 2011). Waterhouse et al. (2002) demonstrated that the best fit between δ18O values of precipitation reconstructed from tree ring cellulose oxygen isotope ratios and observed values occurred only when pex and εbio were assumed to be 46% and 30‰, respectively, values which are different from the average observed values of 42% and 27‰ in (Eqn 1) (Cernusak et al., 2005). In addition, when a plant is under water stress, either from a lack of water or salinity, the oxygen isotope ratio of its biomass, including cellulose, is often lower than that expected according to the above model (Verheyden et al., 2004; Roden et al., 2005; Zhou, 2005). Any reconstruction of paleoclimate, when there is a possibility of salinity or drought stress, will have to take these effects into account.
In this study, we have attempted to gain a better understanding of salinity effects in the recording of oxygen isotope ratios of stem and leaf water in δ18OCELL. First, we hypothesize that salt stress will change the amount of oxygen exchange between carbohydrates and source water (pex) and/or change εbio during cellulose synthesis. Second, we hypothesize that the synthesis of mannitol, a common response by several plant species to salinity, could alter pex and/or εbio, independent of salinity. To test our first hypothesis, we compared Arabidopsis thaliana cultivated hydroponically under salt water and freshwater. To test our second hypothesis, we used different M6PR transgenic lines of A. thaliana that code for mannitol synthesis (Zhifang & Loescher, 2003), and compared the oxygen isotope ratios of stem cellulose from the M6PR transgenic lines with those of wild-type (WT) A. thaliana grown hydroponically under both saline and freshwater.