Measurements of leaf sucrose to explain variability in hydrogen isotope composition of leaf cellulose
Authors: Holloway-Philips Meisha , Baan Jochem , Nelson Daniel , Tcherkez Guillaume , Kahmen Ansgar 
Published: March 2021

&lt;p&gt;The hydrogen isotope composition (&amp;#948;&lt;sup&gt;2&lt;/sup&gt;H) of cellulose has been used to assess ecohydrological processes and carries metabolic information, adding new understanding to how plants respond to environmental change. However, experimental approaches to isolate drivers of &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H variation is limited to the Yakir &amp; DeNiro model (1990), which is difficult to implement and largely unvalidated. Notably, the two biosynthetic fractionation factors in the model, associated with photosynthetic (&amp;#949;&lt;sub&gt;A&lt;/sub&gt;) and post-photosynthetic (&amp;#949;&lt;sub&gt;H&lt;/sub&gt;) processes are currently accepted as constants, and the third parameter &amp;#8211; the extent to which organic molecules exchange hydrogen (f&lt;sub&gt;H&lt;/sub&gt;) with local water &amp;#8211; is usually tuned in order to resolve the difference between modelled and observed cellulose &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values. Thus, by virtue, the metabolically interpretable parameter is only f&lt;sub&gt;H&lt;/sub&gt;, whilst from theory, metabolic flux rates will also impact on the apparent fractionations. To overcome part of this limitation, we measured the &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H of extracted leaf sucrose from fully-expanded leaves of seven species and a phosphoglucomutase &amp;#8216;starchless&amp;#8217; mutant of tobacco to estimate the isotopic offset between sucrose and leaf water (&amp;#949;&lt;sub&gt;sucrose&lt;/sub&gt;). Sucrose &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H explained ~60% of the &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H variation observed in cellulose. In general, &amp;#949;&lt;sub&gt;sucrose&lt;/sub&gt;&amp;#160;was higher (range: -203&amp;#8240; to -114&amp;#8240;; mean: -151 &amp;#177; 21&amp;#8240;) than the currently accepted value of -171&amp;#8240; (&amp;#949;&lt;sub&gt;A&lt;/sub&gt;) reflecting &lt;sup&gt;2&lt;/sup&gt;H-enrichment downstream of triose-phosphate export from the chloroplast, with statistical differences in &amp;#949;&lt;sub&gt;sucrose&lt;/sub&gt;&amp;#160;observed between species estimates. The remaining &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H variation in cellulose was explained by species differences in f&lt;sub&gt;H&amp;#160;&lt;/sub&gt;(estimated by assuming &amp;#949;&lt;sub&gt;H &lt;/sub&gt;= +158&amp;#8240;). We also tested possible links between model parameters and plant metabolism. &amp;#949;&lt;sub&gt;sucrose&lt;/sub&gt;&amp;#160;was positively related to dark respiration (R&lt;sup&gt;2&lt;/sup&gt;=0.27) suggesting an important branch point influencing sugar &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H. In addition, f&lt;sub&gt;H&lt;/sub&gt; was positively related to the turnover time (&amp;#964;) of water-soluble carbohydrates (R&lt;sup&gt;2&lt;/sup&gt;=0.38), but only when estimated using fixed &amp;#949;&lt;sub&gt;A &lt;/sub&gt;= -171&amp;#8240;. To decipher and isolate the &amp;#8220;metabolic&amp;#8221; information contained within &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of cellulose it will be important to assess &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H values of non-structural carbohydrates so that hydrogen isotope fractionation during sugar metabolism can be better understood. This study provides the first attempt at such measurements showing species differences in both source and sink processes are important in understanding &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H variation of cellulose.&lt;/p&gt;