Time-series eco-metabolomics reveals extensive reshuffling in metabolome during transition from cold acclimation to...

Recording environmentally induced variations in the metabolome in plants can be a promising approach for understanding the complex patterns of metabolic regulation and their eco-physiological consequences. Here, we studied metabolome-wide changes and eco-physiological adjustments occurring across the year at high elevation environments in the leaf tissue of Rhododendron anthopogon, an alpine evergreen shrub of the Himalaya. New leaves of R. anthopogon appear after the snow-melt and remain intact even when the plants get covered under snow (November-June). During this whole period, they may undergo several physiological and biochemical adjustments in response to fluctuating temperatures and light conditions. To understand these changes, we analyzed eco-physiological traits, that is, freezing resistance, dry matter content and % of nitrogen and the overall metabolome across 10 different time-points, from August until the snowfall in November 2017, and then from June to August 2018. As anticipated, the freezing resistance increased toward the onset of winters. The leaf tissues exhibited a complete reshuffling of the metabolome during the growth cycle and time-points segregated into four clusters directly correlating with distinct phases of acclimation: non-acclimation (August 22, 2017; August 14, 2018), early cold acclimation (September 12, September 29, October 11, 2017), late cold acclimation (October 23, November 4, 2017), and de-acclimation (June 15, June 28, July 14, 2018). Cold acclimation involved metabolic progression (101 metabolites) with an increase of up to 19.4-fold (gentiobiose), whereas de-acclimation showed regression (120 metabolites) with a decrease of up to 30-fold (sucrose). The changes in the metabolome during de-acclimation were maximum and were not just a reversal of cold acclimation. Our results provided insights into the direction and magnitude of physiological changes in Rhododendron anthopogon that occurred across the year.