Time-series RNA-Seq transcriptome profiling reveals novel insights about cold acclimation and de-acclimation processes in an evergreen shrub of high altitude

The high-altitude alpine regions are characterized by highly variable and harsh environmental conditions. However, relatively little is known about the diverse mechanisms adopted by alpine plants to adapt to these stressful conditions. Here, we studied variation in transcriptome and physiological adjustments occurring across the year at high elevation environments in the leaf tissue of Rhododendron anthopogon, an evergreen shrub of Himalaya. The samples were collected at 12 different time-points, from August until snowfall in November 2017, and then from June to September 2018. It was observed that with a drop in both ambient air temperature and photoperiod towards onset of winter, the freezing resistance of plants increased, resulting in ‘cold acclimation’. Further, ‘de-acclimation’ was associated with a decrease in freezing resistance and increase in photosynthetic efficiency of leaves during spring. A considerable amount of variation was observed in the transcriptome in a time-dependent sequential manner, with a total of 9,881 differentially expressed genes. Based on gene expression profiles, the time-points could be segregated into four clusters directly correlating with the distinct phases of acclimation: non-acclimation (22-August-2017, 14-August-2018, 31-August-2018), early cold acclimation (12-September-2017, 29-September-2017), late cold acclimation (11-October-2017, 23-October-2017, 04-November-2017, 18-September-2018) and de-acclimation (15-June-2018, 28-June-2018, 14-July-2018). Cold acclimation was a gradual process, as indicated by presence of an intermediate stage (early acclimation). However, the plants can by-pass this stage when sudden decrease in temperature is encountered. The maximum variation in expression levels of genes occurred during the transition to de-acclimation, hence was ‘transcriptionally’ the most active phase. The similar or higher expression levels of genes during de-acclimation in comparison to non-acclimation suggested that molecular functionality is re-initiated after passing through the harsh winter conditions.