Figure 2. Kinetics of gene expression in response to low temperature as a function of time, as well as at low and high altitude in the leaf tissue of Caragana jubata.

For panel ‘a’, plants were shifted from 25±3°C (control, CO) to 4±3°C (low temperature, LT) and the samples were harvested at different time intervals as shown in the figure. Gene expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) that was standardized to get amplification in the exponential phase. RT-PCR was also performed for the plants housed at 25°C at the same time intervals as performed for those kept at LT (data not shown since there was no change in gene expression over a span of 48 h). Name of the gene is shown in each panel and the corresponding accession number is shown in Supplementary Table S2. Details of primers and PCR conditions are mentioned in Supplementary Table S7. For panel ‘b’, leaves of two populations (P1 and P2) of C. jubata growing in niche environment of high altitude in Kibber-Gete area (Lahaul and Spiti district, Himachal Pradesh, India) were collected during September/October in two separate years (year 1 and year 2). Samples were also collected for the plants growing at 25±3°C (CO) at low altitude in the institute (altitude, 1300 m; Palampur, Himachal Pradesh, India). Leaf tissues were harvested, stored immediately in liquid nitrogen on site and stored at (−) 80°C till further use. 26S rRNA served as internal control for both panel ‘a’ and ‘b’. Gel figures for RT-PCR are shown in Supplementary Fig. S2a,b. *To calculate integrated density value (IDV), PCR products were separated on 1.2% agarose gel, stained with ethidium bromide and quantified using Alpha DigiDoc 1000 software supplied along with gel documentation system (Alpha DigiDocTM, Alpha Innotech, USA). IDV thus obtained was divided by 1,000 for ease of writing on the y axis; thus, the IDV needs to be multiplied by 1,000 to get the original value of gene expression.