Temperature-regulated anatomical and gene-expression changes in Sinopodophyllum hexandrum seedlings

Sinopodophyllum hexandrum is a high-altitude perennial medicinal plant that produces the economically-valued anticancer metabolite, podophyllotoxin (PPT). While gene profiling under elevated altitude and low temperature conditions have identified transcriptions associated with increased PPT accumulation, changes in cell structure, with adaptive low-temperature conditions have yet to be identified. In this study, anatomical features including leaf micro- and ultra-structure were investigated by monitoring traits when S. hexandrum seedlings were grown under a cooler temperature (15 °C). Differences in leaf micro- and ultra-structure parameters including stomatal density and shape, chloroplast density as well as the abundance of starch grain, osmiophilic granule and thylakoid grana were observed with the lower temperature. Greater chlorophyll content and higher tissue amounts (e.g. leaf area, number of petioles and root size) were also observed at the cooler temperature. These anatomical and morphological changes were associated with elevated PPT levels on a dry weight and per plant basis, as well increased whole plant fresh and dry weights. To link anatomical and morphological changes with elevated PPT levels, mRNA transcripts encoding for photosynthesis, glyco-metabolism, stress response and PPT biosynthesis select genes were monitored and found to be differentially regulated at 15 versus 22 °C. These cool-temperature anatomical/morphological changes are consistent with S. hexandrum's capacity to biosynthesize and accumulate elevated PPT in cooler environments.