Molecular Evidence For Parallel Evolution Of Translucent Bracts Of Two "Glasshouse" Plants Rheum Alexandrae And R.Nobile

Parallel evolution provides an excellent framework to infer the genetic bases of adaptive traits and understand importance of the natural selection in shaping the current biodiversity. The upper leaves of the "glasshouse plants" transform into translucent bracts that show numerous adaptions in alpine habitats. It remains unknown whether similar molecular changes occur under the parallel bract evolution of different "glasshouse" species. In this study, we compared the results on phenotypic and physiological differences and presented the results of cDNA-AFLP analyses of transcriptional changes between translucent bracts and normal leaves of the two "glasshouse plants" Rheum, R. alexandrae and R. nobile respectively. These two species belong to different clades in the phylogenetic analyses of the total genus and are together distributed in Qinghai-Tibet Plateau. We mainly aimed to examine the homologous candidate genes with the same expression changes between the bracts and normal leaves of two species. The main results of the study were as follow:1. We found that the chloroplast pigments of the translucent bracts are reduced dramatically in R. nobile if compared with the normal leaves. Chlorophyll a/b contents of the translucent bracts are just about4%of the normal leaves, and the carotenoid contents are only about12.4%of the normal leaves. The translucent bracts of R. alexandrae are similar to those of the R. nobile in anatomical features but with a little difference:its chlorophyll contents are also significant decreased; however, chlorophyll a contents are reduced much more, equivalent to about18%of the normal leaves while chlorophyll b contents are reduced to less than half of normal leaves (46.9%); and the carotenoid contents are almost not changed, equivalent to about99.2%of the normal leaves.2. The comparison of chloroplast ultrastructure between translucent bracts and normal leaves of two species suggested that the numbers and sizes of chloroplasts are decreased and the chloroplast structures have degenerated in the bracts. The average number of discs per grana stack of chloroplasts in the bracts was about one third to those of the normal leaves. The stroma membranes became obscure in the bracts while they are well-developed in the normal leaves. In addition, the average number of the discs per grana stack of the bracts in R. alexandrae is more than that of the bracts of R. nobile.3. The cDNA-AFLP analyses were further conducted on RNA samples of bracts and normal leaves in both R. nobile and R. alexandrae, respectively. We detected323differentially expressed transcript-derived fragments (TDFs) from approximately6000visualized bands amplified by128pairs of the primer combinations in R. nobile. After being re-amplified and sequenced, a total of110TDFs were obtained. We also detected420differentially expressed TDFs between bracts and normal leaves of R. alexandrae and a total of171TDFs were obtained by the same procedure.4. We compared all differentially expressed TDFs obtained from both R. alexandrae and R. nobile. A total of13homologous TDFs were found between the two species. BLAST identifications in NCBI database suggested that eight of them were found to be involved in stress and defense responses, three were related to photosynthesis, and the other two were not functionally annotated.Overall, we found that the bracts of the two "glasshouse plants" are similar in anatomical features despite their independent origins. The chlorophyll contents are decreased dramatically and the chloroplast structures have degenerated, suggesting that foliar photosynthetic functions in bracts of both species have been reduced or totally altered. In addition, the up-or down-regulations of those candidate genes were highly congruent with anatomical characteristics and adaptive functions of the bracts found for’glasshouse’plants. These findings suggested that the’glasshouse’ phenotypes may have some common molecular bases underlying their parallel evolution of the similar adaptive functions and highlighted the importance of the natural selection in producing such phenotypes.