Characterizing the role of ATP-binding cassette transporters in triterpenoid wax bloom production

Abstract

Some plants have developed lineage-specific mechanisms to increase water retention during drought conditions, including thick epicuticular wax coatings called wax blooms. However, the process underlying synthesis and especially the transport of wax bloom compounds to the leaf surface have yet to be fully elucidated. Numerous ATP-binding cassette (ABC) transporters have been characterized in model systems and crops, and some members of the G transporter subfamily are linked to the production of epicuticular wax blooms. Many studies utilizing model systems focus on long-chain aliphatic wax compounds, even though numerous species produce wax bloom composed primarily of polycyclic triterpenoid compounds. Relative to aliphatic compounds, next to nothing is known about the production of triterpenoid wax blooms, including whether their transport to the plant surface is mediated by ABC transporters with specificity for triterpenoids rather than aliphatics. Kalanchoe is a genus of succulent plants that vary in their ability to produce wax blooms, with some species producing the triterpenoid wax blooms of interest. Here, I investigated the role of ABC transporters in triterpenoid wax bloom production. RNA samples from three Kalanchoe species that produce triterpenoid-rich wax blooms to varying degrees (K. thrysiflora, K. fedtschenkoi, and K. blossfeldiana) were collected and sequenced. Gene expression data and wax chemistry were compared among replicates, varying: (i) plants grown in high light intensity v. low light intensity, (ii) bloom-producing v. bloomless plants, and (iii) leaf epidermal tissue v. mesophyll tissue. I assembled transcriptomes for each species and identified candidate ABC transporter genes using the comparisons described above. I then characterized the transporter candidate genes using heterologous expression in Nicotiana benthamiana and subsequent gas chromatography-mass spectrometry analysis. The data indicated that the ratio of triterpenoids to aliphatic compounds increased when certain transporters are co-infiltrated with a triterpenoid synthase gene. This strongly suggests that Kalanchoe has triterpenoid-specific transporters, and that other triterpenoid-rich wax bloom producing species could as well.