New insights into the influence of NHX-type Cation/H+ antiporter on flower color in Phalaenopsis orchids

Vacuolar sodium/proton Na+(K+)/H+ exchanger (NHX) influence color formation because of their effects on cellular pH and Na+/K+ homeostasis. Research regarding NHXs has mainly focused on the vacuolar NHX family members. However, the NHX functions related to Phalaenopsis flower coloration remain relatively uncharac-terized. In this study, we cloned and characterized PeNHX1, a vacuolar cation/H+ antiporter-encoding gene that is highly expressed in the Phalaenopsis equestris (orchid) flower lip. Phylogenetic and sequence analyses showed that PeNHX1 is a vacuolar NHX protein family member that is similar to other known vacuolar antiporters. The PeNHX1-GFP fusion protein was clearly localized to the vacuolar membrane in a transient transfection assay. A quantitative real-time PCR analysis revealed the increased expression of PeNHX1 in different flower develop-mental stages. Moreover, it was more highly expressed in the lip than in the other flower organs. On the basis of virus-induced gene silencing, we determined that decreased PeNHX1 expression significantly reduces P. equestris petal coloration. Furthermore, the overexpression of PeNHX1 in Phalaenopsis Big Chili caused the pH to increase and the petal color to change from red to blue. The results indicate that NHX1 may mediates the Na + or K+/H+ exchange, thereby regulating the vacuolar pH to promote blue coloration. This research provides a theoretical basis for the development of orchid varieties with blue flowers.