The vacuole pH-related gene RcNHX2 affects flower color shift and Na+ homeostasis in roses

Rose (Rosa spp.) is one of the most famous ornamental plants in the world, and its commodity value largely depends on its flower color. The color of roses mainly depends on the composition and state of anthocyanins, and the vacuolar pH value is an important factor affecting the stability and state of anthocyanins. The vacuolar sodium/proton antiporters (NHXs) play important roles in the maintenance of cellular ion homeostasis and petal vacuolar pH. However, the NHX functions related to rose flower coloration remain relatively uncharacterized. In this study, we cloned and characterized the vacuolar pH-related gene RcNHX2, which encoded a vesicular cation/H⁺ antiporter protein. Phylogenetic sequence analysis revealed that RcNHX2 belongs to the vesicular NHX family of proteins. It is localized in the vesicular membrane, where it exerts its function. RcNHX2 was significantly differentially expressed in different color-presenting types of petals of roses, and it was particularly highly expressed in the blue-purple petals. The overexpression of RcNHX2 in Rosa hybrida 'Florentina' caused the pH to increase and the petal color to change from red to blue-purple. On the basis of virus-induced gene silencing, we determined that decreased RcNHX2 expression significantly reduces R. hybrida 'Blue For You' petal coloration. We indicated that RcNHX2 might be involved in the color shift to blue in roses. Moreover, it was observed that in the cells of the rose plants in which RcNHX2 was silenced, the Na⁺ homeostasis was affected. The results suggest that the vesicular Na⁺/H⁺ transporter, RcNHX2 gene, likely plays a crucial role in the blue color change and the maintenance of cellular Na⁺ homeostasis in roses. These findings offer valuable insights for the cultivation of blue rose.