CmCLCa Plays a Key Role in the Storage of Nitrate in Chrysanthemum Leaf Vacuoles

Nitrogen (N) is very important for chrysanthemum yield and quality. Nitrate (NO3−) is the main form of N absorbed by chrysanthemum. The NO3− accumulated in the leaf vacuoles can be released for redistribution and reutilization within the plant when the external N source is insufficient. Therefore, understanding the storage mechanism of NO3− in the vacuoles of leaves is essential for the cultivation of chrysanthemum varieties with high N-use efficiency and high N-starvation tolerance. In this study, we found that the transcript level of CmCLCa was significantly increased by exogenous NO3−. We generated CmCLCa-RNA interference transgenic lines with knocked-down CmCLCa expression. In 5 mM NO3− hydroponic culture, the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities and vacuolar NO3− content were decreased in CmCLCa-RNAi transgenic lines compared with wild-type (WT) chrysanthemum. Next, WT and CmCLCa-RNAi transgenic plants were grown in hydroponic culture for 28 days with 5 mM NO3− and then subjected to a 7-day N-starvation treatment. The CmCLCa-RNAi transgenic lines were more sensitive to N-starvation than were WT plants and showed significantly decreased vacuolar NO3− content in the leaves. Our results indicate that inhibition of CmCLCa results in reduced NO3− accumulation in the leaf vacuole and lower tolerance to N-starvation. Our findings show that CmCLCa plays a key role in NO3− storage in leaf vacuoles and is a candidate gene for improving the N-starvation tolerance of chrysanthemum.