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.

中文翻译：

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CmCLCa 在菊花叶液泡中硝酸盐的储存中起关键作用

氮（N）对菊花产量和品质非常重要。硝酸盐 (NO3-) 是菊花吸收氮的主要形式。当外部氮源不足时，叶片液泡中积累的 NO3− 可以释放，用于植物内部的重新分配和再利用。因此，了解NO3−在叶片液泡中的储存机制对于培育高氮利用效率和高耐氮饥饿性菊花品种至关重要。在这项研究中，我们发现外源性 NO3- 显着增加了 CmCLCa 的转录水平。我们生成了具有敲低 CmCLCa 表达的 CmCLCa-RNA 干扰转基因品系。在 5 mM NO3− 水培培养中，与野生型 (WT) 菊花相比，CmCLCa-RNAi 转基因株系中的液泡 H+-ATPase (V-ATPase) 和液泡 H+-焦磷酸酶 (V-PPase) 活性和液泡 NO3− 含量降低。接下来，WT 和 CmCLCa-RNAi 转基因植物在具有 5 mM NO3- 的水培培养物中生长 28 天，然后进行 7 天的 N-饥饿处理。CmCLCa-RNAi 转基因株系比 WT 植物对 N 饥饿更敏感，并且叶片中的液泡 NO3− 含量显着降低。我们的结果表明，CmCLCa 的抑制导致叶液泡中 NO3- 积累减少和对 N-饥饿的耐受性降低。我们的研究结果表明，CmCLCa 在叶泡中 NO3− 的储存中起关键作用，并且是提高菊花 N-饥饿耐受性的候选基因。