Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H⁺-ATPase, and MdVHP1, which encodes a vacuolar H⁺-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.

硝酸盐是一种必需的常量营养素，也是调节多种生理过程的信号，过量施用硝酸盐会减少苹果（ Malus Domestica ）果实中苹果酸的积累，但其潜在机制仍知之甚少。在这里，我们证明苹果 BTB/TAZ 蛋白 MdBT2 参与调节苹果酸积累和响应硝酸盐的液泡 pH 值。体外和体内测定表明，MdBT2 通过泛素/26S 蛋白酶体途径直接与 bHLH 转录因子 MdCIbHLH1 相互作用并泛素化，以响应硝酸盐。这种泛素化导致 MdCIbHLH1 蛋白降解，并减少参与苹果酸积累和液泡酸化的 MdCIbHLH1 靶向基因的转录，包括编码液泡 H ⁺ -ATPase 的MdVHA - A和编码液泡 H ⁺ - ATPase 的MdVHP1 。焦磷酸酶，以及编码铝激活苹果酸转运蛋白的MdALMT9 。对苹果材料（包括果实、幼苗和愈伤组织）进行的一系列转基因分析表明，MdBT2 通过调节 MdCIbHLH1 蛋白水平，至少部分（如果不是完全）控制硝酸盐介导的苹果酸积累和液泡 pH 值。综上所述，这些发现表明，MdBT2 通过泛素化来调节 MdCIbHLH1 对硝酸盐的稳定性，从而依次转录降低苹果酸相关基因的表达，从而控制高硝酸盐供应下苹果中的苹果酸积累和液泡酸化。