National Key Laboratory of Crop Biology, Shandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, Shandong 271018, China.
Department of Horticulture, Cornell University, 134A Plant Science, Ithaca, New York 14853.
Plant Physiol. 2020 Jun;183(2):750-764. doi: 10.1104/pp.20.00208. Epub 2020 Apr 2.
Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple () 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 -, which encodes a vacuolar H-ATPase, and , which encodes a vacuolar H-pyrophosphatase, as well as , 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.
过量施用硝酸盐会降低苹果果实中苹果酸的积累，但硝酸盐如何调节苹果酸积累的机制尚不清楚。本研究发现，苹果 BTB/TAZ 蛋白 MdBT2 参与了硝酸盐响应调控苹果酸积累和液泡 pH 的过程。体外和体内实验表明，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，并使其发生泛素化，从而导致 MdCIbHLH1 蛋白降解，并降低与苹果酸积累和液泡酸化相关的 MdCIbHLH1 靶基因的转录，包括编码液泡 H+-ATP 酶的基因、编码液泡 H+-焦磷酸酶的基因以及编码铝激活的苹果酸转运蛋白的基因。在包括果实、幼苗和愈伤组织在内的一系列苹果材料中的转基因分析表明，MdBT2 通过调节 MdCIbHLH1 蛋白水平，至少部分地（如果不是完全地）控制硝酸盐介导的苹果酸积累和液泡 pH。综上所述，这些发现表明，MdBT2 通过泛素化响应硝酸盐来调节 MdCIbHLH1 的稳定性，从而转录降低与苹果酸相关的基因的表达，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。
