BTB-TAZ domain protein MdBT2 modulates malate accumulation by targeting a bHLH transcription factor for degradation in response to nitrate

Abstract

AbstractExcessive 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 MdVHA-A encoding a vacuolar H+-ATPase gene, and MdVHP1 encoding a vacuolar H+-pyrophosphatase gene, as well as MdALMT9 encoding a aluminum-activated malate transporter gene. 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.