BTB-TAZ Domain Protein MdBT2 Modulates Malate Accumulation and Vacuolar Acidification in Response to Nitrate.

Quan-Yan Zhang,Kai-Di Gu,Lailiang Cheng,Da-Gang Hu,Jian-Qiang Yu,Yu-Jin Hao,Jia-Hui Wang,Xiao-Fei Wang,Chun-Xiang You

doi:10.1104/pp.20.00208

Quan-Yan Zhang, Kai-Di Gu + Show 7 more

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https://doi.org/10.1104/pp.20.00208

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Abstract

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.

Highlights

Malate, as a key metabolite, plays a vital role in plant metabolism, pH homeostasis, nutrient uptake, osmotic adjustment, and abiotic stress resistance (Fernie and Martinoia, 2009; Finkemeier and Sweetlove, 2009; Bai et al, 2015; Hu et al., 2017)
To determine if nitrate has any effect on malate accumulation in apple, we provided apple calli and plantlets with different nitrate concentrations (0 to 5 mM)
5A), whereas the protein was more stable in the protein extract of 35S::anti-MdBT2 RNAi transgenic apple calli (35S)::anti-MdBT2 compared to the WT (Fig. 5A). These results suggest that MdBT2 promotes the degradation of the MdCIbHLH1 protein

Summary

Introduction

As a key metabolite, plays a vital role in plant metabolism, pH homeostasis, nutrient uptake, osmotic adjustment, and abiotic stress resistance (Fernie and Martinoia, 2009; Finkemeier and Sweetlove, 2009; Bai et al, 2015; Hu et al., 2017). Malate metabolism can alter fruit malate levels (Sweetman et al, 2009; Centeno et al, 2011), it appears that transport of malate from the cytosol into the vacuole is the step that largely controls malate accumulation (Etienne et al, 2013; Hu et al, 2016a; Ma et al, 2019). The resulting low pH protonates any malate that crosses the tonoplast from the cytosol, effectively trapping malate in the acid form. This “acid trap” mechanism maintains the malate concentration gradient across the tonoplast for its facilitated diffusion (Martinoia et al, 2007; Etienne et al, 2013; Eisenach et al, 2014; Hu et al, 2016a).

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