Molecular Mechanism Of Action Of Nitrateresponsive Gene MdBT2 Involved In The Regulation Of Malate Accumulation In Apple

Acidity is one of the most important quality traits in fruits,the levels of acidity directly affect the flavor and processing quality of fruit.At present,16 kinds of organic acids have been detected in apple fruits,among which malate is the most important organic acid accounting for more than 80% of total acid,which is the main contributor to apple acidity.Malate metabolism is a complex biological process involving synthesis,degradation and transport.Among them,the synthesis of malate can be achieved through a variety of metabolic pathways incorporate with participating in glycolysis,TCA cycle,Calvin cycle and glyoxylate cycle.High concentrations of malate are stored in the vacuoles.The vacuolar proton pumps(VATPase and V-PPase)and malate transporters(ALMT9)are critical for malate transport into the vacuole.The vacuolar proton pump establishes a transmembrane electrochemical potential gradient inside and outside the vacuole,acidifies the vacuole and provides energy for malate transporters.Vacuolar proton pumps acidify the vacuoles by pumping protons across the tonoplast,resulting protonates any malate that crosses the tonoplast from the cytosol,effectively trapping malate in the acid form.Transcription factors,located upstream of the vacuolar proton pump and malate transporter,are widely involved in regulating the synthesis and transport of malate.In addition,the MYB transcription factors(TFs)together with b HLH TFs and WD40 proteins that formMBW complexes to transcriptionally activate the expression of vacuolar proton pumps and malate transporter genes,thereby promoting the storage of malate in vacuoles.In apple,the R2R3-MYB transcription factor MdMYB73 promotes the accumulation of malate by directly binding with the promoter of vacuolar proton pump and vacuolar malate channel protein.Moreover,MdCIbHLH1,a b HLH transcription factor,interacts with MdMYB73 and enhances its effects on downstream target genes to regulate malate accumulation.Malate accumulation is also affected by environmental factors and management practices such as nutrient,temperature,and irrigation.Nitrogen is the most essential macronutrient for plant growth and development,plays a vital role in malate accumulation.In order to obtain higher yield and economic benefit,fruit growers abuse chemical fertilizer,mainly nitrogen fertilizer,which leads to the decline of fruit quality.Excessive application of nitrogen can reduce the accumulation of malate in apple fruits,however,the specific mechanism is not clear,which is a scientific problem to be solved in this study.In this study,apple experimental material was taken to explore the molecular mechanism of malate accumulation regulated by nitrate.The main results were as follows:1.Exogenous application of high concentration nitrate reduced malate accumulation.The accumulation of malate in apple calli,plantlets and fruits were examined after treatment with different concentration of nitrate,such as 0,0.5,and 5 mM.The results showed that high concentration of nitrate significantly reduced the malate accumulation in apple.Further,the expression levels of vacuolar proton pumps genes,the vacuolar malate channel MdALMT9,and the malate-associated MYB TF MdMYB73,were significantly decreased when high concentration of nitrate.In addition,the hydrolytic and proton-pumping activities of V-ATPase and V-PPase were also reduced and average vacuolar pH increased when high concentration of nitrate.2.Nitrate-responsive Gene MdBT2 decreased malate accumulation in apple.MdBT2 transgenic apple plantlets were treated with nitrate to detect malate content,the expression levels of malate-related genes,the hydrolytic and proton-pumping activities of V-ATPase and V-PPase,and the average vacuolar pH.Results verified that MdBT2 inhibited the malate accumulation in response to nitrate.3.MdBT2 interacted with MdCIbHLH1 and MdMYB73.Possible MdBT2-interacting proteins were screened using MdBT2 through yeast cDNA library.It was found that MdBT2 could interact with b HLH transcription factor MdCIbHLH1 and MYB transcription factor MdMYB73.The interactions about MdBT2-MdCIbHLH1 and MdBT2-MdMYB73 were further verified by yeast two hybrid,bimolecular fluorescence complementation and pull down assays.4.MdBT2 promoted the ubiquitination and degradation of MdCIbHLH1 and MdMYB73.The cell-free degradation assays in vivo and in vitro showed that MdBT2 could affect the stability of MdCIbHLH1 and MdMYB73.Furthermore,MdBT2 facilitated the degradation of MdCIbHLH1 and MdMYB73 via the ubiquitin/26 S proteasome pathway.5.MdBT2 negatively regulated MdALMT9 transcriptional activity via MdCIbHLH1 and MdMYB73,thus inhibited the malate accumulation.The transient injection of apple fruits and GUS analysis of apple calli were examined.Results showed that MdBT2 negatively regulated malate accumulation at least partially,via MdCIbHLH1 and MdMYB73,inhibiting the expression level of MdALMT9,reducing the activity of the hydrolytic and proton-pumping activities of V-ATPase and V-PPase.This study sheds light on the molecular mechanism of nitrate regulating malate accumulation through ‘MdBT2-MdCIbHLH1/MdMYB73-MdALMT9’ module,providing an important link between two key metabolites in plant metabolism,furnishing valuable insights into post-translational regulatory networks of malate accumulation.These findings not only help us understand the complex regulatory network of malate accumulation but also are of great significance in guiding apple molecular breeding programs and fruit quality improvement.