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Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification

Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate... aBIOTECH https://doi.org/10.1007/s42994-023-00115-7 aBIOTECH RESEARCH ARTICLE Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification 1 1 1 1 Xiao-Yu Huang , Ying Xiang , Yu-Wen Zhao , Chu-Kun Wang , 1 1 1 1& Jia-Hui Wang , Wen-Yan Wang , Xiao-Long Liu , Quan Sun , 1& Da-Gang Hu National Research Center for Apple Engineering and Technology, Shandong Collaborative Innovation Center of Fruit and Vegetable Quality and Efficient Production, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong, China Received: 16 June 2023 / Accepted: 17 August 2023 Abstract As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH. Keywords Apple, Malate accumulation, P-ATPase, MdPH5, MdMYB73 INTRODUCTION et al. 2013). Additionally, it can also relieve angioscle- rosis, facilitate absorption of calcium, iron, and other Organic acids affect the acidity of fleshy fruits and play elements, and stimulate secretion by digestive glands. an important role in the regulation of osmotic pressure, In fruit cells, malate is mainly synthesized in the pH homeostasis, stress resistance, and sensory quality cytoplasm, via phosphoenolpyruvate carboxylation of fruits (Huang et al. 2021). Malate is one of the most (PEP), and is typically catalyzed by phosphoenolpyru- important organic acids in fruits, and accounts for 90% vate carboxylase (PEPC) and malate dehydrogenase of total organic acids in apple. Malate can generate ATP, (MDH) (Sweetman et al. 2009). Malate is then trans- resist oxidation, and relieve oxidative stress (Etienne ported into vacuoles for storage. Although malate accumulation in cells is controlled by both metabolism and vacuolar storage, the transport process from Xiao-Yu Huang and Ying Xiang contributed equally to this work. the cytoplasm into vacuoles may have a determining effect on malate accumulation. Therefore, it is necessary & Correspondence: sunquan@sdau.edu.cn (Q. Sun), to thoroughly investigate this transport process. As a fap_296566@163.com (D.-G. Hu) The Author(s) 2023 aBIOTECH matter of fact, this process involves multiple vacuolar can affect the transcriptional activity of malate trans- transporters, ion channels and carriers, among which porters and proton pumps to regulate malate accumu- vacuolar transporters and channels play a dominant lation and vacuolar acidification (Hu et al. 2016, 2017; role in the transport of organic acids, such as Jia et al. 2021). CrMYB73, which is homologous to the tonoplast dicarboxylate transporter (tDT) (Emmer- MdMYB73, leads to increased citrate accumulation in lich et al. 2003) and an aluminium-activated malate citrus plants, whereas its downstream target genes transporter (ALMT) (such as ALMT6 and ALMT9/Ma1) remain unclear (Li et al. 2015). In addition, PhPH4 in (Emmerlich et al. 2003; Meyer et al. 2011; Cohen et al. petunia is an R2R3-MYB TF that plays a similar role to 2014; Martinoia 2018). In addition, some proton pumps VvMYB5a and VvMYB5b in grape for the regulation of on the tonoplast also play a key role in this process by citrate accumulation. Specifically, they both can activate transporting H into vacuoles and acidifying vacuoles. expression of the downstream genes PH1 and PH5, thus In this way, the proton electrochemical gradients that acidifying vacuoles (Cavallini et al. 2014; Kasajima et al. serve as the driving force for the transport of malate 2016; Amato et al. 2019). In soybean petals, GmPH4, into vacuoles are generated. which is an R2R3-MYB TF, is also involved in vacuolar To date, three types of proton pumps, including acidification as it directly regulates the expression of ? ? vacuolar H -ATPase (V-ATPase), vacuolar H -PPase (V- GmPH5,aP -type ATPase gene (Sundaramoorthy et al. 3A PPase), and vacuolar P-type ATPase (P-ATPase), have 2020). However, the specific regulators of MdPH5 in been identified in fruits (Hurth et al. 2005; Kovermann apple have not yet been identified. et al. 2007; Martinoia et al. 2007). As a novel proton In this study, the role of MdPH5 in regulating malate pump thoroughly investigated in recent years, the P- accumulation and vacuolar acidification in apple was ATPase was first identified in petunia and may be investigated and its transcriptional regulation by MYB associated with vacuole acidification. In plants, TF MdMYB73 was verified. This study provides a ref- P-ATPase consists of five major evolutionarily related erence for understanding the molecular factors involved subfamilies (P1-P5). Among them, ATPases in the P3 in fruit quality. subfamily are responsible for energizing the electro- chemical gradient serving as the driving force of sec- ondary transport (Pedersen et al. 2012). The P-type MATERIALS AND METHODS ATPases (PhPH5 and PhPH1) found in petunia are involved in the vacuolar acidification of petal cells, Plant materials and growth conditions whereas only PhPH5 shows independent proton trans- port activity (Verweij et al. 2008; Faraco et al. 2014;Li ‘Orin’ apple calli obtained from young embryos were et al. 2016). Moreover, their homologous genes were subcultured at 3-week intervals on MS medium sup- also identified in fruits. In citrus, the homologous gene, plemented with 1.5 mg/L 2,4-dichlorophenoxyacetic CitPH5, is closely related to the generation of super- acid (2,4-D) and 0.4 mg/L 6-benzylaminopurine (6-BA) acidified fruit (Strazzer et al. 2019). In pear, PbPH5 is at 25 C, in the dark. Subsequently, these calli were localized to the vacuolar membrane and can promote subcultured, at half-month intervals, three times before malate accumulation in fruits (Song et al. 2022). As a being used for further studies. homologue of PhPH5 in apple, MdPH5 is slightly ‘Royal Gala’ apples were collected at 41, 70, 94, upregulated in transgenic MdMYB73 callus, which is 128 days post-flowering. ‘Royal Gala’ apple were col- responsible for malate accumulation and vacuolar lected at 120 days post-flowering and stored in air- acidification (Hu et al. 2017), suggesting that MdPH5 conditioned tanks. Batch of apples of similar size, color, may also be associated with malate accumulation in maturity, disease-free, insect-free, and without apple. Nevertheless, the specific function of MdPH5 in mechanical damage were selected for storage for apple fruits remains unclear. 120 days, and samples were taken every 30 days. These The regulation of organic acid transporters and pro- apples were frozen in liquid nitrogen. ton pumps involves complex gene regulatory networks. Indeed, transcriptional regulation is one of the most Bioinformatics analysis of the MdPH5 gene common events. Among them, MYB transcription factor (TF) families comprise plant-specific R2R3-MYB TFs, The basic information of the MdPH5 sequence was many of which are involved in the transport of organic obtained from the NCBI database (https://www.ncbi. acids by activating or inhibiting gene expression of nlm.nih.gov/). The MdPH5 secondary structure predic- transporters and proton pumps. Remarkably, MdMYB1, tion was adopted from SOPMA (https://npsaprabi.ibcp. MdMYB44, and MdMYB73, which are present in apple, fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The Author(s) 2023 aBIOTECH Phylogenetic analysis of PH5 proteins Viral vector-mediated transient expression in apple skins The MEGA_X based on the neighbor-joining method and bootstrap analysis with 1000 replications was used to Apple skin injection assays were performed as descri- construct the phylogenetic tree of MdPH5 and Ara- bed previously. MdPH5-TRV (TRV1 ? MdPH5-TRV2) bidopsis thaliana P subfamily. was suppression expression vector. MdPH5-IL60 (IL60- 3A 1 ? MdPH5-IL60-2) was overexpression vector, Analysis of the MdPH5 promoter MdMYB73-IL60 and so on. IL60 (IL60-1 ? IL60-2) and TRV (TRV1 ? TRV2) were empty vectors and used as The cis element in the MdPH5 promoter (1500 bp references. MdPH5-TRV2 ? MdMYB73-IL60 was mixed upstream of the transcription initiation site) was ana- with MdPH5-TRV (TRV1 ? MdPH5-TRV2) and lyzed with the online software PlantCARE (http://bioin MdMYB73-IL60 (IL60-1 ? MdMYB73-IL60-2) and formatics.psb.ugent.be/webtools/plantcare/html/). injected into apple fruit. IL60 ? TRV (IL60-1 ? IL60- 2 ? TRV1 ? TRV2) was empty vector and used as Construction of the MdPH5 gene expression reference. vector and genetic transformation of apple calli Measurement of vacuolar pH The ORF sequence of MdPH5 was cloned into pRI-101 to obtain the overexpression vector, and the non-con- Isolation of protoplasts from apple calli and measure- served regions of MdPH5’s ORF were reversely cloned ment of vacuolar pH were carried out, as previously into pRI-101 to obtain the antisense vector. Transgenic described by Hu et al. (2016). The vacuolar pH was apple calli were obtained by Agrobacterium-mediated detected with the cell-permeant and pH-sensitive fluo- 0 0 transformation. rescent dye, 2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluo- rescein (BCECF)-AM, while vacuolar pH was quantified Quantitative real-time-PCR (RT-qPCR) analysis by the ratio of pH-dependent (488 nm) and pH-inde- pendent (458 nm) excitation wavelengths from a cali- Plant RNA was extracted with an RNA extraction kit bration curve, and ratio images were generated with the (TIANGEN, Beijing, China) and single-stranded cDNA ion concentration tool of Zeiss LSM confocal software. was obtained with a reverse transcription kit (TaKaRa, Shiga, Japan). The RT-qPCR analyses were executed with Determination of malate content three biological and technical replications to test the expression levels of MdPH5, which were performed with Malate content was measured by high-performance the methods as described by Hu et al. (2016). The liquid chromatography, as previously described by Hu -DDCT quantitative analysis of results used the 2 method. et al. (2016). Analysis of subcellular localization EMSA The full-length coding sequences of MdPH5 were fused EMSA was conducted according to Xie et al. (2012). to the GFP protein, to construct the fusion expression MdMYB73 was cloned into the expression vector vector 35S:MdPH5-GFP, and the resulting plasmid was pGEX4T-1. The MdMYB73-GST recombinant protein was transformed into Agrobacterium strain LBA3101. The expressed in Escherichia coli strain BL21. An oligonu- constructed vector was injected into tobacco (Nicotiana cleotide probe of the MdMYB73 promoter was labeled benthamiana) epidermal cells and cultured in the dark using an EMSA probe biotin labeling kit (Beyotime) for 3 days. The AtCBL-red fluorescent protein (RFP) was according to the manufacturer’s instructions. The used as a vacuolar membrane marker (Ma et al. 2019) recombined protein of MdMYB73-GST was incubated and was co-transformed with 35S:MdPH5-GFP. Fluores- with 10 9 binding buffer, 1 lg/lL poly (dI-dC), and 400 fmol of biotin-labeled double-stranded binding consen- cence images were obtained at 488 nm with a high- resolution laser confocal microscope (LSM880, Zeiss, sus oligonucleotides (total volume 20 lL) using a Meta, Jena, Germany). LightShift Chemiluminescent EMSA Kit (Thermo Scien- tific). The binding reaction was performed at room temperature for 20 min. The DNA–protein complexes were separated on 6.5% non-denaturing polyacrylamide gels, electrotransferred, and detected following the The Author(s) 2023 aBIOTECH manufacturer’s instructions. The binding specificity was 70 DAB, 70 DAB vs. 94 DAB, and 94 DAB vs. 128 DAB, also examined by competition with a fold excess of were 1746 and 4426, 1289 and 1876, and 2435 and unlabeled oligonucleotides. 4939, respectively (Fig. S2A). Figures S2B and S2C are Venn diagrams reflecting the overlap of differential LUC assay genes in different cases. As observed, the malate content decreased regularly and the expression of genes that The apple MdPH5 promoter fragments were amplified might be associated with malate showed changes by PCR and cloned into the pGreenII 0800-LUC vector to according to the transcriptomic data, as shown in the construct the LUC reporter vector (MdPH5pro-LUC). heat maps. Figure S2D shows the expression levels of 32 The full-length coding sequences of MdMYB73 were malate-related genes, at different developmental stages. cloned into the effector vector pGreenII 62-SK. Individ- According to the heat maps of the expression levels of ual combinations of reporter vectors and effector vec- malate-related genes, the malate content decreased tors were transformed into Agrobacterium strain significantly from 41 to 71 DAB. As observed, genes GV3101 cells alongside the pSOUP vector. The such as MD03G1155400, MD13G1044200, and Agrobacterium strains were used to tobacco (Nicotiana MD17G1155800 were significantly downregulated benthamiana) epidermal cells. A live-imaging apparatus (Fig. S3A). To visualize changes of malate-related genes was used to measure luminescence after 2 days. and identify genes with the highest correlation with malate, three volcano maps (41 DAB vs. 70 DAB, 41 DAB Chromatin immunoprecipitation qPCR analysis vs. 94 DAB, and 41 DAB vs. 128 DAB) were developed. The number of downregulated genes in the volcano 35S:MdMYB73-GFP and 35S::GFP transgenic apple cul- maps were 4, 6, and 6, respectively. Among these genes, tures were used for the ChIP-qPCR analysis. The anti- MD17G1155800 was the only malate-related gene pre- GFP antibody (Beyotime) was used for chromatin sent in all data sets (Fig. 1B; Fig. S3B, C). As malate was immunoprecipitation (ChIP), as described by Xie et al. significantly downregulated in 41 DAB vs. 70 DAB, 41 (2012). The resultant samples were used as templates DAB vs. 94 DAB, and 41 DAB vs. 128 DAB, for qPCR assay. MD17G1155800 may be positively correlated with malate. It has been reported that Md17G1155800 is a Data presentation and statistical analysis P-type proton pump (MdPH5), and its homologous gene, PbPH5, in petunia can acidify vacuoles and change The data obtained in this study were analyzed by DPS petal color (Faraco et al 2014). Software (Enfield, UK), with P \ 0.05 considered as indicative of significant differences. Correlation of MdPH5 expression with malate content during apple fruit ripening and post- harvest RESULTS To further clarify the role of MdPH5 in apple, the level of Analysis of transcriptome and multiple MdPH5 expression in the transcriptome dataset was metabolites in apple fruit at different monitored. The results showed that its expression level developmental stages decreased (Fig. 1C). Correlation analysis of MdPH5 and different carbohydrates and acid substances revealed To clarify the trends of different metabolites in apple that MdPH5 expression was highly correlated with fruits after flowering, the contents of major carbohy- malate (R = 0.8787; P \ 0.05) (Fig. 1D). Unlike for drates and malate in apple fruits, 41, 70, 94, and 128 the acids, the contents of galactose, starch, sorbitol, days after blooming (DAB), were determined by gas maltose, and glucose had low correlations with the chromatography–mass spectrometry (Fig. S1A). These expression of MdPH5 (Fig. S4A–E). Although the corre- results showed that the malate content decreased lation between MdPH5 expression and fructose content gradually and regularly after flowering (Fig. 1A). The was relatively high (R = 0.8159) (Fig. S4F), unlike the levels of both galactose and sorbitol were minimized by correlation between MdPH5 expression and malate 70 DAB, whereas the level of starch was maximized by content, the correlation between them was negative. 70 DAB (Fig. S1B). These results indicated that MdPH5 is positively corre- A total of 12 samples, collected at 4 stages were lated with malate accumulation in apple plants. investigated, using transcriptome analysis. The number To clarify the impacts of MdPH5 on malate content, of upregulated and downregulated genes, in 41 DAB vs. during storage, Gala apple fruit stored for 0, 30, 60, 90, The Author(s) 2023 aBIOTECH Fig. 1 Correlation of MdPH5 expression with malate content in apple fruit during fruit development and storage. A Malate contents in apple fruit on different DAB. B Volcanic maps of malate-related genes in the 41 DAB vs. 70 DAB group. C The expression levels of MdPH5 on different DAB. D Correlation of the MdPH5 expression with malate content in apple at different developmental stages. E Malate contents at different storage periods. F Expression of MdPH5 in different storage periods determined by RT-qPCR. G Correlation of MdPH5 expression with malate content at different storage periods. Herein, letters indicate significant differences (P \ 0.05) among the culture media for the various parameters, based on the two-way ANOVA, followed by Duncan’s multiple range test. Bars are SE (n =3) and 120 days were employed as test samples. These As MdPH5 encodes a P-ATPase, pCaMV35S::MdPH5- results demonstrated that the malate content decreased GFP fusion vectors were developed and visualized by gradually with the extension of storage (Fig. 1E). Sub- transient expression in leaves of N. benthamiana, with sequently, RT-qPCR analysis of samples, at different pCaMV35S::GFP as a negative control, so as to determine post-harvest stages, showed that the expression of the subcellular localization of MdPH5. Herein, different MdPH5 also decreased gradually with increasing storage profiles of the vacuolar membrane-labeled red fluores- time (Fig. 1F), indicating a positive correlation of cence and the green fluorescence of the MdPH5-GFP MdPH5 expression with the malate content protein were observed. Additionally, the fluorescence (R = 0.8421; P \ 0.05) (Fig. 1G). signal of MdPH5-GFP overlapped with that of the AtCBL- labeled vacuolar membrane marker (Fig. 2D). There- Bioinformatics analysis and subcellular fore, it could be concluded that MdPH5 is localized localization of MdPH5 protein to the vacuolar membrane. The cDNA of MdPH5 was 2850 bp in full length and Role of MdPH5 in regulating malate encodes for 950 amino acids. As shown in Fig. 2A, the accumulation and vacuolar pH in apple cDNA of MdPH5 comprised three conserved domains. Herein, the secondary protein structure of MdPH5 was Transgenic calli with overexpression or silencing of predicted. The results showed that random coils MdPH5 were acquired to investigate the functions of the (69.88%), alpha-helices (30.00%), extended-strands gene (Fig. 3A, B). The expression of MdPH5 in the (17.05%), and beta-turns (5.58%) were dominant in the overexpression or silencing groups was significantly secondary protein structure of MdPH5 (Fig. 2B). higher and lower than that in the control group, A phylogenetic tree was established, using MEGA-X, respectively, indicating successful preparation of the to investigate the genetic correlation of MdPH5 with the gene overexpression or silencing materials (Fig. 3C). P subfamily ATPases in Arabidopsis thaliana. These Compared with the control group, the overexpression or 3A results indicated that MdPH5 (MD17G1155800) had the silencing groups exhibited increased and decreased closest genetic correlation with At1G17260 (Fig. 2C). malate contents (Fig. 3D), respectively, indicating that MdPH5 favors malate accumulation in calli. The Author(s) 2023 aBIOTECH Fig. 2 Bioinformatics analysis of the MdPH5 gene and subcellular localization of the MdPH5 protein. A Conserved sequence of the MdPH5 gene; a1 refers to ATPase-N, a2 refers to E1-E2_ ATPase, and a3 refers to hydrolase. B Predicted secondary structures of the MdPH5 protein. The numbers denote the length of amino acids. C Phylogenetic tree of the MdPH5 protein and ATPase of the Arabidopsis thaliana P subfamily. D Subcellular localization of the MdPH5 protein with a AtCBL tonoplast marker. Herein, lines and boxes highlight the 3A position of vacuoles exhibiting green and red fluorescence, respectively. 35S::GFP refers to the control group, scale bar = 10 lm, and B represents a bright field The effects of MdPH5 on vacuolar pH were investi- the average malate content in the MdPH5-IL60 group 0 0 gated on the basis of BCECF [2 ,7 -Bis(2-carboxyethyl)- increased by 0.671 mg/g. Similarly, the average malate 5(6)-carboxyfluorescein], which is a ratiometric fluo- content in the MdPH5-TRV group decreased by rescent pH indicator. The average vacuolar pH value of 0.670 mg/g, which was consistent with that in WT apple calli was 3.66, whereas that of MdPH5 over- the transgenic calli (Fig. 3I). expression or silencing apple calli was 3.96 and 3.41, respectively (Fig. 3E, F). Overall, it could be inferred Role of MdMYB73 in MdPH5 expression that MdPH5 regulates malate accumulation and vacuolar pH in apple calli. Previous studies have shown that some TFs can directly To further verify the effects of MdPH5 on malate, the activate the expression of several vacuolar proton pump expression of MdPH5 in apple was tuned by a virus- subunit genes (Hu et al. 2016, 2017). To elucidate the vector-based transformation method. Two virus con- transcriptional regulatory mechanism of MdPH5 in structors (MdPH5-IL60 and MdPH5-TRV) were injected apple, cis-acting element analysis of its promoter was into the fruit by agrobacterium transformation, with conducted and the results demonstrated the presence of empty vectors as the control (Fig. 3G). The expression abundant MYB-binding cis elements in the MdPH5 pro- level of MdPH5 was aligned with expectation by a 7-day moter. This may be attributed to the presence of some incubation in darkness (Fig. 3H). Then the malate con- MYB TFs that are located upstream of MdPH5 and reg- tent in the MdPH5-IL60 group was measured and ulate its expression. After that, the MYB TFs interacting compared with that in the control group. As indicated, with MdPH5 were identified by yeast one hybrid assays. The Author(s) 2023 aBIOTECH Fig. 3 Functions of MdPH5. A, B Transgenic callus materials obtained. C The gene expression of MdPH5 in the wild-type (WT) and MdPH5 transgenic apple calli (overexpression and antisense) determined by RT-qPCR. D Malate contents in MdPH5-OVX and MdPH5-RNAi transgenic apple calli. E Emission intensities of protoplast vacuoles in WT and transgenic calli MdPH5-OVX and MdPH5-RNAi loaded with wit20,70-bis-(2-carbox-yethyl)-5-(6)-carboxyfluorescein at 488 nm (first column) and 458 nm (second column). The pseudo-color scale on the right indicates the fluorescence intensity. Scale bar = 10 lm. Lowercase letters indicate significant differences at P \ 0.05. All values are the mean ± SD of three independent replicates. F Quantification of the luminal pH in vacuoles of WT and transgenic calli MdPH5-OVX and MdPH5-RNAi. Error bars represent the SE of 5 measurements from 30 individual intact vacuoles. Lowercase letters indicate significant differences at P \ 0.05. G Apple fruit injected with plasmid mixtures (IL60: IL60-1 ? IL60-2; MdPH5-IL60: IL60- 1 ? MdPH5-IL60-2). An empty IL60 vector was used as the control group. A solution containing agrobacterium cells (TRV: TRV1 ? TRV2; MdPH5-TRV: TRV1 ? MdPH5-TRV2) was injected into the apple tissue, with an empty TRV vector serving as the control group. Scale bar = 2 cm. Measured expression levels of MdPH5 (H) and malate contents (I) at the injection sites. Significant differences are indicated by the use of lowercase letters if P \ 0.05. All values are the mean ± SD (n = 5) These results demonstrated that MdMYB73, which was the MYB-binding element in other three regions was not previously identified as a major TF regulating malate, is enriched (Fig. 4A, B). The results provided in vivo evi- indeed a candidate partner. dence for the binding of MdMYB73 to the MdPH5 Chromatin immunoprecipitation (ChIP)-PCR assays promoter. were employed to investigate the in vivo binding of An electrophoretic mobility shift assay (EMSA) was MdMYB73 to the MdPH5 promoter. Herein, performed to investigate the in vitro binding of 35S::MdMYB73-GFP and 35S::GFP transgenic apple cul- MdMYB73 to the MdPH5 promoter. As observed, tures were used. The MYB-binding site element (CAA- MdMYB73 bound to the MdPH5 promoter fragments CAG) in Region S4 of the MdPH5 promoter was enriched containing the CAACAG motifs, and the level of a specific in the 35S::MdMYB73-GFP transgenic cultures, though DNA-MdMYB73 protein complex decreased with the The Author(s) 2023 aBIOTECH Fig. 4 MdMYB73 regulates malate accumulation by binding to MdPH5. A The putative MYB-binding element on the MdPH5 promoters; the numbers represent the integrated position. B ChIP-qPCR results indicating the enrichments of the target gene promoters in the 35S::MdMYB73-GFP transgenic apple seedlings compared with the 35S::GFP transgenic apple seedlings. C Results of electrophoretic mobility shift assays of the interaction between MdMYB73 and labeled DNA probes in the MdPH5 promoters. Lane 1 of each blot shows the labeled DNA probes without the MdMYB73 protein; lane 2 shows the labeled DNA probes and the MdMYB73 protein without a competitor. Different amounts (9 5 and 9 10) of unlabeled DNA fragments were added as cold competitors. D Results of LUC activity assays indicating that MdMYB73 enhances the basal activity of the MdPH5 promoters. E The expression level of MdPH5 in MdMYB73 transgenic apple seedlings. A significant difference is indicated by distinct lowercase letters at P \ 0.05. All values are the mean ± SD (n =3) increase in the number of unlabeled MYB-competing complexes were not observed if the CAACAG motif was probes with the same sequence. However, these changed to the AAAAAA motif (Fig. 4C). These results The Author(s) 2023 aBIOTECH provided in vitro evidence for the binding of MdMYB73 that in the injection area of MdPH5-TRV, but slightly to the MdPH5 promoter. lower than that in the injection area of the control The luciferase (LUC) transactivation assay was group. In other words, MdPH5-TRV may partially offset employed to clarify the effects of MdMYB73 on the the effects of MdMYB73-IL60 in the injection area of activity of the MdPH5 promoter. As indicated, promoter- MdMYB73-IL60 ? MdPH5-TRV (Fig. 5D). In summary, LUC reporter plasmids were expressed, transiently, in MdMYB73 is genetically upstream of MdPH5 in the the leaves of N. benthamiana, via transfection with regulation of malate accumulation in apple fruit. Agrobacterium tumefaciens strain GV3101. The infli- trated leaves containing the sequences of MdPH5 pro- moter showed higher LUC activity (Fig. 4D). In DISCUSSION summary, these findings support the notion that MdPH5 transcription is activated by MdMYB73. Acidity is one of the most important quality-related In addition, the expression levels of MdMYB73 and features of apple, which directly affects fruit flavor and MdPH5 in 35S::MdMYB73-GFP transgenic apple plantlets quality. Apple contains abundant organic acids, among were investigated. As indicated, the expression levels of which malate is the dominant species, as it accounts for both genes in the transgenic apple plantlets were sig- more than 90% of the total organic acids (Yu et al. nificantly higher than those in the control group 2021). Widely distributed in various plant tissues and (Fig. 4E). organs, malate has various important functions in the life cycle of plants (Fernie et al. 2004; Noguchi and Role of MdMYB73 as an upstream gene of MdPH5 Yoshida 2008; Sweetman et al. 2009; Bai et al. 2015;Hu in regulating malate accumulation et al. 2017; Dong et al. 2018). Vacuolar proton pumps and malate transporters are essential key factors influ- To clarify the regulatory role of MdMYB73 and MdPH5 in encing the transport of malate into vacuoles (Terrier malate accumulation, four virus builders (IL60 ? TRV, et al. 2001; Bai et al. 2015;Maetal. 2015, 2019;Hu MdPH5-TRV, MdMYB73-IL60, and MdPH5-TRV ? et al. 2016). In this study, MdPH5, a P-type ATPase, was MdMYB73-IL60) were used for fruit injection tests shown to promote vacuolar acidification and malate (Fig. 5A). The results of RT-qPCR showed that, com- accumulation in apple calli and fruit. As an upstream pared with IL60 ? TRV, the relative transcription levels MYB TF of MdPH5, MdMYB73 binds to the MdPH5 of MdMYB73 and MdPH5 were consistent with expec- promoter and activates its expression, thereby facili- tations (Fig. 5B, C) and the transcription level of MdPH5 tating malate accumulation. In addition, the expression decreased with downregulated expression of MdMYB73. of MdPH5 was positively correlated with malate content, Subsequently, the malate content was determined. As at both the developmental and post-harvest stages. indicated, the malate concentration in the injection area Vacuolar proton pumps have significant effects on of MdMYB73-IL60 was higher than that in the control fruit acidity, as they can deliver H to acidifying vac- group, whereas that in the injection area of MdPH5-TRV uoles, thereby facilitating the accumulation of organic was lower than that in the injection area of the control acids (Emmerlich et al. 2003). As MdPH5 acidifies apple group. Additionally, the malate content in the injection vacuoles, its homolog PhPH5 acidifies petal cell vac- area of MdMYB73-IL60 ? MdPH5-TRV was higher than uoles. Nevertheless, PhPH5 affects petal color rather Fig. 5 Verification of the upstream and downstream relationship of MdMYB73 and MdPH5 genes. A Injected apples kept in darkness for a week. Scale bar = 2 cm. B, C Expression of related genes in apple fruit after injection. D Malate contents in different groups. Error bars represent the SE of five measurements from at least independent biological replicates The Author(s) 2023 aBIOTECH than the accumulation of organic acids (Faraco et al. 2014). P-type ATPases promote the accumulation of organic acids and play an important role in fruits. Ma10, a candidate gene for the acidity of apple fruit, encodes a P -type ATPase proton pump that promotes the 3A transport of malate into vacuoles and vacuole acidifi- cation (Ma et al. 2019). In pears, the PH5 gene also promotes malate accumulation (Song et al. 2021). Overall, these genes exhibit similar functions to MdPH5 in apple. However, downregulated expression of CitPH5 led to the formation of some low-acid varieties of some fruits, including lemon, orange, pummelo, and rangpur lime (Strazzer et al. 2019). Despite that citric acid is the dominant organic acid in these fruits, this study focused on the effects of MdPH5 on malate accumulation, but not citric acid accumulation in apple. In addition, the cis element of MdPH5 promoter was further analyzed using Fig. 6 Working model showing that MdMYB73 binds to the the PlantCARE. As shown in Supplemental Table 2, there MdPH5 promoter to regulate malate accumulation in apple are many cis elements involved in light responsiveness, drought inducibility, and hormone responsiveness (jas- MdMYB73 can also simultaneously regulate both types monic acid, salicylic acid, and auxin). These findings of proton pumps. further suggest that the MdPH5-mediated malate Besides MYB TFs, WRKY, bHLH, ERF, and other TFs metabolism may be induced by various signals, such as are also involved in the regulation of organic acid plant hormones, stress, and light. accumulation and vacuole acidification. An InDel in the Malate content is subjected to the synergistic effects SlALMT9 promoter disrupts a W-box binding site, of environmental factors, developmental and metabolic thereby preventing the binding of the WRKY TFs (Ye signaling pathways, and corresponding TFs. TFs can et al. 2017). In apple, MdbHLH3 (bHLH TF) directly regulate malate accumulation either individually or in regulates the expression of MdcyMDH, which is a the form of complexes (Etienne et al. 2013;Huetal. cytosolic malate dehydrogenase gene, to mediate car- 2016, 2017;Li etal. 2020). In apple, the transcription bohydrate allocation and malate accumulation (Yu et al. activities of malate transporters and proton pumps can 2021). CitERF13 (citrus TF) regulates citrate accumu- be significantly tuned by regulating MdMYB1, lation by directly activating CitVHA-c4, which is a vac- MdMYB44, and MdMYB73, which can effectively regu- uolar proton pump gene (Li et al. 2016). Also, the late malate accumulation and vacuolar acidification (Hu formation of MBW complexes can directly regulate the et al. 2016, 2017; Jia et al. 2021). However, MdMYB1 expression of downstream acid-related target genes. As and MdMYB73 are positive regulators, whereas reported, MdbHLH3, MdbHLH49, and MdCIbHLH1 MdMYB44 is a negative regulator, and they have dif- interact with MdMYB1, MdMYB44, and MdMYB73, ferent downstream target genes. Indeed, the direct respectively, and enhance the activities of corresponding downstream target genes of MdMYB1 are MdVHA-B1, MYB TFs to further regulate the activities of down- MdVHA-E, MdVHF1, and MdtDT, whereas MdMYB44 stream genes, including malate transporters and vac- inhibits the promoter activity of MdVHA-A3, MdVHA-D2, uolar proton pumps (Xie et al. 2012; Hu et al. Ma10, and MdALMT9. In contrast, MdMYB73 directly 2016, 2017; Jia et al. 2021). Therefore, there may be activates the expression of MdVHA-A, MdVHP1, and more complex regulatory relationships in addition to MdALMT9. In this study, MdPH5 was identified to be the proposed MdMYB73-MdPH5 malate regulatory another downstream target gene of MdMYB73. Simi- pathway. In addition, the cis element of MdMYB73 pro- larly, MdMYB73 could regulate MdPH5 to promote moter was further analyzed using the PlantCARE. As malate accumulation (Hu et al. 2016, 2017; Jia et al. shown in Supplemental Table 3, there are many cis 2021;Fig. 6). Notably, MdMYB44 has been demon- elements involved in light responsiveness and drought strated to regulate both V-ATPase and P-ATPase genes inducibility. These results further suggest that the pro- (Jia et al. 2021). To date, only a few downstream posed MdMYB73-MdPH5 malate regulatory pathway V-ATPase target genes for MdMYB73 TF have been may be induced by light and drought signals. reported. MdPH5 is a P -type ATPase, suggesting that 3A The Author(s) 2023 aBIOTECH Fruit quality is one of the most concerned features in References fruit breeding. It has been demonstrated that organic Amato A, Cavallini E, Walker AR, Pezzotti M, Bliek M, Quattrocchio acids significantly affect fruit flavor. Malate is one of the F (2019) The MYB5-driven MBW complex recruits a WRKY most important organic acids in apple, while the genetic factor to enhance the expression of targets involved in basis for its accumulation in apple vacuoles remains vacuolar hyper-acidification and trafficking in grapevine. unclear (Wu et al. 2007; Zhang et al. 2012). In this study, Plant J 99:1220–1241. https://doi.org/10.1111/tpj.14419 Bai Y, Dougherty L, Cheng L, Zhong GY, Xu K (2015) Uncovering co- MdPH5, a P -type vacuolar proton pump, was 3A expression gene network modules regulating fruit acidity in demonstrated to promote malate accumulation and diverse apples. BMC Genom 16:612. https://doi.org/10. vacuolar acidification, and MdMYB73, an upstream MYB 1186/s12864-015-1816-6 TF of MdPH5, can facilitate malate accumulation by Cavallini E, Zenoni S, Finezzo L, Guzzo F, Zamboni A, Avesani L (2014) Functional diversification of grapevine MYB5a and binding to its promoter and activating its expression MYB5b in the control of flavonoid biosynthesis in a petunia (Fig. 6). This study provides a new method for acidity anthocyanin regulatory mutant. Plant Cell Physiol regulation of apple fruit, and may facilitate the devel- 55:517–534. https://doi.org/10.1093/pcp/pct190 opment of new varieties with improved flavor and Cohen S, Itkin M, Yeselson Y, Tzuri G, Portnoy V, Harel-Baja R (2014) The PH gene determines fruit acidity and contributes stress resistance. to the evolution of sweet melons. Nat Commun 5:4026. https://doi.org/10.1038/ncomms5026 Supplementary InformationThe online version contains Dong H, Bai L, Zhang Y, Zhang G, Mao Y, Min L, Xiang FY, Qian DD, supplementary material available at https://doi.org/10.1007/ Zhu XH, Song CL (2018) Modulation of guard cell turgor and s42994-023-00115-7. drought tolerance by a peroxisomal acetate-malate shunt. Mol Plant 11:1278–1291. https://doi.org/10.1016/j.molp.2018. Acknowledgements We would like to thank Prof. Takaya Mor- 07.008 iguchi of National Institute of Fruit Tree Science, Japan, for ‘Orin’ Emmerlich V, Linka N, Reinhold T, Ekkehard Neuhaus H (2003) apple calli. This work was supported by grants from the National The plant homolog to the human sodium/dicarboxylic Key Research and Development Program of China cotransporter is the vacuolar malate carrier. Proc Natl Acad (2022YFD2100102), National Natural Science Foundation of Sci USA 100:11122–11126. https://doi.org/10.1073/pnas. China (32122080, 31972375), and Shandong Province (ZR2020YQ25). Etienne A, Genard M, Lobit P, Mbeguie AMD, Bugaud C (2013) What controls fleshy fruit acidity? A review of malate and Author contributions DGH and QS conceived, designed, and citrate accumulation in fruit cells. J Exp Bot 64:1451–1469. supervised the project. XYH, YX, YWZ, CKW, JHW, WYW, and XLL https://doi.org/10.1093/jxb/ert035 performed the experiments and analyzed the data. DGH and XYH Faraco M, Spelt C, Bliek M, Verweij W, Hoshino A, Espen L, Prinsi B, wrote the paper. All authors discussed the results and commented Jaarsma R, Tarhan E, Sansebastiano GPD et al (2014) on the manuscript. Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower Data availability The data that support the findings of this study color. Cell Rep 6:32–43. https://doi.org/10.1016/j.celrep. are available from the corresponding author upon reasonable 2013.12.009 request. 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Plant Mol Biol Rep 8:87–99. https://doi.org/10.1016/j.mito.2007.09.003 30:539–546 Pedersen CN, Axelsen KB, Harper JF, Palmgren MG (2012) Evolution of plant p-type ATPases. Front Plant Sci 3:31. https://doi.org/10.3389/fpls.2012.00031 The Author(s) 2023 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png aBIOTECH Springer Journals

Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification

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Abstract

aBIOTECH https://doi.org/10.1007/s42994-023-00115-7 aBIOTECH RESEARCH ARTICLE Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification 1 1 1 1 Xiao-Yu Huang , Ying Xiang , Yu-Wen Zhao , Chu-Kun Wang , 1 1 1 1& Jia-Hui Wang , Wen-Yan Wang , Xiao-Long Liu , Quan Sun , 1& Da-Gang Hu National Research Center for Apple Engineering and Technology, Shandong Collaborative Innovation Center of Fruit and Vegetable Quality and Efficient Production, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong, China Received: 16 June 2023 / Accepted: 17 August 2023 Abstract As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH. Keywords Apple, Malate accumulation, P-ATPase, MdPH5, MdMYB73 INTRODUCTION et al. 2013). Additionally, it can also relieve angioscle- rosis, facilitate absorption of calcium, iron, and other Organic acids affect the acidity of fleshy fruits and play elements, and stimulate secretion by digestive glands. an important role in the regulation of osmotic pressure, In fruit cells, malate is mainly synthesized in the pH homeostasis, stress resistance, and sensory quality cytoplasm, via phosphoenolpyruvate carboxylation of fruits (Huang et al. 2021). Malate is one of the most (PEP), and is typically catalyzed by phosphoenolpyru- important organic acids in fruits, and accounts for 90% vate carboxylase (PEPC) and malate dehydrogenase of total organic acids in apple. Malate can generate ATP, (MDH) (Sweetman et al. 2009). Malate is then trans- resist oxidation, and relieve oxidative stress (Etienne ported into vacuoles for storage. Although malate accumulation in cells is controlled by both metabolism and vacuolar storage, the transport process from Xiao-Yu Huang and Ying Xiang contributed equally to this work. the cytoplasm into vacuoles may have a determining effect on malate accumulation. Therefore, it is necessary & Correspondence: sunquan@sdau.edu.cn (Q. Sun), to thoroughly investigate this transport process. As a fap_296566@163.com (D.-G. Hu) The Author(s) 2023 aBIOTECH matter of fact, this process involves multiple vacuolar can affect the transcriptional activity of malate trans- transporters, ion channels and carriers, among which porters and proton pumps to regulate malate accumu- vacuolar transporters and channels play a dominant lation and vacuolar acidification (Hu et al. 2016, 2017; role in the transport of organic acids, such as Jia et al. 2021). CrMYB73, which is homologous to the tonoplast dicarboxylate transporter (tDT) (Emmer- MdMYB73, leads to increased citrate accumulation in lich et al. 2003) and an aluminium-activated malate citrus plants, whereas its downstream target genes transporter (ALMT) (such as ALMT6 and ALMT9/Ma1) remain unclear (Li et al. 2015). In addition, PhPH4 in (Emmerlich et al. 2003; Meyer et al. 2011; Cohen et al. petunia is an R2R3-MYB TF that plays a similar role to 2014; Martinoia 2018). In addition, some proton pumps VvMYB5a and VvMYB5b in grape for the regulation of on the tonoplast also play a key role in this process by citrate accumulation. Specifically, they both can activate transporting H into vacuoles and acidifying vacuoles. expression of the downstream genes PH1 and PH5, thus In this way, the proton electrochemical gradients that acidifying vacuoles (Cavallini et al. 2014; Kasajima et al. serve as the driving force for the transport of malate 2016; Amato et al. 2019). In soybean petals, GmPH4, into vacuoles are generated. which is an R2R3-MYB TF, is also involved in vacuolar To date, three types of proton pumps, including acidification as it directly regulates the expression of ? ? vacuolar H -ATPase (V-ATPase), vacuolar H -PPase (V- GmPH5,aP -type ATPase gene (Sundaramoorthy et al. 3A PPase), and vacuolar P-type ATPase (P-ATPase), have 2020). However, the specific regulators of MdPH5 in been identified in fruits (Hurth et al. 2005; Kovermann apple have not yet been identified. et al. 2007; Martinoia et al. 2007). As a novel proton In this study, the role of MdPH5 in regulating malate pump thoroughly investigated in recent years, the P- accumulation and vacuolar acidification in apple was ATPase was first identified in petunia and may be investigated and its transcriptional regulation by MYB associated with vacuole acidification. In plants, TF MdMYB73 was verified. This study provides a ref- P-ATPase consists of five major evolutionarily related erence for understanding the molecular factors involved subfamilies (P1-P5). Among them, ATPases in the P3 in fruit quality. subfamily are responsible for energizing the electro- chemical gradient serving as the driving force of sec- ondary transport (Pedersen et al. 2012). The P-type MATERIALS AND METHODS ATPases (PhPH5 and PhPH1) found in petunia are involved in the vacuolar acidification of petal cells, Plant materials and growth conditions whereas only PhPH5 shows independent proton trans- port activity (Verweij et al. 2008; Faraco et al. 2014;Li ‘Orin’ apple calli obtained from young embryos were et al. 2016). Moreover, their homologous genes were subcultured at 3-week intervals on MS medium sup- also identified in fruits. In citrus, the homologous gene, plemented with 1.5 mg/L 2,4-dichlorophenoxyacetic CitPH5, is closely related to the generation of super- acid (2,4-D) and 0.4 mg/L 6-benzylaminopurine (6-BA) acidified fruit (Strazzer et al. 2019). In pear, PbPH5 is at 25 C, in the dark. Subsequently, these calli were localized to the vacuolar membrane and can promote subcultured, at half-month intervals, three times before malate accumulation in fruits (Song et al. 2022). As a being used for further studies. homologue of PhPH5 in apple, MdPH5 is slightly ‘Royal Gala’ apples were collected at 41, 70, 94, upregulated in transgenic MdMYB73 callus, which is 128 days post-flowering. ‘Royal Gala’ apple were col- responsible for malate accumulation and vacuolar lected at 120 days post-flowering and stored in air- acidification (Hu et al. 2017), suggesting that MdPH5 conditioned tanks. Batch of apples of similar size, color, may also be associated with malate accumulation in maturity, disease-free, insect-free, and without apple. Nevertheless, the specific function of MdPH5 in mechanical damage were selected for storage for apple fruits remains unclear. 120 days, and samples were taken every 30 days. These The regulation of organic acid transporters and pro- apples were frozen in liquid nitrogen. ton pumps involves complex gene regulatory networks. Indeed, transcriptional regulation is one of the most Bioinformatics analysis of the MdPH5 gene common events. Among them, MYB transcription factor (TF) families comprise plant-specific R2R3-MYB TFs, The basic information of the MdPH5 sequence was many of which are involved in the transport of organic obtained from the NCBI database (https://www.ncbi. acids by activating or inhibiting gene expression of nlm.nih.gov/). The MdPH5 secondary structure predic- transporters and proton pumps. Remarkably, MdMYB1, tion was adopted from SOPMA (https://npsaprabi.ibcp. MdMYB44, and MdMYB73, which are present in apple, fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The Author(s) 2023 aBIOTECH Phylogenetic analysis of PH5 proteins Viral vector-mediated transient expression in apple skins The MEGA_X based on the neighbor-joining method and bootstrap analysis with 1000 replications was used to Apple skin injection assays were performed as descri- construct the phylogenetic tree of MdPH5 and Ara- bed previously. MdPH5-TRV (TRV1 ? MdPH5-TRV2) bidopsis thaliana P subfamily. was suppression expression vector. MdPH5-IL60 (IL60- 3A 1 ? MdPH5-IL60-2) was overexpression vector, Analysis of the MdPH5 promoter MdMYB73-IL60 and so on. IL60 (IL60-1 ? IL60-2) and TRV (TRV1 ? TRV2) were empty vectors and used as The cis element in the MdPH5 promoter (1500 bp references. MdPH5-TRV2 ? MdMYB73-IL60 was mixed upstream of the transcription initiation site) was ana- with MdPH5-TRV (TRV1 ? MdPH5-TRV2) and lyzed with the online software PlantCARE (http://bioin MdMYB73-IL60 (IL60-1 ? MdMYB73-IL60-2) and formatics.psb.ugent.be/webtools/plantcare/html/). injected into apple fruit. IL60 ? TRV (IL60-1 ? IL60- 2 ? TRV1 ? TRV2) was empty vector and used as Construction of the MdPH5 gene expression reference. vector and genetic transformation of apple calli Measurement of vacuolar pH The ORF sequence of MdPH5 was cloned into pRI-101 to obtain the overexpression vector, and the non-con- Isolation of protoplasts from apple calli and measure- served regions of MdPH5’s ORF were reversely cloned ment of vacuolar pH were carried out, as previously into pRI-101 to obtain the antisense vector. Transgenic described by Hu et al. (2016). The vacuolar pH was apple calli were obtained by Agrobacterium-mediated detected with the cell-permeant and pH-sensitive fluo- 0 0 transformation. rescent dye, 2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluo- rescein (BCECF)-AM, while vacuolar pH was quantified Quantitative real-time-PCR (RT-qPCR) analysis by the ratio of pH-dependent (488 nm) and pH-inde- pendent (458 nm) excitation wavelengths from a cali- Plant RNA was extracted with an RNA extraction kit bration curve, and ratio images were generated with the (TIANGEN, Beijing, China) and single-stranded cDNA ion concentration tool of Zeiss LSM confocal software. was obtained with a reverse transcription kit (TaKaRa, Shiga, Japan). The RT-qPCR analyses were executed with Determination of malate content three biological and technical replications to test the expression levels of MdPH5, which were performed with Malate content was measured by high-performance the methods as described by Hu et al. (2016). The liquid chromatography, as previously described by Hu -DDCT quantitative analysis of results used the 2 method. et al. (2016). Analysis of subcellular localization EMSA The full-length coding sequences of MdPH5 were fused EMSA was conducted according to Xie et al. (2012). to the GFP protein, to construct the fusion expression MdMYB73 was cloned into the expression vector vector 35S:MdPH5-GFP, and the resulting plasmid was pGEX4T-1. The MdMYB73-GST recombinant protein was transformed into Agrobacterium strain LBA3101. The expressed in Escherichia coli strain BL21. An oligonu- constructed vector was injected into tobacco (Nicotiana cleotide probe of the MdMYB73 promoter was labeled benthamiana) epidermal cells and cultured in the dark using an EMSA probe biotin labeling kit (Beyotime) for 3 days. The AtCBL-red fluorescent protein (RFP) was according to the manufacturer’s instructions. The used as a vacuolar membrane marker (Ma et al. 2019) recombined protein of MdMYB73-GST was incubated and was co-transformed with 35S:MdPH5-GFP. Fluores- with 10 9 binding buffer, 1 lg/lL poly (dI-dC), and 400 fmol of biotin-labeled double-stranded binding consen- cence images were obtained at 488 nm with a high- resolution laser confocal microscope (LSM880, Zeiss, sus oligonucleotides (total volume 20 lL) using a Meta, Jena, Germany). LightShift Chemiluminescent EMSA Kit (Thermo Scien- tific). The binding reaction was performed at room temperature for 20 min. The DNA–protein complexes were separated on 6.5% non-denaturing polyacrylamide gels, electrotransferred, and detected following the The Author(s) 2023 aBIOTECH manufacturer’s instructions. The binding specificity was 70 DAB, 70 DAB vs. 94 DAB, and 94 DAB vs. 128 DAB, also examined by competition with a fold excess of were 1746 and 4426, 1289 and 1876, and 2435 and unlabeled oligonucleotides. 4939, respectively (Fig. S2A). Figures S2B and S2C are Venn diagrams reflecting the overlap of differential LUC assay genes in different cases. As observed, the malate content decreased regularly and the expression of genes that The apple MdPH5 promoter fragments were amplified might be associated with malate showed changes by PCR and cloned into the pGreenII 0800-LUC vector to according to the transcriptomic data, as shown in the construct the LUC reporter vector (MdPH5pro-LUC). heat maps. Figure S2D shows the expression levels of 32 The full-length coding sequences of MdMYB73 were malate-related genes, at different developmental stages. cloned into the effector vector pGreenII 62-SK. Individ- According to the heat maps of the expression levels of ual combinations of reporter vectors and effector vec- malate-related genes, the malate content decreased tors were transformed into Agrobacterium strain significantly from 41 to 71 DAB. As observed, genes GV3101 cells alongside the pSOUP vector. The such as MD03G1155400, MD13G1044200, and Agrobacterium strains were used to tobacco (Nicotiana MD17G1155800 were significantly downregulated benthamiana) epidermal cells. A live-imaging apparatus (Fig. S3A). To visualize changes of malate-related genes was used to measure luminescence after 2 days. and identify genes with the highest correlation with malate, three volcano maps (41 DAB vs. 70 DAB, 41 DAB Chromatin immunoprecipitation qPCR analysis vs. 94 DAB, and 41 DAB vs. 128 DAB) were developed. The number of downregulated genes in the volcano 35S:MdMYB73-GFP and 35S::GFP transgenic apple cul- maps were 4, 6, and 6, respectively. Among these genes, tures were used for the ChIP-qPCR analysis. The anti- MD17G1155800 was the only malate-related gene pre- GFP antibody (Beyotime) was used for chromatin sent in all data sets (Fig. 1B; Fig. S3B, C). As malate was immunoprecipitation (ChIP), as described by Xie et al. significantly downregulated in 41 DAB vs. 70 DAB, 41 (2012). The resultant samples were used as templates DAB vs. 94 DAB, and 41 DAB vs. 128 DAB, for qPCR assay. MD17G1155800 may be positively correlated with malate. It has been reported that Md17G1155800 is a Data presentation and statistical analysis P-type proton pump (MdPH5), and its homologous gene, PbPH5, in petunia can acidify vacuoles and change The data obtained in this study were analyzed by DPS petal color (Faraco et al 2014). Software (Enfield, UK), with P \ 0.05 considered as indicative of significant differences. Correlation of MdPH5 expression with malate content during apple fruit ripening and post- harvest RESULTS To further clarify the role of MdPH5 in apple, the level of Analysis of transcriptome and multiple MdPH5 expression in the transcriptome dataset was metabolites in apple fruit at different monitored. The results showed that its expression level developmental stages decreased (Fig. 1C). Correlation analysis of MdPH5 and different carbohydrates and acid substances revealed To clarify the trends of different metabolites in apple that MdPH5 expression was highly correlated with fruits after flowering, the contents of major carbohy- malate (R = 0.8787; P \ 0.05) (Fig. 1D). Unlike for drates and malate in apple fruits, 41, 70, 94, and 128 the acids, the contents of galactose, starch, sorbitol, days after blooming (DAB), were determined by gas maltose, and glucose had low correlations with the chromatography–mass spectrometry (Fig. S1A). These expression of MdPH5 (Fig. S4A–E). Although the corre- results showed that the malate content decreased lation between MdPH5 expression and fructose content gradually and regularly after flowering (Fig. 1A). The was relatively high (R = 0.8159) (Fig. S4F), unlike the levels of both galactose and sorbitol were minimized by correlation between MdPH5 expression and malate 70 DAB, whereas the level of starch was maximized by content, the correlation between them was negative. 70 DAB (Fig. S1B). These results indicated that MdPH5 is positively corre- A total of 12 samples, collected at 4 stages were lated with malate accumulation in apple plants. investigated, using transcriptome analysis. The number To clarify the impacts of MdPH5 on malate content, of upregulated and downregulated genes, in 41 DAB vs. during storage, Gala apple fruit stored for 0, 30, 60, 90, The Author(s) 2023 aBIOTECH Fig. 1 Correlation of MdPH5 expression with malate content in apple fruit during fruit development and storage. A Malate contents in apple fruit on different DAB. B Volcanic maps of malate-related genes in the 41 DAB vs. 70 DAB group. C The expression levels of MdPH5 on different DAB. D Correlation of the MdPH5 expression with malate content in apple at different developmental stages. E Malate contents at different storage periods. F Expression of MdPH5 in different storage periods determined by RT-qPCR. G Correlation of MdPH5 expression with malate content at different storage periods. Herein, letters indicate significant differences (P \ 0.05) among the culture media for the various parameters, based on the two-way ANOVA, followed by Duncan’s multiple range test. Bars are SE (n =3) and 120 days were employed as test samples. These As MdPH5 encodes a P-ATPase, pCaMV35S::MdPH5- results demonstrated that the malate content decreased GFP fusion vectors were developed and visualized by gradually with the extension of storage (Fig. 1E). Sub- transient expression in leaves of N. benthamiana, with sequently, RT-qPCR analysis of samples, at different pCaMV35S::GFP as a negative control, so as to determine post-harvest stages, showed that the expression of the subcellular localization of MdPH5. Herein, different MdPH5 also decreased gradually with increasing storage profiles of the vacuolar membrane-labeled red fluores- time (Fig. 1F), indicating a positive correlation of cence and the green fluorescence of the MdPH5-GFP MdPH5 expression with the malate content protein were observed. Additionally, the fluorescence (R = 0.8421; P \ 0.05) (Fig. 1G). signal of MdPH5-GFP overlapped with that of the AtCBL- labeled vacuolar membrane marker (Fig. 2D). There- Bioinformatics analysis and subcellular fore, it could be concluded that MdPH5 is localized localization of MdPH5 protein to the vacuolar membrane. The cDNA of MdPH5 was 2850 bp in full length and Role of MdPH5 in regulating malate encodes for 950 amino acids. As shown in Fig. 2A, the accumulation and vacuolar pH in apple cDNA of MdPH5 comprised three conserved domains. Herein, the secondary protein structure of MdPH5 was Transgenic calli with overexpression or silencing of predicted. The results showed that random coils MdPH5 were acquired to investigate the functions of the (69.88%), alpha-helices (30.00%), extended-strands gene (Fig. 3A, B). The expression of MdPH5 in the (17.05%), and beta-turns (5.58%) were dominant in the overexpression or silencing groups was significantly secondary protein structure of MdPH5 (Fig. 2B). higher and lower than that in the control group, A phylogenetic tree was established, using MEGA-X, respectively, indicating successful preparation of the to investigate the genetic correlation of MdPH5 with the gene overexpression or silencing materials (Fig. 3C). P subfamily ATPases in Arabidopsis thaliana. These Compared with the control group, the overexpression or 3A results indicated that MdPH5 (MD17G1155800) had the silencing groups exhibited increased and decreased closest genetic correlation with At1G17260 (Fig. 2C). malate contents (Fig. 3D), respectively, indicating that MdPH5 favors malate accumulation in calli. The Author(s) 2023 aBIOTECH Fig. 2 Bioinformatics analysis of the MdPH5 gene and subcellular localization of the MdPH5 protein. A Conserved sequence of the MdPH5 gene; a1 refers to ATPase-N, a2 refers to E1-E2_ ATPase, and a3 refers to hydrolase. B Predicted secondary structures of the MdPH5 protein. The numbers denote the length of amino acids. C Phylogenetic tree of the MdPH5 protein and ATPase of the Arabidopsis thaliana P subfamily. D Subcellular localization of the MdPH5 protein with a AtCBL tonoplast marker. Herein, lines and boxes highlight the 3A position of vacuoles exhibiting green and red fluorescence, respectively. 35S::GFP refers to the control group, scale bar = 10 lm, and B represents a bright field The effects of MdPH5 on vacuolar pH were investi- the average malate content in the MdPH5-IL60 group 0 0 gated on the basis of BCECF [2 ,7 -Bis(2-carboxyethyl)- increased by 0.671 mg/g. Similarly, the average malate 5(6)-carboxyfluorescein], which is a ratiometric fluo- content in the MdPH5-TRV group decreased by rescent pH indicator. The average vacuolar pH value of 0.670 mg/g, which was consistent with that in WT apple calli was 3.66, whereas that of MdPH5 over- the transgenic calli (Fig. 3I). expression or silencing apple calli was 3.96 and 3.41, respectively (Fig. 3E, F). Overall, it could be inferred Role of MdMYB73 in MdPH5 expression that MdPH5 regulates malate accumulation and vacuolar pH in apple calli. Previous studies have shown that some TFs can directly To further verify the effects of MdPH5 on malate, the activate the expression of several vacuolar proton pump expression of MdPH5 in apple was tuned by a virus- subunit genes (Hu et al. 2016, 2017). To elucidate the vector-based transformation method. Two virus con- transcriptional regulatory mechanism of MdPH5 in structors (MdPH5-IL60 and MdPH5-TRV) were injected apple, cis-acting element analysis of its promoter was into the fruit by agrobacterium transformation, with conducted and the results demonstrated the presence of empty vectors as the control (Fig. 3G). The expression abundant MYB-binding cis elements in the MdPH5 pro- level of MdPH5 was aligned with expectation by a 7-day moter. This may be attributed to the presence of some incubation in darkness (Fig. 3H). Then the malate con- MYB TFs that are located upstream of MdPH5 and reg- tent in the MdPH5-IL60 group was measured and ulate its expression. After that, the MYB TFs interacting compared with that in the control group. As indicated, with MdPH5 were identified by yeast one hybrid assays. The Author(s) 2023 aBIOTECH Fig. 3 Functions of MdPH5. A, B Transgenic callus materials obtained. C The gene expression of MdPH5 in the wild-type (WT) and MdPH5 transgenic apple calli (overexpression and antisense) determined by RT-qPCR. D Malate contents in MdPH5-OVX and MdPH5-RNAi transgenic apple calli. E Emission intensities of protoplast vacuoles in WT and transgenic calli MdPH5-OVX and MdPH5-RNAi loaded with wit20,70-bis-(2-carbox-yethyl)-5-(6)-carboxyfluorescein at 488 nm (first column) and 458 nm (second column). The pseudo-color scale on the right indicates the fluorescence intensity. Scale bar = 10 lm. Lowercase letters indicate significant differences at P \ 0.05. All values are the mean ± SD of three independent replicates. F Quantification of the luminal pH in vacuoles of WT and transgenic calli MdPH5-OVX and MdPH5-RNAi. Error bars represent the SE of 5 measurements from 30 individual intact vacuoles. Lowercase letters indicate significant differences at P \ 0.05. G Apple fruit injected with plasmid mixtures (IL60: IL60-1 ? IL60-2; MdPH5-IL60: IL60- 1 ? MdPH5-IL60-2). An empty IL60 vector was used as the control group. A solution containing agrobacterium cells (TRV: TRV1 ? TRV2; MdPH5-TRV: TRV1 ? MdPH5-TRV2) was injected into the apple tissue, with an empty TRV vector serving as the control group. Scale bar = 2 cm. Measured expression levels of MdPH5 (H) and malate contents (I) at the injection sites. Significant differences are indicated by the use of lowercase letters if P \ 0.05. All values are the mean ± SD (n = 5) These results demonstrated that MdMYB73, which was the MYB-binding element in other three regions was not previously identified as a major TF regulating malate, is enriched (Fig. 4A, B). The results provided in vivo evi- indeed a candidate partner. dence for the binding of MdMYB73 to the MdPH5 Chromatin immunoprecipitation (ChIP)-PCR assays promoter. were employed to investigate the in vivo binding of An electrophoretic mobility shift assay (EMSA) was MdMYB73 to the MdPH5 promoter. Herein, performed to investigate the in vitro binding of 35S::MdMYB73-GFP and 35S::GFP transgenic apple cul- MdMYB73 to the MdPH5 promoter. As observed, tures were used. The MYB-binding site element (CAA- MdMYB73 bound to the MdPH5 promoter fragments CAG) in Region S4 of the MdPH5 promoter was enriched containing the CAACAG motifs, and the level of a specific in the 35S::MdMYB73-GFP transgenic cultures, though DNA-MdMYB73 protein complex decreased with the The Author(s) 2023 aBIOTECH Fig. 4 MdMYB73 regulates malate accumulation by binding to MdPH5. A The putative MYB-binding element on the MdPH5 promoters; the numbers represent the integrated position. B ChIP-qPCR results indicating the enrichments of the target gene promoters in the 35S::MdMYB73-GFP transgenic apple seedlings compared with the 35S::GFP transgenic apple seedlings. C Results of electrophoretic mobility shift assays of the interaction between MdMYB73 and labeled DNA probes in the MdPH5 promoters. Lane 1 of each blot shows the labeled DNA probes without the MdMYB73 protein; lane 2 shows the labeled DNA probes and the MdMYB73 protein without a competitor. Different amounts (9 5 and 9 10) of unlabeled DNA fragments were added as cold competitors. D Results of LUC activity assays indicating that MdMYB73 enhances the basal activity of the MdPH5 promoters. E The expression level of MdPH5 in MdMYB73 transgenic apple seedlings. A significant difference is indicated by distinct lowercase letters at P \ 0.05. All values are the mean ± SD (n =3) increase in the number of unlabeled MYB-competing complexes were not observed if the CAACAG motif was probes with the same sequence. However, these changed to the AAAAAA motif (Fig. 4C). These results The Author(s) 2023 aBIOTECH provided in vitro evidence for the binding of MdMYB73 that in the injection area of MdPH5-TRV, but slightly to the MdPH5 promoter. lower than that in the injection area of the control The luciferase (LUC) transactivation assay was group. In other words, MdPH5-TRV may partially offset employed to clarify the effects of MdMYB73 on the the effects of MdMYB73-IL60 in the injection area of activity of the MdPH5 promoter. As indicated, promoter- MdMYB73-IL60 ? MdPH5-TRV (Fig. 5D). In summary, LUC reporter plasmids were expressed, transiently, in MdMYB73 is genetically upstream of MdPH5 in the the leaves of N. benthamiana, via transfection with regulation of malate accumulation in apple fruit. Agrobacterium tumefaciens strain GV3101. The infli- trated leaves containing the sequences of MdPH5 pro- moter showed higher LUC activity (Fig. 4D). In DISCUSSION summary, these findings support the notion that MdPH5 transcription is activated by MdMYB73. Acidity is one of the most important quality-related In addition, the expression levels of MdMYB73 and features of apple, which directly affects fruit flavor and MdPH5 in 35S::MdMYB73-GFP transgenic apple plantlets quality. Apple contains abundant organic acids, among were investigated. As indicated, the expression levels of which malate is the dominant species, as it accounts for both genes in the transgenic apple plantlets were sig- more than 90% of the total organic acids (Yu et al. nificantly higher than those in the control group 2021). Widely distributed in various plant tissues and (Fig. 4E). organs, malate has various important functions in the life cycle of plants (Fernie et al. 2004; Noguchi and Role of MdMYB73 as an upstream gene of MdPH5 Yoshida 2008; Sweetman et al. 2009; Bai et al. 2015;Hu in regulating malate accumulation et al. 2017; Dong et al. 2018). Vacuolar proton pumps and malate transporters are essential key factors influ- To clarify the regulatory role of MdMYB73 and MdPH5 in encing the transport of malate into vacuoles (Terrier malate accumulation, four virus builders (IL60 ? TRV, et al. 2001; Bai et al. 2015;Maetal. 2015, 2019;Hu MdPH5-TRV, MdMYB73-IL60, and MdPH5-TRV ? et al. 2016). In this study, MdPH5, a P-type ATPase, was MdMYB73-IL60) were used for fruit injection tests shown to promote vacuolar acidification and malate (Fig. 5A). The results of RT-qPCR showed that, com- accumulation in apple calli and fruit. As an upstream pared with IL60 ? TRV, the relative transcription levels MYB TF of MdPH5, MdMYB73 binds to the MdPH5 of MdMYB73 and MdPH5 were consistent with expec- promoter and activates its expression, thereby facili- tations (Fig. 5B, C) and the transcription level of MdPH5 tating malate accumulation. In addition, the expression decreased with downregulated expression of MdMYB73. of MdPH5 was positively correlated with malate content, Subsequently, the malate content was determined. As at both the developmental and post-harvest stages. indicated, the malate concentration in the injection area Vacuolar proton pumps have significant effects on of MdMYB73-IL60 was higher than that in the control fruit acidity, as they can deliver H to acidifying vac- group, whereas that in the injection area of MdPH5-TRV uoles, thereby facilitating the accumulation of organic was lower than that in the injection area of the control acids (Emmerlich et al. 2003). As MdPH5 acidifies apple group. Additionally, the malate content in the injection vacuoles, its homolog PhPH5 acidifies petal cell vac- area of MdMYB73-IL60 ? MdPH5-TRV was higher than uoles. Nevertheless, PhPH5 affects petal color rather Fig. 5 Verification of the upstream and downstream relationship of MdMYB73 and MdPH5 genes. A Injected apples kept in darkness for a week. Scale bar = 2 cm. B, C Expression of related genes in apple fruit after injection. D Malate contents in different groups. Error bars represent the SE of five measurements from at least independent biological replicates The Author(s) 2023 aBIOTECH than the accumulation of organic acids (Faraco et al. 2014). P-type ATPases promote the accumulation of organic acids and play an important role in fruits. Ma10, a candidate gene for the acidity of apple fruit, encodes a P -type ATPase proton pump that promotes the 3A transport of malate into vacuoles and vacuole acidifi- cation (Ma et al. 2019). In pears, the PH5 gene also promotes malate accumulation (Song et al. 2021). Overall, these genes exhibit similar functions to MdPH5 in apple. However, downregulated expression of CitPH5 led to the formation of some low-acid varieties of some fruits, including lemon, orange, pummelo, and rangpur lime (Strazzer et al. 2019). Despite that citric acid is the dominant organic acid in these fruits, this study focused on the effects of MdPH5 on malate accumulation, but not citric acid accumulation in apple. In addition, the cis element of MdPH5 promoter was further analyzed using Fig. 6 Working model showing that MdMYB73 binds to the the PlantCARE. As shown in Supplemental Table 2, there MdPH5 promoter to regulate malate accumulation in apple are many cis elements involved in light responsiveness, drought inducibility, and hormone responsiveness (jas- MdMYB73 can also simultaneously regulate both types monic acid, salicylic acid, and auxin). These findings of proton pumps. further suggest that the MdPH5-mediated malate Besides MYB TFs, WRKY, bHLH, ERF, and other TFs metabolism may be induced by various signals, such as are also involved in the regulation of organic acid plant hormones, stress, and light. accumulation and vacuole acidification. An InDel in the Malate content is subjected to the synergistic effects SlALMT9 promoter disrupts a W-box binding site, of environmental factors, developmental and metabolic thereby preventing the binding of the WRKY TFs (Ye signaling pathways, and corresponding TFs. TFs can et al. 2017). In apple, MdbHLH3 (bHLH TF) directly regulate malate accumulation either individually or in regulates the expression of MdcyMDH, which is a the form of complexes (Etienne et al. 2013;Huetal. cytosolic malate dehydrogenase gene, to mediate car- 2016, 2017;Li etal. 2020). In apple, the transcription bohydrate allocation and malate accumulation (Yu et al. activities of malate transporters and proton pumps can 2021). CitERF13 (citrus TF) regulates citrate accumu- be significantly tuned by regulating MdMYB1, lation by directly activating CitVHA-c4, which is a vac- MdMYB44, and MdMYB73, which can effectively regu- uolar proton pump gene (Li et al. 2016). Also, the late malate accumulation and vacuolar acidification (Hu formation of MBW complexes can directly regulate the et al. 2016, 2017; Jia et al. 2021). However, MdMYB1 expression of downstream acid-related target genes. As and MdMYB73 are positive regulators, whereas reported, MdbHLH3, MdbHLH49, and MdCIbHLH1 MdMYB44 is a negative regulator, and they have dif- interact with MdMYB1, MdMYB44, and MdMYB73, ferent downstream target genes. Indeed, the direct respectively, and enhance the activities of corresponding downstream target genes of MdMYB1 are MdVHA-B1, MYB TFs to further regulate the activities of down- MdVHA-E, MdVHF1, and MdtDT, whereas MdMYB44 stream genes, including malate transporters and vac- inhibits the promoter activity of MdVHA-A3, MdVHA-D2, uolar proton pumps (Xie et al. 2012; Hu et al. Ma10, and MdALMT9. In contrast, MdMYB73 directly 2016, 2017; Jia et al. 2021). Therefore, there may be activates the expression of MdVHA-A, MdVHP1, and more complex regulatory relationships in addition to MdALMT9. In this study, MdPH5 was identified to be the proposed MdMYB73-MdPH5 malate regulatory another downstream target gene of MdMYB73. Simi- pathway. In addition, the cis element of MdMYB73 pro- larly, MdMYB73 could regulate MdPH5 to promote moter was further analyzed using the PlantCARE. As malate accumulation (Hu et al. 2016, 2017; Jia et al. shown in Supplemental Table 3, there are many cis 2021;Fig. 6). Notably, MdMYB44 has been demon- elements involved in light responsiveness and drought strated to regulate both V-ATPase and P-ATPase genes inducibility. These results further suggest that the pro- (Jia et al. 2021). To date, only a few downstream posed MdMYB73-MdPH5 malate regulatory pathway V-ATPase target genes for MdMYB73 TF have been may be induced by light and drought signals. reported. MdPH5 is a P -type ATPase, suggesting that 3A The Author(s) 2023 aBIOTECH Fruit quality is one of the most concerned features in References fruit breeding. It has been demonstrated that organic Amato A, Cavallini E, Walker AR, Pezzotti M, Bliek M, Quattrocchio acids significantly affect fruit flavor. Malate is one of the F (2019) The MYB5-driven MBW complex recruits a WRKY most important organic acids in apple, while the genetic factor to enhance the expression of targets involved in basis for its accumulation in apple vacuoles remains vacuolar hyper-acidification and trafficking in grapevine. unclear (Wu et al. 2007; Zhang et al. 2012). In this study, Plant J 99:1220–1241. https://doi.org/10.1111/tpj.14419 Bai Y, Dougherty L, Cheng L, Zhong GY, Xu K (2015) Uncovering co- MdPH5, a P -type vacuolar proton pump, was 3A expression gene network modules regulating fruit acidity in demonstrated to promote malate accumulation and diverse apples. BMC Genom 16:612. https://doi.org/10. vacuolar acidification, and MdMYB73, an upstream MYB 1186/s12864-015-1816-6 TF of MdPH5, can facilitate malate accumulation by Cavallini E, Zenoni S, Finezzo L, Guzzo F, Zamboni A, Avesani L (2014) Functional diversification of grapevine MYB5a and binding to its promoter and activating its expression MYB5b in the control of flavonoid biosynthesis in a petunia (Fig. 6). This study provides a new method for acidity anthocyanin regulatory mutant. Plant Cell Physiol regulation of apple fruit, and may facilitate the devel- 55:517–534. https://doi.org/10.1093/pcp/pct190 opment of new varieties with improved flavor and Cohen S, Itkin M, Yeselson Y, Tzuri G, Portnoy V, Harel-Baja R (2014) The PH gene determines fruit acidity and contributes stress resistance. to the evolution of sweet melons. Nat Commun 5:4026. https://doi.org/10.1038/ncomms5026 Supplementary InformationThe online version contains Dong H, Bai L, Zhang Y, Zhang G, Mao Y, Min L, Xiang FY, Qian DD, supplementary material available at https://doi.org/10.1007/ Zhu XH, Song CL (2018) Modulation of guard cell turgor and s42994-023-00115-7. drought tolerance by a peroxisomal acetate-malate shunt. Mol Plant 11:1278–1291. https://doi.org/10.1016/j.molp.2018. Acknowledgements We would like to thank Prof. Takaya Mor- 07.008 iguchi of National Institute of Fruit Tree Science, Japan, for ‘Orin’ Emmerlich V, Linka N, Reinhold T, Ekkehard Neuhaus H (2003) apple calli. This work was supported by grants from the National The plant homolog to the human sodium/dicarboxylic Key Research and Development Program of China cotransporter is the vacuolar malate carrier. Proc Natl Acad (2022YFD2100102), National Natural Science Foundation of Sci USA 100:11122–11126. https://doi.org/10.1073/pnas. China (32122080, 31972375), and Shandong Province (ZR2020YQ25). Etienne A, Genard M, Lobit P, Mbeguie AMD, Bugaud C (2013) What controls fleshy fruit acidity? A review of malate and Author contributions DGH and QS conceived, designed, and citrate accumulation in fruit cells. J Exp Bot 64:1451–1469. supervised the project. XYH, YX, YWZ, CKW, JHW, WYW, and XLL https://doi.org/10.1093/jxb/ert035 performed the experiments and analyzed the data. DGH and XYH Faraco M, Spelt C, Bliek M, Verweij W, Hoshino A, Espen L, Prinsi B, wrote the paper. All authors discussed the results and commented Jaarsma R, Tarhan E, Sansebastiano GPD et al (2014) on the manuscript. Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower Data availability The data that support the findings of this study color. Cell Rep 6:32–43. https://doi.org/10.1016/j.celrep. are available from the corresponding author upon reasonable 2013.12.009 request. Fernie AR, Carrari F, Sweetlove LJ (2004) Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron trans- Declarations port. Curr Opin Plant Biol 7:254–261. https://doi.org/10. 1016/j.pbi.2004.03.007 Conflict of interest The authors declare that they have no con- Hu DG, Sun CH, Ma QJ, You CX, Cheng L, Hao YJ (2016) MdMYB1 flict of interest. regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples. Plant Open Access This article is licensed under a Creative Commons Physiol 170:1315–1330. https://doi.org/10.1104/pp.15. Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or for- Hu DG, Li YY, Zhang QY, Li M, Sun CH, Yu JQ, Hao YJ (2017) The mat, as long as you give appropriate credit to the original R2R3-MYB transcription factor MdMYB73 is involved in author(s) and the source, provide a link to the Creative Commons malate accumulation and vacuolar acidification in apple. licence, and indicate if changes were made. The images or other Plant J 91:443–454. https://doi.org/10.1111/tpj.13579 third party material in this article are included in the article’s Huang XY, Wang CK, Zhao YW, Sun CH, Hu DG (2021) Mechanisms Creative Commons licence, unless indicated otherwise in a credit and regulation of organic acid accumulation in plant vacuoles. line to the material. 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Journal

aBIOTECHSpringer Journals

Published: Dec 1, 2023

Keywords: Apple; Malate accumulation; P-ATPase; MdPH5; MdMYB73

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