Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2022 Oct 22;191(1):414–427. doi: 10.1093/plphys/kiac491

Low-acidity ALUMINUM-DEPENDENT MALATE TRANSPORTER4 genotype determines malate content in cultivated jujube

Chunmei Zhang 1,#, Yanqiu Geng 2,#, Hanxiao Liu 3, Mengjia Wu 4, Jingxin Bi 5, Zhongtang Wang 6, Xiaochang Dong 7, Xingang Li 8,✉
PMCID: PMC9806563  PMID: 36271866

Abstract

Jujube (Ziziphus jujuba Mill.), the most economically important fruit tree in Rhamnaceae, was domesticated from sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). During domestication, fruit sweetness increased and acidity decreased. Reduction in organic acid content is crucial for the increase in sweetness of jujube fruit. In this study, the determination of malate content among 46 sour jujube and 35 cultivated jujube accessions revealed that malate content varied widely in sour jujube (0.90–13.31 mg g−1) but to a lesser extent in cultivated jujube (0.33–2.81 mg g−1). Transcriptome sequencing analysis showed that the expression level of Aluminum-Dependent Malate Transporter 4 (ZjALMT4) was substantially higher in sour jujube than in jujube. Correlation analysis of mRNA abundance and fruit malate content and transient gene overexpression showed that ZjALMT4 participates in malate accumulation. Further sequencing analyses revealed that three genotypes of the W-box in the promoter of ZjALMT4 in sour jujube associated with malate content were detected, and the genotype associated with low malate content was fixed in jujube. Yeast one-hybrid screening showed that ZjWRKY7 binds to the W-box region of the high-acidity genotype in sour jujube, whereas the binding ability was weakened in jujube. Transient dual-luciferase and overexpression analyses showed that ZjWRKY7 directly binds to the promoter of ZjALMT4, activating its transcription, and thereby promoting malate accumulation. These findings provide insights into the mechanism by which ZjALMT4 modulates malate accumulation in sour jujube and jujube. The results are of theoretical and practical importance for the exploitation and domestication of germplasm resources.

As the target gene of transcription factor ZjWRKY7, the variation of W-box in the promoter of ZjALMT4 determines malate accumulation in wild jujube and its cultivar fruits.

Introduction

Jujube (Ziziphus jujuba Mill.) is the most economically important tree species in the Rhamnaceae and is a traditional fruit tree cultivated in China for more than 7,000 years (Qu and Wang, 1993). Jujube fruits are of high nutritional value and, in particular, the contents of soluble sugar, ascorbic acid, cyclic nucleotide, triterpenic acid, iron, and zinc are higher than those of many other fruits (Liu et al., 2020). Jujube was domesticated from wild/sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). The greatest difference in flavor between the fruit of jujube and sour jujube is that jujube fruits are high in sugar content and taste sweet, whereas sour jujube fruits taste very sour. Organic acids, collectively responsible for acidity, are important components that affect fruit flavor and consumer preference. Therefore, the mechanism of organic acid accumulation in jujube and sour jujube fruit is considered to be an important determinant of fruit quality and pivotal in the domestication of jujube from sour jujube.

Malate is an important organic acid that affects plant growth and development and is involved in the regulation of pH balance in the cytoplasm and stress responses (Hurth et al., 2005; Saito and Matsuda, 2010; Ye et al., 2017). Most of the malate accumulated by plant cells is transported to the vacuole for storage. In fruit crops, malate affects fruit quality and flavor, such as in apple (Malus pumila Mill.), tomato (Solanum lycopersicum L.), and strawberry (Fragaria × ananassa (Weston) Duchesne ex Rozier) (Ye et al., 2017; Hu et al., 2017). In jujube, malic acid is the predominant organic acid in the fruit (Gao et al., 2012), and the malate content of sour jujube fruit is several times higher than that of cultivated jujube (Zhao et al., 2016; Huang et al., 2016).

Malate accumulation is affected by synthesis, degradation, and transport. Previous studies have shown that Nicotinamide Adenine Dinucleotide (NAD)-dependent malate dehydrogenase (MDH) promotes the transformation of oxaloacetic acid to malic acid in the mitochondria or cytoplasm (Miller et al., 1998; Yu et al., 2021); the Nicotinamide Adenine Dinucleotide Phosphate (NADP)-dependent MDH in chloroplasts converts oxaloacetate to malate using NADPH as a cofactor (Scheibe, 2004). Phosphoenolpyruvate carboxylase is a similarly crucial enzyme in malate synthesis that promotes oxaloacetic acid accumulation. In the cytoplasm, the NADP-dependent malic enzyme is an important malate-degrading enzyme that catalyzes the conversion of malate to pyruvate (Beruter, 2004; Yao et al., 2009). In addition, malate accumulation is determined by intracellular transport between the vacuole and cytoplasm (Sweetman et al., 2009; Etienne et al., 2013).

The tonoplast dicarboxylic acid transporter (tDT), one type of malate transporter, was identified in Arabidopsis (Arabidopsis thaliana) and is involved in the transport of malic acid between the vacuole and cytoplasm, as well as in the regulation of pH balance in the cytoplasm and stress responses (Hurth et al. 2005). In addition to tDT, aluminum-dependent malate transporters (ALMTs) are involved in transmembrane transport of organic acids and many participate in malate accumulation in the fruit (Rongala, 2008; Xu et al., 2018; Chen et al., 2019). Thirteen ALMTs have been identified in Arabidopsis, among which AtALMT4, AtALMT5, AtALMT6, and AtALMT9 are localized to the tonoplast (Kovermann et al., 2007). Of these ALMTs, AtALMT6 and AtALMT9 are considered to participate in malic acid transport to the vacuole (Kovermann et al., 2007; Meyer et al., 2011). In tomato, SlALMT4 and SlALMT5 are expressed during fruit development and overexpression of SlALMT5 alters the organic acid content in the seed (Sasaki et al., 2016). SlALMT9 is considered to be responsible for the increase in malate content in the fruit during tomato domestication (Ye et al., 2017). Apple Ma1 (ALMT9) is a crucial malate transporter that is responsible for differences in malic acid content in the fruit between wild and cultivated accessions (Ma et al., 2015). In apricot fruit, the downregulated expression of ALMT9 at the ripening stage is strongly associated with flavor enhancement (Sakr et al., 2018). In addition, vacuolar proton pumps, which provide energy for organic acid transport (Shiratake and Martinoia, 2007), have been reported involved in malate accumulation (Hu et al., 2016; Ma et al., 2019).

In recent decades, several transcription factors have been shown to participate in malate synthesis and transport. In Arabidopsis, WRKY46 functions as a transcriptional repressor of ALMT1 to regulate aluminum-induced malate secretion (Ding et al., 2013). In tomato fruit, SlWRKY42 is considered to negatively regulate malate accumulation (Ye et al., 2017). Apple basic helix–loop–helix protein MdbHLH3 regulates the transcription of cytoplasm MDH (MdcyMDH) and thereby influences malate accumulation (Yu et al., 2021). In addition, R2R3 MYB transcription factors MdMYB73 and MdMYB44 directly bind to the cis-elements of MdVHA-A, MdVHP1, and MdALMT9 to modulate vacuolar acidification (Hu et al., 2017; Jia et al., 2021). Apple MdWRKY126 modulates malate accumulation in the fruit by regulating a cytosolic MDH encoded by MdMDH5 (Zhang et al., 2022). In the previous studies of jujube and sour jujube, several domesticated candidate genes associated with organic acid accumulation were identified based on genome sequence analysis (Huang et al., 2016; Shen et al., 2021; Zhang et al. 2021). However, the molecular mechanism of malate accumulation in fruit of jujube and sour jujube has not been elucidated.

In this study, we investigated the contribution of ZjALMT4 to malate accumulation in jujube and sour jujube fruit. A single-nucleotide polymorphism (SNP) between jujube and sour jujube was detected in the cis-element binding site (W-box) in the promoter of ZjALMT4, which influenced the expression level of the gene. In addition, ZjWRKY7 regulated the expression of ZjALMT4 by binding to the W-box to modulate malate accumulation. The present findings provide insights into the molecular changes that affected fruit quality during domestication of jujube.

Results

Malate content and pH of cultivated jujube and sour jujube fruit

The malate content was measured at five critical stages of fruit development in cultivated jujube “Jinsi No. 4” and sour jujube “Taiansuanzao” (Figure 1, A and B). In sour jujube, malate accumulated sharply from the white mature stage and the highest content (15.5 mg g−1) was attained at the half-red stage. In cultivated jujube, malate accumulation was not detected during fruit ripening. The final malate content in ripe fruit of cultivated jujube “Jinsi No. 4” was ˂1/12 in ripe fruit of sour jujube “Taiansuanzao.” To confirm the difference in malate content, fruit from 35 cultivated jujube and 46 sour jujube accessions were sampled at the half-red stage to measure malate accumulation (Figure 1C). The malate content of cultivated jujube fruit ranged from 0.33 to 2.81 mg g−1 fresh weight (FW) with an average of 1.54 mg g−1 FW. The fruit of most sour jujube accessions were high in malate with contents ranging from 3.20 to 13.31 mg g−1 FW, but in several accessions, the malate content ranged from 0.90 to 3.06 mg g−1 FW. The average content of the high-malate sour jujube accessions was as much as 7.51 mg g−1 FW and that of low-malate accessions was 2.16 mg g−1 FW. In addition, we measured fruit pH (Figure 1D) for 68 individuals using the same samples. The average pH of sour jujube was 3.61, which differed significantly from that of cultivated jujube (mean pH 4.81). Considering all individuals tested, the Pearson correlation coefficient between malate content and pH value was −0.70 (P < 0.01) (Figure 1E).

Figure 1.

Figure 1

Open in a new tab

Malate content in fruit of sour jujube and cultivated jujube. Phenotypic characteristics (A) and malate accumulation (B) of sour jujube and cultivated jujube fruit at five developmental stages. Y, young stage; E, enlargement stage; W, white mature stage; R, red stage; FR, full red stage. The fruit images of cultivated jujube (up) and sour jujube (below) in (A) were digitally extracted for comparison. Values in (B) are presented as the mean ± sd on the basis of three biological replicates. C, Malate content in fruit of 35 cultivated jujube and 46 sour jujube accessions. Values are presented as the mean ± sd on the basis of three biological replicates. Asterisks mean the samples used for transcriptome sequencing. D, pH values of the cultivated jujube and sour jujube fruits. Each data was from three biological replicates with three technical repetitions. Significant differences between two populations were detected by one-way analysis of variance (ANOVA). **P < 0.01. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. E, Correlation analysis of malate content and pH.

Expression of ZjAlMT4 is positively correlated with malate accumulation in jujube fruit

To investigate the genetic basis of malate accumulation in jujube and its wild progenitor sour jujube, samples from five sour jujube accessions and five cultivated jujube accessions (Figure 1C) were selected for RNA-sequencing (RNA-seq) to identify the crucial genes associated with malate accumulation. A total of 205.33 × 109 clean reads were generated from 30 RNA libraries with the effective data volume for each sample ranging from 5.70 × 109 to 7.95 × 109. By comparing reads with the jujube “Junzao” reference genome, 88.12%–92.86% (and 77.8% in one library) of the clean reads were aligned. Comparison of the transcriptome between the jujube cultivars and sour jujube accessions was conducted to identify differentially expressed genes (DEGs). A total of 5,692 DEGs were detected, comprising 4,130 upregulated genes and 1,562 downregulated genes (Supplemental Table S1). Among these DEGs, the genes associated with malate accumulation were selected to generate a heat map (Figure 2A). Notably, one ALMT gene (ZjALMT4, Zj.jz000565069) was significantly upregulated in the sour jujube accessions and thus may be associated with malate accumulation in the fruit. Concomitantly, one V-type proton ATPase subunit F (Zj.jz034597015) and a pyrophosphate-energized vacuolar membrane proton pump 1 (Zj.jz023027019) exhibited higher Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) values in sour jujube than in the cultivars, which may provide increased energy for malic acid transport.

Figure 2.

Figure 2

Open in a new tab

Identification of ZjALMT4 association with malate accumulation in jujube and sour jujube. A, Hierarchical clustering of genes associated with malate accumulation among five cultivated jujube and five sour jujube accessions based on RNA-seq analysis. Log2FC means Log2foldchange. B, Expression levels of ZjALMT4 in different tissues. C-fruit means the cultivated fruit; S-fruit means the sour jujube fruit. Values are presented as the mean ± sd on the basis of three biological replicates. Significant differences were detected by Student’s t test. Different letters indicate significant differences among different tissues at P < 0.05. C, Correlation analysis of malate content and ZjALMT4 mRNA abundance.

To gain insight into the nature of ZjALMT4 expression in cultivated jujube and sour jujube fruit, we first estimated the expression levels of ZjALMT4 in different tissues by reverse transcription–quantitative PCR (RT–qPCR). The transcripts were more abundant in the fruit, especially in sour jujube fruit, and were weakly expressed in the leaf, flower, root, and phloem (Figure 2B). In addition, we quantified the expression levels in the fruit of sour jujube and cultivated jujube accessions. The average expression level of ZjALMT4 in sour jujube was twice that in cultivated jujube accessions (Supplemental Figure S1). The ZjALMT4 mRNA levels were significantly correlated with malate content (Pearson’s r = 0.71, P < 0.01) (Figure 2C). Thus, ZjALMT4 expression was indicated to be positively correlated with malate accumulation in jujube fruit.

Overexpression of ZjALMT4 and the vacuolar membrane localization of its product

To clarify the effect of ZjALMT4 on malate accumulation, we generated the 35S::ZjALMT4 construct to transiently transform white mature fruit of jujube and strawberry. The expression level of ZjALMT4 in the overexpression group was substantially higher than that of the control in jujube fruit (Figure 3A). In strawberry, ZjALMT4 was only expressed in the transgenic group and expression was negligible in the control group (Figure 3B). The malate content was 1.6 and 1.4 times higher in ZjALMT4 transgenic jujube and strawberry fruit, respectively, than that in the corresponding control groups (Figure 3, C and D). These results confirmed that overexpression of ZjALMT4 increased malate accumulation.

Figure 3.

Figure 3

Open in a new tab

Transient overexpression of ZjALMT4. The relative expression levels of ZjALMT4 (A, B) and malate content around the injection sites in fruit of jujube and strawberry (C and D), respectively. Values are presented as the mean ± sd on the basis of three biological replicates. Significant differences were detected by Student’s t test. Different letters indicate significant differences at P < 0.05. MT-OE, ZjALMT4 over-expressed in positive vector; MT-CK, control vector without ZjALMT4. E, Subcellular colocalization of the ZjALMT4-GFP fusion protein with a AtCBL3 tonoplast marker. Arrows and boxes highlight the position of vacuoles exhibiting green and red fluorescence.

The subcellular localization of the pCaMV35S::ZjAlMT4-GFP fusion protein was visualized by transient expression in leaves of Nicotiana benthamiana. As emphasized with arrows and boxes (Figure 3E), we observed the distinct outline of the vacuolar membrane from the red fluorescence of the vacuolar membrane marker and the green fluorescence of the ZjALMT4-GFP fusion protein. The outline of the vacuolar membrane was not visible under bright-field optics. In the merged image, the ZjAlMT4-GFP fluorescence signal overlapped with that of the AtCBL3-labeled vacuolar membrane marker. Therefore, we concluded that ZjAlMT4 was localized to the vacuolar membrane.

Different genotypes in the W-box in the promoter of ZjALMT4

In the course of domestication of cultivated jujube, the malate content of the fruit decreased. We speculated that selective domestication of the associated genes had occurred. To dissect the genetic basis of malate accumulation, we accessed the selective-sweep data calculated by Shen et al. (2021), and observed that ZjALMT4 (Zijuj10G0147600) and ZjALMT9 (Zijuj10G0147500) were located in one selective region. In combination with the transcriptomic analysis results (Figure 2), ZjALMT4 was indicated to be the candidate gene involved in malate accumulation. Comparative sequence analysis revealed that the coding sequences of ZjALMT4 in sour jujube were consistent with those of cultivated jujube “Junzao” and “Dongzao” (Supplemental Figure S2).

To test whether the differences in transcript levels were regulated by natural variation in the promoter region, we sequenced 1,300 bp in the upstream (5′) promoter of ZjALMT4 for 18 cultivated jujube and 22 sour jujube accessions randomly. Small irregular insertions/deletions or base-pair substitutions were detected among the sequences. It was notable that, among these mutations, the SNP at position −1,054 bp resulted in variation of the W box (G-GTCAA, forward: TTGAC-C), which is known to be the WRKY transcription factor-binding site. In sour jujube, three genotypes were detected (G-GTCAA, C-GTCAA, and T-GTCAA), whereas in cultivated jujube only the genotype T-GTCAA was detected (Figure 4, A and B; Supplemental Figure S3). In combination with the malate content data, these results showed that the “G/C” genotype was associated with high malate content, whereas “T” genotype was associated with low malate content (Figure 4C; Supplemental Table S2).

Figure 4.

Figure 4

Open in a new tab

Identification of different genotypes of ZjALMT4 and interaction between ZjALMT4 and ZjWRKY7. A, Schematic diagram of the W-box sequence in different genotypes. B, The proportion of three genotypes in jujube and sour jujube, respectively. C, Fruit malate content in different genotypes of jujube and sour jujube. “TT,” homozygous genotype at position 1,054 bp upstream of the ZjALMT4 initiation codon; “TG/C,” heterozygous genotypes containing “TG” or “TC” at position 1,054-bp upstream of the ZjALMT4 initiation codon; “GG/CC/GC,” genotypes containing “G” or “C” at position 1,054-bp upstream of the ZjALMT4 initiation codon. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. D, Y1H assay of ZjWRKY7/20 binding to ZjALMT4 promoter fragments. The bait vectors S(C)ZjALMT4pro, S(G)ZjALMT4pro, and Z(T)ZjALMT4pro fragments containing the regions bordering the W-Box at position 1,054-bp upstream of the ZjALMT4 initiation codon with the C/G/T mutation, and the prey vector containing ZjWRKY7 or ZjWRKY20 were introduced into yeast strain Y1H. Yeast cells were grown on SD/–Leu medium supplemented with different concentrations of ABA (0, 50, 200, or 400 ng mL−1). The bait vectors (S(C)ZjALMT4 pro, S(G)ZjALMT4pro, and Z(T)ZjALMT4pro) + pGADT7 were transformed into Y1H as a negative control.

ZjWRKY7 positively regulates the expression of ZjALMT4

To functionally determine the major expression-regulating regions of ZjALMT4, a prey cDNA library was constructed for yeast one-hybrid (Y1H) screening. The 20-bp DNA fragments bordering the W-box from G-GTCAA, C-GTCAA, or T-GTCAA were fused separately to the pAbAi vector to construct the bait strain. The library vectors were cloned in the strains to screen the binding transcripts. Two WRKY transcription factors (ZjWRKY7 and ZjWRKY20) were screened from the prey strains of sour jujube. In addition, one-on-one bait–prey interactions were conducted on SD/−Leu/medium supplemented with different concentrations of aureobasidin A (ABA) (Figure 4D). The Y1H assays revealed that, when incubated with ZjWRKY20, yeast cells transformed with T-GTCAA grew similarly to those transformed with the G-GTCAA sequence with increase in ABA concentration, whereas yeast cells transformed with C-GTCAA grew more slowly and growth declined with increase in ABA concentration. When incubated with ZjWRKY7, yeast cells transformed with T-GTCAA grew more slowly and showed weaker activity than those transformed with G/C-GTCAA with increase in ABA concentration. Given that the fruit malate content of C-type sour jujube was similar to that of G-type sour jujube (Figure 4C), the binding of ZjWRKY7 was consistent with the malate content. We concluded that ZjWRKY7, rather than ZjWRKY20, was the crucial transcription factor responsible for the differential expression of ZjALMT4 between cultivated jujube and sour jujube.

To verify the interaction, DNA fragments from high-malate sour jujube and low-malate cultivated jujube accessions were fused to the firefly luciferase (LUC) gene to construct promoter–LUC reporter plasmids. Together with the ZjWRKY7/20 effector constructs, the promoter–LUC reporter plasmids were expressed transiently in N. benthamiana leaves through transfection with Agrobacterium tumefaciens strain GV3101. The infiltrated leaves harboring the sour jujube promoter sequences showed stronger LUC activity than the leaves infiltrated with the cultivated jujube promoter sequences. These results suggested that the G/C-GTCAA sequence is crucial for transcription of ZjALMT4 and is regulated by ZjWRKY7 (Figure 5).

Figure 5.

Figure 5

Open in a new tab

Binding of ZjWRKY7/20 to the promoter of ZjALMT4 from jujube (proZjALMT4) or sour jujube (proSZjALMT4) assayed using a transient dual-LUC system. The ZjWRKY7/20 open reading frame was cloned into the effector vector (pGreenII 62-SK), and the ZjALMT4 promoter fragments of sour jujube and cultivated jujube were inserted into the reporter vector (pGreenII 0800-LUC). Both effector and reporter vectors were transformed into Agrobacterium cells and used to infiltrate N. benthamiana leaves.

To further verify the interaction of ZjWRKY7 and ZjALMT4, we used a viral vector-based transformation method to modify the expression level of ZjWRKY7 in jujube. Two viral constructs, IL60-WRKY7 and TRV-WRKY7, were generated and infiltrated separately into jujube fruit; the empty vectors were used as controls. After incubation for 5 days, the expression levels of ZjWRKY7 and ZjALMT4 were quantified. In the group infiltrated with IL60-WRKY7, the expression level of ZjWRKY7 was 1.6 times higher than that of the control and, consistently, the abundance of ZjALMT4 transcripts was significantly increased. In addition, the malate content was double that of the control. Similarly, in the group infiltrated with TRV-WRKY7, consistent with the downregulation of ZjWRKY7 expression, the expression level of ZjALMT4 decreased significantly (Figure 6), the malate content was not detected. We sprayed exogenous malate solution (3%, w/v) onto jujube leaves, and the control group was treated with the same volume of distilled water. The expression of ZjWRKY7 was determined by RT–qPCR analysis. The ZjWRKY7 expression level increased by 60% in malate-treated leaves compared with that of the control group.

Figure 6.

Figure 6

Open in a new tab

ZjWRKY7 activates ZjALMT4 expression and promotes malate accumulation. RT-qPCR analysis of the relative expression levels of ZjWRKY7 (A) and ZjALMT4 (B) around the injection site of jujube fruit via ZjWRKY7-IL60 overexpression vector-based transformation. C, Malate content of the injected jujube fruit in (A). Transient expression of ZjWRKY7 (D) and ZjALMT4 (E) in the injected jujube fruit via ZjWRKY7-TRV antisense expression vector-based transformation. The empty vectors were used as controls. F, RT–qPCR analysis of the relative expression levels of ZjWRKY7 in response to spray application of 3% malate solution. Values are presented as the mean ± sd on the basis of three repetitions. Significant differences were detected by Student’s t test. Different letters indicate significant differences at P < 0.01.

Discussion

ZjALMT4 plays a crucial role in malate accumulation

Most currently cultivated fruit species have been artificially selected or domesticated from wild progenitors, which has often been accompanied by flavor improvement, such as decreased acidity and increased sweetness of the fruit (Fernie and Yan, 2019). Sour jujube, as the wild progenitor of cultivated jujube, is named as such because the fruit taste is very sour. Malate, as an important organic acid, affects the flavor of many fruits. In this study, the average malate content was more than 4 times higher in sour jujube fruit (7.51 mg g−1 FW) than that in cultivated jujube fruit (1.54 mg g−1 FW). Consistent with malate accumulation, the pH of sour jujube fruit was significantly lower than that of cultivated jujube. In addition, the malate content was significantly negatively correlated with the pH for all tested sour jujube and cultivated jujube fruits. Among all tested samples, the malate content varied widely in sour jujube (0.90–13.31 mg g−1) but to a lesser extent in cultivated jujube (0.33–2.81 mg g−1). For cultivated jujube, the loss of malate accumulation in fruit may be the result of natural selection or artificial domestication of a low-acid type of wild jujube.

Members of the ALMT protein family participate in fruit malate accumulation. For example, the apple homolog Ma1 (MdALMT9), tomato homolog SlALMT9, and PpALMT1 of peach (Prunus persica (L.) Batsch) control malate transport and accumulation in the fruit (Bai et al., 2012; Ye et al., 2017; Zhang et al., 2020; Yu et al., 2021). In this study, among the 12 putative orthologs of ALMTs identified in the public jujube genome database, using transcriptome sequence analysis of diverse sour jujube and cultivated jujube accessions, ZjALMT4 was identified as being associated with fruit malate accumulation. Furthermore, correlation analysis, tissue differential expression, and overexpression showed that ZjALMT4 increased malate content in the fruit. These results confirmed that ZjALMT4 contributed to malate accumulation. In addition, vacuolar proton pumps, including V-type ATPase and V-type pyrophosphorylase, hydrolyze ATP, and pyrophosphate transport H+ to drive vacuolar acidification, and at the same time provide energy for malic acid transport (Shiratake and Martinoia, 2007). In this study, the vacuolar proton pumps Zj.jz034597015 and Zj.jz023027019 had higher FPKM values in sour jujube fruit than in cultivated jujube fruit and thus would power greater ZjALMT4 transport activity in the former fruit.

Phylogenetic analysis revealed that ZjALMT4 is most closely related to MdALMT9/Ma1, and then to AtALMT4, AtALMT5, and AtALMT6 of Arabidopsis and SlALMT9 of tomato (Supplemental Figure S4). The proteins AtALMT4 and AtALMT6 are considered to play roles in guard cells to control the stomatal aperture (Meyer et al., 2011; Eisenach et al., 2017). Therefore, we speculate that the domains and functions of ZjALMT4 are conserved with those of MdALMT9 and SlALMT9, whereas the other functions reported for the model plant Arabidopsis should be studied further. Based on the results of composite-likelihood-ratio selection peaks in the jujube and sour jujube genomes (Shen et al., 2021), we observed that ZjALMT4 was located within the selected regions, which may have participated in jujube domestication. We speculate that similar to grain yield in graminaceous crops (Chen et al., 2022), ZjALMT4, as well as MdALMT9 and SlALMT9, experienced convergent selection for fruit malate content during domestication.

Genetic variation in the promoter of ZjALMT4 determines fruit malate content, and the low-acidity genotype of ZjALMT4 is fixed in cultivated jujube

Cis-acting elements upstream of structural genes regulate transcription efficiency through specific binding of the corresponding transcription factors (Kumar et al., 2017; Endo et al., 2018). Accordingly, genetic variation of the cis-element is usually responsible for the variation in related agronomic traits. In a study of tomato domestication, Ye et al. (2017) observed that a deletion in the W-box in the upstream promoter of SlALMT9 enhanced malic acid transport activity, leading to the enhancement of fruit flavor. Domestication and selection of the upstream promoter region of the Tonoplast Sugar Transporter ClTST2 is strongly associated with an increase in the sugar content in vacuoles of watermelon (Citrullus lanatus) pulp (Ren et al., 2018). In this study, we sequenced the upstream region of ZjALMT4 of 40 jujube and sour jujube accessions and detected stable linked-genetic variation at position −1,054 bp (G/C/T) associated with fruit malate accumulation. We suggest that natural variation in the promoter of ZjALMT4 is responsible for the variation in malate content between cultivated jujube and sour jujube.

Regarding the aforementioned variation in the ZjALMT4 promoter, all genotype combinations were detected in the sour jujube population, whereas only the T:T genotype was detected in the jujube population (Supplemental Table S2). This result was in accordance with the broad variation in malate accumulation in sour jujube fruit, which ranged from 0.90 to 13.31 mg g−1 FW, whereas cultivated jujube showed a narrow range from 0.33 to 2.81 mg g−1 FW. According to the genotype frequency from the population genetics (Nei and Kumar 2000), ZjALMT4 is subject to natural or domesticated selection. We speculate that in sour jujube the T-type arose as a result of mutation from some unknown factors and during subsequent domestication or genetic improvement the T:T genotype conferring low malate content was fixed in the cultivated species. This also explains why the genetic diversity of cultivated jujube is lower than that of wild relatives (Huang et al., 2016). However, inconsistent with this suggestion, the fruit malate content of sour jujube accessions with the T:T genotype was higher than that in cultivated jujube. Given that organic acid accumulation is not controlled by a single gene, additional genes (such as the vacuolar proton pumps Zj.jz034597015 and Zj.jz023027019) other than ZjALMT4 may participate in malate accumulation in sour jujube fruit.

ZjWRKY7 plays a crucial role in regulating the expression of ZjALMT4

The WRKY transcription factor subfamily participates in diverse biological processes, such as plant growth and development, secondary metabolism, and abiotic stress response, by binding to the W-box (C/TTGACC/T) in the promoter of the target gene to regulate gene expression. In Arabidopsis, WRKYs promote aluminum tolerance via activation of ALMT expression (Wang et al., 2020; Ding et al., 2013). Recent studies indicate that WRKY transcription factors play an essential role in regulating acidification. For example, Li et al. (2018) concluded that WRKY22 was involved in citrate secretion by regulating the expression of OsFRDL4 in rice (Oryza sativa L.). Ye et al. (2017) reported that SlWRKY42 can bind to the upstream promoter of SlALMT9 and alters malate accumulation in tomato fruit. Zhang et al. (2022) reported that in apple fruit MdWRKY126 activated cytosolic MDH to contribute to malate accumulation. In addition, PbWRKY40 from Pyrus betulaefolia functions positively in salt tolerance and modulates organic acid accumulation by regulating PbVHA-B1 expression (Lin et al., 2022). In this study, the SNP in the W-box of ZjALMT4 was associated with malate accumulation. Using a Y1H library screening system, we determined that ZjWRKY7 directly targets the variable region in the W-box cis-element in sour jujube. To confirm that ZjWRKY7 functions to regulate ZjALMT4, we performed transient dual-LUC, overexpression, and viral vector-based transfection experiments, all of which demonstrated that ZjWRKY7 positively regulated ZjALMT4 expression and altered malate accumulation in jujube fruit.

Many other transcription factors may also promote malate accumulation by activation of malate transporters. For example, MdCIbHLH1, MdMYB73, and MYB44 target MdALMT9 in apple (Hu et al., 2017; Zhang et al., 2020; Jia et al., 2021), and SENSITIVE TO PROTON RHIZOTOXICITY1 mediates aluminum-induced expression of ALMT1 in Arabidopsis (Tokizawa et al., 2021). We also detected MYB and MYC cis-elements (the binding sites of basic helix-loop-helix protein (bHLH) transcription factors) in the promoter of ZjALMT4 (Supplemental Figure S5), which may be associated with the regulation of malic acid accumulation. However, no relevant differences were detected in these sites between the jujube and sour jujube populations. Therefore, we speculate that ZjWRKY7 is involved in the regulation of malic acid accumulation in jujube and sour jujube.

Herein, we propose a hypothetical model to explain the molecular basis for the difference in fruit malate content between jujube and sour jujube (Figure 7). In high-malate sour jujube fruit, ZjWRKY7 can bind to the W-box cis-element in the promoter of ZjALMT4 to positively regulate its transcription and promote the accumulation of malic acid. Mutation of the W-box site weakens the binding ability of ZjWRKY7 and, as a result, reduces malic acid accumulation in the fruit. During subsequent domestication, this low-acid mutation was fixed in cultivated jujube.

Figure 7.

Figure 7

Open in a new tab

Working model for ZjALMT4 function in malate accumulation in jujube and sour jujube. The model proposes that the jujube transcription factor ZjWRKY7 activates transcription of ZjALMT4 by binding to the W-box in the promoter and increases the malate content of the fruit. When the W-box is mutated, the ability of ZjWRKY7 to bind to the promoter of ZjALMT4 is diminished and malate accumulation is reduced. In addition, WRKY7 is promoted by malate.

Materials and methods

Plant material

Fruit from cultivated jujube (Z. jujuba Mill.) “Jinsi No. 4” and sour jujube (Z. jujuba var. spinosa (Bunge) Hu ex H.F.Chow.) “Taiansuanzao” were collected at five developmental stages for determination of malate accumulation. In addition, leaves and ripe fruit from 35 cultivated jujube and 46 sour jujube accessions were collected for population analysis. All jujube cultivars and “Taiansuanzao” were sampled from the Jujube Germplasm Resource Nursery (36.15° N, 117.07° E) at the Shandong Fruit Research Institute, Tai’an, China. The other sour jujube samples were gathered from low mountains mostly in or around Shandong Province, China (Supplemental Table S2). The sampled fruits were cut into small pieces, immediately frozen in liquid nitrogen, and stored at −80°C for subsequent analysis. The sampled leaves were desiccated in silica gel for DNA extraction.

Identification and quantification of malic acid

Malic acid was extracted and determined essentially as described by Gao et al. (2011). For each sample, 0.5-g frozen tissue was added to 3 mL of 0.01-M monopotassium and directly ground with a precooled mortar. The homogenate was transferred to a 10-mL centrifuge tube, ultrasonicated for 20 min, and then centrifuged for 10 min at 12,000 rpm at 4°C. The supernatant was transferred to a new centrifuge tube, the residue was resuspended with monopotassium solution, ultrasonicated, and centrifuged as performed previously. The supernatant was diluted with 0.01-mL monopotassium to 10-mL volume and used for organic acid determination. All samples were extracted and analyzed in triplicate.

A high-performance liquid chromatography system with an ultraviolet detector, Chromeleon software (Dinex), and a reverse C18 column (5 µm × 250 mm) was used. The mobile phase comprised 5% methanol and 95% 0.01 ML monopotassium solution (v/v) (pH 2.7). The flux was set to 1 mL min−1 and the injection volume was 10 µL. The column temperature was set at 30°C. Spectra were acquired at wavelengths between 200 and 400 nm, and malic acid quantification was performed at 210 nm with three technical repetitions. The malic acid standard curve was generated using a standard with concentrations of 1–500 µg mL−1 using the same method. All samples were extracted and analyzed in triplicate.

Determination of fruit pH

For each sample, 0.5-g frozen mixed fruit tissue was added to 10-mL ddH2O and ground with a precooled mortar. The homogenate was transferred to a 10-mL centrifuge tube and allowed to stand at room temperature for 2 h. A PHB-1 portable pH meter was used to determine the pH of the homogenate. All samples were extracted and analyzed in triplicate.

RNA-seq and data analysis

Total RNA was extracted using a Plant RNA Extraction Kit (Foregene, Chengdu, China). The RNA was submitted to the Shanghai OE Biotechnology Company for the construction and sequencing of cDNA libraries as described in our previous study (Dong et al., 2022). Protein-coding gene expression was calculated on the basis of the reads per kilobase per million reads value. Three biological replicates were analyzed per sample. The genes differentially expressed between sour jujube and cultivated jujube were identified with DESeq software (Anders and Huber, 2010). Statistically significant differences were detected using the Benjamini–Hochberg adjusted P-values (P < 0.05). All sequence reads have also been deposited in the NCBI online database (PRJNA822549). The reference genome and gene annotation information of this study was from Huang et al. (2016) (http://dx.doi.org/10.5061/dryad.83fr7).

Correlation analysis between gene expression characteristics of ZjALMT4 and malate content

Expression levels of ZjALMT4 in jujube and sour jujube accessions were analyzed using RT–qPCR. Total RNA of each sample was extracted using a Plant RNA Extraction Kit (Foregene). Synthesis of cDNA and RT–qPCR were performed in accordance with our previous study (Zhang et al., 2018). All samples were extracted and analyzed in triplicate. The primers used are listed in Supplemental Table S3. Correlation analysis between the gene expression characteristics of ZjALMT4 and malate content was analyzed using SPSS version 16.0 software (SPSS, Chicago, IL, USA).

Protein subcellular localization

Vector construction followed the method described by Jiang et al. (2017). The coding sequence of ZjALMT4 was connected downstream of the CaMV35S promoter in the pVBG2300-GFP vector. The positive and control vector were transiently expressed in N. benthamiana leaves by Agrobacterium-mediated infiltration using strain GV3101. The pRT101-AtCBL3-red fluorescent protein (RFP) was used as a vacuole membrane marker and was cotransformed with CaMV35S-ZjALMT4-GFP. The N. benthamiana plants were incubated at 25°C for 48–72 h. The fluorescent protein signal was visualized using a high-resolution laser confocal microscope (ZEISS LSM710) at excitation wavelengths of 488 nm for GFP (laser, OPLS 488; intensity, 5%; gain, 100) and 520 nm for mCherry (laser, OPLS 552; intensity, 2%; gain, 800).

Cloning and sequencing of the ZjALMT4 promoter region of jujube and sour jujube

Total genomic DNA was extracted using a Plant DNA Extraction Kit (Takara, Dalian, China) in accordance with the manufacturer’s instructions from 18 cultivated jujube and 22 sour jujube randomly selected accessions. The primers were designed in accordance with the ZjALMT4 promoter sequence of ∼1,300 bp in the 5′-upstream region and are listed in Supplemental Table S3. The PCR was performed using Tks Gflex DNA Polymerase (Takara, Dalian, China) following the manufacturer’s instructions. The promoter sequences were then cloned into TA/Blunt-Zero vector to generate recombinant constructs, and transformed into Escherichia coli strain DH5a using a 5 min TA/Blunt-Zero Cloning Kit (Vazyme, Nanjing, China). The positive clones were sequenced and comparatively analyzed. The regions bordering the SNP sites in jujube and sour jujube were used for prediction of transcription factor binding sites using the PlantCare database (Lescot et al., 2002) (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Y1H assay

Total RNA was extracted from mixed fruit of jujube and sour jujube at the ripening stage. The cDNA was synthesized using the Make Your Own “Mate & Plate” Library System (Takara, Dalian, China)). A cDNA Normalization Kit (Clontech, Takara) was used to normalize the cDNA process. After fractional separation and purification, the cDNA was recombined with the linearized PGADT7-Rec vector to construct the library plasmids. The library plasmids were transformed into yeast (Saccharomyces cerevisiae) strain Y187 to construct the yeast activation domain (AD) library of samples.

Three tandem copies of the W-box motifs in the promoter (Supplemental Table S4) of ZjALMT4 from sour jujube and the SNP regions from cultivated jujube were inserted into the pAbAi plasmid to construct the pBait-AbAi vector. The recombined vector was transferred to yeast strain Gold1 competent cells and cultured on SD/−Ura medium. The bait strains were cultured on SD/−Ura medium supplemented with different concentrations of ABA to detect the minimum inhibitory concentration. The cDNA AD library vectors were transferred to yeast strains containing the pBait-AbAi vector and cultured on SD/−Leu/+ABA medium. After incubation for 3 days, the positive strains were picked and cultured again to eliminate false positives. The AD vectors from positive strains were sequenced to detect the binding transcripts. For one-on-one bait–prey interactions, the open reading frames of binding transcripts were cloned into the pGADT7 vector, transferred to yeast strain Gold1 competent cells containing the pBait-AbAi vector, and cultured on SD/−Leu/medium supplemented with different concentrations of ABA.

Transient dual-LUC assay

The ZjALMT4 promoter fragments of jujube and sour jujube were amplified by PCR and cloned into the pGreenII 0800-LUC vector to construct the LUC reporter vector (ZjALMT4pro-LUC) in accordance with the method of An et al. (2017). The full-length coding sequences of ZjWRKY7 and ZjWRKY20 were cloned into the effector vector pGreenII 62-SK. Individual combinations of reporter vectors and effector vectors were transformed into Agrobacterium strain GV3101 cells alongside the pSoup vector. The Agrobacterium strains were used to infiltrate N. benthamiana leaves. A live-imaging apparatus was used to measure luminescence after ∼2 days.

Construction of viral vectors and transient expression in jujube

The ZjWRKY7 open reading frame was inserted into the IL-60 vector to construct the overexpression vector IL-60-ZjWRKY7. The IL-60-ZjWRKY7 and IL-60 vectors were transformed into Agrobacterium strain GV3101 cells, and then injected into two sides of jujube fruit. The full-length of ZjWRKY7 open reading frame was cloned into the Tobacco rattle virus (TRV) vector to construct the antisense orientation vector TRV-ZjWRKY7 and then injected into jujube fruit; the TRV vector was used as the control. The injected jujube fruits were incubated for 5 days in an incubator under a 12-h/12-h (light/dark) photoperiod before gene expression analysis. Sequence information of the above genes/proteins is shown in Supplemental Table S5.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers PRJNA306374, with the gene models available in the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.83fr7. All RNA-seq data have been deposited in the NCBI online database (PRJNA822549).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. The relative expression level of ZjALMT4 in 46 sour jujube and 35 cultivated jujube accessions.

Supplemental Figure S2. Protein sequences of ZjALMT4 from sour jujube (KAH7516556.1) and cultivated jujube “Junzao” (Zj.jz000565069) and “Dongzao” (XP_015895669.1).

Supplemental Figure S3. Three promoter sequences of ZjALMT4 around the W-box region.

Supplemental Figure S4. Phylogenetic analysis of ZjALMT4 and homologous proteins from Malus × domestica, S. lycopersicum, and A. thaliana.

Supplemental Figure S5. Prediction of cis-acting elements in the ZjALMT4 promoter of jujube and sour jujube.

Supplemental Table S1. Summary of DEGs between five sour jujube and five cultivated jujube accessions based on RNA-seq.

Supplemental Table S2. Summary of 40 jujube accessions used for cloning and sequencing of the ZjALMT4 promoter.

Supplemental Table S3. Primer sequences used in this study.

Supplemental Table S4. Promoter sequences used for Y1H screening.

Supplemental Table S5. Sequences information of the major genes/proteins mentioned in this study.

Supplementary Material

kiac491_Supplementary_Data
Click here for additional data file. (1.9MB, zip)

Acknowledgments

Z.C. thanks her postdoctoral co-supervisor, Professor Hao Yu-jin, who passed away on 15 March 2021, for valuable advice on a draft of this manuscript. Professor Hao is the beacon of our scientific research road. We will always cherish him.

Funding

This work was supported by The National Key Research and Development Program of China (2018YFD1000607), the China Postdoctoral Science Foundation (Grant No. 2019M662416), the Natural Science Foundation of Shandong Province (ZR2019BC029), and The Introduction and Training Plan of Young Creative Talents at Universities in Shandong Province: Research Group of Forest Tree Biotechnology.

Conflict of interest statement. The authors declare no conflicts of interests.

Contributor Information

Chunmei Zhang, State Forestry and Grassland Administration Key Laboratory of Silviculture in downstream areas of the Yellow River, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Yanqiu Geng, State Forestry and Grassland Administration Key Laboratory of Silviculture in downstream areas of the Yellow River, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Hanxiao Liu, State Forestry and Grassland Administration Key Laboratory of Silviculture in downstream areas of the Yellow River, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Mengjia Wu, State Forestry and Grassland Administration Key Laboratory of Silviculture in downstream areas of the Yellow River, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Jingxin Bi, State Forestry and Grassland Administration Key Laboratory of Silviculture in downstream areas of the Yellow River, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Zhongtang Wang, Shandong Institute of Pomology, Tai’an, 271000, China.

Xiaochang Dong, Shandong Institute of Pomology, Tai’an, 271000, China.

Xingang Li, College of Forestry, Northwest A&F University, Yangling, China.

 

X.L. and C.Z. designed the project. Y.G., C.Z., H.L., and M.W. collected the samples and performed the experiments. C.Z., J.B., and X.L. analyzed the data. C.Z. and X.D. drafted the manuscript. Z.W., X.D., and X.L. discussed and modified the manuscript.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) are Xingang Li (xingangle@nwsuaf.edu.cn) and Chunmei Zhang (zhangcm2017@sdau.edu.cn).

References

  1. An JP, Qu FJ, Yao JF, Wang XN, You CX, Wang XF, Hao YJ (2017) The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic Res 4: 17023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11: R106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bai Y, Dougherty L, Li MJ, Fazio G, Cheng LL, Xu KN (2012) A natural mutation-led truncation in one of the two aluminium-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple. Mol Genet Genomics 287: 663–678 [DOI] [PubMed] [Google Scholar]
  4. Beruter J (2004) Carbohydrate metabolism in two apple genotypes that differ in malate accumulation. J Plant Physiol 161: 1011–1029 [DOI] [PubMed] [Google Scholar]
  5. Chen Q, Xu X, Xu D, Zhang H, Zhang C, Li G (2019) WRKY18 and WRKY53 coordinate with HISTONE ACETYLTRANSFERASE1 to regulate rapid responses to sugar. Plant Physiol 180: 2212–2226 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen WK, Chen L, Zhang X, Yang N, Guo JH, Wang M, Ji SH, Zhao XY, Yin PF, Cai LC, et al. (2022) Convergent selection of a WD40 protein that enhances grain yield in maize and rice. Science 375: eabg7985 [DOI] [PubMed] [Google Scholar]
  7. Ding ZJ, Yan JY, Xu XY, Li GX, Zheng SJ (2013) WRKY46 functions as a transcriptional repressor of ALMT1, regulating aluminum-induced malate secretion in Arabidopsis. Plant J 76: 825–835 [DOI] [PubMed] [Google Scholar]
  8. Dong XC, Tang HX, Zhang Q, Zhang CM, Wang ZT (2022) Transcriptomic analyses provide new insights into jujube fruit quality affected by water deficit stress. Sci Hortic 291: 110558 [Google Scholar]
  9. Eisenach C, Baetz U, Huck NV, Zhang JB, De Angeli A, Beckers GJM, Martinoia E (2017) ABA-lnduced stomatal closure involves ALMT4, a phosphorylation-dependent vacuolar anion channel of Arabidopsis. Plant cell 29: 2552–2569 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Endo S, Iwamoto K, Fukuda H (2018) Overexpression and cosuppression of xylem-related genes in an early xylem differentiation stage-specific manner by the AtTED4 promoter. Plant Biotechnol J 16: 451–458 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Etienne A, Genard M, Lobit P, Mbeguie AMD, Bugaud C (2013) What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. J Exp Bot 64: 1451–1469 [DOI] [PubMed] [Google Scholar]
  12. Fernie AR, Yan J (2019) De novo domestication: an alternative route toward new crops for the future. Mol Plant 12: 615–631 [DOI] [PubMed] [Google Scholar]
  13. Gao QH, Wu PT, Liu JR, Wu CS, Parry JW, Wang M (2011) Physico-chemical properties and antioxidant capacity of different jujube (Ziziphus jujuba Mill.) cultivars grown in Loess Plateau of China. Sci Hortic Amsterdam 130: 67–72 [Google Scholar]
  14. Gao QH, Wu CS, Yu JG, Wang M, Ma YJ, Li CL (2012) Textural characteristic, antioxidant activity, sugar, organic acid, and phenolic profiles of 10 promising jujube (Ziziphus jujuba Mill.) selections. J Food Sci 77: C1218–C1225 [DOI] [PubMed] [Google Scholar]
  15. Hu DG, , SunCH, , MaQJ, , YouC-X, , ChengL, , Hao YJ (2016) MdMYB1 regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples. Plant Physiol 170: 1315–1330 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hu DG, Li YY, Zhang QY, Li M, Sun CH, Yu JQ, Hao YJ (2017) The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple. Plant J 91: 443–454 [DOI] [PubMed] [Google Scholar]
  17. Huang J, Zhang CM, Zhao X, Fei ZJ, Wan KK, Zhang Z, Pang XM, Yin X, Bai Y, Sun XQ, et al. (2016) The jujube genome provides insights into genome evolution and the domestication of sweetness/acidity taste in fruit trees. PLoS Genet 12: e1006433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurth MA, Suh SJ, Kretzschmar T, Geis T, Bregante M, Gambale F, Martinoia E, Neuhaus HE (2005) Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol 137: 901–910 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jia D, Wu P, Shen F, Li W, Zheng X, Wang Y, Yuan Y, Zhang X, Han Z (2021) Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.). Plant Physiol 186: 549–568 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jiang Y, Liu C, Yan D, Wen X, Liu Y, Wang H, Dai J, Zhang Y, Liu Y, Zhou B (2017) MdHB1 down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar ‘Granny Smith’. J Exp Bot 68: 1055–1069 [DOI] [PubMed] [Google Scholar]
  21. Kovermann P, Meyer S, Hortensteiner S, Picco C, Scholz-Starke J, Ravera S, Lee Y, Martinoia E (2007) The Arabidopsis vacuolar malate channel is a member of the ALMT family. Plant J 52: 1169–1180 [DOI] [PubMed] [Google Scholar]
  22. Kumar V, Saha D, Thakare DR, Jajoo A, Jain PK, Bhat SR, Srinivasan R (2017) A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants. Plant Sci 264: 138–148 [DOI] [PubMed] [Google Scholar]
  23. Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30: 325–327 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li GZ, Wang ZQ, Yokosho K, Ding B, Fan W, Gong QQ, Li GX, Wu YR., Yang JL, Ma JF, et al. (2018) Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa). New Phytol 219: 149–162 [DOI] [PubMed] [Google Scholar]
  25. Lin LK, Yuan KL, Huang YD, Dong HZ, Qiao QH, Xing CH., Huang X S, Zhang SL (2022) A WRKY transcription factor PbWRKY40 from Pyrus betulaefolia functions positively in salt tolerance and modulating organic acid accumulation by regulating PbVHA-B1 expression. Environ Exp Bot 196: 104–782 [Google Scholar]
  26. Liu M, Wang J, Wang L, Liu P, Zhao J, Zhao Z, Yao S, Stanica F, Liu Z, Wang L, et al. (2020) The historical and current research progress on jujube-a superfruit for the future. Hortic Res 7: 119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ma B, Liao L, Zheng H, Chen J, Wu B, Ogutu C, Li S, Korban SS, Han Y (2015) Genes encoding aluminum-activated malate transporter II and their association with fruit acidity in apple. Plant Genome 8: 1–14 [DOI] [PubMed] [Google Scholar]
  28. Ma BQ, Liao L, Fang T, Peng Q, Ogutu C, Zhou H, Ma FW, Han YP (2019) A Ma10 gene encoding P-type ATPase is involved in fruit organic acid accumulation in apple. Plant Biotechnol J 17: 674–686 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyer S, Scholz-Starke J, De Angeli A, Kovermann P, Burla B, Gambale F, Martinoia E (2011) Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation. Plant J 67: 247–257 [DOI] [PubMed] [Google Scholar]
  30. Miller SS, Driscoll BT, Gregerson RG, Gantt JS, Vance CP (1998) Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH. Plant J 15: 173–184 [DOI] [PubMed] [Google Scholar]
  31. Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York, NY [Google Scholar]
  32. Qu Z, Wang Y (1993) Fruit tree records of China, Chinese jujube volume. China Forestry Publ. House, Beijing, China [Google Scholar]
  33. Ren Y, Guo SG, Zhang J, He HJ, Sun HH, Tian SW, Gong GY, Zhang HY, Levi A, Tadmor Y, Xu Y (2018) A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon. Plant Physiol 176: 836–850 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rongala J (2008) Identification and Localization of Vacuolar Organic Acid Carriers Ingrapevine Berries. The University of Adelaide, Adelaide, Australia
  35. Saito K, Matsuda F (2010) Metabolomics for functional genomics, systems biology, and biotechnology. Annu Rev Plant Biol 61: 463–489 [DOI] [PubMed] [Google Scholar]
  36. Sakr S, Wang M, Dédaldéchamp F, Perez-Garcia MD, Ogé L, Hamama L, Atanassova R (2018) The sugar-signaling hub: overview of regulators and interaction with the hormonal and metabolic network. Int J Mol Sci 19: 2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sasaki T, Tsuchiya Y, Ariyoshi M, Nakano R, Ushijima K, Kubo Y, Mori IC, Higashiizumi E, Galis I, Yamamoto Y (2016) Two members of the aluminumactivated malate transporter family, SlALMT4 and SlALMT5, are expressed during fruit development, and the overexpression of SlALMT5 alters organic acid contents in seeds in tomato (Solanum lycopersicum). Plant Cell Physiol 57: 2367–2379 [DOI] [PubMed] [Google Scholar]
  38. Scheibe R. (2004) Malate valves to balance cellular energy supply. Physiol Plant 120: 21–26 [DOI] [PubMed] [Google Scholar]
  39. Shen LY, Luo H, Wang XL, Wang XM, Qiu XJ, Liu H, Zhou SS, Jia KH, Nie S, Bao YT, et al. (2021) Chromosome-scale genome assembly for Chinese sour jujube and insights into its genome evolution and domestication signature. Front Plant Sci 12: 773090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shiratake K, Martinoia E. (2007) Transporters in fruit vacuoles. Plant Biotechnol 24: 127–133 [Google Scholar]
  41. Sweetman C, Deluc LG, Cramer GR, Ford CM, Soole KL (2009) Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry 70: 1329–1344 [DOI] [PubMed] [Google Scholar]
  42. Tokizawa M, Enomoto T, Ito H, Wu LJ, Kobayashi Y, Mora-Macias J, Armenta-Medina D, Iuchi S, Kobayashi M, Nomoto M, et al. (2021) High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis. J Exp Bot 72: 2769–2789 [DOI] [PubMed] [Google Scholar]
  43. Wang ZR, Liu L, Su H, Guo LQ, Zhang JL, Li YF, Xu JY, Zhang XC, Guo YD, Zhang N (2020) Jasmonate and aluminum crosstalk in tomato: identification and expression analysis of WRKYs and ALMTs during JA/Al-regulated root growth. Plant Physiol Bioc 154: 409–418 [DOI] [PubMed] [Google Scholar]
  44. Xu LL, Qiao X, Zhang MY, Zhang SL (2018) Genome-Wide analysis of aluminum-activated malate transporter family genes in six rosaceae species, and expression analysis and functional characterization on malate accumulation in Chinese white pear. Plant Sci 274: 451–465 [DOI] [PubMed] [Google Scholar]
  45. Yao YX, Li M, Liu Z, You CX, Wang DM, Zhai H, Hao YJ (2009) Molecular cloning of three malic acid related genes MdPEPC, MdVHA-A, MdcyME and their expression analysis in apple fruits. Sci Hortic 122: 404–408 [Google Scholar]
  46. Ye J, Wang X, Hu T, Zhang F, Wang B, Li C, Yang T, Li H, Lu Y, Giovannoni JJ, et al. (2017) An InDel in the promoter of Al-ACTIVATED MALATE TRANSPORTER9 selected during tomato domestication determines fruit malate contents and aluminum tolerance. Plant Cell 29: 2249–2268 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yu JQ, Gu KD, Sun CH, Zhang QY, Wang JH, Ma FF, You CX, Hu DG, Hao YJ (2021) The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate. Plant Biotechnol J 19: 285–299 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang C, Bian Y, Hou S, Li X (2018) Sugar transport played a more important role than sugar biosynthesis in fruit sugar accumulation during Chinese jujube domestication. Planta 248: 1187–1199 [DOI] [PubMed] [Google Scholar]
  49. Zhang LH, Ma BQ, Wang CZ, Chen XY, Ruan YL, Yuan YY, Ma FW, Li MJ (2022) MdWRKY126 modulates malate accumulation in apple fruit by regulating cytosolic malate dehydrogenase (MdMDH5). Plant Physiol 188: 2059–2072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhang QY, Gu KD, Cheng LL, Wang JH, Yu JQ, Wang XF, You CX, Hu DG, Hao YJ (2020) BTB-TAZ domain protein MdBT2 modulates malate accumulation and vacuolar acidification in response to nitrate(1) (OPEN). Plant Physiol 183: 750–764 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhang Z, Shi Q, Wang B, Ma A, Wang Y, Xue Q, Shen B, Hamaila H, Tang T, Qi X, et al. (2021) Jujube metabolome selection determined the edible properties acquired during domestication. Plant J 109:1116–1133 [DOI] [PubMed] [Google Scholar]
  52. Zhao A, Xue X, Wang Y, Sui, Ren H, Li D (2016) Characteristic analysis of sugars and organic acids components and contents of Chinese jujube and wild jujube fruits. J Tarim Univ 28: 29–36 [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kiac491_Supplementary_Data
Click here for additional data file. (1.9MB, zip)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES