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Article

Identification and Functional Analysis of Key Genes Regulating Organic Acid Metabolism in Jujube Fruit

by
Panpan Tong
1,2,†,
Dengyang Lu
3,4,†,
Guanglian Liao
5,
Cuiyun Wu
1,2 and
Jiangbo Wang
1,2,*
1
College of Horticulture and Forestry, Tarim University, Alar 843300, China
2
National-Local Joint Engineering Laboratory of High Efficiency and Superior Quality Cultivation and Fruit Deep Processing Technology on Characteristic Fruit Trees, Alar 843300, China
3
College of Life Science and Technology, Tarim University, Alar 843300, China
4
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Alar 843300, China
5
National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(11), 2515; https://doi.org/10.3390/agronomy14112515
Submission received: 11 September 2024 / Revised: 22 October 2024 / Accepted: 22 October 2024 / Published: 26 October 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
Organic acids are crucial indicators of fruit flavor quality, but the metabolic characteristics and regulatory genes of organic acids during jujube fruit development remain largely unexplored. In this study, the cultivar ‘Heigeda’ with a high organic acid content was used as the experimental material. The organic acid content was quantified, and key candidate genes were identified through transcriptome analysis. The results indicated that malic acid and citric acid were the main organic acid content in jujube fruit and increased gradually with fruit development. Transcriptome analysis identified nine genes associated with malic acid and seven with citric acid, with four genes co-regulating malic acid and citric acid. Functional assays by transient overexpression and silencing of these four genes in the jujube fruits revealed that overexpression significantly upregulated the malic and citric acid content. However, only the silencing of aconitase1 (ZjACO1) and aconitase3 (ZjACO3) significantly downregulated the content of malic and citric acids. Therefore, aconitase1 (ZjACO1) and aconitase3 (ZjACO3) are considered the key genes that regulate the metabolism of citric acid and malic acid in jujube fruits. Our study can enrich the regulation mechanism of the organic acid metabolism of jujube fruit and provide theoretical support for the efficient cultivation of jujube fruit.

1. Introduction

Ziziphus jujuba Mill. is the largest and most economically important dried fruit tree in China [1]. Jujube fruit is not only rich in trace elements, carbohydrates, amino acids, and other nutrients, but it is also rich in vitamin C, flavonoids, cyclic adenosine phosphate triterpenoids, and other active substances [2,3], which together give jujube fruit a unique flavor. Among these components, organic acids play a crucial role in determining fruit flavor [4]. However, the molecular mechanism of organic acid metabolism in jujube fruit is rarely reported. Therefore, an in-depth understanding of the differences and the dynamic changes in the organic acid content during the ripening process of jujube fruits as well as the regulatory mechanisms affecting their accumulation can provide crucial reference information for the genetic improvement of jujube fruit.
Organic acids are essential substances for photosynthesis, respiration, and other processes in plants, and they play an important role in the growth and development of plant cells [5,6]. The organic acid content in most jujube fruits gradually accumulates throughout fruit development, and reaches the highest content at the ripening stage, with malic acid, citric acid, and quinic acid being the main acids [7,8]. In apples, malic acid is the main organic acid. It is mainly synthesized by malate dehydrogenase (MdMDH) in the cytoplasm and degraded by NADP-malic enzymes (MdNADP-MEs) [9]. In citrus fruits, citric acid is the main organic acid. It is mainly synthesized by citrate synthase (Cscyt-CS) in the cytoplasm and degraded by citrate lyase (CsCL) and NADP-isocitrate dehydrogenase (CsNADP-ICDH) [10]. With the development of omics technology, transcriptome analysis has been successfully applied to study the regulation mechanism of the accumulation of flavor substances in fruits [11,12,13]. For example, transcriptome techniques revealed the gene expression characteristics and patterns of organic acid synthesis and accumulation in the flesh of Litchi chinensis [14]. It also revealed the differences in sugar accumulation between wild watermelons and cultivated watermelons during development. Mainly, it was found that seven UDP-sugar transferase genes are associated with the glycosylation of cucurbitacin [15]. Combined with the WGCNA and metabolomics analysis, the transcription factors highly correlated with organic acids in fruit were also identified, including Hypoxia-Associated Protein 3 (ZjHAP3), Teosinte Branched1/Cycloidea/PCF14 (ZjTCP14), and Myeloblastosis 78 (ZjMYB78) [16]. Moreover, in Prunus avium L., several key differentially expressed genes (DEGs) involved in sugar metabolism, organic acid metabolism, and flavonoid metabolism were identified. Among them, the key genes involved in the flavonoid pathway may play an important role in regulating the increase in anthocyanin content during fruit development [17]. At present, many genes related to organic acid metabolism have been reported, such as malate synthase (McMS), isocitrate lyase (McICL) [18], and aluminum-dependent malate transporter 4 (ZjALMT4) [19]. However, there are few reports on the regulation of organic acid metabolism in jujube fruit, and the key genes regulating acid metabolism in jujube fruit are still poorly understood.
Therefore, in this study, jujube fruits with high organic acid were used as materials to identify the accumulation patterns and components of organic acids. At the same time, key genes were identified through transcriptome analysis, and their functions were validated using transient overexpression and silencing techniques. The results of this study can further enrich the theory of the organic acid regulation of jujube fruit and can provide theoretical support for the efficient cultivation technology of jujube fruit.

2. Materials and Methods

2.1. Materials and Growth Conditions

The jujube cultivar ‘Heigeda’ (Alar, China) is distinguished by its thin skin, thick flesh, and small pit, with a notably high organic acid content of over 0.60%, making it an ideal material for studying organic acid metabolism [20]. In this study, cv ‘Heigeda’ fruit samples were collected from five fruit development stages, including the young fruit stage (YF, 20 DAF, jujube fruits developed to a short columnar shape), the early white maturation stage (EF, 40 DAF), the white maturation stage (WM, 60 DAF, jujube fruit is fully developed and mainly white), the half-red fruit stage (HR, 100 DAF, coloring area of jujube peel reaches 50%), and the full-red fruit stage (FR, 120 DAF, jujube fruits turn completely red). Nine individual plants of the ‘Heigeda’ cultivar were cultivated at the Tarim University Jujube Germplasm Resource Repository located at 40°54′ N, 81.30′ E. The soil at the test site was of a sandy type. The site is distinguished by a long duration of sunlight exposure, a significant temperature difference between day and night, and scarce precipitation. Moreover, weeds at the site are cleared every month. The orchard was flat, and the conditions of the soil, fertilizer, and water management were maintained consistently throughout the study. The experiments were performed using a one-way completely randomized design. Three plants were randomly selected as biological replicates, with a total of three biological replicates. Adopting this method can ensure the reliability and reproducibility of the results. From each plant, 15 fruits were collected. The collected samples were treated with liquid nitrogen at once and stored at −80 °C until use.

2.2. Determination of Organic Acid Components

The extraction process and the derivatization procedure were performed following the techniques outlined in earlier research studies [21]. After grinding the samples with liquid nitrogen, 0.5 g of the sample was weighed into a 10 mL centrifuge tube. Then, 7 mL of 80% methanol was added, followed by incubation at 70 °C for 30 min. After cooling, ultrasonic extraction was performed for 90 min, followed by centrifugation at 4000 rpm for 10 min. A volume of 2 mL of the upper liquid phase was carefully moved to a separate centrifuge tube and subjected to centrifugation at 12, stirred at a speed of 000 rpm for 15 min, and then 0.5 mL of the supernatant was taken and dried to dryness using a vacuum rotary evaporator at 60 °C. The dried sample was then subjected to a derivatization reaction by adding 0.8 mL of hydroxylamine hydrochloride solution and reacting at 70 °C for 1 h. After cooling, 0.4 mL of hexamethyldisilazane (HMDS) and 0.2 mL of trimethylchlorosilane (TMCS) were sequentially added and heated at 70 °C for 2 h. Finally, 0.5 mL of the supernatant was transferred to a 2 mL auto-sampler vial for GC-FID analysis, and qualitative analysis was performed based on the retention times of each component. The content of organic acid components was determined by gas chromatography using ISQII (GC-MS, Thermo, MA, USA) [22]. Gas chromatography was performed using a 5%-Phenyl-methyl polysiloxane column (Agilent, CA, USA), with high-purity nitrogen as the carrier gas at a flow rate of 45 mL/min. The injector temperature, detector temperature, and flow rates for air and hydrogen were set using their default operational parameters [23].

2.3. Transcriptome Analysis

The fruit samples collected at five developmental stages (three biological replicates in each stage) were sent to Beijing Novogene Biotechnology Co., Ltd. (Beijing, China), for sequencing using the HiSeq 2500 platform. Briefly, using fastqc for quality control of raw data and fastp to filter low-quality data, clean data were obtained. Next, the clean data were mapped to the winter jujube genome (PRJNA251714) [24] by HISAT2, and SAMtools 1.21 sorted the SAM file to obtain the short generated BAM files. Finally, FPKM values were calculated using an R 4.3.0 script. Principal component analysis (PCA) was conducted using the FactoMineR 2.11 and factoextra 1.0.7 packages. Differential gene expression between the two groups was identified using the DESeq2 1.38.0 package. EnhancedVolcano 1.22.0 was used for volcano plot visualization, the VennDiagram package was used to create Venn diagrams 1.7.3, and WGCNA analysis was performed using the WGCNA 1.73 package.

2.4. Transient Overexpression and Virus-Induced Gene Silencing (VIGS) Assays

The key candidate genes were cloned and constructed into a 1300-GUS-Flag vector. A VIGS tool of Sol Genomics was used to design the VIGS primers and cloned the 300 bp fragment of candidate genes to the pTRV2 vector. The key candidate gene primers are listed in Supplementary Table S1. The plasmids were confirmed by sequencing and transformed into Agrobacterium strains GV3101. Half-red stage ‘Dongzao’ fruits were injected, with an empty vector injection serving as the control. Following the injection, the fruits were kept in darkness for three days [21] then immediately treated with liquid nitrogen and stored at −80 °C.

2.5. qRT-PCR

Following the methodology previously outlined, the internal reference gene selected for qRT-PCR analysis was Ubiquitin (ZjUBQ) [25]. The 10 μL reaction mix comprised the following components: 0.5 μL of the cDNA template, 0.2 μL each of forward and reverse primers (gene primers are provided in Supplementary Table S1), and 5 μL of PowerUp™ SYBR™ Green Master Mix, with the remaining volume made up of 4.1 μL of double-distilled water (ddH2O). The relative levels of gene expression were calculated using the 2−∆∆CT method.

2.6. Statistical Analysis

All experiments were conducted using three independent biological replicates. The correlation value was detected using Microsoft Excel 2016 and presented as a Pearson value. Statistical significance was analyzed by a t-test using SPSS IBM 26 software.

3. Results

3.1. Changes in Organic Acid Components in Jujube Fruit During Fruit Development

The analysis of organic acid components during fruit development revealed that malic acid content gradually increased, ranging from 0.18 to 2.85 mg/g. Citric acid content initially decreased slightly before rapidly increasing, with a variation range of 0.58 to 2.66 mg/g. The contents of malic acid and citric acid both reach the highest level at the full-red fruit stage and occupy the main acid components. At this stage, the content of malic acid is slightly higher than that of citric acid, and the contents of malic acid and citric acid are significantly higher than that of quinic acid. In addition, quinic acid content remained low, ranging from 0.09 to 0.14 mg/g (Figure 1).

3.2. Identification of Key Genes Regulating Malic Acid and Citric Acid by Transcriptome Analysis

A total of 148.50 GB of clean data were obtained by transcriptome sequencing, with a mean Q30 of 93.04% (Supplementary Table S2), and PCA showed that three biological replicates in each period were good (Figure 2A), which could be used for subsequent analysis. There were 2755 and 3782 differentially expressed genes in WM (white mature stage) vs. HR (half-ripe stage) and WM (white mature stage) vs. FR (fully ripe stage) (Figure 2B,C, Supplementary Tables S3 and S4), and these genes were distributed in multiple biological functions of GO terms, including malate dehydrogenase activity (GO:0016616) and malate transmembrane transporter activity (GO:0010286) (Figure 2D, Supplementary Table S5). Among these differential genes, 2313 differential genes were shared between WM vs. HR and WM vs. FR (Figure 2E), among which 623 genes were significantly associated with malic acid, 978 genes were significantly associated with citric acid, and 486 genes were significantly associated with both malic acid and citric acid (Supplementary Table S6). Through the analysis of a gene expression time trend, the 486 core DEGs could be divided into two clusters (Figure 2F).
Finally, 492 DEGs related to citric acid, 137 DEGs related to malic acid, and 487 DEGs co-related to malic acid and citric acid were selected for WGCNA analysis, with a total of 1115 DEGs. These genes were divided into 12 modules and were assigned different colors. Among them, the turquoise module contains the largest number of genes, and the tan module contains the fewest number of genes (Figure 2G). The analysis of module–trait relationships shows that eight modules are positively correlated with malic acid and citric acid, and four modules are negatively correlated with malic acid and citric acid. Most modules are significantly correlated with malic acid and citric acid contents (p < 0.05), with the blue module being the most significant (p < 0.01; Figure 2H). Based on the GO annotation of DEGs, seven key candidate genes related to citric acid metabolism and nine key candidate genes related to malic acid metabolism were identified. Of these, four genes were found to be involved in the metabolism of both citric acid and malic acid (Supplementary Table S7).

3.3. Real-Time Fluorescence Quantitative (qRT-PCR) Analysis

To verify the accuracy of RNA-seq results, twelve key candidate genes were randomly selected for qRT-PCR analysis. The qRT-PCR data and transcriptome data of these genes showed an extremely significant positive correlation (Figure 3), with an average correlation coefficient of 0.929, indicating the reliability of the transcriptome data. Additionally, aconitase1 (ZjACO1), aconitase3 (ZjACO3), and heat stress-associated protein 32 (ZjHSA32) exhibited r values below 0.900, which may be attributed to factors such as primer efficiency, PCR amplification conditions, or the presence of homologous genes.

3.4. Transient Overexpression and Silencing Analysis

The aconitase1 (ZjACO1), aspartate2 (ZjATT2), aconitase3 (ZjACO3), and 2-Oxoglutarate Dehydrogenase1 (ZjOGDH1) genes not only showed a correlation of over 0.920 with both malic acid and citric acid content, but they are also likely involved in the metabolic pathways of both acids. Therefore, it is essential to conduct a functional analysis of these genes. The expression levels of four key candidate genes were significantly upregulated following transient overexpression in jujube fruit (Figure 4A–D), accompanied by a significant increase in malic and citric acid contents. The highest increase in malic acid was observed after an overexpression of aconitase3 (ZjACO3). Meanwhile, the malic acid content also increased significantly after an overexpression of other genes (Figure 4E,F). Conversely, gene expression levels were significantly downregulated after transient silencing in jujube fruits (Figure 4G–J), leading to a significant reduction in malic and citric acid contents upon the silencing of aconitase1 (ZjACO1) and aconitase3 (ZjACO3). Notably, the silencing of 2-Oxoglutarate Dehydrogenase1 (ZjOGDH1) resulted in a significant increase in malic and citric acid contents. However, the silencing of aspartate2 (ZjATT2) did not affect the content of citric acid (Figure 4K,L).

4. Discussion

Fruit flavor quality is an important economic index affecting consumers to buy fruit [26,27], and the components and content of organic acids are one of the important factors determining fruit flavor [20]. The organic acids of jujube fruit changed significantly during the process from the young fruit stage to the fully ripe stage. Consistent with previous studies [8,28,29], this study found that the main organic acids of jujube fruits included malic acid, citric acid, and quinic acid. Among these acids, malic acid and citric acid were the main components in the late stage of fruit development. However, the main organic acids in the fruit of the new cultivar ‘Mazao’ were higher in the young fruit stage and lower in the mature stage [16]. Our study results are different from these findings, highlighting that different cultivars may have distinct metabolic pathways and regulatory mechanisms.
Combined transcriptomic analysis is an important technique for studying fruit flavor quality. By sequencing and analyzing the transcriptomes of fruits at different developmental stages, the gene expression patterns and regulatory networks in fruits can be revealed, and then the synthesis and accumulation mechanisms of fruit flavor substances can be understood. In fruits such as kiwifruits [30] and apples [12], combined transcriptomic analysis has achieved many important research results regarding related phenotypes. However, few reports have been used to identify the key genes for the accumulation of sugar and acid during the development of jujube fruit. To reveal the flavor differences in jujube fruits caused by different acid accumulation, this study conducted transcriptome sequencing on the main developmental stages of jujube fruits, identified a series of genes related to organic acid metabolism, and further enriched the research on the mechanism of the organic acid metabolism of jujube fruits. Elucidating the underlying molecular regulatory mechanisms of organic acid changes is a key step [31]. Fruit flavor is controlled by environment, development, and metabolic signals, and transcription factors play an important role in these processes. In tomatoes, overexpression of the ABA-responsive element-binding protein1 (SlAREB1) can effectively promote the accumulation of citric acid, malic acid, and glutamate in the fruit [32]. In apples, basic helix-loop-helix3 (MdbHLH3) directly activates cytosolic malate dehydrogenase (MdcyMDH) to encourage the accumulation of malic acid in apple fruits, and overexpression of basic helix-loop-helix3 (MdbHLH3) can also improve the photosynthetic capacity and carbohydrate content of leaves, thus increasing the carbohydrate accumulation in apple fruits [33]. In this study, we identified genes related to the accumulation of malic acid and citric acid by WGCNA analysis. We screened a series of transcription factors, which can be co-expressed with related structural genes, and are likely to play an important role in the regulation of organic acid content in jujube fruit.
The accumulation of organic acids in fruits is regulated by metabolism and transport. Citric acid can be produced by the tricarboxylic acid cycle or the glyoxylic acid cycle, while malic acid is mainly produced by the oxaloacetic acid reduction pathway in the tricarboxylic acid cycle [34]. In this study, aconitase1 (ZjACO1), aspartate2 (ZjATT2), aconitase3 (ZjACO3), and 2-Oxoglutarate Dehydrogenase1 (ZjOGDH1) were identified as candidate genes regulating malic acid and citric acid accumulation. In this study, the overexpression and instantaneous silencing of aconitase1 (ZjACO1) and aconitase3 (ZjACO3) significantly affected the organic acid content of jujube fruits, suggesting that they may be key functional genes in regulating organic acid accumulation in jujube fruits. However, aconitase3 (CitACO3) is considered to be a key gene in citric acid degradation in citrus fruits [35], which is most likely caused by the specific mechanism of citric acid metabolism regulation in different plants. ATT2 regulates the synthesis of citric acid and malic acid by regulating the supply of acetyl-CoA and influencing its entry into the tricarboxylic acid cycle. At the same time, OGDH can catalyze the conversion of alpha-ketoglutaric acid to succinyl-CoA, which further affects the synthesis of citric acid and malic acid [36]. Interestingly, we found that the malic acid content was significantly reduced after the instantaneous silencing of aspartate2 (ZjATT2), but the citric acid content did not change significantly, which may indicate that multiple recovery mechanisms or regulatory networks are working together after the silencing of aspartate2 (ZjATT2), resulting in a relatively stable level of citric acid. This also reflects that with the loss of function of 2-Oxoglutarate Dehydrogenase1 (ZjOGDH1), the re-regulation of metabolic pathways may lead to an increase in the content of major organic acids [36,37]. The key gene functions verified in this study can be employed in breeding programs to cultivate jujube varieties with high or low organic acid contents, thereby enriching the nutritional value and taste of jujube fruits. These genes can also play a significant role in the field of food processing. For example, by regulating key genes, processed jujube products with specific acidity and flavor can be produced to meet the needs of different consumers and provide product innovation directions for food processing enterprises. In terms of agricultural sustainable development, molecular marker-assisted selection technology can be developed for precision agricultural management to improve planting efficiency. Finally, the regulatory mechanisms of these genes are complex and may involve interactions with other genes or environmental factors. Future research directions may include investigating the interactions between these candidate genes and other genes involved in organic acid metabolism and then combining them with environmental factors to cultivate jujube varieties with stronger stress resistance. Exploring the epigenetic regulation of these genes could provide additional insights into the control of organic acid accumulation in jujube fruits.

5. Conclusions

Fruit organic acids are the key factors affecting fruit quality. In this study, the content of acid components in jujube fruit during its development was determined, and malic acid and citric acid were the main components of jujube fruit. Combined with transcriptome analysis, seven key candidate genes related to citric acid metabolism and nine key candidate genes related to malic acid metabolism were identified, of which 4 genes were related to both malic acid metabolism and citric acid metabolism. After transient overexpression of these genes on jujube fruits, malic acid and citric contents were significantly upregulated, but only silencing of aconitase1 (ZjACO1) and aconitase3 (ZjACO3) significantly downregulated the malic acid and citric acid content, indicating that 2-Oxoglutarate Dehydrogenase1 (ZjOGDH1) may have feedback regulation for homologous genes. In general, this study has identified the main organic acid components and their key metabolic genes during the development of jujube fruits, providing an important basis for an in-depth understanding of the organic acid metabolism mechanism of jujube fruits and improving the quality of jujube fruits through gene regulation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14112515/s1: Table S1: Primers used in this study. Table S2: Data statistics of transcriptome sequencing. Table S3: Identification of differentially expressed genes in WM and HR. Table S4: Identification of differentially expressed genes in WM and FR. Table S5: GO analysis. Table S6: Genes for Venn analysis. Table S7: WGCNA analysis.

Author Contributions

P.T.: sample collection and writing the original draft. D.L.: data measurement. J.W. and G.L.: reviewing and editing. C.W.: supervision and conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This research received financial support from the National Natural Science Foundation of China under grant number 32260729, allocated to J.W., as well as from the Key Industry Support Plan Project of XPCC, grant number 2017DB006, also designated to J.W. The agencies that provided financial support did not influence the design of the study, the process of data gathering, the analysis and interpretation, or the writing of the manuscript.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Determination of organic acid components during fruit development. YF, young fruit stage, 20 DAF; FE, fruit expanded stage, 40 DAF; WM, white mature stage, 60 DAF; HR, half-ripe stage, 100 DAF; FR, fully ripe stage, 120 DAF. The same lowercase letter means that there is no significant difference between different stages of the same component.
Figure 1. Determination of organic acid components during fruit development. YF, young fruit stage, 20 DAF; FE, fruit expanded stage, 40 DAF; WM, white mature stage, 60 DAF; HR, half-ripe stage, 100 DAF; FR, fully ripe stage, 120 DAF. The same lowercase letter means that there is no significant difference between different stages of the same component.
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Figure 2. Comprehensive transcriptome analysis of differentially expressed genes and their association with organic acid metabolism. (A) PCA; (B) volcanic analysis of WM vs. HR; (C) volcanic analysis of WM vs. FR; (D) GO analysis of co-expression of differential genes; (E) Venn diagram analysis of co-expression of differential genes and malic acid- and citric acid-related genes; (F) time trend analysis of core co-expression genes; (G,H) WGCNA analysis.
Figure 2. Comprehensive transcriptome analysis of differentially expressed genes and their association with organic acid metabolism. (A) PCA; (B) volcanic analysis of WM vs. HR; (C) volcanic analysis of WM vs. FR; (D) GO analysis of co-expression of differential genes; (E) Venn diagram analysis of co-expression of differential genes and malic acid- and citric acid-related genes; (F) time trend analysis of core co-expression genes; (G,H) WGCNA analysis.
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Figure 3. qRT-PCR analysis of some key candidate genes. The bar chart represents transcriptome data, and the line chart represents qRT-PCR data. The r value represents the correlation coefficient. ** represents that the p value of the comparison between transcriptome data and qRT-PCR data is less than 0.01.
Figure 3. qRT-PCR analysis of some key candidate genes. The bar chart represents transcriptome data, and the line chart represents qRT-PCR data. The r value represents the correlation coefficient. ** represents that the p value of the comparison between transcriptome data and qRT-PCR data is less than 0.01.
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Figure 4. Transient overexpression and silence analysis. (A–D) qRT-PCR analysis of overexpression of ZjACO1, ZjATT2, ZjACO3, and ZjOGDH1; (E,F) malic acid content and citric acid content after overexpression of four key genes; (G–J) qRT-PCR analysis of silencing of ZjACO1, ZjATT2, ZjACO3, and ZjOGDH1; and (K,L) malic acid content and citric acid content after silencing of four key genes. * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significance.
Figure 4. Transient overexpression and silence analysis. (A–D) qRT-PCR analysis of overexpression of ZjACO1, ZjATT2, ZjACO3, and ZjOGDH1; (E,F) malic acid content and citric acid content after overexpression of four key genes; (G–J) qRT-PCR analysis of silencing of ZjACO1, ZjATT2, ZjACO3, and ZjOGDH1; and (K,L) malic acid content and citric acid content after silencing of four key genes. * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significance.
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MDPI and ACS Style

Tong, P.; Lu, D.; Liao, G.; Wu, C.; Wang, J. Identification and Functional Analysis of Key Genes Regulating Organic Acid Metabolism in Jujube Fruit. Agronomy 2024, 14, 2515. https://doi.org/10.3390/agronomy14112515

AMA Style

Tong P, Lu D, Liao G, Wu C, Wang J. Identification and Functional Analysis of Key Genes Regulating Organic Acid Metabolism in Jujube Fruit. Agronomy. 2024; 14(11):2515. https://doi.org/10.3390/agronomy14112515

Chicago/Turabian Style

Tong, Panpan, Dengyang Lu, Guanglian Liao, Cuiyun Wu, and Jiangbo Wang. 2024. "Identification and Functional Analysis of Key Genes Regulating Organic Acid Metabolism in Jujube Fruit" Agronomy 14, no. 11: 2515. https://doi.org/10.3390/agronomy14112515

APA Style

Tong, P., Lu, D., Liao, G., Wu, C., & Wang, J. (2024). Identification and Functional Analysis of Key Genes Regulating Organic Acid Metabolism in Jujube Fruit. Agronomy, 14(11), 2515. https://doi.org/10.3390/agronomy14112515

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