Research Progress and Biosynthetic Mechanisms of Nutritional Compounds Obtained from Various Organs During the Developmental Stages of a Medicinal Plant (Chinese Jujube).

The fruit of the jujube tree is high in nutrients and has various health benefits. China is a major producer of jujube, and it is now cultivated all around the world. Numerous studies have demonstrated the nutritional value and potential health advantages of bioactive compounds found in the jujube tree. Furthermore, the jujube tree has a remarkable 7000-year agricultural history. The jujube plant has developed a rich gene pool, making it a valuable resource for germplasm. Different studies have focused on the developmental stages of jujube fruits to identify the optimal time for harvest and to assess the changes in their bioactive natural compounds or products during the process of development but the molecular mechanism underlying the production of bioactive natural products in Z. jujuba is still poorly understood. Moreover, the potential differential expressed genes (DEGs) identified as responsible for the synthesis of these compounds should be further functionally verified. It has been noticed that the contents of total flavonoids, total phenolic, and vitamin C increase significantly during the ripening process, while the contents of soluble sugars and organic acids decrease gradually. In this review, we have also scrutinized the challenges that hinder the utilization of jujube fruit resources and suggested potential areas for further research. As such, our review serves as a valuable resource for the future development of jujube-based nutritional compounds and the incorporation of their nutritional elements into the functional foods industry.

Anatomical observation and transcriptome analysis of branch-twisted mutations in Chinese jujube.

BACKGROUND: Plant organs grow in a certain direction and organ twisted growth, a rare and distinctive trait, is associated with internal structure changes and special genes. The twisted branch mutant of Chinese jujube jujube, an important fruit tree native to China and introduced to nearly 50 countries, provides new typical materials for exploration of plant twisted growth. RESULTS: In this study, the cytological characteristics and related genes of twisted branches in Chinese jujube were revealed by microscopy observation and transcriptome analysis. The unique coexistence of primary and secondary structures appeared in the twisted parts of branches, and special structures such as collateral bundle, cortical bundles, and internal phloem were formed. Ninety differentially expressed genes of 'Dongzao' and its twisted mutant were observed, in which ZjTBL43, ZjFLA11, ZjFLA12 and ZjIQD1 were selected as candidate genes. ZjTBL43 was homologous to AtTBL43 in Arabidopsis, which was involved in the synthesis and deposition of cellular secondary wall cellulose. The attbl43 mutant showed significant inflorescence stem bending growth. The transgenic lines of attbl43 with overexpression of ZjTBL43 were phenotypically normal.The branch twisted growth may be caused by mutations in ZjTBL43 in Chinese jujube. AtIQD10, AtFLA11 and AtFLA12 were homologous to ZjIQD1, ZjFLA11 and ZjFLA12, respectively. However, the phenotype of their function defect mutants was normal. CONCLUSION: In summary, these findings will provide new insights into the plant organ twisted growth and a reference for investigation of controlling mechanisms of plant growth direction.

Isolation and characterization of endophytic bacteria for controlling root rot disease of Chinese jujube.

AIM: Fusarium oxysporum is the primary pathogen causing root rot disease that severely affects cultivation of jujube fruit in the Xinjiang province of China. The aim of this study was to identify endophytic bacteria in healthy jujube organs that could effectively suppress F. oxysporum growth. METHODS AND RESULTS: Different plant organs (leaves, twigs and roots) were collected from healthy Chinese jujube cultivated in southern Xinjiang province of China. The endophytic bacterium Brevibacterium halotolerans JZ7 was selected for its strong antagonistic activity and growth-promoting characteristics. Gas-chromatography mass-spectrometry analysis showed that acetoin, 2,3-butanediol and fenretinide were the three dominant volatile organic compounds produced by strain JZ7. Fenretinide strongly suppressed spore germination of F. oxysporum in vitro. Pot experiments showed that strain JZ7 colonized both the roots and rhizosphere soil of Chinese jujube and significantly reduced F. oxysporum level in jujube rhizosphere soil. CONCLUSION: We demonstrated that B. halotolerans JZ7 can be developed into a biological control agent to combat root rot disease of Chinese jujube in the Xinjiang province of China. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested strategy for biological control of jujube root rot disease is fully in accordance with the current principles of sustainability.

Genome-wide identification of CNGC genes in Chinese jujube (Ziziphus jujuba Mill.) and ZjCNGC2 mediated signalling cascades in response to cold stress.

BACKGROUNDS: Cyclic nucleotide gated channels (CNGCs) play multifaceted roles in plant physiological processes, especially with respect to signalling processes, plant development, and responses to environmental stresses. However, little information is known about the CNGC family in the large cosmopolitan family Rhamnaceae, which has strong tolerance to biotic and abiotic stresses. RESULTS: In the current study, a total of 15 ZjCNGCs which located on 7 chromosomes were firstly identified in Chinese jujube (Ziziphus jujuba Mill.), the most important species of Rhamnaceae in terms of economic and ecological values. Phylogenetic analysis showed that these ZjCNGCs could be classified into four groups, ZjCNGC12 belonged to group IVA, and ZjCNGC13, 14, 15 belonged to group IVB. In addition, the paralogous and orthologous homology duplication of ZjCNGC15 occurred during the evolutionary process. The characteristics of ZjCNGCs regarding to exon-intron numbers and post-translational modifications showed diversified structures and functions. Motif composition and protein sequence analysis revealed that the phosphate-binding cassette and hinge regions were conserved among ZjCNGCs. Prediction of the cis-acting regulatory elements and expression profiles by real-time quantitative PCR analysis showed that some of the ZjCNGCs responded to environmental changes, especially ZjCNGC2, which was significantly downregulated in response to cold stress, and ZjCNGC4 was highly induced in response to cold, salt and alkaline stresses. ZjCNGC13 and 14 were highly induced in the phytoplasma-resistant cultivar and downregulated in the susceptible cultivar. Furthermore, ZjCNGC2 could be regulated by cAMP treatment, microtubule changes and interact with ZjMAPKK4, which suggested that cAMP and microtubule might play important roles in ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress. CONCLUSIONS: The identification and classification analysis of ZjCNGCs were firstly reported, and some key individual ZjCNGCs might play essential roles in the response to biotic and abiotic stresses, especially ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress. This systematic analysis could provide important information for further functional characterization of ZjCNGCs with the aim of breeding stress-resistant cultivars.

Genome-wide identification of MAPKKK genes and their responses to phytoplasma infection in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to a wide range of biotic and abiotic stresses. In a previous study, we identified ten ZjMAPKs and five ZjMAPKKs in the Chinese jujube genome. We found that some members of ZjMAPKs and ZjMAPKKs may play key roles in the plant's response to phytoplasma infection. However, how these ZjMAPKKs are modulated by ZjMAPKKKs during the response process has not been elucidated. Little information is available regarding MAPKKKs in Chinese jujube. RESULTS: A total of 56 ZjMAPKKKs were identified in the jujube genome. All of these kinases contain the key S-TKc (serine/threonine protein kinase) domain, which is distributed among all 12 chromosomes. Phylogenetic analyses show that these ZjMAPKKKs can be classified into two subfamilies. Specifically, 41 ZjMAPKKKs belong to the Raf subfamily, and 15 belong to the MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures. Only one pair of ZjMAPKKKs (15/16, on chromosome 5) was found to be tandemly duplicated. Using qPCR, the expression profiles of these MAPKKKs were investigated in response to infection with phytoplasma. In the three main infected tissues (witches' broom leaves, phyllody leaves, and apparently normal leaves), ZjMAPKKK26 and - 45 were significantly upregulated, and ZjMAPKKK3, - 43 and - 50 were significantly downregulated. ZjMAPKKK4, - 10, - 25 and - 44 were significantly and highly induced in sterile cultivated tissues infected by phytoplasma, while ZjMAPKKK6, - 7, - 17, - 18, - 30, - 34, - 35, - 37, - 40, - 41, - 43, - 46, - 52 and - 53 were significantly downregulated. CONCLUSIONS: For the first time, we present an identification and classification analysis of ZjMAPKKKs. Some ZjMAPKKK genes may play key roles in the response to phytoplasma infection. This study provides an initial understanding of the mechanisms through which ZjMAPKKKs are involved in the response of Chinese jujube to phytoplasma infection.

Genome-wide analysis of the bZIP gene family in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Among several TF families unique to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important. Chinese jujube (Ziziphus jujuba Mill.) is a popular fruit tree species in Asia, and its fruits are rich in sugar, vitamin C and so on. Analysis of the bZIP gene family of jujube has not yet been reported. In this study, ZjbZIPs were identified firstly, their expression patterns were further studied in different tissues and in response to various abiotic and phytoplasma stresses, and their protein-protein interactions were also analyzed. RESULTS: At the whole genome level, 45 ZjbZIPs were identified and classified into 14 classes. The members of each class of bZIP subfamily contain a specific conserved domain in addition to the core bZIP conserved domain, which may be related to its biological function. Relative Synonymous Codon Usage (RSCU) analysis displayed low values of NTA and NCG codons in ZjbZIPs, which would be beneficial to increase the protein production and also indicated that ZjbZIPs were at a relative high methylation level. The paralogous and orthologous events occurred during the evolutionary process of ZjbZIPs. Thirty-four ZjbZIPs were mapped to but not evenly distributed among 10 pseudo- chromosomes. 30 of ZjbZIP genes showed diverse tissue-specific expression in jujube and wild jujube trees, indicating that these genes may have multiple functions. Some ZjbZIP genes were specifically analyzed and found to play important roles in the early stage of fruit development. Moreover, some ZjbZIPs that respond to phytoplasma invasion and abiotic stress environmental conditions, such as salt and low temperature, were found. Based on homology comparisons, prediction analysis and yeast two-hybrid, a protein interaction network including 42 ZjbZIPs was constructed. CONCLUSIONS: The bioinformatics analyses of 45 ZjbZIPs were implemented systematically, and their expression profiles in jujube and wild jujube showed that many genes might play crucial roles during fruit ripening and in the response to phytoplasma and abiotic stresses. The protein interaction networks among ZjbZIPs could provide useful information for further functional studies.

Genome-wide analysis of the WRKY gene family and their positive responses to phytoplasma invasion in Chinese jujube.

BACKGROUND: The WRKY gene family is one of the most important families in higher plants. As transcription factors, they actively respond to biotic and abiotic stress and are also involved in plant development. Chinese jujube (Ziziphus jujuba Mill.) is the largest type of dried fruit tree in China in terms of production, but its production is largely limited by phytoplasma infection, and the information about the role of WRKY genes under phytoplasma stress was still limited. RESULTS: We identified 54 ZjWRKYs in the jujube genome and classified them into three subgroups according to conserved WRKY domains and zinc-finger structure. 41 ZjWRKYs were distributed on 11 of 12 pseudo chromosomes in Chinese jujube. The majority of ZjWRKYs were highly expressed in the seven examined tissues, indicating that they play multiple roles in these vegetative and reproductive organs. Transcriptome data showed that most of the characterised ZjWRKYs were highly expressed at later stages of fruit development. RT-qPCR demonstrated that the expression of 23 ZjWRKYs changed following phytoplasma infection, suggesting that they are involved in signalling pathways that respond to phytoplasma stress. Then, STRING analysis and yeast two-hybrid screening proved that some ZjWRKY proteins were interacting with ZjMAPKK proteins, which were also involved in phytoplasma invasion. Moreover, their differential expressions were further confirmed in resistant and susceptible jujube varieties under phytoplasma stress. These results suggest that ZjWRKYs play significant roles in phytoplasma tolerance and should be crucial candidate genes for jujube-phytoplasma interaction. CONCLUSIONS: 54 ZjWRKYs in Chinese jujube were identified and classified into three subgroups. 41 ZjWRKYs were unevenly distributed along the chromosomes. The majority of ZjWRKYs were highly expressed in various tissues. Most of the ZjWRKYs were positive responses to phytoplasma invasion, and that provided candidate genes for the future studies of jujube-phytoplasma interaction.

Genome-wide analysis of the bHLH gene family in Chinese jujube (Ziziphus jujuba Mill.) and wild jujube.

BACKGROUND: The bHLH (basic helix-loop-helix) transcription factor is one of the largest families of transcription factors in plants, containing a large number of members with diverse functions. Chinese jujube (Ziziphus jujuba Mill.) is the species with the highest economic value in the family Rhamnaceae. However, the characteristics of the bHLH family in the jujube genome are still unclear. Hence, ZjbHLHs were first searched at a genome-wide level, their expression levels under various conditions were investigated systematically, and their protein-protein interaction networks were predicted. RESULTS: We identified 92 ZjbHLHs in the jujube genome, and these genes were classified into 16 classes according to bHLH domains. Ten ZjbHLHs with atypical bHLH domains were found. Seventy ZjbHLHs were mapped to but not evenly distributed on 12 pseudo- chromosomes. The domain sequences among ZjbHLHs were highly conserved, and their conserved residues were also identified. The tissue-specific expression of 37 ZjbHLH genes in jujube and wild jujube showed diverse patterns, revealing that these genes likely perform multiple functions. Many ZjbHLH genes were screened and found to be involved in flower and fruit development, especially in earlier developmental stages. A few genes responsive to phytoplasma invasion were also verified. Based on protein-protein interaction prediction and homology comparison, protein-protein interaction networks composed of 92 ZjbHLHs were also established. CONCLUSIONS: This study provides a comprehensive bioinformatics analysis of 92 identified ZjbHLH genes. We explored their expression patterns in various tissues, the flowering process, and fruit ripening and under phytoplasma stress. The protein-protein interaction networks of ZjbHLHs provide valuable clues toward further studies of their biological functions.

Transcript analyses reveal a comprehensive role of abscisic acid in modulating fruit ripening in Chinese jujube.

BACKGROUND: Chinese jujube (Ziziphus jujuba Mill.) is a non-climacteric fruit; however, the underlying mechanism of ripening and the role of abscisic acid involved in this process are not yet understood for this species. RESULTS: In the present study, a positive correlation between dynamic changes in endogenous ABA and the onset of jujube ripening was determined. Transcript analyses suggested that the expression balance among genes encoding nine-cis-epoxycarotenoid dioxygenase (ZjNCED3), ABA-8'-hydroxylase (ZjCYP707A2), and beta-glucosidase (ZjBG4, ZjBG5, ZjBG8, and ZjBG9) has an important role in maintaining ABA accumulation, while the expression of a receptor (ZjPYL8), protein phosphatase 2C (ZjPP2C4-8), and sucrose nonfermenting 1-related protein kinase 2 (ZjSnRK2-2 and ZjSnRK2-5) is important in regulating fruit sensitivity to ABA applications. In addition, white mature 'Dongzao' fruit were harvested and treated with 50 mg L- 1 ABA or 50 mg L- 1 nordihydroguaiaretic acid (NDGA) to explore the role of ABA in jujube fruit ripening. By comparative transcriptome analyses, 1103 and 505 genes were differentially expressed in response to ABA and NDGA applications on the 1st day after treatment, respectively. These DEGs were associated with photosynthesis, secondary, lipid, cell wall, and starch and sugar metabolic processes, suggesting the involvement of ABA in modulating jujube fruit ripening. Moreover, ABA also exhibited crosstalk with other phytohormones and transcription factors, indicating a regulatory network for jujube fruit ripening. CONCLUSIONS: Our study further elucidated ABA-associated metabolic and regulatory processes. These findings are helpful for improving strategies for jujube fruit storage and for gaining insights into understand complex non-climacteric fruit ripening processes.

Genome-wide identification, characterization, and expression analysis of the expansin gene family in Chinese jujube (Ziziphus jujuba Mill.).

Main conclusion 30 expansin genes were identified in the jujube genome. Phylogenetic analysis classified expansins into 17 subgroups. Closely related expansins share a conserved gene structure. ZjEXPs had different expression patterns in different tissues. Plant-specific expansins were first discovered as pH-dependent cell-wall-loosening proteins involved in diverse physiological processes. No comprehensive analysis of the expansin gene family has yet been carried out at the whole genome level in Chinese jujube (Ziziphus jujuba Mill.). In this study, 30 expansin genes were identified in the jujube genome. These genes, which were distributed with varying densities across 10 of the 12 jujube chromosomes, could be divided into four subfamilies: 19 ZjEXPAs, 3 ZjEXPBs, 1 ZjEXLA, and 7 ZjEXLBs. Phylogenetic analysis of expansin genes in Arabidopsis, rice, apple, grape, and jujube classified these genes into 17 subgroups. Members of the same subfamily and subgroup shared conserved gene structure and motif compositions. Homology analysis identified 20 homologous gene pairs between jujube and Arabidopsis. Further analysis of ZjEXP gene promoter regions uncovered various growth, development and stress-responsive cis-acting elements. Expression analysis and transcript profiling revealed that ZjEXPs had different expression patterns in different tissues at various developmental stages. ZjEXPA4 and ZjEXPA6 were highly expressed in young fruits, ZjEXPA3 and ZjEXPA5 were significantly expressed in flowers, and ZjEXPA7 was specifically expressed in young leaves. The results of this study, the first systematic analysis of the jujube expansin gene family, can serve as a strong foundation for further elucidation of the physiological functions and biological roles of jujube expansin genes.

Phytochemical Profiling and Fingerprint Analysis of Chinese Jujube (Ziziphus jujuba Mill.) Leaves of 66 Cultivars from Xinjiang Province.

Foliage of jujube (Ziziphus jujuba Mill.) as a byproduct of agriculture, is a traditional nutraceutical material in China. Previous studies have shown that it is a rich resource of polyphenols. However, information on its complete phenolic profile and the difference between cultivars is still limited. This study investigated and compared phytochemical profiles of leaves of 66 Chinese jujube cultivars. Forty-two compounds, including 22 flavonols, two flavanols, one flavanone, 13 derivatives of phenolic acids, three simple acids, and one unknown hexoside were identified/tentatively identified using high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry. Eight major flavonols were quantified by HPLC coupled with an ultraviolet (UV) detector. The contents of total flavonoids ranged from 2.6-25.1 mg/g dry weight (DW). Differences between cultivars were analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA). This study presents a systematic study on the phenolic compounds in Chinese jujube leaves of different cultivars.

Sugar transport played a more important role than sugar biosynthesis in fruit sugar accumulation during Chinese jujube domestication.

MAIN CONCLUSION: Sugar transport, including the symplasmic pathway in plasmodesmata and apoplasmic pathway mediated by sugar transporters, accelerated sugar accumulation in cultivated jujube, while sugar metabolism-related genes played weak roles in jujube domestication. The fruit of Chinese jujube (Ziziphus jujuba Mill.) is high in sugar concentration. By contrast, wild type-sour jujube (Z. jujuba Mill. var. spinosa Hu) contains markedly less sugar. It is unknown whether sugar transport or sugar metabolism drove sugar accumulation during jujube domestication. Using a combination of ultrastructural observations, phylogenetic analysis, testing for soluble sugars, and transcriptional analysis, the sugar accumulation mechanism was studied in the developmental stages of cultivated jujube and sour jujube. Our results indicate that the symplasmic transport pathway in plasmodesmata is present in cultivated jujube, but not in sour jujube. Sugar transporter genes have higher frequencies of duplication than sugar metabolism-related genes. Gene expression patterns indicate that sugar transporter genes, especially ZjSUT2, ZjSWEET1, ZjSWEET7, ZjSWEET11, ZjSTP3, and ZjSTP13a, rather than sugar metabolism-related genes showed higher expression levels in cultivated jujube versus sour jujube during fruit sugar accumulation. These findings suggest that sugar transport, including apoplasmic and symplasmic transport, rather than sugar biosynthesis, is associated with the difference in sugar accumulation between jujube and sour jujube, and that it may drive jujube domestication. This study provides valuable genetic information for jujube improvement, and offers new insights into fruit tree domestication related to sugar accumulation.

Comparative Analysis of Pigments, Phenolics, and Antioxidant Activity of Chinese Jujube (Ziziphus jujuba Mill.) during Fruit Development.

Chinese jujube (Ziziphus jujuba Mill.) fruit are much admired for their unique flavor and high nutritional value. This study tracks changes in skin color and antioxidant activity over six stages (S1 … S6) of fruit development in two cultivars of jujube, 'Junzao' and the color mutant 'Tailihong'. The study records the changing levels of chlorophylls, carotenoids, anthocyanins, and phenolic compounds during fruit development. Contents of chlorophylls, ß-carotenes and anthocyanins decreased throughout the developmental stages in two jujube cultivars, while lutein contents decreased at first and then increased to a maximum at S6. The levels of total phenolics, total flavonoids, total flavanols, total anthocyanins, procyanidin B1, procyanidin B2, procyanidin B3, (+)-catechin, quercetin, and ferulic acid are significantly higher in 'Tailihong' than in 'Junzao' before the onset ripening (before S3). However, after S3 the level differences of these components in the two cultivars are not significant. In both cultivars, antioxidant activity reduces gradually throughout fruit development. Our findings indicate how the skin color of jujube fruit during maturation is due to changes in the levels of flavonoids, carotenoids, and anthocyanins. The color changes are also associated with changes in antioxidant activity.

Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Chinese jujube (Ziziphus jujuba Mill.) is one of the most important members in the Rhamnaceae family. The whole genome sequence and more than 30,000 proteins of Chinese jujube have been obtained in 2014. Mitogen-activated protein kinase cascades are universal signal transduction modules in plants, which is rapidly activated under various biotic and abiotic stresses. To date, there has been no comprehensive analysis of the MAPK and MAPKK gene family in Chinese jujube at the whole genome level. RESULTS: By performing a series of bioinformatics analysis, ten MAPK and five MAPKK genes were identified from the genome database of Chinese jujube, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that ZjMAPKs was classified into four known groups, including A, B, C and D. ZjMAPKs contains five members of the TEY phosphorylation site and five members with the TDY motif. The ZjMAPKK family was subsequently divided into three groups, A, B and D. The gene structure, conserved motifs, functional annotation and chromosome distribution of ZjMAPKs and ZjMAPKKs were also predicted. ZjMAPKs and ZjMAPKKs were distributed on nine pseudo-chromosomes of Chinese jujube. Subsequently, expression analysis of ZjMAPK and ZjMAPKK genes using reverse transcription PCR and quantitative real-time PCR was carried out. The majority of ZjMAPK and ZjMAPKK genes were expressed in all tested organs/tissues with considerable differences in transcript levels indicating that they might be constitutively expressed. Moreover, ZjMKK5 was specific expressed in early development stage of jujube flower bud, indicating it plays some roles in reproductive organs development. The transcript expression of most ZjMAPK and ZjMAPKK genes was down-regulated in response to plant growth regulators, darkness treatment and phytoplasma infection. CONCLUSIONS: We identified ten ZjMAPK and five ZjMAPKK genes from the genome database of Chinese jujube, the research results shown that ZjMPKs and ZjMKKs have the different expression patterns, indicating that they might play different roles in response to various treatments. The results provide valuable information for the further elucidation of physiological functions and biological roles of jujube MAPKs and MAPKKs.

Ameliorating effect of Chinese jujube polyphenol on blood glucose oxidative stress in type 2 diabetic rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a common metabolic disease characterized by insulin resistance and insufficient relative insulin secretion, leading to elevated blood sugar and the development of diabetic complications. T2DM not only seriously affects people's health and quality of life, but also brings a heavy burden to society and economy. At present, the treatment of T2DM mainly relies on drug therapy, but these drugs often have problems such as side effects, resistance and high cost, and can not fully meet the needs and expectations of patients. Therefore, it is of great significance and value to find safe and effective natural medicines or functional foods to assist the treatment and prevention of T2DM. OBJECTIVE: Chinese jujube are a common fruit that contain abundant polyphenolic compounds, which exhibit multiple physiological activities, such as antioxidation, anti-inflammation, and blood glucose lowering. The objective of this study was to explore the impact of red date polyphenols on glycemic control and oxidative stress status in patients with type 2 diabetes mellitus (T2DM).

ZjWRKY23 and ZjWRKY40 Promote Fruit Size Enlargement by Targeting and Downregulating Cytokinin Oxidase/Dehydrogenase 5 Expression in Chinese Jujube.

Fruit size is crucial for fruit trees, as it contributes to both quality and yield. However, the underlying mechanism of fruit size regulation remains largely unknown. Taking advantage of using a fruit double-sized bud mutant of Chinese jujube, "Jinkuiwang" and its wild type, "Jinsixiaozao", we carried out a comprehensive study on the mechanism of fruit size development in jujube. Using weighted gene coexpression network analyses, a number of candidate regulators for fruit size including those involved in hormonal signaling pathways, transcription factors, and heat shock proteins were identified. A hub gene named cytokinin oxidase/dehydrogenase 5 (ZjCKX5), responsible for cytokinin degradation, was found to play a negative role in regulating fruit size development, and overexpressing ZjCKX5 in tomato and Arabidopsis resulted in much smaller fruits and dwarf plants. Furthermore, another two hub genes, ZjWRKY23 and ZjWRKY40 transcription factors, were found to participate in fruit size regulation by targeting and downregulating the ZjCKX5 expression. Overexpressing ZjWRKY23 or ZjWRKY40 in tomato led to much larger fruits and promoted plant architecture. Based on these results, a molecular framework for jujube fruit size regulation, namely, ZjWRKY-ZjCKX5 module, was proposed. This study provides a new insight into the molecular networks underlying fruit size regulation.

Genomic C-Value Variation Analysis in Jujube (Ziziphus jujuba Mill.) in the Middle Yellow River Basin.

Chinese jujube (Ziziphus jujuba Mill.) originated in the Yellow River basin (YRB) of the Shanxi-Shaanxi region. The genomic C-value is a crucial indicator for plant breeding and germplasm evaluation. In this study, we used flow cytometry to determine the genomic C-values of jujube germplasms in the YRB of the Shanxi-Shaanxi region and evaluated their differences in different sub-regions. Of the 29 sub-regions, the highest and lowest variations were in Linxian and Xiaxian, respectively. The difference between jujube germplasms was highly significant (F = 14.89, p < 0.0001) in Linxian. Cluster analysis showed that both cluster 2 and 4 belonged to Linxian, which were clearly separated from other taxa but were cross-distributed in them. Linxian County is an important gene exchange center in the YRB of the Shanxi-Shaanxi region. Principal component analysis showed that cluster 1 had low genomic C-values and single-fruit weights and cluster 2 had high genomic C-values and vitamin C contents. The genomic C-value was correlated with single-fruit weight and vitamin C content. In addition, the genomic C-value was used to predict fruit agronomic traits, providing a reference for shortening the breeding cycle and genetic diversity-related studies of jujube germplasm.

Three Novel Adenylate Cyclase Genes Show Significant Biological Functions in Plant.

Adenylate cyclase is the key enzyme solely synthesizing cAMP which participates in cell metabolism regulations and functions as an intracellular second messenger. However, the biological functions of plant ACs have not been elucidated clearly for their poor conservative sequences and low detectable cAMP. We performed a systematic study of plant ACs by using Chinese jujube, whose fruit exhibits the highest cAMP content among plants. Three novel ACs were identified from Chinese jujube, and two types of methods including in vitro and in vivo were used to certificate ZjAC1-3 which can catalyze the conversion of ATP into cAMP. The biological functions of significant accelerations of seed germination, root growth, and flowering were found via overexpression of these AC genes in Arabidopsis, and these functions of ACs were further demonstrated by treating the AC-overexpressing transgenic lines and wild type Arabidopsis with bithionol and dibutyryl-cAMP. At last, transcriptome data revealed that the underlying mechanism of the biological functions of ACs might be regulation of the key genes involved in the circadian rhythm pathway and the hormone signal transduction pathway. This research established a foundation for further investigating plant AC genes and provided strong evidence for cAMP serving as a signaling molecule in plants.

Metabolomics and transcriptomics analyses for characterizing the alkaloid metabolism of Chinese jujube and sour jujube fruits.

Introduction: Jujube is an important economic forest tree whose fruit is rich in alkaloids. Chinese jujube (Ziziphus jujuba Mill.) and sour jujube (Ziziphus spinosa Hu.) are the two most important species of the jujube genus. However, the mechanisms underlying the synthesis and metabolism of alkaloids in jujube fruits remain poorly understood. Methods: In this study, the fruits of Ziziphus jujuba 'Hupingzao' and Ziziphus spinosa 'Taigusuanzao' in different harvest stages were used as test materials, we first integrated widely targeted metabolomics and transcriptomics analyses to elucidate the metabolism of alkaloids of jujube fruits. Results: In the metabolomics analysis, 44 alkaloid metabolites were identified in 4 samples, 3 of which were unique to sour jujube fruit. The differential alkaloid metabolites (DAMs) were more accumulated in sour jujube than in Chinese jujube; further, they were more accumulated in the white ripening stage than in the red stage. DAMs were annotated to 12 metabolic pathways. Additionally, transcriptomics data revealed 259 differentially expressed genes (DEGs) involved in alkaloid synthesis and metabolism. By mapping the regulatory networks of DAMs and DEGs, we screened out important metabolites and 11 candidate genes. Discussion: This study preliminarily elucidated the molecular mechanism of jujube alkaloid synthesis. The candidate genes regulated the synthesis of key alkaloid metabolites, but the specific regulation mechanism is unclear. Taken together, our results provide insights into the metabolic networks of alkaloid synthesis in Chinese jujube and sour jujube fruits at different harvest stages, thereby providing a theoretical reference for further research on the regulatory mechanism of jujube alkaloids and their development and utilization.

Virus-Induced Gene Silencing (VIGS) in Chinese Jujube.

Virus-induced gene silencing (VIGS) is a fast and efficient method for assaying gene function in plants. At present, the VIGS system mediated by Tobacco rattle virus (TRV) has been successfully practiced in some species such as cotton and tomato. However, little research of VIGS systems has been reported in woody plants, nor in Chinese jujube. In this study, the TRV-VIGS system of jujube was firstly investigated. The jujube seedlings were grown in a greenhouse with a 16 h light/8 h dark cycle at 23 °C. After the cotyledon was fully unfolded, Agrobacterium mixture containing pTRV1 and pTRV2-ZjCLA with OD600 = 1.5 was injected into cotyledon. After 15 days, the new leaves of jujube seedlings showed obvious photo-bleaching symptoms and significantly decreased expression of ZjCLA, indicating that the TRV-VIGS system had successfully functioned on jujube. Moreover, it found that two injections on jujube cotyledon could induce higher silencing efficiency than once injection. A similar silencing effect was then also verified in another gene, ZjPDS. These results indicate that the TRV-VIGS system in Chinese jujube has been successfully established and can be applied to evaluate gene function, providing a breakthrough in gene function verification methods.