The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses.

BACKGROUND: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear. RESULTS: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity. CONCLUSIONS: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

Genome-wide screening of the RNase T2 gene family and functional analyses in jujube (Ziziphus jujuba Mill.).

BACKGROUND: Ribonuclease (RNase T2) plays crucial roles in plant evolution and breeding. However, there have been few studies on the RNase T2 gene family in Ziziphus jujuba Mill., one of important dried fruit tree species. Recently, the released sequences of the reference genome of jujube provide a good chance to perform genome-wide identification and characterization of ZjRNase gene family in the jujube. RESULTS: In this study, we identified four members of RNase T2 in jujube distributed on three chromosomes and unassembled chromosomes. They all contained two conserved sites (CASI and CASII). Analysis of the phylogenetic relationships revealed that the RNase T2 genes in jujube could be divided into two groups: ZjRNase1 and ZjRNase2 belonged to class I, while ZjRNase3 and ZjRNase4 belonged to class II. Only ZjRNase1 and ZjRNase2 expression were shown by the jujube fruit transcriptome analysis. So ZjRNase1 and ZjRNase2 were selected functional verification by overexpression transformation of Arabidopsis. The overexpression of these two genes led to an approximately 50% reduction in seed number, which deserve further attention. Moreover, the leaves of the ZjRNase1 overexpression transgenic lines were curled and twisted. Overexpression of ZjRNase2 resulted in shortened and crisp siliques and the production of trichomes, and no seeds were produced. CONCLUSION: In summary, these findings will provide new insights into the molecular mechanisms of low number of hybrid seeds in jujube and a reference for the future molecular breeding of jujube.

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.

Hsa_circ_0007334 Promotes the Osteogenic Differentiation and Proliferation of Human Bone Marrow Mesenchymal Stem Cells by Sponging miR-144-3p.

This study aimed to identify the possible function and the molecular mechanism of hsa_circ_0007334 in human bone marrow mesenchymal stem cells (hBMSCs) osteogenic differentiation. The level of hsa_circ_0007334 was detected by means of quantitative real-time polymerase chain reaction (RT-qPCR). Alkaline phosphatase (ALP), RUNX2, osterix (OSX), and osteocalcin (OCN) were monitored to analyze the degree of osteogenic differentiation under routine culture or under the control of hsa_circ_0007334. The proliferation of hBMSCs was tested with a cell counting kit-8 (CCK-8) assay. The migration of hBMSCs was tested using the Transwell assay. Bioinformatics analysis was used to predict the possible targets of hsa_circ_0007334 or miR-144-3p. Dual-luciferase reporter assay system was used to analyze the combination between hsa_circ_0007334 and miR-144-3p. Hsa_circ_0007334 was upregulated in osteogenic differentiation of hBMSCs. Osteogenic differentiation increased by hsa_circ_0007334 in vitro was confirmed with levels of ALP and bone markers (RUNX2, OCN, OSX). hsa_circ_0007334 overexpression promoted osteogenic differentiation, proliferation, and migration of hBMSCs, and knockdown of hsa_circ_0007334 has the opposite effects. miR-144-3p was identified as the target of hsa_circ_0007334. The targeting genes of miR-144-3p are involved in osteogenic-differentia-tion-related biological processes (such as bone development, epithelial cell proliferation, and mesenchymal cell apoptotic prosess) and pathways (including FoxO and VEGF signaling pathway). Hsa_circ_0007334, therefore, presents itself as a promising biological for osteogenic differentiation.

UCHL3 promotes cervical cancer development and metastasis by stabilizing NRF2 via deubiquitination.

Cervical cancer is one of the most lethal gynaecological malignancies in females. The deubiquitylase UCHL3 has been studied as an oncogenic factor in multiple cancers. However, the expression pattern and function profile of UCHL3 in cervical cancer hasn't been fully characterized. Here, we revealed that UCHL3 was highly expressed in cervical cancer and overexpressed UCHL3 predicted a poor survival probability in cervical cancer patients. Our findings showed that knockdown of UCHL3 inhibited cell growth, migration and invasion in cervical cancer cells while UCHL3 knockdown inhibited cervical cancer development and metastasis in vivo in mouse models. Mechanistically, co-immunoprecipitation assay showed that UCHL3 directly interacted with NRF2. Knockdown of UCHL3 decreased NRF2 expression while overexpression of UCHL3 stabilized NRF2 via deubiquitination. In addition, overexpression of UCHL3 with C92A mutation didn't affect NRF2 stability. Moreover, we revealed that overexpression of NRF2 could antagonize the function of UCHL3 knockdown in cervical cancer cells. Collectively, our findings suggest that UCHL3 promotes cervical cancer development and metastasis by stabilizing NRF2 via deubiquitination. Thus, UCHL3/NRF2 axis could be utilized to develop efficient treatments for cervical cancer patients.

The genome of Candidatus phytoplasma ziziphi provides insights into their biological characteristics.

Phytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches' broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete 'Candidatus Phytoplasma ziziphi' chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.

Microwell Confined Electro-Coalescence for Rapid Formation of High-Throughput Droplet Array.

Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This work proposes and validates a method for rapid formation of uniform droplet array based on microwell confined droplets electro-coalescence of screen-printed emulsion droplets, namely electro-coalescence droplet array (ECDA). The electro-coalescence of droplets is according to the polarization induced electrostatic and dielectrophoretic forces, and the dielectrowetting effect. The photolithographically fabricated microwells are highly regular and reproducible, ensuring identical volume and physical confinement to achieve uniform droplet array, and meanwhile the microwell isolation protects the paired water droplets from further fusion and broadens its feasibility to different fluidic systems. Under optimized conditions, a droplet array with an average diameter of 85 µm and a throughput of 106 in a 10 cm × 10 cm chip can be achieved within 5 s at 120 Vpp and 50 kHz. This ECDA chip is validated for various microwell geometries and functional materials. The optimized ECDA are successfully applied for digital viable bacteria counting, showing comparable results to the plate culture counting. Such an ECDA chip, as a digitizable and high-throughput platform, presents excellent potential for high-throughput screening, analysis, absolute quantification, etc.

Lung microbiota and potential treatment of respiratory diseases.

The unique microbiome found in the lungs has been studied and shown to be associated with both pulmonary homeostasis and lung diseases. The lung microbiome has the potential to produce metabolites that modulate host-microbe interactions. Specifically, short-chain fatty acids (SCFAs) produced by certain strains of the lung microbiota have been shown to regulate immune function and maintain gut mucosal health. In response, this review described the distribution and composition of the microbiota in lung diseases and discussed the impact of the lung microbiota on health and lung disease. In addition, the review further elaborated on the mechanism of microbial metabolites in microbial-host interaction and their application in the treatment of lung diseases. A better understanding of the interaction between the microbiota, metabolites, and host will provide potential strategies for the development of novel methods for the treatment of pulmonary microbial induced lung diseases.

An integrated model of CDCA5 and FOXM1 expression combined with a residual disease that predicts prognosis in ovarian cancer patients.

Ovarian cancer (OC) is the most prevalent type of gynecologic cancer, leading to global death. Unfortunately, less than half of patients diagnosed with this cancer survive for up to five years. The factor forkhead box M1 (FOXM1) is a crucial oncoprotein in ovarian cancer and is currently recognized as a potential therapeutic target. The role of the Cell division cycle-associated 5 (CDCA5) is critical for advancing different types of cancers. However, the significance of CDCA5 in OC from a clinical perspective is not well comprehended. This study aimed to build a risk prognosis model and assess the data supporting the prognostic usefulness of CDCA5 and FOXM1 expression in patients with OC. In OC, we found that CDCA5 and FOXM1 were expressed. To establish the existence of variables that were independently related to PFS and OS, Cox regression, data from clinics, and Kaplan-Meier analysis were used. A risk score model and nomogram were created using the independent prognostic parameters. The accuracy of the model's predictions was then evaluated using decision curve analysis (DCA), calibration curve, and receiver operating characteristic(ROC) analysis. Finally, the patients were separated into groups based on their cut-off value, and then the differences in survival were investigated. Significant correlations were found between OC and CDCA5, and FOXM1 expression levels (P <0.0001). Serous ovarian tumors (P=0.025) and even specific subgroups of high-grade serous ovarian tumors were shown to have elevated CDCA5 expression levels. In our database, FOXM1 expression levels were discovered to be related to intestinal metastases (P=0.014). In OC, the expression of FOXM1 was positively correlated with the overexpression of CDCA5 (rs=0.46, P<0.0001). The results of the multivariate analysis indicated that residual disease (RD) (P=0.005), CDCA5 expression level (P=0.028), and FOXM1 expression level (P<0.0001) were identified as independent prognostic factors for PFS. Additionally, RD (P=0.023) and FOXM1 expression level (P<0.0001) were identified as independent prognostic factors for OS. While the prediction model's performance with RD was poor (AUC=0.645 for PFS, AUC=0.650 for OS), the model's performance with tissue biomarkers was enhanced (AUC=0.797 for PFS, AUC=0.741 for OS). The nomogram and risk score method showed a benefit for prognosis prediction. In summary, poor outcomes are predicted by CDCA5, which is overexpressed in OC patients and has a positive correlation with the level of FOXM1 expression. An aid to prognosis prediction in patients with OC and a resource for therapy planning is a risk prognosis model based on CDCA5 and FOXM1 expression with RD.

Relationship between serum CHIP levels with cardiovascular disease in maintenance haemodialysis patients.

Many preclinical studies reported that the carboxyl terminus of Hsp70-interacting protein (CHIP) has cardiovascular protective effects. This study was designed to explore whether CHIP is related with cardiovascular disease (CVD) in maintanence haemodialysis (MHD) patients. 217 MHD patients and 150 healthy controls were recruited, serum CHIP concentration and clinical characteristics were measured. MHD patients were followed-up for 36 months and their cardiovascular events (CVEs) and survival conditions were recorded. Here, the data shows that serum CHIP concentrations in MHD patients were lower than those in healthy controls (31.69 ± 18.2 pg/mL vs 84.53 ± 22.1 pg/mL, p < 0.05). CHIP negatively correlated with age, C-reactive protein, B-type brain natriuretic peptide, phosphorus, parathyroid hormone, carotid intima-media thickness (CIMT) and left ventricular septal thickness (LVSTd), whereas it positively associated with albumin, haemoglobin, creatinine, Kt/V and ejection fraction (p < 0.05, respectively). Partial correlation and multiple linear regression analysis verified the negative relationship between CHIP with CIMT or LVSTd (p < 0.05, respectively). Using quartile method and Kaplan-Meier survival function, it indetified that the lower serum CHIP concentration predicted risk of CVEs, CVD and all-cause death (p < 0.001). Cox regression analysis manifested CHIP was negatively associated with CVEs (HR = 0.914, 95%CI 0.880-0.950, p < 0.001), CVD mortality (HR = 0.747, 95%CI 0.651-0.857, p < 0.001) and all-cause death (HR = 0.769, 95%CI 0.696-0.850, p < 0.001). In conclusion, the data of this study revealed that serum CHIP level is significantly correlated with multiple risk factors of CVD and may be one of the predictors of CVD risk and death in MHD patients.

Contribution of the LAC Genes in Fruit Quality Attributes of the Fruit-Bearing Plants: A Comprehensive Review.

Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that provides structure and toughness to the cell walls of numerous fruit plants. The LAC genes that encode laccase enzymes play vital roles in plant physiology, including the synthesis of pigments like PA that contribute to the colors of fruits, and in defending against pathogens and environmental stresses. They are crucial for fruit development, ripening, structural maintenance in plants, and adaptation to various environmental factors. As such, these genes and enzymes are essential for plant growth and development, as well as for various biotechnological applications in environmental remediation and industrial processes. This review article emphasizes the significance of genes encoding laccase enzymes during fruit growth, specifically pertaining to the strengthening of the endocarp through lignification. This process is crucial for ensuring fruit defense and optimizing seed scattering. The information gathered in this article will aid breeders in producing future fruit-bearing plants that are resistant to disease, cost-effective, and nutrient-rich.

Spatial and Temporal Distribution of Phenolic and Flavonoid Compounds in Sour Jujube (Ziziphus. Acidojujuba Cheng et Liu) and Their Antioxidant Activities.

In order to comprehensively analyze the antioxidant substances in sour jujube, total phenolic content (TPC) and total flavonoids contents (TFC) in different organs, including stem, leaf, flower, fruit pulp, and seed were analyzed for their contents and antioxidant activities. The results showed that leaves possessed significantly higher TPC and TFC (20.4 and 20.5 mg/g, respectively) than the other organs and have the highest antioxidant activity, which were also higher than the wild blueberry (A well-known for its high TPC). Subsequently, the variations in the antioxidant content and antioxidant activity of leaves were analyzed during leaf development. TPC in leaves sampled in may and august were significantly higher than that in other months, while the highest one was found in may. The n-hexane, ethyl acetate, n-butanol, and water fractions obtained from the main methanol extract of sour jujube leaves were evaluated for TPC and TFC and their antioxidant activity and it was found that ethyl acetate fraction displayed the highest TPC and TFC (184.5 and 193.3 mg/g, respectively), as well as the best antioxidant activity. In addition, using LC-MS and HPLC, ethyl acetate fraction was analyzed from qualitative and quantitative aspects; 31-one phenolic compounds, including catechin (33.0 mg/g), epigallocatechin (15.3 mg/g), quercetin 3-O-glucoside (11.4 mg/g), naringenin (6.7 mg/g), esculetin (4.8 mg/g), and chlorogenic acid (4.6 mg/g) were identified. Catechin, esculetin, epigallocatechin, chlorogenic acid, quercetin 3-O-glucoside, and naringenin exhibited high antioxidant activity. These results provide a theoretical basis for further study and utilization of flavonoid and polyphenols in sour jujube.

Codon usage patterns across seven Rosales species.

BACKGROUND: Codon usage bias (CUB) analysis is an effective method for studying specificity, evolutionary relationships, and mRNA translation and discovering new genes among various species. In general, CUB analysis is mainly performed within one species or between closely related species and no such study has been applied among species with distant genetic relationships. Here, seven Rosales species with high economic value were selected to conduct CUB analysis. RESULTS: The results showed that the average GC1, GC2 and GC3 contents were 51.08, 40.52 and 43.12%, respectively, indicating that the A/T content is more abundant and the Rosales species prefer A/T as the last codon. Neutrality plot and ENc plot analysis revealed that natural selection was the main factor leading to CUB during the evolution of Rosales species. All 7 Rosales species contained three high-frequency codons, AGA, GTT and TTG, encoding Arg, Val and Leu, respectively. The 7 Rosales species differed in high-frequency codon pairs and the distribution of GC3, though the usage patterns of closely related species were more consistent. The results of the biclustering heat map among 7 Rosales species and 20 other species were basically consistent with the results of genome data, suggesting that CUB analysis is an effective method for revealing evolutionary relationships among species at the family or order level. In addition, chlorophytes prefer using G/C as ending codon, while monocotyledonous and dicotyledonous plants prefer using A/T as ending codon. CONCLUSIONS: The CUB pattern among Rosales species was mainly affected by natural selection. This work is the first to highlight the CUB patterns and characteristics of Rosales species and provides a new perspective for studying genetic relationships across a wide range of species.

Genomic constitution and relationships of Ziziphus species collected from Malakand Division, Pakistan.

Polyploidization has played a major role in plant evolution and can alter plant morphology, phenology, and ecology within only one or a few generations. Ziziphus species are economically as well as nutritionally important fruit-yielding trees. Identification of genotypes with unique traits or those with higher ploidy levels or a broader genetic base could lead to further improvements within the species. The current study has assessed the ploidy levels in the Ziziphus species (Ziziphus jujuba Mill. and Ziziphus nummularia (Burm. f. Wight & Arn) with phenotypic traits, flow cytometry, and chromosomal count as well as with SSRs markers. Morphological traits were inferred to be the most important drivers of trait variations among the investigated genotypes. The total sugar, total cAMPs, titratable acid, and chlorophyll (a, b, and total) were also significantly different in contrast with diploid plants, which showed that tetraploid Ziziphus had the potential to increase nutritional contents. Out of twenty (20), five (5) Z. jujuba genotypes (ZJL-9, ZJL-12, ZJL-17, ZJL-18, and ZJL-19) were found tetraploid 2n = 4x = 48, with genome size ranging from 965.9 to1238.8 Mb that was significantly higher than the tetraploid Z. jujuba Mill. variety Dongzao. Similarly, Z. nummularia ZNL-07 to ZNL-15 have found tetraploid 2n = 4x = 72 with genomic sizes ranging from 1152.2 to 1746.8 Mb respectively. Simple sequence repeats (SSRs) marker was applied to assess the genetic relationship within Ziziphus genotypes. To the best of our understanding, this is the first report on the identification of naturalized random tetraploids within the Pakistani Ziziphus species. This study provides important insights into the genomic architecture of Ziziphus species with implications for classification, conservation, and improvements of Ziziphus germplasm resources.

ZjSEP3 modulates flowering time by regulating the LHY promoter.

BACKGROUND: SEPALLATA3 (SEP3), which is conserved across various plant species, plays essential and various roles in flower and fruit development. However, the regulatory network of the role of SEP3 in flowering time at the molecular level remained unclear. RESULTS: Here, we investigated that SEP3 in Ziziphus jujuba Mill. (ZjSEP3) was expressed in four floral organs and exhibited strong transcriptional activation activity. ZjSEP3 transgenic Arabidopsis showed an early-flowering phenotype and altered the expression of some genes related to flowering. Among them, the expression of LATE ELONGATED HYPOCOTYL (AtLHY), the key gene of circadian rhythms, was significantly suppressed. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assays (EMSAs) further verified that ZjSEP3 inhibited the transcription of AtLHY by binding to the CArG-boxes in its promoter. Moreover, ZjSEP3 also could bind to the ZjLHY promoter and the conserved binding regions of ZjSEP3 were found in the LHY promoter of various plant species. The ectopic regulatory pathway of ZjSEP3-AtLHY was further supported by the ability of 35S::AtLHY to rescue the early-flowering phenotype in ZjSEP3 transgenic plants. In ZjSEP3 transgenic plants, total chlorophyll content and the expression of genes involved in chlorophyll synthesis increased during vegetative stages, which should contribute to its early flowering and relate to the regulatory of AtLHY. CONCLUSION: Overall, ZjSEP3-AtLHY pathway represents a novel regulatory mechanism that is involved in the regulation of flowering time.

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.

Evaluation of Cholinesterase Inhibitory Potential of Different Genotypes of Ziziphus nummularia, Their HPLC-UV, and Molecular Docking Analysis.

Ziziphus nummularia is an important source of valuable phytoconstituents, which are widely used in traditional medicine system of Indo-Pak sub-continent. In this study we investigated the distribution of phenolic compounds in the fruit pericarps of six different genotypes (ZNP01-06) of Z. nummularia growing in the unexplored hilly areas of Pakistan. The methanolic extracts of these genotypes were screened for total phenolic content (TPC), total flavonoid content (TFC), antioxidant, and cholinesterase inhibitory potentials. The observed biological potentials were explained in terms of the outcome of molecular docking and HPLC analyses. Among them, genotype ZNP02 displayed high TPC (88.50 ± 1.23 µg/mL) and showed potent scavenging activity against DPPH (67.03 ± 1.04 µg/mL) and ABTS (65.3 ± 1.74 µg/mL) in comparison to ascorbic acid (68.7 ± 0.47 µg/mL). Moreover, genotypes ZNP01, ZNP02, and ZNP04 displayed potent inhibition against acetyl and butyryl cholinesterases (AChE and BChE) with IC50 values of 21.2, 20.5, and 23.7 µg/mL (AChE) and 22.7, 24.4, and 33.1 µg/mL (BChE), respectively. Furthermore, the individual compounds in the most potent species ZNP01 responsible for potent enzyme inhibition (identified through HPLC-UV analysis), were computed via docking simulation software to the enzyme structures. Among these compounds rutin exhibited significant binding affinity with value of -9.20 kcal/mol. The differences amongst the phytochemical compositions of the selected genotypes highlighted the genotypic variations in them. Based on our results it was concluded that the selected plant can be used as remedy of oxidative stress and neurodegenerative diseases. However, further studies are needed to isolate responsible compounds and test the observed potential in vivo, along with toxicological evaluations in animal models.

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.