Comprehensive study of volatile compounds and transcriptome data providing genes for grape aroma.

BACKGROUND: Fruit aroma is an important quality with respect to consumer preference, but the most important aroma compounds and their genetic regulatory mechanisms remain elusive. RESULTS: In this study, we qualitatively analysed volatile compounds in the pulp and skin of five table grape cultivars with three aroma types (muscat, strawberry, and neutral) using solid-phase microextraction gas chromatography/mass spectrometry. We identified 215 aroma compounds, including 88 esters, 64 terpenes, and 29 alcohols, and found significant differences in the number of compounds between the pulp and skin, especially for terpenes. Skin transcriptome data for the five grape cultivars were generated and subjected to aroma compound-gene correlation analysis. The combined transcriptomic analysis and terpene profiling data revealed 20 candidate genes, which were assessed in terms of their involvement in aroma biosynthetic regulation, including 1 VvCYP (VIT_08s0007g07730), 2 VvCCR (VIT_13s0067g00620, VIT_13s0047g00940), 3 VvADH (VIT_00s0615g00010, VIT_00s0615g00030, VIT_ 00s0615g00020), and 1 VvSDR (VIT_08s0040g01200) in the phenylpropanoids synthesis pathway, and 1 VvDXS (VIT_05s0020g02130) and 6 VvTPS (VIT_13s0067g00370, Vitis_vinifera_newGene_3216, VIT_13s0067g00380, VIT_13s0084g00010, VIT_00s0271g00010, and VIT_13s0067g00050) in the methylerythritol phosphate pathway (involved in the production and accumulation of aromatic compounds). Additionally, 2 VvMYB (VIT_17s0000g07950, VIT_03s0063g02620) and 1 VvGATA (VIT_15s0024g00980) transcription factor played important regulatory roles in the accumulation of key biosynthetic precursors of these compounds in grapes. Our results indicated that downstream genes, specifically 1 VvBGLU (VIT_03s0063g02490) and 2 VvUGT (VIT_17s0000g07070, VIT_17s0000g07060) are involved in regulating the formation and volatilization of bound compounds in grapes. CONCLUSIONS: The results of this study shed light on the volatile compounds and "anchor points" of synthetic pathways in the pulp and skin of muscat and strawberry grapes, and provide new insight into the regulation of different aromas in grapes.

Expression Analysis of AUX/IAA Family Genes in Apple Under Salt Stress.

Members of the auxin/indoleacetic acid (Aux/IAA) gene family in plants are primary auxin-responsive genes that play important roles in many aspects of plant development and in responses to abiotic stress. Recently, 33 Aux/IAA have been identified in the apple genome. The biological responses of MdIAAs to salt stress are still unknown. In this study, Malus zumi, Malus baccata, and Malus × domestica 'Fuji' plantlets were subjected to salt stress by supplementing hydroponic media with NaCl at various concentrations. M. zumi showed the strongest salt resistance, followed by 'Fuji', and M. baccata was the most sensitive to salt stress. Tissue-specific expression profiles of MdIAAs were determined by quantitative real-time polymerase chain reaction. When apple plantlets were subjected to salt stress, most of salt-responsive MdIAAs were up-regulated by 1 h, 3 h, and 6 h in roots, shoot tips, and leaves, respectively. Highly expressed MdIAAs in roots, especially for M. zumi, consisted with the salt tolerance of apple rootstocks. Transgenic apple calli were tolerant to salt stress when over-expressed salt-responsive genes, MdIAA8, -9, and -25. These results provide clues about salt resistance in these three Malus species, which helps apple breeding of salt tolerance by genetic transformation.

Functional Characterization of MdTAC1a Gene Related to Branch Angle in Apple (Malus x domestica Borkh.).

The Tiller Angle Control 1 (TAC1) gene belongs to the IGT family, which mainly controls plant branch angle, thereby affecting plant form. Two members of MdTAC1 are identified in apple; the regulation of apple branch angle by MdTAC1 is still unclear. In this study, a subcellular localization analysis detected MdTAC1a in the nucleus and cell membrane, but MdTAC1b was detected in the cell membrane. Transgenic tobacco by overexpression of MdTAC1a or MdTAC1b showed enlarged leaf angles, the upregulation of several genes, such as GA 2-oxidase (GA2ox), and a sensitive response to light and gravity. According to a qRT-PCR analysis, MdTAC1a and MdTAC1b were strongly expressed in shoot tips and vegetative buds of weeping cultivars but were weakly expressed in columnar cultivars. In the MdTAC1a promoter, there were losses of 2 bp in spur cultivars and 6 bp in weeping cultivar compared with standard and columnar cultivars. An InDel marker specific to the MdTAC1a promoter was developed to distinguish apple cultivars and F1 progeny. We identified a protein, MdSRC2, that interacts with MdTAC1a, whose encoding gene which was highly expressed in trees with large branch angles. Our results indicate that differences in the MdTAC1a promoter are major contributors to branch-angle variation in apple, and the MdTAC1a interacts with MdSRC2 to affect this trait.

SEFPN: Scale-Equalizing Feature Pyramid Network for Object Detection.

Feature Pyramid Network (FPN) is used as the neck of current popular object detection networks. Research has shown that the structure of FPN has some defects. In addition to the loss of information caused by the reduction of the channel number, the features scale of different levels are also different, and the corresponding information at different abstract levels are also different, resulting in a semantic gap between each level. We call the semantic gap level imbalance. Correlation convolution is a way to alleviate the imbalance between adjacent layers; however, how to alleviate imbalance between all levels is another problem. In this article, we propose a new simple but effective network structure called Scale-Equalizing Feature Pyramid Network (SEFPN), which generates multiple features of different scales by iteratively fusing the features of each level. SEFPN improves the overall performance of the network by balancing the semantic representation of each layer of features. The experimental results on the MS-COCO2017 dataset show that the integration of SEFPN as a standalone module into the one-stage network can further improve the performance of the detector, by ∼1AP, and improve the detection performance of Faster R-CNN, a typical two-stage network, especially for large object detection APL∼2AP.

Genome-Wide Identification of the Aux/IAA Family Genes (MdIAA) and Functional Analysis of MdIAA18 for Apple Tree Ideotype.

The Aux/IAA (auxin/indole-3-acetic acid) gene family is one of the early auxin-responsive gene families, which play a central role in auxin response. Few reports are involved in Aux/IAA genes in fruit trees, especially in apple (Malus × domestica Borkh.). A total of 33 MdIAA members were identified, of which 27 members contained four conserved domains, whereas the others lost one or two conserved domains. Several cis-elements in promoters of MdIAAs were predicted responsive to hormones and abiotic stress. Tissue-specific expression patterns of MdIAAs in different apple tree ideotypes were investigated by quantitative real-time PCR. A large number of MdIAAs were highly expressed in leaf buds and reproductive organs, and MdIAAs clustered in same group showed similar expression profiles. Overexpression of MdIAA18 in Arabidopsis resulted in compact phenotype. These results indicated that MdIAA genes may be involved in vegetative and reproductive growth of apple. Taken together, the results provide useful clues to reveal the function of MdIAAs in apple and control apple tree architecture by manipulation of MdIAAs.

Comparative Transcriptome Analysis of Genes Involved in Anthocyanin Biosynthesis in Red and Green Walnut (Juglans regia L.).

Fruit color is an important economic trait. The color of red walnut cultivars is mainly attributed to anthocyanins. The aim of this study was to explore the differences in the molecular mechanism of leaf and peel color change between red and green walnut. A reference transcriptome of walnut was sequenced and annotated to identify genes related to fruit color at the ripening stage. More than 290 million high-quality reads were assembled into 39,411 genes using a combined assembly strategy. Using Illumina digital gene expression profiling, we identified 4568 differentially expressed genes (DEGs) between red and green walnut leaf and 3038 DEGs between red and green walnut peel at the ripening stage. We also identified some transcription factor families (MYB, bHLH, and WD40) involved in the control of anthocyanin biosynthesis. The trends in the expression levels of several genes encoding anthocyanin biosynthetic enzymes and transcription factors in the leaf and peel of red and green walnut were verified by quantitative real-time PCR. Together, our results identified the genes involved in anthocyanin accumulation in red walnut. These data provide a valuable resource for understanding the coloration of red walnut.

How does workplace ostracism affect employee innovation behavior: An analysis of chain mediating effect.

This study seeks to examine the relationship between workplace ostracism and innovation behavior while considering the mediating role of knowledge hiding and organizational identification. The study also tests the moderating role of task interdependence in these relationships. The study collected data through structured questionnaires from 409 participants (i.e., employees) working in the small to medium-sized enterprise of big cities of China. The study adopted a structured equation modeling technique for data analysis. Significantly, the study results suggest that workplace ostracism is negatively associated with innovation behavior, both directly and indirectly via knowledge hiding and organizational identification. We also find that task interdependence weakens the positive relationship between workplace ostracism and knowledge hiding. Current study has tested the negative relationship between workplace ostracism and innovation behavior unlike most of the previous investigations that have focused on positive factors. Our study from a rational perspective to explore the influence mechanism between workplace ostracism and innovation behavior is addition to the previous research and the rich, in revelation managers motivate employees to implement knowledge sharing activities at the same time, pay attention to take measures to restrain negative knowledge such as knowledge hidden activities, to activate the creativity of organization staff of intellectual resources. This paper contributes to innovation behavior literature which is an important part of innovation management based on both conservation of resources theory and social network theory.

Does small and medium enterprise differential leadership increase subordinate knowledge hiding? Evidences from job insecurity, territorial consciousness and leadership performance expectation.

Leadership is considered as a significant antecedent of knowledge hiding in SMEs (small and medium enterprises), but the differential dimension of leadership has been evidently neglected in both theoretical and empirical areas. Drawing on conservation of resource theory and social cognitive theory, this research investigates whether and how SME differential leadership influences subordinate knowledge hiding. Specifically, we analyze the underlying mechanisms of a chain-mediator-job insecurity and territorial consciousness and a boundary condition-leadership performance expectation. Multi-wave and multi-source data were collected from a sample of 704 Chinese SME employees and 140 relevant leaders and applied HLM meso-mediational frameworks, and Bootstrap technique with non-parametric percentile residuals for deviation correction. The results show that differential leadership plays a potential role in promoting subordinate knowledge hiding through the serial intervening mechanism of job Insecurity and territorial consciousness in SMEs. Furthermore, the positive relationship between SME differential leadership and job insecurity becomes stronger among subordinates under higher leadership performance expectation; the positive indirect relationship between SME differential leadership and subordinate knowledge hiding is stronger with higher levels of leadership performance expectation. This study contributes to the existing academic literature by empirically analyzing the under-investigated correlation between differential leadership and subordinate knowledge hiding in SMEs, and by exploring the underlying mechanisms and a boundary condition.

The Impact of Network Embeddedness on the Innovation Performance of New Generation of Employees in the Post-COVID-19 Era-The Mediating Role of Psychological Contract.

The innovation activities of new generation of employees have the characteristics of double network embeddedness, and the degree of psychological contract fulfilment is an important factor that affects their innovation performance. Based on the attributes of internal network embeddedness and external network embeddedness, this paper builds a hypothesis model of the relationship between network embeddedness, psychological contract and innovation performance. It explores the impact and mechanism of network embeddedness on the innovation performance of new generation of employees and the mediating role of the psychological contract. Empirical research shows that network embeddedness has a positive effect on the innovation performance of new generation of employees. The psychological contract has a mediating role in network embeddedness on innovation performance of new generation of employees. These conclusions continue and deepen the research on network embeddedness and innovation performance and further enrich and expand the application of social networks in the research of individual innovation performance of new generation of employees.

"Lubricant" or "Stumbling Block"?: The Paradoxical Association Between Team Authoritarian Leadership and Creative Deviance.

Recently, creative deviance has been lauded to be an innovation-enhancing approach with applications in many new and high-tech domains. Previous study on antecedents to creative deviance remains scattered and vague. Our research conceptualizes creative deviance from the perspective of independent innovation and explores its antecedents, mechanisms, as well as conditions. Team authoritarian leadership is conceptualized as a contradictory unity as it mixes advantages and disadvantages. However, it is surprising to find that there are very few researches that have examined its relevant influence mechanisms and boundary conditions for authoritarian leadership. Contributing to an advanced understanding of authoritarian leadership in research and development teams, we investigated whether team authoritarian leadership is positively or negatively related to creative deviance. Drawing on social information processing theory and regulatory focus theory, we supposed that team authoritarian leadership facilitates creative deviance when the degree is low and inhibits it when the degree is high; dual occupational stress and prevention regulatory focus play mediation roles between team authoritarian leadership and creative deviance respectively, both variables play a chain mediation role in that relationship; and the mindfulness characteristic of an individual moderates the inverted-U team authoritarian leadership-creative deviance association, such that this association is weaker with low individual mindfulness. With two-phase questionnaire data collected from 433 members in 82 R&D teams of high-tech enterprises in electronic information technology, new material technology, new medical technology, resource and environment technology and advanced manufacturing technology randomly selected from five provinces in eastern China, these hypotheses are supported empirically. Overall, we find that, our study broadens antecedents and the relevant occurrence mechanisms of creative deviance when studied through a leadership management lens. Moreover, our research enriches the cognate studies on authoritarian leadership by empirically demonstrating that team authoritarian leadership may function as an double-edged sword of creative deviance in the R&D workplace. These above findings offer insightful thoughts to scholars in the field of authoritarian leadership and bring practical suggestions for team superiors who seek to implement best innovation practice.

O-GlcNAcylation and stablization of SIRT7 promote pancreatic cancer progression by blocking the SIRT7-REGγ interaction.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and its dismal prognosis indicates the urgent need to elucidate the potential oncogenic mechanisms. SIRT7 is a classic NAD+-dependent deacetylase that stabilizes the transformed state of cancer cells. However, its functional roles in PDAC are still unclear. Here, we found that SIRT7 expression is upregulated and predicts poor prognosis in PDAC. Then we screened the new interacting proteins of SIRT7 by mass spectrometry and the results showed that SIRT7 can interact with O-GlcNAc transferase (OGT). O-GlcNAcylation stabilizes the SIRT7 protein by inhibiting its interaction with REGγ to prevent degradation, and hyper-O-GlcNAcylation in pancreatic cancer cells leads to hypoacetylation of H3K18 via SIRT7, which promotes transcriptional repression of several tumour suppressor genes. In addition, SIRT7 O-GlcNAcylation at the serine 136 residue (S136) is required to maintain its protein stability and deacetylation ability. In vivo and in vitro experiments showed that blocking SIRT7 O-GlcNAcylation at S136 attenuates tumour progression. Collectively, we demonstrate that O-GlcNAcylation is an important post-translational modification of SIRT7 in pancreatic cancer cells, and elucidating this mechanism of SIRT7 is expected to pave the way for the development of novel therapeutic methods in the future.

Erythrocyte-Camouflaged Mesoporous Titanium Dioxide Nanoplatform for an Ultrasound-Mediated Sequential Therapies of Breast Cancer.

BACKGROUND: The hypoxic microenvironment promotes tumor resistance to most treatments, especially highly oxygen-dependent sonodynamic therapy (SDT). METHOD AND RESULTS: In view of the aggravation of hypoxia by oxygen consumption during SDT, a biomimetic drug delivery system was tailored to integrate SDT with hypoxia-specific chemotherapy. In this system, mesoporous titanium dioxide nanoparticles (mTNPs) were developed to deliver the hypoxia-activated prodrug AQ4N with high loading efficiency. Subsequently, a red blood cell (RBC) membrane was coated onto the surface of mTNP@AQ4N. RBC-mTNPs@AQ4N inherited the immune escape ability from RBC membranes, thus efficiently reducing the immunological clearance and improving the work concentration. Upon activation by ultrasound (US), mTNPs as sonosensitizers generate reactive oxide species (ROS), which not only induce apoptosis and necrosis but also disrupt RBC membranes to achieve the US-mediated on-demand release of AQ4N. The released AQ4N was activated by hypoxia to convert into toxic products, which effectively supplemented the inefficiency of SDT in hypoxic tissues. Importantly, SDT-aggravated hypoxia further potentiated this hypoxia-specific chemotherapy of AQ4N. CONCLUSION: Based on the sequential strategy, RBC-mTNPs@AQ4N exhibited an excellent synergistic therapeutic effect, thus potentially advancing the development of SDT in cancer treatments.

Exosomes Derived from Pancreatic Cancer Cells Induce Osteoclast Differentiation Through the miR125a-5p/TNFRSF1B Pathway.

BACKGROUND: Pancreatic cancer (PC) was regarded as the 4th principal cause of cancer-related fatalities in the United States and patients usually suffered from severe nutrition deficiency, muscle wasting, as well as bone loss. In our previous research, we have found that PC-derived exosomes potentially initiate insulin resistance in skeletal muscle cells. However, the role of exosomes in the PC-related bone loss remains unknown. METHODS: The effect of PC-derived exosomes on the osteoclast differentiation and femoral bone structure in the orthotopic xenograft mouse model were investigated. MiRNA expression profiles were detected and a dual luciferase experiment was conducted to identify the direct target of miRNA. RESULTS: Our data showed that PC-derived exosomes significantly induced osteoclast differentiation and increased expression of NFAT2, TRAP, CTSK and MMP-9. The bone volume fraction and trabecular thickness of femur significantly reduced in osteoporotic model. Microarray analyses and luciferase reporter assay showed that the process was, at least partially, mediated by the miR-125a-5p/TNFRSF1B signaling pathways. CONCLUSION: According to the results, novel insights have been claimed the effect of exosomes derived from PC on bone deterioration and explained correlation between PC and cancer-related bone loss.

A New Fusion Peptide Targeting Pancreatic Cancer and Inhibiting Tumor Growth.

BACKGROUND: Pancreatic cancer is a highly malignant tumor of the digestive system. Early pancreatic cancer is often difficult to diagnosis due to its atypical clinical symptoms. Patients with pancreatic cancer have a very poor prognosis because they have lost the opportunity for radical surgical tumor resection and they are less sensitive to the clinically used radiotherapy and chemotherapy. METHODS: In this study, a peptide targeting pancreatic cancer cells was screened by phage display technology, and its targeting property was evaluated in vitro using PANC1 cells by fluorescence imaging and flow cytometry. Furthermore, the targeting peptide was conjugated to the pro-apoptotic KLAKLAKKLAKLAK (KLA), the fusion peptide and its targeting ability that allowing KLA to specifically enter pancreatic tumor cells in vitro and in vivo was confirmed by fluorescence imaging and in vivo imaging system (IVIS). Its mechanism was determined using flow cytometry, mitochondrial membrane potential evaluation and Western blot. The inhibitory effect on pancreatic tumor growth and toxic effects were evaluated by animal experiment. RESULTS: Due to the internalization facilitated by the targeting mechanism of the targeting peptide, KLA specifically entered pancreatic cancer cells, destroyed mitochondria and induced apoptosis. The fusion peptide and its targeting ability that allowing KLA to specifically enter pancreatic tumor cells and exert a significant inhibitory effect on pancreatic tumor growth with reduced toxic effects. CONCLUSION: This approach possesses potential advantages in the clinical diagnosis and treatment of pancreatic cancer.

Nitric oxide-induced stromal depletion for improved nanoparticle penetration in pancreatic cancer treatment.

Abundant desmoplastic stroma, which typically exists in pancreatic ductal adenocarcinoma (PDAC), can act as a natural protective physical barrier rendering insufficient drug delivery and penetration. To address this issue, we herein report a two-step sequential delivery strategy for enhanced pancreatic cancer therapy. In this sequential strategy, the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) loaded liposomes (Lip-SNAP) were firstly delivered to pancreatic stellate cells (PSCs) in tumor tissue to inhibit the production of dense stroma, by inhibiting the expression of TGF-ß1 and its downstream profibrotic signal transduction. Therefore, the PSC-mediated desmoplastic reaction could be suppressed by inhibiting the expression of fibronectin, α-SMA and collagen. The gemcitabine (GEM) loaded liposomes (Lip-GEM) were administrated subsequently. The enhanced intratumoral penetration of Lip-GEM was then achieved due to the stromal disruption in consequence of NO treatment, thus significantly improving the drug delivery efficiency. The tumor growth inhibition of the two-step sequential delivery of Lip-SNAP and Lip-GEM was investigated on both subcutaneous and orthotopic tumor mouse models, to show the remarkably improved therapeutic efficacy of GEM. Such NO-induced stromal depletion provides a general strategy to overcome the blockage of desmoplastic stroma on other therapeutic agents.

FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors.

Pancreatic ductal adenocarcinoma (PDAC) is intractable due to its strong invasiveness and metastatic ability. Epithelial-mesenchymal transition (EMT) is the pivotal driver of tumor invasion and metastasis. The four-and-a-half LIM domain (FHL) family is involved in regulating transforming growth factor (TGF)-ß and Ras signaling, which might control the EMT process. In this study, we found that higher expression of four-and-a-half LIM domains 3 (FHL3) predicted poor prognosis in PDAC. The decreasing of FHL3 changed the EMT phenotype by blocking the TGFß/Atk/GSK3ß/ubiquitin pathways. Interestingly, the GSK3ß inhibitor could abrogate the role of FHL3 in the regulation of snail1 and twist1 expression, which implied that GSK3ß plays a pivotal role in the FHL3-mediated EMT process. Furthermore, we found that FHL3 can directly bind to GSK3ß, which weakened the interaction between GSK3ß and snail1/twist1. We also found that the LIM-3 domain of FHL3 was required for the binding of FHL3 to GSK3ß. Collectively, our study implied that FHL3, as a binding partner of GSK3ß, promoted tumor metastasis in PDAC through inhibiting the ubiquitin-degradation of snail1 and twist1.

Lycium ruthenicum studies: Molecular biology, Phytochemistry and pharmacology.

Lycium ruthenicum has been used as ethnic medicine and nutraceutical food. Existing studies of L. ruthenicum can be classifies into three areas: (1) those in which molecular biology methods were used to study its origin, genetic variation and relationships with other species; (2) those in which phytochemical methods were used to extract, isolate, and identify compounds; and (3) those in which pharmacological methods were used to study active compounds. The purpose of this paper is to provide an overview of L. ruthenicum studies. This review will provide a useful bibliography for further investigations and applications of L. ruthenicum in medicines and foods.

Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3.

One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut.

Integrated microRNA and mRNA expression profiling reveals a complex network regulating pomegranate (Punica granatum L.) seed hardness.

The breeding of new soft-seeded pomegranate cultivars provides new products for the market and increases farmers' incomes, yet the genetic architecture mediating seed hardness is largely unknown. Here, the seed hardness and hundred-seed weights of 26 cultivars were determined in 2 successive years. We conducted miRNA and mRNA sequencing to analyse the seeds of two varieties of Punica granatum: soft-seeded Tunisia and hard-seeded Sanbai, at 60 and 120 d after flowering. Seed hardness was strongly positively correlated with hundred-seed weight. We detected 25 and 12 differentially expressed miRNA-mRNA pairs with negative regulatory relationships between the two genotypes at 60 and 120 d after flowering, respectively. These miRNA-mRNA pairs mainly regulated seed hardness by altering cell wall structure. Transcription factors including NAC1, WRKY and MYC, which are involved in seed hardness, were targeted by differentially expressed mdm-miR164e and mdm-miR172b. Thus, seed hardness is the result of a complex biological process regulated by a miRNA-mRNA network in pomegranate. These results will help us understand the complexity of seed hardness and help to elucidate the miRNA-mediated molecular mechanisms that contribute to seed hardness in pomegranate.