Functional analysis of two caffeoyl-coenzyme 3 a-o-methyltransferase involved in pear lignin metabolism.

Caffeoyl-coenzyme 3 A-O-methyltransferase (CCoAOMT) plays a crucial role in the lignin synthesis in many higher plants. In this study, nine PbCCoAOMT genes in total were identified from pear, and classified into six categories. We treated pear fruits with hormones abscisic acid (ABA) and methyl jasmonate (MeJA) and salicylic acid (SA) and observed differential expression levels of these genes. Through qRT-PCR, we also preliminarily identified candidate PbCCoAOMT gene, potentially involved in lignin synthesis in pear fruits. Additionally, the overexpression of PbCCoAOMT1/2 in Arabidopsis and pear fruits increased in lignin content. Enzymatic assays showed that recombinant PbCCoAOMT1/2 proteins have similar enzymatic activity in vitro. The Y1H (Yeast one-hybrid) and dual luciferase (dual-LUC) experiments demonstrated that PbMYB25 can bind to the AC elements in the promoter region of the PbCCoAOMT1 gene. Our findings suggested that the PbCCoAOMT1 and PbCCoAOMT2 genes may contribute to the synthesis of lignin and provide insights into the mechanism of lignin biosynthesis and stone cell development in pear fruits.

Application of citric acid can enhance the accuracy for 13C-urea breath tests in the diagnosis of Helicobacter pylori infection in Chinese patients.

Previous published data have confirmed that the addition of a citric acid meal improves the accuracy of the 13C-urea breath test (13C-UBT). However, some studies have suggested that a citric acid test meal may not be necessary. Thus, the aim of this study was to evaluate the combination of a 13C-UBT with a citric acid meal for the diagnosis of Helicobacter pylori (Hp) infection in a Chinese population, particularly for patients with results in the gray zone. In this paired self-controlled study, all subjects had previously undergone 13C-UBTs without citric acid meals and were randomly divided into two groups based on different doses of citric acid (a low-dose citric acid group and a high-dose citric acid group, comprising meals with 0.68 g and 3.84 g citric acid powder, respectively). Positive rapid urease test (CLO) test and histology results were considered the 'gold standard'. The mean delta over baseline (DOB) value, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were compared between the two groups, particularly for patients with results in the gray zone. In total, 285 patients were tested. Of these patients, 189 were included in the low-dose citric acid group, and 96 were included in the high-dose citric acid group. Among patients with a positive 13C-UBT result without citric acid [delta over baseline (DOB) value ≥ 4‰, n = 174] and a negative 13C-UBT result without citric acid (DOB value < 4‰, n = 111), 8.0% (14/174) were false positive, and 0.9% (1/111) was false negative as determined by gold standard. Of 14 patients with false positive, 78.6% (11/14) false positive were in the gray zone of 4-10‰. However, there were no false positive 13C-UBT results with citric acid in the the gray zone of 4-10‰. In the comparison of the commercial 13C-UBT with the 13C-UBT in the low-dose citric acid group, the sensitivity, specificity, PPV, NPV and accuracy at 15 min were as follows: 99.1% vs. 99.1%, 97.5% vs. 88.9%, 98.2% vs. 92.2%, 98.8% vs. 98.6% and 98.4% vs. 94.7%, respectively. In the the gray zone of 4.0-10.0‰, the comparison of the commercial 13C-UBT with the 13C-UBT in the low-dose citric acid group, the sensitivity, specificity, PPV, and accuracy at 15 min were as follows: 94.4% vs. 100.0%, 100.0% vs. 0%, 100.0% vs. 75.0% and 95.8% vs. 75.0%, respectively. No significant difference was observed between the 15-min and 30-min measurement intervals in the low- and high-dose citric acid groups, including patients with results in the gray zone. The low-dose citric acid test, with an optimal measurement interval of 15 min, was highly accurate in the diagnosis of Hp infection in the Chinese population, especially for individuals with results in the gray zone.

Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

CDK12 regulates angiogenesis of advanced prostate cancer by IGFBP3.

Prostate cancer (PCa) is a prevalent malignancy among men, with a majority of patients presenting with distant metastases at the time of initial diagnosis. These patients are at a heightened risk of developing more aggressive castration­resistant PCa following androgen deprivation therapy, which poses a greater challenge for treatment. Notably, the inhibition of tumor angiogenesis should not be considered an ineffective treatment strategy. The regulatory role of CDK12 in transcriptional and post­transcriptional processes is essential for the proper functioning of various cellular processes. In the present study, the expression of CDK12 was first knocked down in cells using CRISPR or siRNA technology. Subsequently, RNA­seq analysis, co­immunoprecipitation, western blotting, reverse transcription­quantitative polymerase chain reaction and the LinkedOmics database were employed to reveal that CDK12 inhibits insulin like growth factor binding protein 3 (IGFBP3). Western blot analysis also demonstrated that CDK12 promoted VEGFA expression by inhibiting IGFBP3, which involves the Akt signaling pathway. Then, CDK12 was found to promote PCa cell proliferation, cell migration and angiogenesis by inhibiting IGFBP3 through cell proliferation assays, cell migration assays and tube formation assays, respectively. Finally, animal experiments were performed for in vivo validation. It was concluded that CDK12 promoted PCa and its angiogenesis by inhibiting IGFBP3.

DoAP2/ERF89 activated the terpene synthase gene DoPAES in Dendrobium officinale and participated in the synthesis of ß-patchoulene.

Dendrobium officinale Kimura et Migo is a tonic plant that has both ornamental and medicinal properties. Terpenoids are significant and diverse secondary metabolites in plants, and are one of the important natural active ingredients in D. officinale. The AP2/ERF gene family plays a major role in primary and secondary metabolism. However, the AP2/ERF transcription factor family has not been identified in D. officinale, and it is unclear if it is involved in the regulation of terpenoid biosynthesis. This study identified a sesquiterpene synthetase-ß-patchoulene synthase (DoPAES) using transcriptome and terpenic metabolic profile analyses. A total of 111 members of the AP2/ERF family were identified through the whole genome of D. officinale. The tissue-specific expression and gene co-expression pattern of the DoAP2/ERF family members were analyzed. The results showed that the expression of DoPAES was highly correlated with the expression of DoAP2/ERF89 and DoAP2/ERF47. The yeast one-hybrid (Y1H) assays and dual-luciferase experiments demonstrated that DoAP2/ERF89 and DoAP2/ERF47 could regulate the expression of DoPAES. The transcriptional regulatory effects were examined using homologous transient expression of DoAP2/ERF89 in protocorms of D. officinale. DoAP2/ERF89 positively regulated the biosynthesis of ß-patchoulene. This study showed that DoAP2/ERF89 can bind to the promoter region of DoPAES to control its expression and further regulate the biosynthesis of ß-patchoulene in D. officinale. These results provide new insights on the regulation of terpenoid biosynthesis.

Transcription factor PbMYB80 regulates lignification of stone cells and undergoes RING finger protein PbRHY1-mediated degradation in pear fruit.

The Chinese white pear (Pyrus bretschneideri) fruit carries a high proportion of stone cells, adversely affecting fruit quality. Lignin is a main component of stone cells in pear fruit. In this study, we discovered that a pear MYB transcription factor, PbMYB80, binds to the promoters of key lignin biosynthesis genes and inhibits their expression. Stable overexpression of PbMYB80 in Arabidopsis showed that lignin deposition and secondary wall thickening were inhibited, and the expression of the lignin biosynthesis genes in transgenic Arabidopsis was decreased. Transient overexpression of PbMYB80 in pear fruit inhibited lignin metabolism and stone cell development, and the expression of some genes in the lignin metabolism pathway was reduced. In contrast, silencing PbMYB80 with VIGS increased the lignin and stone cell content in pear fruit, and increased expression of genes in the lignin metabolism pathway. By screening a pear fruit cDNA library in yeast, we found that PbMYB80 binds to a RING finger (PbRHY1) protein. We also showed that PbRHY1 exhibits E3 ubiquitin ligase activity and degrades ubiquitinated PbMYB80 in vivo and in vitro. This investigation contributes to a better understanding of the regulation of lignin biosynthesis in pear fruit, and provides a theoretical foundation for increasing pear fruit quality at the molecular level.

Functional and kinetics of two efficient phenylalanine ammonia lyase from Pyrus bretschneideri.

BACKGROUND: The enzyme phenylalanine ammonia lyase (PAL) controls the transition from primary to secondary metabolism by converting L-phenylalanine (L-Phe) to cinnamic acid. However, the function of PAL in pear plants (Pyrus bretschneideri) has not yet been fully elucidated. RESULTS: We identified three PAL genes (PbPAL1, PbPAL2 and PbPAL3) from the pear genome by exploring pear genome databases. The evolutionary tree revealed that three PbPALs were classified into one group. We expressed PbPAL1 and PbPAL2 recombinant proteins, and the purified PbPAL1 and PbPAL2 proteins showed strict substrate specificity for L-Phe, no activity toward L-Tyr in vitro, and modest changes in kinetics and enzyme characteristics. Furthermore, overexpression of PbAL1 and PbPAL1-RNAi, respectively, and resulted in significant changes in stone cell and lignin contents in pear fruits. The results of yeast one-hybrid (Y1H) assays that PbWLIM1 could bind to the conserved PAL box in the PbPAL promoter and regulate the transcription level of PbPAL2. CONCLUSIONS: Our findings not only showed PbPAL's potential role in lignin biosynthesis but also laid the foundation for future studies on the regulation of lignin synthesis and stone cell development in pear fruit utilizing molecular biology approaches.

Functional analysis of a dirigent protein AtsDIR23 in Acorustatarinowii.

Acorus tatarinowii (A. tatarinowii) is a medicinal plant of the Araceae family. Currently, pharmacology focuses on the study of volatile oils, but there are few reports of another important secondary metabolite, lignan. Dirigent protein is thought to play an important role in plant secondary metabolism and responds to a variety of biotic and abiotic stresses. However, the DIR gene family of A. tatarinowii has not been systematically analyzed, and it is unknown whether it affects lignan synthesis. In this study, a total of 27 AtsDIRs were identified by comprehensive analysis of the genome of the medicinal plant A. tatarinowii, and the candidate gene AtsDIR23 that may be involved in lignan synthesis was screened through bioinformatics and transcriptome analysis. It is worth noting that AtsDIR23 is significantly expressed in rhizomes and is a member of the DIR-a subfamily. Subsequently, subcellular localization revealed that AtsDIR23 was localized in chloroplasts. The functional verification of AtsDIR23 b y the transient transformation of A. tatarinowii and the stable transformation of Arabidopsis thaliana showed that the content of lignans in overexpressed plants increased. Co-expression analysis screening revealed the MYB transcription factor (AtsMYB91) that is highly correlated with AtsDIR23 expression, while yeast one-hybrid assays and double luciferase experiments showed that AtsMYB91 negatively regulated the expression of AtsDIR23 b y binding to the AtsDIR23 promoter. In conclusion, AtsDIR23 can promote the accumulation of lignans, which provides a reference for further research on the regulation of lignans by DIR genes.

Two monolignoid biosynthetic genes 4-coumarate:coenzyme A ligase (4CL) and p-coumaric acid 3-hdroxylase (C3H) involved in lignin accumulation in pear fruits.

One of the most important factors impacting the quality of pear fruit is the presence of stone cells and lignin. Lignin is the main component of stone cells in pear fruits. Two monolignoid biosynthetic genes 4-coumarate:coenzyme A ligase (4CL) and p-coumaric acid 3-hdroxylase (C3H) are involved in lignin accumulation in pear fruits. However, the functions of these genes in lignin biosynthesis were excluded in pear. In our study, we isolated and cloned Pb4CL11 (GenBank: KM455955.1) and PbC3H1 (GenBank: KM373790.1) from pear, which contained 1644 bp encoded 54 amino acids (AA), and 1539 bp encoded 513 AA, respectively. The expression of Pb4CL11 and PbC3H1 in Arabidopsis thaliana led to an increase in cell wall thickness for intervascular fibers and xylem cells and lignin content. Overexpression of Pb4CL11 and PbC3H1 in A. thaliana can significantly increase the expression of AtPAL, AtC4H, AtHCT, AtC3H, AtCCOMT, AtCCR, AtF5H, AtCOMT, AtCAD4 and AtCAD5 with promotion of lignin biosynthesis. Taken together, our study's findings not only demonstrated the probable function of Pb4CL11 and PbC3H1 in lignin biosynthesis but also laid the groundwork for future studies using molecular biological methods to control lignin production and the formation of stone cells in pear fruits.

Secreted protease PRSS35 suppresses hepatocellular carcinoma by disabling CXCL2-mediated neutrophil extracellular traps.

Hepatocytes function largely through the secretion of proteins that regulate cell proliferation, metabolism, and intercellular communications. During the progression of hepatocellular carcinoma (HCC), the hepatocyte secretome changes dynamically as both a consequence and a causative factor in tumorigenesis, although the full scope of secreted protein function in this process remains unclear. Here, we show that the secreted pseudo serine protease PRSS35 functions as a tumor suppressor in HCC. Mechanistically, we demonstrate that active PRSS35 is processed via cleavage by proprotein convertases. Active PRSS35 then suppresses protein levels of CXCL2 through targeted cleavage of tandem lysine (KK) recognition motif. Consequently, CXCL2 degradation attenuates neutrophil recruitment to tumors and formation of neutrophil extracellular traps, ultimately suppressing HCC progression. These findings expand our understanding of the hepatocyte secretome's role in cancer development while providing a basis for the clinical translation of PRRS35 as a therapeutic target or diagnostic biomarker.

Effects on stone cell development and lignin deposition in pears by different pollinators.

Introduction: The pear pulp is formed by the development of the ovary wall, which is the somatic cell of the female parent, and its genetic traits are identical to those of the female parent, so that its phenotypic traits should also be identical to those of the female parent. However, the pulp quality of most pears, especially the stone cell clusters (SCCs) number and degree of polymerization (DP), were significantly affected by the paternal type. Stone cells are formed by the deposition of lignin in parenchymal cell (PC) walls. Studies on the effect of pollination on lignin deposition and stone cell formation in pear fruit have not been reported. Methods: In this study, 'Dangshan Su' (P. bretschneideri Rehd.) was selected as the mother tree, while 'Yali' (P. bretschneideri Rehd.) and 'Wonhwang' (P. pyrifolia Nakai.) were used as the father trees to perform cross-pollination. We investigated the effects of different parents on SCCs number and DP, and lignin deposition by microscopic and ultramicroscopic observation. Results and Discussion: The results showed that the formation of SCCs proceeds was consistent in DY and DW, but the SCC number and DP in DY were higher than that in DW. Ultramicroscopy revealed that the lignification process of DY and DW were all from corner to rest regions of the compound middle lamella and the secondary wall, with lignin particles deposited along the cellulose microfibrils. They were alternatively arranged until they filled up the whole cell cavity to culminate in the formation of stone cells. However, the compactness of the wall layer of cell wall was significantly higher in DY than in DW. We also found that the pit of stone cell was predominantly single pit pair, they transported degraded material from the PCs that were beginning to lignify out of the cells. Stone cell formation and lignin deposition in pollinated pear fruit from different parents were consistent, but the DP of SCCs and the compactness of the wall layer were higher in DY than that in DW. Therefore, DY SCC had a higher ability to resist the expansion pressure of PC.

Single cell sequencing revealed the mechanism of CRYAB in glioma and its diagnostic and prognostic value.

Background: We explored the characteristics of single-cell differentiation data in glioblastoma and established prognostic markers based on CRYAB to predict the prognosis of glioblastoma patients. Aberrant expression of CRYAB is associated with invasive behavior in various tumors, including glioblastoma. However, the specific role and mechanisms of CRYAB in glioblastoma are still unclear. Methods: We assessed RNA-seq and microarray data from TCGA and GEO databases, combined with scRNA-seq data on glioma patients from GEO. Utilizing the Seurat R package, we identified distinct survival-related gene clusters in the scRNA-seq data. Prognostic pivotal genes were discovered through single-factor Cox analysis, and a prognostic model was established using LASSO and stepwise regression algorithms. Moreover, we investigated the predictive potential of these genes in the immune microenvironment and their applicability in immunotherapy. Finally, in vitro experiments confirmed the functional significance of the high-risk gene CRYAB. Results: By analyzing the ScRNA-seq data, we identified 28 cell clusters representing seven cell types. After dimensionality reduction and clustering analysis, we obtained four subpopulations within the oligodendrocyte lineage based on their differentiation trajectory. Using CRYAB as a marker gene for the terminal-stage subpopulation, we found that its expression was associated with poor prognosis. In vitro experiments demonstrated that knocking out CRYAB in U87 and LN229 cells reduced cell viability, proliferation, and invasiveness. Conclusion: The risk model based on CRYAB holds promise in accurately predicting glioblastoma. A comprehensive study of the specific mechanisms of CRYAB in glioblastoma would contribute to understanding its response to immunotherapy. Targeting the CRYAB gene may be beneficial for glioblastoma patients.

Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

Branched-chain amino acid (BCAA) catabolism is related to tumorigenesis. However, the underlying mechanism and specific contexts in which BCAAs affect tumor progression remain unclear. Here, we demonstrate that BCAA catabolism is activated in liver cancer cells without glutamine. Enhanced BCAA catabolism leads to BCAA-derived carbon and nitrogen flow toward nucleotide synthesis, stimulating cell-cycle progression and promoting cell survival. Mechanistically, O-GlcNAcylation increases under glutamine-deprivation conditions and stabilizes the PPM1K protein, leading to dephosphorylation of BCKDHA and enhanced decomposition of BCAAs. Dephosphorylation of BCKDHA and high expression of PPM1K promote tumorigenesis in vitro and in vivo and are closely related to the poor prognosis of clinical patients with hepatocellular carcinoma (HCC). Inhibition of BCAA and glutamine metabolism can further retard HCC growth in vivo. These results not only elucidate a mechanism by which BCAA catabolism affects tumorigenesis but also identify pBCKDHA and PPM1K as potential therapeutic targets and predictive biomarkers.

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense.

Dendrobium huoshanense is a kind of precious herb with important medicinal and edible value in China, which is widely used in traditional Chinese medicine for various diseases. Recent studies have paid close attention to the genetic expression of the biosynthetic pathway of the main active components (polysaccharides, alkaloids, and flavonoids), and real-time polymerase chain reaction (qPCR) is one of the most widely used methods for doing so. However, so far, no reference gene selections have been reported in D. huoshanense. In this study, 15 reference gene candidates (GAPDH, eIF, EF-1α, PP2A, UBCE, RPL5, TBP, APT1, MDH, PTBP3, PEPC, CYP71, NCBP2, TIP41, and F-box) were selected and evaluated for their expression stability in D. huoshanense under various experimental conditions, including in different tissues (root, stem, and leaf), abiotic stresses (oxidative, drought, cold, and UV), and hormone treatment (methyl jasmonate) using three statistical programs (geNorm, NormFinder, and BestKeeper). Then, the RefFinder program was employed to comprehensively validate the stability of the selected reference genes. Finally, the expression profiles of the CESA and GMPP genes were further analyzed, and these results indicated that TBP, NCBP2, and CYP71 were the top three most stable reference genes after comprehensive comparison, which could be used as stable reference genes for normalizing the genes expression in D. huoshanense. This study described here provides the first data regarding on reference gene selection in D. huoshanense, which will be extremely beneficial for future research on the gene expression normalization in D. huoshanense.

Transcriptomic analysis of genes related to alkaloid biosynthesis and the regulation mechanism under precursor and methyl jasmonate treatment in Dendrobium officinale.

Dendrobium officinale is both a traditional herbal medicine and a plant of high ornamental and medicinal value. Alkaloids, especially terpenoid indole alkaloids (TIAs), with pharmacological activities are present in the tissues of D. officinale. A number of genes involved in alkaloid biosynthetic pathways have been identified. However, the regulatory mechanisms underlying the precursor and methyl jasmonate (MeJA)-induced accumulation of alkaloids in D. officinale are poorly understood. In this study, we collected D. officinale protocorm-like bodies (PLBs) and treated them with TIA precursors (tryptophan and secologanin) and MeJA for 0 (T0), 4 (T4) and 24 h (T24); we also established control samples (C4 and C24). Then, we measured the total alkaloid content of the PLBs and performed transcriptome sequencing using the Illumina HiSeq 2,500 system. The total alkaloid content increased significantly after 4 h of treatment. Go and KEGG analysis suggested that genes from the TIA, isoquinoline alkaloid, tropane alkaloid and jasmonate (JA) biosynthetic pathways were significantly enriched. Weighted gene coexpression network analysis (WGCNA) uncovered brown module related to alkaloid content. Six and seven genes related to alkaloid and JA bisosynthetic pathways, respectively, might encode the key enzymes involved in alkaloid biosynthesis of D. officinale. Moreover, 13 transcription factors (TFs), which mostly belong to AP2/ERF, WRKY, and MYB gene families, were predicted to regulate alkaloid biosynthesis. Our data provide insight for studying the regulatory mechanism underlying TIA precursor and MeJA-induced accumulation of three types of alkaloids in D. officinale.

Transcriptomics and metabolomics changes triggered by exogenous 6-benzylaminopurine in relieving epicotyl dormancy of Polygonatum cyrtonema Hua seeds.

Polygonatum cyrtonema Hua is one of the most useful herbs in traditional Chinese medicine and widely used in medicinal and edible perennial plant. However, the seeds have the characteristics of epicotyl dormancy. In this study, the molecular basis for relieving epicotyl dormancy of P. cyrtonema seeds under exogenous 6-benzylaminopurine (6-BA) treatment was revealed for the first time through transcriptome and metabolomics analysis. We determined the elongation of epicotyl buds as a critical period for dormancy release and found that the content of trans-zeatin, proline, auxin and gibberellin was higher, while flavonoids and arginine were lower in the treatment group. Transcriptome analysis showed that there were significant differences in gene expression in related pathways, and the expression patterns were highly consistent with the change of metabolites in corresponding pathways. Co-expression analysis showed that cytokinin dehydrogenase of P. cyrtonema (PcCKXs) and pelargonidin in flavonoid biosynthesis, as well as L-proline, L-ornithine, and L-citrulline in arginine and proline metabolism form network modules, indicating that they have related regulatory roles. Above all, our findings provide new insight into the exogenous 6-BA relieving epicotyl dormancy of P. cyrtonema seeds.

PbUGT72AJ2-Mediated Glycosylation Plays an Important Role in Lignin Formation and Stone Cell Development in Pears (Pyrus bretschneideri).

Glycosylation is necessary for many processes of plant secondary metabolism. It can maintain plant homeostasis and is of great significance to normal plant growth and development. At present, the significance of glycosylation for lignin biosynthesis has been proven in some plants, but it has not yet been reported in pears. We used in situ hybridization, in vitro expression, substrate catalysis, transgenic Arabidopsisthaliana, and transient transformation of pear fruit in our investigation, which was predicated on the identification of a gene PbUGT72AJ2 that may be involved in lignin monolignol glycosylation according to our previous work. These results revealed that PbUGT72AJ2 transcripts were localized to some pulp cell walls, lignin deposition, and stone cell areas of pear fruit. The recombinant PbUGT72AJ2-pGEX4T-1 protein had activity against coniferyl alcohol and sinapyl alcohol, and its catalytic efficiency against coniferyl alcohol was higher than that against sinapyl alcohol. When PbUGT72AJ2 was transferred into Arabidopsisthaliana mutants, it was found that some characteristics of Arabidopsisthalianaugt72e3 mutants were restored. In Arabidopsisthaliana, overexpression of PbUGT72AJ2 enhanced the contents of coniferin and syringin, whereas lignification did not change significantly. Transient transformation of pear fruit showed that when PbUGT72AJ2 in pear fruit was silenced by RNA interference, the content of lignin and stone cells in pear fruit increased, whereas the gene PbUGT72AJ2 was overexpressed in pear fruit, and there was almost no change in the pear fruit compared with the control. Lignin deposition in pear fruit was closely related to stone cell development. In this study, we proved that PbUGT72AJ2 plays an important role in lignin deposition and stone cell development in pear fruit, which provides a molecular biological basis for improving pear fruit quality at the molecular level.

Comparative genomic analysis of the COBRA genes in six Rosaceae species and expression analysis in Chinese white pear (Pyrus bretschneideri).

COBRA-Like (COBL) genes encode a glycosylphosphatidylinositol (GPI) anchoring protein unique to plants. In current study, 87 COBRA genes were identified in 6 Rosaceae species, including Pyrus bretschneideri (16 genes), Malus domestica (22 genes), Fragaria vesca (13 genes), Prunus mume (11 genes), Rubus occidentalis (13 genes) and Prunus avium (12 genes). We revealed the evolution of the COBRA gene in six Rosaceae species by phylogeny, gene structure, conservative sequence, hydrophobicity analysis, gene replication events and sliding window analysis. In addition, based on the analysis of expression patterns in pear fruit combined with bioinformatics, we identified PbCOBL12 and PbCOBL13 as potential genes regulating secondary cell wall (SCW) formation during pear stone cell development. This study aimed to understand the evolutionary relationship of the COBRA gene in Rosaceae species, clarify the potential function of COBRA in pear fruit development, and provide essential theoretical basis and gene resources for improving pear fruit quality through genetical modification mechanism.

Comparative Genomic Analysis of SAUR Gene Family, Cloning and Functional Characterization of Two Genes (PbrSAUR13 and PbrSAUR52) in Pyrus bretschneideri.

The SAUR (small auxin-up RNA) gene family is the biggest family of early auxin response genes in higher plants and has been associated with the control of a variety of biological processes. Although SAUR genes had been identified in several genomes, no systematic analysis of the SAUR gene family has been reported in Chinese white pear. In this study, comparative and systematic genomic analysis has been performed in the SAUR gene family and identified a total of 116 genes from the Chinese white pear. A phylogeny analysis revealed that the SAUR family could be classified into four groups. Further analysis of gene structure (introns/exons) and conserved motifs showed that they are diverse functions and SAUR-specific domains. The most frequent mechanisms are whole-genome duplication (WGD) and dispersed duplication (DSD), both of which may be important in the growth of the SAUR gene family in Chinese white pear. Moreover, cis-acting elements of the PbrSAUR genes were found in promoter regions associated with the auxin-responsive elements that existed in most of the upstream sequences. Remarkably, the qRT-PCR and transcriptomic data indicated that PbrSAUR13 and PbrSAUR52 were significantly expressed in fruit ripening. Subsequently, subcellular localization experiments revealed that PbrSAUR13 and PbrSAUR52 were localized in the nucleus. Moreover, PbrSAUR13 and PbrSAUR52 were screened for functional verification, and Dangshan pear and frandi strawberry were transiently transformed. Finally, the effects of these two genes on stone cells and lignin were analyzed by phloroglucinol staining, Fourier infrared spectroscopy, and qRT-PCR. It was found that PbrSAUR13 promoted the synthesis and accumulation of stone cells and lignin, PbrSAUR52 inhibited the synthesis and accumulation of stone cells and lignin. In conclusion, these results indicate that PbrSAUR13 and PbrSAUR52 are predominantly responsible for lignin inhibit synthesis, which provides a basic mechanism for further study of PbrSAUR gene functions.