Transcription factor-based biosensor: A molecular-guided approach for advanced biofuel synthesis.

As a sustainable and renewable alternative to petroleum fuels, advanced biofuels shoulder the responsibility of energy saving, emission reduction and environmental protection. Traditional engineering of cell factories for production of advanced biofuels lacks efficient high-throughput screening tools and regulating systems, impeding the improvement of cellular productivity and yield. Transcription factor-based biosensors have been widely applied to monitor and regulate microbial cell factory products due to the advantages of fast detection and in-situ screening. This review updates the design and application of transcription factor-based biosensors tailored for advanced biofuels and related intermediates. The construction and genetic parts selection principle of biosensors are discussed. Strategies to enhance the performance of biosensor, including regulating promoter strength and RBS strength, optimizing plasmid copy number, implementing genetic amplifier, and modulating the structure of transcription factor, have also been summarized. We further review the application of biosensors in high-throughput screening of new metabolic engineering targets, evolution engineering, confirmation of protein function, and dynamic regulation of metabolic flux for higher production of advanced biofuels. At last, we discuss the current limitations and future trends of transcription factor-based biosensors.

Ocean acidification and desalination increase the growth and photosynthesis of the diatom Skeletonema costatum isolated from the coastal water of the Yellow Sea.

Global climate changes induce substantial alterations in the marine system, including ocean acidification (OA), desalination and warming of surface seawater. Here, we examined the combined effects of OA and reduced salinity under different temperatures on the growth and photosynthesis of the diatom Skeletonema costatum. After having been acclimated to 2 CO2 concentrations (400 µatm, 1000 µatm) and 2 salinity levels (20 psu, 30 psu) at temperature levels of 10 °C and 20 °C, the diatom showed enhanced growth rate at the lowered salinity and elevated pCO2 irrespective of the temperature. The OA treatment increased the net photosynthetic rate and biogenic silica (Bsi) contents. Increasing the temperature from 10 to 20 °C raised the net photosynthetic rate by over twofold. The elevated pCO2 increased the net and gross photosynthetic rates by 20%-40% and by 16%-32%, respectively, with the higher enhancement observed at the higher levels of salinity and temperature. Our results imply that OA and desalination along with warming to the levels tested can enhance S. costatum's competitiveness in coastal phytoplankton communities under influence of future climate changes.

Unraveling the Role of RNase L Knockout in Alleviating Immune Response Activation in Mice Bone Marrow after Irradiation.

Ionizing radiation (IR) induces severe hematopoietic injury by causing DNA and RNA damage as well as activating the immune responses, necessitating the development of effective therapeutic strategies. Ribonuclease L (RNase L) as an innate immune response pathway is triggered by exogenous and endogenous abnormal dsRNA under viral infection and dyshomeostasis, thereby activating the immune responses. Thus, we investigated the effect of RNase L on irradiation-induced bone marrow damage using RNase L knockout (RNase L-/-) mice. Phenotypic analysis revealed that RNase L knockout mitigates irradiation-induced injury in the bone marrow. Further investigation into the mechanism of RNase L by RNA-seq, qRT-PCR, and CBA analysis demonstrated that RNase L deficiency counteracts the upregulation of genes related to immune responses induced by irradiation, including cytokines and interferon-stimulated genes. Moreover, RNase L deficiency inhibits the increased levels of immunoglobulins in serum induced by irradiation. These findings indicate that RNase L plays a role in the immune response induced by irradiation in the bone marrow. This study further enhances our understanding of the biological functions of RNase L in the immune response induced by irradiation and offers a novel approach for managing irradiation-induced bone marrow injury through the regulation of RNase L activation.

Integrated plasma proteomics and N-glycoproteomics reveals alterations in the N-glycosylation in Chinese hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is a life-threatening disease that presents diagnostic challenges due to the absence of reliable biomarkers. Recently, plasma proteomics and glycoproteomics have emerged as powerful tools for identifying potential diagnostic biomarkers for various diseases. In this study, we conducted a comprehensive proteomic and glycoproteomic analysis of plasma samples from 11 HCC patients and 11 healthy control (HC) individuals. We identified 20 differentially expressed (DE) proteins and 32 DE intact glycosylated peptides (IGPs) that can effectively differentiate between HCC patients and HC samples. Our findings demonstrate that IGP profiles had better predictive power than protein profiles for screening HCC. Pathways associated with DE proteins and IGPs were identified. It was reported that the protein expression level of galectin 3 binding protein (LGALS3BP) and its N-linked glycosylation at the N398 and N551 sites might serve as valuable candidates for HCC diagnosis. These results highlight the importance of N-glycoproteomics in advancing our understanding of HCC and suggest possible candidates for the future diagnosis of this disease.

Biomarkers and Tourette syndrome: a systematic review and meta-analysis.

Objective: This research aims to investigate whether peripheral biomarkers might differentiate individuals with Tourette syndrome (TS) from those without the condition. Methods: A broad range of databases was searched through November 2022. This study employed a systematic literature review and subsequent meta-analysis of case-control studies that assessed the aberration of biomarkers of patients with TS and controls. Results: A total of 81 studies were identified, out of which 60 met the eligibility criteria for inclusion in the meta-analysis. Following a meticulous screening procedure to determine the feasibility of incorporating case-control studies into the meta-analysis, 13 comparisons were statistically significant [CD3+ T cell, CD4+ T cell, CD4+ T cell to CD8+ T cell ratio, NK-cell, anti-streptolysin O antibodies, anti-DNase antibodies, glutamic acid (Glu), aspartic acid (Asp), ferritin (Fe), zinc (Zn), lead (Pb), vitamin D, and brain-derived neurotrophic factor (BDNF)]. Publication bias was found for anti-streptolysin O antibodies. Suggestive associations were evidenced for norsalsolinol (NSAL), neuron-specific enolase (NSE), and S100B. Conclusion: In this study, we present empirical evidence substantiating the link between several peripheral biomarkers and the early diagnosis of TS. Larger and more standardized studies are necessary to replicate the observed results, elucidate the specificity of the biomarkers for TS, and evaluate their precision for use in clinical settings.

Disentangled self-attention neural network based on information sharing for click-through rate prediction.

With the exponential growth of network resources, recommendation systems have become successful at combating information overload. In intelligent recommendation systems, the prediction of click-through rates (CTR) plays a crucial role. Most CTR models employ a parallel network architecture to successfully capture explicit and implicit feature interactions. However, the existing models ignore two aspects. One limitation observed in most models is that they focus only on the interaction of paired term features, with no emphasis on modeling unary terms. The second issue is that most models input characteristics indiscriminately into parallel networks, resulting in network input oversharing. We propose a disentangled self-attention neural network based on information sharing (DSAN) for CTR prediction to simulate complex feature interactions. Firstly, an embedding layer transforms high-dimensional sparse features into low-dimensional dense matrices. Then, the disentangled multi-head self-attention learns the relationship between different features and is fed into a parallel network architecture. Finally, we set up a shared interaction layer to solve the problem of insufficient information sharing in parallel networks. Results from experiments conducted on two real-world datasets demonstrate that our proposed method surpasses existing methods in predictive accuracy.

Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, 'Yunnan' quince (Cydonia oblonga Mill.) had a dwarfing effect on 'Zaosu' pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising 'Zaosu' (scion) grafted onto 'Yunnan' quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 down-regulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

Polymorphism in IGFALS gene and its association with scrotal circumference in Hu lambs.

Scrotal circumference is an important reproductive index of breeding rams, which has a high genetic correlation with ejaculation volume and semen quality. In this study, the scrotal circumference of 1353 male Hu sheep at different stages of development was measured and descriptive statistical analysis was performed. The results showed that the coefficient of variation of scrotal circumference at each stage was greater than 10%, and its heritability were moderately to high, ranging from 0.318 to 0.719. We used PCR amplification and Sanger sequencing to scan the polymorphisms of the IGFALS gene, and performed association analysis with the circumference of the scrotum at different stages. We identified a synonymous mutation g.918 G > C in exon 1 of the IGFALS gene, and this mutation was significantly associated with scrotal circumference at 100, 120, 140, 160 and 180 days (p < 0.05). Therefore, IGFALS gene polymorphism can be used as a molecular marker affecting scrotal circumference of Hu sheep, which can provide a reference for future molecular marker-assisted selection of scrotal circumference in sheep.

Radiation-induced nasopharyngeal ulcers after re-irradiation with intensity-modulated radiotherapy in locoregional recurrent nasopharyngeal carcinoma patients: a dose-volume-outcome analysis.

OBJECTIVE: To analyze the interrelation between radiation dose and radiation-induced nasopharyngeal ulcer (RINU) in locoregional recurrent nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). METHODS: Clinical data were collected from 363 patients with locoregional recurrent NPC who received re-irradiated with definitive IMRT from 2009 to 2017. Twenty-nine patients were diagnosed with RINU. Univariate and multivariate analyses were used to re-evaluate the first and second radiotherapy plans and to identify predictive dosimetric factors. RESULTS: All dosimetric parameters were notably associated with the progression to RINU (p < 0.01) using paired samples Wilcoxon signed rank tests. Multivariate analysis showed that EQD2_ [Formula: see text]D80 (dose for 80 percent volume of the unilateral nasopharynx lesion) was an independent prognostic factor for RINU (p = 0.001). The area under the ROC curve for EQD2_ [Formula: see text]D80 was 0.846 (p < 0.001), and the cutoff point of 137.035 Gy could potentially be the dose tolerance of the nasopharyngeal mucosa. CONCLUSIONS: The sum of equivalent dose in 2 Gy fractions (EQD2) in the overlapping volumes between initial and re-irradiated nasopharyngeal mucosal tissue can be effective in predicting the hazard of developing RINU in NPC patients undergoing radical re­irradiation with IMRT and we propose a EQD2_ [Formula: see text]D80 threshold of 137.035 Gy for the nasopharynx.

Development of cancer biomarker heat shock protein 90α certified reference material using two different isotope dilution mass spectrometry techniques.

Heat shock protein 90α (HSP90α) has been regarded as an important indicator for judging tumor metastasis and prognosis due to its significant upregulation in various tumors. Therefore, the accurate quantification of HSP90α is of great significance for clinical diagnosis and therapy of cancers. However, the lack of HSP90α certified reference material (CRM) leads to the accuracy and consistency of quantification methods not being effectively evaluated. Besides, quantitative results without traceability make comparisons between different studies difficult. In this study, an HSP90α solution CRM was developed from the recombinant protein raw material. The recombinant protein is a dimer, and the purity of the CRM candidate reached 96.71%. Both amino acid analysis-isotope dilution mass spectrometry (AAA-IDMS) and unique peptide analysis-isotope dilution mass spectrometry (UPA-IDMS) were performed to measure the content of HSP90α in the solution CRM candidate, and the certified value was assessed to be 66.2 ± 8.8 µg/g. Good homogeneity of the CRM was identified, and the stability examination suggested that the CRM was stable for at least 4 months at - 80 °C and for 7 days at 4 °C. With traceability to SI unit (kg), this CRM has potential to help establish a metrological traceability chain for quantification of HSP90α, which will make the quantification results standardized and comparable regardless of the quantitative methods.

Identification of AGO2 and PLEC genes polymorphisms in Hu sheep and their relationship with body size traits.

The body size traits are major traits in livestock, which intuitively displays the development of the animal's bones and muscles. This study used PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR) to analyze the Single-nucleotide polymorphism and expression characteristics of Argonaute RISC catalytic component 2 (AGO2) and Plectin (PLEC) genes in Hu sheep. Two intron mutations were found in Hu sheep, which were AGO2 g.51700 A > C and PLEC g.23157 C > T, respectively. Through association analysis of two mutation sites and body size traits, it was found that AGO2 g.51700 A > C mainly affects the chest and cannon circumference of Hu sheep of while PLEC g.23157 C mainly affects body height and body length. The combined genotypes of AGO2 and PLEC genes with body size traits showed SNPs at the AGO2 g.51700 A > C and PLEC g.23157 C > T loci significantly improved the body size traits of Hu sheep. In addition, the AGO2 gene has the highest expression levels in the heart, rumen, and tail fat, and the PLEC gene is highly expressed in the heart. These two loci can provide new research ideas for improving the body size traits of Hu sheep.

Ultrahigh UV Responsivity Quasi-Two-Dimensional BixSn1-xO2 Films Achieved through Surface Reaction.

In this study, quasi-two-dimensional BixSn1-xO2 (BTO) thin films were fabricated using a liquid metal transfer method. The ultraviolet (UV) photodetector based on BTO thin films was constructed, and the ultrahigh responsivity of 589 A/W was observed at 300 nm UV light illumination. Interestingly, by dropping ethanol during light-off period, the recovery time induced by the persistent photoconductivity (PPC) effect is reduced from 1.65 × 103 s to 5.71 s. Furthermore, the recovery time can also be reduced by dropping methanol, propylene glycol, NaNO2, and Na2SO3 after light termination. The working mechanisms are attributed to the rapid consumption of holes stored in BTO thin films by reaction with those solutions. This work demonstrates that the BTO thin films have potential applications in high-performance UV detectors and present an innovation route to weaken the PPC effects in semiconductors by introducing chemical liquids on their surface.

PbBPC4 involved in a xylem-deficient dwarf phenotype in pear by directly regulating the expression of PbXND1.

Dwarfing is an important agronomic trait in fruit breeding. At present, dwarf cultivars or dwarfing rootstocks are used for high-density planting. Although some dwarf rootstocks have been used in the cultivation of pear (Pyrus bretschneideri Rehd), the breeding of dwarf pear rootstocks or cultivars is still sorely lacking. A previous study reported that PbXND1 results in a xylem-dwarf phenotype in pear trees. However, the regulatory mechanism upstream of PbXND1 is unclear. In this study, we identified PbBPC4 as an upstream regulatory factor of PbXND1 in yeast one-hybrid assays. In ß-glucuronidase staining and dual-luciferase assays, PbBPC4 enhanced the activity of the PbXND1 promoter. Tobacco plants overexpressing PbBPC4 showed decreased plant height because of a reduced xylem size. Similar changes in the xylem was observed in transgenic pear roots; those overexpressing PbBPC4 showed reduced xylem size, and those with silencing PbBPC4 expression showed increased xylem size, greater density of xylem vessels, and a larger proportion of the xylem out of the total cross-section area. Expression analyses showed that PbBPC4 increases the transcription of PbXND1, leading to reduced transcript levels of genes involved in the positive regulation of xylem development, ultimately resulting in a xylem-deficient dwarf phenotype. Taken together, our results reveal the mechanism by which PbBPC4 participates in the regulation of xylem development via directly altering the expression of PbXND1, thus leading to the dwarf phenotype in pear. These findings have reference value for the breeding of dwarf pear trees.

Precise Control of Trypsin Immobilization by a Programmable DNA Tetrahedron Designed for Ultrafast Proteome Digestion and Accurate Protein Quantification.

In proteomics research, with advantages including short digestion times and reusable applications, immobilized enzyme reactors (IMERs) have been paid increasing attention. However, traditional IMERs ignore the reasonable spatial arrangement of trypsin on the supporting matrixes, resulting in the partial overlapping of the active domain on trypsin and reducing digesting efficiency. In this work, a DNA tetrahedron (DNA TET)-based IMER Fe3O4-GO-AuNPs-DNA TET-Trypsin was designed and prepared. The distance between vertices of DNA TETs effectively controls the distribution of trypsin on the nanomaterials; thus, highly efficient protein digestion and accurate quantitative results can be achieved. Compared to the in-solution digestion (12-16 h), the sequence coverage of bovine serum albumin was up to 91% after a 2-min digestion by the new IMER. In addition, 3328 proteins and 18,488 peptides can be identified from HeLa cell protein extract after a 20-min digestion. For the first time, human growth hormone reference material was rapidly and accurately quantified after a 4-h digestion by IMER. Therefore, this new IMER has great application potential in proteomics research and SI traceable quantification.

Boosting photo-induced antimicrobial activity of lignin nanoparticles with curcumin and zinc oxide.

Lignin nanoparticles have gained increasing attention as a potential antimicrobial agent due to their biocompatibility, biodegradability, and low toxicity. However, the limited ability of lignin to act as an antibacterial is a major barrier to its widespread use. Thus, it is crucial to develop novel approaches to amplify lignin's biological capabilities in order to promote its effective utilization. In this study, we modified lignin nanoparticles (LNPs) with photo-active curcumin (Cur), zinc oxide (ZnO), or a combination of both to enhance their antimicrobial properties. The successful modifications of LNPs were confirmed using comprehensive characterization techniques. The antimicrobial efficacy of the modified LNPs was assessed against both gram-positive and gram-negative bacterial strains. The results showed that the modification of LNPs with Cur and ZnO have much higher antibacterial and antibiofilm activities than unmodified LNPs. Moreover, photo illumination resulted in even higher antibacterial activity. Furthermore, atomic force microscopy revealed bacterial cells lysis and membrane damage by ZnO/Cur modified LNPs. Our research demonstrates that ZnO/Cur modified LNPs can serve as novel hybrid materials with enhanced antimicrobial capabilities. In addition, the photo-induced enhancement in antibacterial activity not only demonstrated the versatility of this hybrid material but also opened up interesting potential for bioinspired therapeutics agents.

Relationship between hindgut microbes and feed conversion ratio in Hu sheep and microbial longitudinal development.

Feed efficiency is an important indicator in the sheep production process, which plays an important role in improving economic benefits and strengthening energy conservation and emission reduction. Compared with the rumen, the fermentation of the hindgut microorganisms can also provide part of the energy for the host, and the composition of the hindgut microorganisms will affect the feed efficiency. Therefore, we hope to find new ways to regulate sheep feed efficiency by studying the sheep gut microbes. In this study, male Hu sheep with the same birth date were raised under the same conditions until 180 d old. The sheep were divided into high and low groups according to the feed conversion ratio (FCR) at 80 to 180 d old, and the differences in rectal microorganisms between the two groups were compared. The permutational multivariate analysis (PERMANOVA) test showed that there were differences in microorganisms between the two groups (P < 0.05). Combined with linear fitting analysis, a total of six biomarkers were identified, including Ruminobacter, Eubacterium_xylanophilum_group, Romboutsia, etc. Functional enrichment analysis showed that microorganisms may affect FCR through volatile fatty acids synthesis and inflammatory response. At the same time, we conducted a longitudinal analysis of the hindgut microbes, sampling nine-time points throughout the sheep birth to market stages. The microbiota is clearly divided into two parts: before weaning and after weaning, and after weaning microbes are less affected by before weaning microbial composition.

The level of feed efficiency determines the input of sheep production costs and the income of economic benefits. Improving sheep feed efficiency can effectively save energy and reduce emissions. Gut microbes play an important role in the process of feed fermentation. In this study, biomarkers associated with feed efficiency were identified by exploring the relationship between microbes and feed conversion ratio. At the same time, the longitudinal development of microorganisms was explored. It provides a basis for the regulation of intestinal microbes in sheep.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Machine learning model identification and prediction of patients' need for ICU admission: A systematic review.

BACKGROUND: The emergency department (ED) triage process serves as a crucial first step for patients seeking acute care, This initial assessment holds crucial implications for patient survival and prognosis. In this study, a systematic review of the existing literature was performed to investigate the performance of machine learning (ML) models in recognizing and predicting the need for intensive care among ED patients. METHODS: Four prominent databases (PubMed, Embase, Cochrane Library and Web of Science) were searched for relevant literature published up to April 28, 2023. The Prediction model study Risk of Bias Assessment Tool (PROBAST) was employed to evaluate the risk of bias and feasibility of prediction models. RESULTS: In ten studies, the main algorithms used were Gradient Boostin, Logistic Regressio, Neural Network, Support Vector Machines, Random Forest. The performance of each model was as follows: Gradient Boosting had a sensitivity range of 0.3 to 0.96, specificity range of 0.6 to 0.99, accuracy range of 0.37 to 0.99, precision range of 0.3 to 0.96, and AUC value range of 0.68 to 0.93; Logistic Regression had a sensitivity range of 0.46 to 0.97, specificity range of 0.28 to 0.99, accuracy range of 0.66 to 0.97, precision range of 0.27 to 0.63, and AUC value range of 0.72 to 0.97; Neural Networks had a sensitivity range of 0.45 to 0.96, specificity range of 0.58 to 0.99, accuracy range of 0.36 to 0.97, precision range of 0.27 to 0.96, and AUC value range of 0.67 to 0.91; Support Vector Machines had a sensitivity range of 0.49 to 0.83, specificity range of 0.94 to 0.98, accuracy range of 0.33 to 0.97, precision range of 0.53 to 0.94, and AUC values were not reported; Random Forests had a sensitivity range of 0.75 to 0.91, specificity range of 0.77 to 0.94, accuracy range of 0.35 to 0.77, precision range of 0.36 to 0.94, and AUC value of 0.83. CONCLUSION: ML models have demonstrated good performance in identifying and predicting critically ill patients in ED triage. However, because of the limited number of studies on each model, further high-quality prospective research is needed to validate these findings.

Dihydrochalcone glycoside biosynthesis in Malus is regulated by two MYB-like transcription factors and is required for seed development.

Dihydrochalcones (DHCs) including phlorizin (phloretin 2'-O-glucoside) and its positional isomer trilobatin (phloretin 4'-O-glucoside) are the most abundant phenylpropanoids in apple (Malus spp.). Transcriptional regulation of DHC production is poorly understood despite their importance in insect- and pathogen-plant interactions in human physiology research and in pharmaceuticals. In this study, segregation in hybrid populations and bulked segregant analysis showed that the synthesis of phlorizin and trilobatin in Malus leaves are both single-gene-controlled traits. Promoter sequences of PGT1 and PGT2, two glycosyltransferase genes involved in DHC glycoside synthesis, were shown to discriminate Malus with different DHC glycoside patterns. Differential PGT1 and PGT2 promoter activities determined DHC glycoside accumulation patterns between genotypes. Two transcription factors containing MYB-like DNA-binding domains were then shown to control DHC glycoside patterns in different tissues, with PRR2L mainly expressed in leaf, fruit, flower, stem, and seed while MYB8L mainly expressed in stem and root. Further hybridizations between specific genotypes demonstrated an absolute requirement for DHC glycoside production in Malus during seed development which explains why no Malus spp. with a null DHC chemotype have been reported.

PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3.

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.