Search

Pichia pastoris (syn. Komagataella spp.) has attracted extensive attention as an efficient platform for recombinant protein (RP) production. For obtaining a higher protein titer, many researchers have put lots of effort into different areas and made some progress. Here, we summarized the most recent advances of the last 5 years to get a better understanding of its future direction of development. The appearance of innovative genetic tools and methodologies like the CRISPR/Cas9 gene-editing system eases the manipulation of gene expression systems and greatly improves the efficiency of exploring gene functions. The integration of novel pathways in microorganisms has raised more ideas of metabolic engineering for enhancing RP production. In addition, some new opportunities for the manufacture of proteins have been created by the application of novel mathematical models coupled with high-throughput screening to have a better overview of bottlenecks in the biosynthetic process. [ABSTRACT FROM AUTHOR]

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes. [ABSTRACT FROM AUTHOR]

SUMMARY: Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long‐day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti‐parallel alpha‐helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene‐edited mutants developed and flowered normally, but an Mting2‐1 Tnt1 insertion mutant and gene‐edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT‐like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2‐1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene‐edited mutants indicated that it was dispensable to MtING2 function in wild‐type plants. RNA sequencing experiments indicated that >7000 genes are mis‐expressed in the Mting2‐1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP‐seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2‐1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism. Significance Statement: The timing of flowering is important for plant adaptation and crop productivity, but knowledge on how this is regulated in legumes is limited. Here, we report the functional characterization of MtING2 and show that it is important in regulating plant architecture and flowering time in the model legume Medicago truncatula, providing evidence of the physiological role of an ING‐like gene in plants. [ABSTRACT FROM AUTHOR]

High salinity and drought seriously limit the production of many crops worldwide, including apple (Malus x. domestica Borkh). Members of the bZIP family of transcription factors play important roles in abiotic stress in various plants, but there have been few studies in perennial tree species. In our previous study, we conducted a genome-wide survey of bZIP family transcription factor genes in apple. Here, we focused on one of these genes, MdbZIP26, which is induced by high salinity, drought, and exogenous abscisic acid (ABA). The MdbZIP26 promoter contains several apparent cis-acting elements associated with abiotic stress response, such as ABRE/G-box, DRE, GT1, and GMSCAM4. The temporal and spatial expression patterns of MdbZIP26 were consistent with a role in abiotic stress response. Arabidopsis thaliana plants expressing MdbZIP26 showed enhanced tolerance to dehydration and salinity, and this was associated with altered expression of ABA/stress-regulated genes. Considered together, these results suggest that MdbZIP26 plays a role in the resistance of drought and high salinity stress in apple via ABA-mediated signaling. [ABSTRACT FROM AUTHOR]

The MIKCC-type gene family plays important roles in plant growth, development, and tolerance of biotic and abiotic stress, especially during floral organ differentiation. However, there have been no studies of MIKCC-type genes in rose, and functional differentiation of family members has not been explored. In this study, we identified 42 MIKCC-type genes in rose, classified the genes into 12 subfamilies, and constructed a phylogenetic tree. We performed expression analysis of these genes, and found that expression patterns correlated with the predicted subfamily, indicating that the features of MIKCC-type genes were broadly conserved during evolution. Collinear analysis of MIKCC genes among Rosaceae species confirmed the occurrence of whole genome duplications (WGD) and revealed some species-specific MIKCC genes. Transcriptome analysis showed that the expression of some MIKCC-type genes responded to low temperatures (4°C, 24 h) during flower organ differentiation. These conserved, duplicated, and novel expression patterns of MIKCC-type genes may have facilitated the adaptation of rose to various internal and external environmental changes. The results of this study provide a theoretical basis for future functional analysis of the MIKCC genes in rose and investigation of the evolutionary pattern of the MIKCC gene family in the Rosaceae genome. [ABSTRACT FROM AUTHOR]

Malformed fruits depreciate a plant's market value. In tomato (Solanum lycopersicum), fruit malformation is associated with the multi‐locule trait, which involves genes regulating shoot apical meristem (SAM) development. The expression pattern of TOPLESS3 (SlTPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR‐associated nuclease 9 (Cas9)‐mediated gene editing. Lower SlTPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination (DAG5) stage. Differentially expressed genes in the SAM of wild‐type (WT) and SlTPL3‐RNAi plants, identified by transcriptome deep sequencing (RNA‐seq), were enriched in the gibberellin (GA) biosynthesis and plant hormone signaling pathways. Moreover, exogenous auxin and paclobutrazol treatments rescued the multi‐locule phenotype, indicating that SlTPL3 affects SAM size by mediating auxin and GA levels in the SAM. Furthermore, SlTPL3 interacted with WUSCHEL (SlWUS), which plays an important role in SAM size maintenance. We conducted RNA‐seq and DNA affinity purification followed by sequencing (DAP‐seq) analyses to identify the genes regulated by SlTPL3 and SlWUS in the SAM and to determine how they regulate SAM size. We detected 24 overlapping genes regulated by SlTPL3 and SlWUS and harboring an SlWUS‐binding motif in their promoters. Furthermore, functional annotation revealed a notable enrichment for functions in auxin transport, auxin signal transduction, and GA biosynthesis. Dual‐luciferase assays also revealed that SlTPL3 enhances SlWUS‐mediated regulation (repression and activation) of SlPIN3 and SlGA2ox4 transcription, indicating that the SlTPL3–SlWUS module regulates SAM size by mediating auxin distribution and GA levels, and perturbations of this module result in enlarged SAM. These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SlTPL3–SlWUS module as a key regulator. [ABSTRACT FROM AUTHOR]

Key message: Structural variants (SV) of 23 barley inbreds, detected by the best combination of SV callers based on short-read sequencing, were associated with genome-wide and gene-specific gene expression and, thus, were evaluated to predict agronomic traits. In human genetics, several studies have shown that phenotypic variation is more likely to be caused by structural variants (SV) than by single nucleotide variants. However, accurate while cost-efficient discovery of SV in complex genomes remains challenging. The objectives of our study were to (i) facilitate SV discovery studies by benchmarking SV callers and their combinations with respect to their sensitivity and precision to detect SV in the barley genome, (ii) characterize the occurrence and distribution of SV clusters in the genomes of 23 barley inbreds that are the parents of a unique resource for mapping quantitative traits, the double round robin population, (iii) quantify the association of SV clusters with transcript abundance, and (iv) evaluate the use of SV clusters for the prediction of phenotypic traits. In our computer simulations based on a sequencing coverage of 25x, a sensitivity > 70% and precision > 95% was observed for all combinations of SV types and SV length categories if the best combination of SV callers was used. We observed a significant (P < 0.05) association of gene-associated SV clusters with global gene-specific gene expression. Furthermore, about 9% of all SV clusters that were within 5 kb of a gene were significantly (P < 0.05) associated with the gene expression of the corresponding gene. The prediction ability of SV clusters was higher compared to that of single-nucleotide polymorphisms from an array across the seven studied phenotypic traits. These findings suggest the usefulness of exploiting SV information when fine mapping and cloning the causal genes underlying quantitative traits as well as the high potential of using SV clusters for the prediction of phenotypes in diverse germplasm sets. [ABSTRACT FROM AUTHOR]

Background. DNA binding with one finger (Dof) proteins are plant-specific tran- scription factor (TF) that plays a significant role in various biological processes such as plant growth and development, hormone regulation, and resistance to abiotic stress. The Dof genes have been identified and reported in multiple plants, but so far, the whole genome identification and analysis of Dof transcription factors in blueberry (Vaccinium corymbosum L.) have not been reported yet. Methods. Using the Vaccinium genome, we have identified 51 VcDof genes in blueberry. We have further analyzed their physicochemical properties, phylogenetic relationships, gene structure, collinear analysis, selective evolutionary pressure, cis- acting promoter elements, and tissue and abiotic stress expression patterns. Results. Fifty-one VcDof genes were divided into eight subfamilies, and the genes in each subfamily contained similar gene structure and motif ordering. A total of 24 pairs of colinear genes were screened; VcDof genes expanded mainly due to whole-genome duplication, which was subjected to strong purifying selection pressure during the evolution. The promoter of VcDof genes contains three types of cis-acting elements for plant growth and development, phytohormone and stress defense responsiveness. Expression profiles of VcDof genes in different tissues and fruit developmental stages of blueberry indicated that VcDof2 and VcDof45 might play a specific role in anthesis and fruit growth and development. Expression profiles of VcDof genes in different stress indicated that VcDof1, VcDof11, and VcDof15 were highly sensitive to abiotic stress. This study provides a theoretical basis for further clarifying the biological function of Dof genes in blueberry. [ABSTRACT FROM AUTHOR]

SUMMARY: The calmodulin‐binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C‐REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334, which includes the N‐terminal CG‐1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN‐LIKE A1 (EXPL1) and NINE‐CIS‐EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold‐treated plants. Rather, domain‐swapping experiments indicated that the CAMTA3 CG‐1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG‐1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C‐terminal calmodulin‐binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature. Significance Statement: Determining how plants alter gene expression in response to environmental stimuli is fundamental to our understanding of how plants cope with environmental stress. Here, we present results indicating that the N‐terminal DNA binding region of CAMTA3 has a role in regulating its ability to induce the expression of target genes in response to cold temperature. [ABSTRACT FROM AUTHOR]

The article presents a study that discusses effective storage of excess energy in abdominal subcutaneous adipose tissue during periods of overeating that may help attenuate weight-gain-related insulin resistance. The objective of this study was to assess changes in the expression of factors regulating abdominal subcutaneous adipose tissue storage capacity in response to a brief exposure to overeating in non-obese adults.

DNA methylation of both viral and host DNA is one of the major mechanisms involved in the development of Epstein–Barr virus-associated gastric carcinoma (EBVaGC); thus, epigenetic treatment using demethylating agents would seem to be promising. We have verified the effect of MC180295, which was discovered by screening for demethylating agents. MC180295 inhibited cell growth of the EBVaGC cell lines YCCEL1 and SNU719 in a dose-dependent manner. In a cell cycle analysis, growth arrest and apoptosis were observed in both YCCEL1 and SNU719 cells treated with MC180295. MKN28 cells infected with EBV were sensitive to MC180295 and showed more significant inhibition of cell growth compared to controls without EBV infection. Serial analysis of gene expression analysis showed the expression of genes belonging to the role of BRCA1 in DNA damage response and cell cycle control chromosomal replication to be significantly reduced after MC180295 treatment. We confirmed with quantitative PCR that the expression levels of BRCA2, FANCM, RAD51, TOP2A, and CDC45 were significantly decreased by MC180295. LMP1 and BZLF1 are EBV genes with expression that is epigenetically regulated, and MC180295 could up-regulate their expression. In conclusion, MC180295 inhibited the growth of EBVaGC cells by suppressing DNA repair and the cell cycle. [ABSTRACT FROM AUTHOR]

Melon (Cucumis melo L.) seed germination significantly affects its economic value. Cultivation of melon varieties with high germination ability and seedling vigor is beneficial in large-scale melon propagation. In this study, two melon genotypes differing in their germination ability, P5 with low and P10 with high germination ability, were used to identify the optimal seed germination conditions by evaluating different water immersion times and germination temperatures. The germination rate of the P5 and P10 parental genotypes and their segregating population, consisting of 358 F2: 3 families, were evaluated for 2 years to identify their genetic basis. QTL analysis was performed on a high-density geneticmap constructed using specific-locus amplified fragment sequencing (SLAF-seq). The germination rate of F1 and F2 populations treated with water immersion for 8 h at 28 °C and measured at 48 h showed a normal distribution Genetic mapping carried out using the high-density genetic map revealed eight QTLs in chromosomes 2, 4, 5, 6, and 8 that controlmelon seed germination, of which 2020/2021-qsg5.1 was consistently significant in both years of experimentation. qsg5.1 explained 15.13% of the phenotypic variance with a LOD of 4.1. To fine map the candidate region of qsg5.1, eight cleaved amplified polymorphism sequence (CAPS) markers were used to construct a genetic map with another 421 F2 individual fruits. The major QTL qsg5.1 was located between SNP53 and SNP54 within a 55.96 Kb interval containing four genes. qRT-PCR gene expression analysis of the candidate genes showed that MELO3C031219.2 (Phosphorus transporter PHO-5) exhibited a significant difference in gene expression between the parental lines at 24, 32, and 48 h after germination, potentially being the underlying gene controlling melon seed germination. These results provide a theoretical basis for the molecular mechanisms controlling melon seed germination and can practically contribute to further improving germination to increase the propagation effciency of commercial melon cultivars. [ABSTRACT FROM AUTHOR]

Key message: The CsGAI gene, identified by map-based, was involved in regulating seed germination in low temperature via the GA and ABA signaling pathways. Low temperature reduces the percentage of seeds germinating and delays seed germinating time, thus posing a threat to cucumber production. However, the molecular mechanism regulating low temperature germination in cucumber is unknown. We here dissected a major quantitative trait locus qLTG1.1 that controls seed germination at low temperature in cucumber. First, we fine-mapped qLTG1.1 to a 46.3-kb interval, containing three candidate genes. Sequence alignment and gene expression analysis identified Csa1G408720 as the gene of interest that was highly expressed in seeds, and encoded a highly conserved, low temperature-regulated DELLA family protein CsGAI. GUS expression analysis indicated that higher promoter activity underscored higher transcriptional expression of the CsGAI gene. Consistent with the known roles of GAI in ABA and GA signaling during germination, genes involved in the GA (CsGA2ox, CsGA3ox) and ABA biosynthetic pathways (CsABA1, CsABA2, CsAAO3 and CsNCED) were found to be differently regulated in the tolerant and sensitive genotypes under low temperatures, and this was reflected in differences in their ratio of GA-to-ABA. Based on these data, we proposed a working model explaining how CsGAI integrates the GA and ABA signaling pathways, to regulate cucumber seed germination at low temperature, thus providing new insights into this mechanism. [ABSTRACT FROM AUTHOR]

B‐cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto‐regulatory loop with oncogene MYC, drives a MM specific gene‐expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4‐MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4‐MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell‐lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor‐induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half‐life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4. [ABSTRACT FROM AUTHOR]

Simple Summary: Soft tissue sarcomas (STSs) are rare malignant conditions with more than 70 subtypes that are difficult to treat, especially in advanced or metastatic states. Recently, next-generation sequencing technologies have provided comprehensive information and developed personalized medicine for treating cancer in general and STSs in particular. Growing knowledge of diverse gene alterations and biomolecular targets in various subtypes of STSs raises hope for novel treatment approaches and heralds a paradigm shift in the treatment of STSs. Activated cyclin-dependent kinases (CDKs) appear to play a critical role in sarcoma development and represent important targets for sarcoma therapy. This review discusses how CDK signaling influences STS development and its implications for STS prediction and targeted treatment. Soft tissue sarcomas (STSs) are tumors that are challenging to treat due to their pathologic and molecular heterogeneity and their tumor biology that is not yet fully understood. Recent research indicates that dysregulation of cyclin-dependent kinase (CDK) signaling pathways can be a strong driver of sarcogenesis. CDKs are enzyme forms that play a crucial role in cell-cycle control and transcription. They belong to the protein kinases group and to the serine/threonine kinases subgroup. Recently identified CDK/cyclin complexes and established CDK/cyclin complexes that regulate the cell cycle are involved in the regulation of gene expression through phosphorylation of critical components of transcription and pre-mRNA processing mechanisms. The current and continually growing body of data shows that CDKs play a decisive role in tumor development and are involved in the proliferation and growth of sarcoma cells. Since the abnormal expression or activation of large numbers of CDKs is considered to be characteristic of cancer development and progression, dysregulation of the CDK signaling pathways occurs in many subtypes of STSs. This review discusses how reversal and regulation can be achieved with new therapeutics and summarizes the current evidence from studies regarding CDK modulation for STS treatment. [ABSTRACT FROM AUTHOR]

There is growing evidence that synonymous codon variants (SCVs) can cause disease through the disruption of different processes of protein production. The aim of the study is to investigate whether the 14 SCVs reported in the F9 variant database were the pathogenic causes of hemophilia B. The impacts of SCVs on splicing and protein expression were detected using a combination of in silico prediction, in vitro minigene splicing assay and cell expression detection. The splicing transcripts were identified and quantified by co‐amplification fluorescent PCR. The mechanism of splicing was verified by a modified pU1snRNA and pU7snRNA approach. Aberrant splicing patterns were found in eight SCVs. Five of the 8 SCVs produced almost all aberrant splicing isoforms, which were expected to truncate protein, three of them presented a partial defect on both splicing and protein secretion, the overall effects were consistent with the residual Factor IX activity of the affected cases. Neither the pre‐messenger RNA (mRNA) splicing process nor the protein function was impaired in the rest six SCVs. In conclusion, our study firstly revealed the pathogenic mechanism of the 14 F9 SCVs and highlighted the importance of performing mRNA splicing analysis and protein expression studies of SCVs in inherited disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Loss-of-function variants of the RAD51C gene are known to confer a risk of breast and ovarian cancers. In this study, we analyzed the impact of RAD51C variants on splicing, a highly regulated gene expression step by which introns are removed and exons are sequentially joined. Exon recognition is guided by specific sequences, the 3′ and 5′ splice sites, which define the exon boundaries. Variants of these sequences of susceptibility genes may lead to aberrant splicing and abnormal transcripts that may trigger a disease. Splicing can be tested using a biotechnological tool called minigenes, which mimic the human gene of interest. Thus, we checked 20 RAD51C splice-site variants using the minigene mgR51C_ex2-8. We found that they all disrupted the splicing mechanism, and 16 variants could be classified as likely pathogenic. Our findings are clinically actionable, and variant carriers may benefit from tailored prevention protocols and therapies. RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1–4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants. [ABSTRACT FROM AUTHOR]

Barley (Hordeum vulgare) is the fourth most highly produced cereal in the world after wheat, rice and maize and is mainly utilized as malts and for animal feed. Barley, a model crop of the tribe Triticeae, is important in comparative analyses of Poaceae. However, molecular understanding about the developmental processes is limited in barley. Our previous work characterized one of two WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes present in the barley genome: NARROW LEAFED DWARF1 (NLD1). We demonstrated that NLD1 plays a pivotal role in the development of lateral organs. In the present study, we describe a bifurcated palea (bip) mutant of barley focusing on flower and leaf phenotypes. The palea in the bip mutant was split into two and develop towards inside the lemma surrounding the carpels and anthers. The bip mutant is devoid of lodicules, which develop in a pair at the base of the stamen within the lemma in normal barley. bip also exhibited malformations in leaves, such as narrow leaf due to underdeveloped leaf-blade width, and reduced trichome density. Map-based cloning and expression analysis indicated that BIP is identical to another barley WOX3 gene, named HvWOX3. The bip nld1 double mutant presented a more severe reduction in leaf-blade width and number of trichomes. By comparing the phenotypes and gene expression patterns of various WOX3 mutants, we concluded that leaf bilateral outgrowth and trichome development are promoted by both NLD1 and HvWOX3 , but that HvWOX3 serves unique and pivotal functions in barley development that differ from those of NLD1. [ABSTRACT FROM AUTHOR]

Cell plasticity endows differentiated cells with competence to be reprogrammed to other lineages. Although extrinsic factors driving cell-identity conversion have been extensively characterized, it remains elusive which intrinsic epigenetic attributes, including high-order chromatin organization, delineate cell plasticity. By analysing the transcription-factor-induced transdifferentiation from fibroblasts to hepatocytes, we uncovered contiguous compartment-switchable regions (CSRs) as a unique chromatin unit. Specifically, compartment B-to-A CSRs, enriched with hepatic genes, possessed a mosaic status of inactive chromatin and pre-existing and continuous accessibility in fibroblasts. Pre-existing accessibility enhanced the binding of inducible factor Foxa3, which triggered epigenetic activation and chromatin interaction as well as hepatic gene expression. Notably, these changes were restrained within B-to-A CSR boundaries that were defined by CTCF occupancy. Moreover, such chromatin organization and mosaic status were detectable in different cell types and involved in multiple reprogramming processes, suggesting an intrinsic chromatin attribute in understanding cell plasticity. [ABSTRACT FROM AUTHOR]

Salinity causes a detrimental impact on plant growth, particularly when the stress occurs during germination and early development stages. Barley is one of the most salt-tolerant crops; previously we mapped two quantitative trait loci (QTL) for salinity tolerance during germination on the short arm of chromosome 2H using a CM72/Gairdner doubled haploid (DH) population. Here, we narrowed down the major QTL to a region of 0.341 or 0.439 Mb containing 9 or 24 candidate genes belonging to 6 or 20 functional gene families according to barley reference genomes v1 and v3 respectively, using two DH populations of CM72/Gairdner and Skiff/CM72, F2 and F3 generations of CM72/Gairdner/*Spartacus CL. Two Receptorlike kinase 4 (RLPK4) v1 or Receptor-like kinase (RLK) v3 could be the candidates for enhanced germination under salinity stress because of their upregulated expression in salt-tolerant variety CM72. Besides, several insertion/deletion polymorphisms were identified within the 3rd exon of the genes between CM72 and Gairdner. The sequence variations resulted in shifted functional protein domains, which may be associated with differences in salinity tolerance. Two molecular markers were designed for selecting the locus with receptor-like protein kinase 4, and one was inside HORVU2Hr1G111760.1 or HORVU.MOREX.r3.2HG0202810.1. The diagnostic markers will allow for pyramiding of 2H locus in barley varieties and facilitate genetic improvement for saline soils. Further, validation of the genes to elucidate the mechanisms involved in enhancing salinity tolerance at germination and designing RLPK4 specific markers is proposed. For this publication, all the analysis was based on barley reference genome of 2017 (v1), and it was used throughout for consistence. However, the positions of the markers and genes identified were updated according to new genome (v3) for reference. [ABSTRACT FROM AUTHOR]

Cold tolerance is improved by cold stress acclimation (CS-ACC), and the cold tolerance level is 'remembered' by plants. However, the underlying signaling mechanisms remain largely unknown. Here, the CS memory mechanism was studied by bioinformation, plant physiological and photosynthetic parameters, and gene expression. We found that CS-ACC induced the acquisition of CS memory and enhanced the maintenance of acquired cold tolerance (MACT) in cucumber seedlings. The H2O2 content and NADPH oxidase activity encoded by CsRBOH was maintained at higher levels during recovery after CS-ACC and inhibition of RBOH-dependent signaling after CS-ACC resulted in a decrease in the H2O2 content, NADPH oxidase activity, and MACT. CsRBOH2, 3, 4, and 5 showed high expression during recovery after CS-ACC. Many BZR-binding sites were identified in memory-responsive CsRBOHs promoters, and CsBZR1 and 3 showed high expression during recovery after CS-ACC. Inhibition of RBOH-dependent signaling or brassinosteroids affected the maintenance of the expression of these memory-responsive CsRBOHs and CsBZRs. The photosynthetic efficiency (PE) decreased but then increased with the prolonged recovery after CS-ACC, and was higher than the control at 48 h of recovery; however, inhibition of RBOH-dependent signaling resulted in a lower PE. Further etiolated seedlings experiments showed that a photosynthetic capacity was necessary for CS memory. Therefore, photosynthesis mediated by RBOH-dependent signaling is essential for CS memory. [ABSTRACT FROM AUTHOR]

Seed germination is a critical first stage of plant development but can be arrested by factors including dormancy and environmental conditions. Strategies to enhance germination are of interest to plant breeders to ensure the ability to utilize the genetic potential residing inside a dormant seed. In this study, seed germination in two sugarbeet (Beta vulgaris ssp. vulgaris L.) lines F1004 and F1015 through incubating seeds in hydrogen peroxide (H2O2) solution was improved over 70% relative to germinating seeds through water incubation. It was further found that low germination from water incubation was caused by physical dormancy in F1015 seeds with initial seed imbibition blocked by the seed pericarp, and physiological dormancy in F1004 seeds with germination compromised due to the physiological condition of the embryo. To identify genes that are differentially expressed in response to cellular activities promoted by H2O2 during overcoming different type of dormancies, an RNA-Seq study was carried out and found H2O2 treatment during germination accelerated the degradation of seed stored mRNAs that were synthesized before or during seed storage to provide protections and maintain the dormant state. Comparison of transcripts in H2O2-treated seeds between the two sugarbeet lines identified differentially expressed genes (DEGs) that were higher in F1004 for alleviating physiological dormancy were known to relative to gene expression regulation. The research established that H2O2 overcomes both physical and physiological dormancies by hastening the transition of seeds from dormancy into germination. More DEGs related to gene expression regulation were involved in relieving physiological dormancy which provides new knowledge about the role of exogenous H2O2 as a signaling molecule for regulating gene activities during germination. Moreover, the protocol using H2O2 to promote germination will be useful for rescuing plant germplasms with poor germination. [ABSTRACT FROM AUTHOR]

Background: The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion. Results: Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples. Conclusions: The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them. [ABSTRACT FROM AUTHOR]

Key message: Grain disarticulation in wild progenitor of wheat and barley evolved through a local duplication event followed by neo-functionalization resulting from changes in location of gene expression. One of the most critical events in the process of cereal domestication was the loss of the natural mode of grain dispersal. Grain dispersal in barley is controlled by two major genes, Btr1 and Btr2, which affect the thickness of cell walls around the disarticulation zone. The barley genome also encodes Btr1-like and Btr2-like genes, which have been shown to be the ancestral copies. While Btr and Btr-like genes are non-redundant, the biological function of Btr-like genes is unknown. We explored the potential biological role of the Btr-like genes by surveying their expression profile across 212 publicly available transcriptome datasets representing diverse organs, developmental stages and stress conditions. We found that Btr1-like and Btr2-like are expressed exclusively in immature anther samples throughout Prophase I of meiosis within the meiocyte. The similar and restricted expression profile of these two genes suggests they are involved in a common biological function. Further analysis revealed 141 genes co-expressed with Btr1-like and 122 genes co-expressed with Btr2-like, with 105 genes in common, supporting Btr-like genes involvement in a shared molecular pathway. We hypothesize that the Btr-like genes play a crucial role in pollen development by facilitating the formation of the callose wall around the meiocyte or in the secretion of callase by the tapetum. Our data suggest that Btr genes retained an ancestral function in cell wall modification and gained a new role in grain dispersal due to changes in their spatial expression becoming spike specific after gene duplication. [ABSTRACT FROM AUTHOR]

Main conclusion: Exogenous ABA played a positive role in the accumulation and biosynthesis of aroma components of postharvest kiwifruit after low-temperature storage, especially the esters production during ripening. Low-temperature storage (LTS) generally affects the aroma formation associated with the decrease in aroma quality in kiwifruit. In this work, abscisic acid (ABA) treatment after LTS increased the production of aroma components in postharvest kiwifruit and enhanced the related enzyme activity, especially alcohol acyltransferase (AAT), branched amino acid transaminase (BCAT) and hydroperoxide lyase (HPL). Corresponding to the enzyme activity, the gene expression of AchnAAT, AchnADH, AchnBCAT and AchnHPL was significantly up-regulated by ABA. The principal component analysis further illustrated the differences in aroma components between ABA and the control. The positive correlation of aroma accumulation with the expression levels of AchnPDC and AchnLOX and the enzyme activities of BCAT and pyruvate decarboxylase (PDC) was also revealed by correlation analysis. In addition, the promoter sequences of the key genes involved in aroma biosynthesis contained multiple cis-elements (ABRE and G-box) of ABA-responsive proteins. Combining the transcriptome sequencing data, the promoting role of ABA signaling in the regulation of aroma biosynthesis of postharvest kiwifruit after LTS was discussed. This study would provide a reference for improving aroma quality of postharvest kiwifruit after LTS, as well the molecular mechanism of kiwifruit aroma fading after LTS. [ABSTRACT FROM AUTHOR]

Plant tissue culture constitutes an indispensable tool in the advancement of agricultural sciences and modern agriculture today. However, its applications remain limited due to the genotype-dependent culture response as well as the recalcitrant species, such as sesame (Sesamum indicum L.). In the present study, reproducible and high-efficiency plant regeneration from cotyledonary explants was achieved in different genotypes of sesame using thymol as the supplement to culture media. We found that removal of NH4NO3 from Murashige and Skoog (MS) basal salt prevents browning of the explants, and supplement of thymol to the MS medium promotes callus induction and shoot regeneration, as well as improves adventitious root formation. Moreover, we showed that the occurrence of shoot regeneration implicates 91 differential expression genes (DEGs) and is coincident with endogenous abscisic acid (ABA) and salicylic acid (SA) reduction in the explants. The DEGs are mostly enriched in functions related to disaccharide and oligosaccharide metabolic processes, and response to red or far-red light, as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including pentose phosphate pathway, carbon fixation in photosynthetic organisms, and plant MAPK signaling pathway. Finally, we confirmed the regulatory role of thymol on the relevant gene expression underlying in vitro shoot regeneration. Thus, thymol may offer a promising prospect of broad application in plant tissue culture. [ABSTRACT FROM AUTHOR]

Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology. [ABSTRACT FROM AUTHOR]

Cold stress is one of the most limiting factors for plant growth and development. Cold stress adversely affects plant physiology, molecular and biochemical processes by determining oxidative stress, poor nutrient and water uptake, disorganization of cellular membranes and reduced photosynthetic efficiency. Therefore, to recover impaired plant functions under cold stress, the application of bio-stimulants can be considered a suitable approach. Melatonin (MT) is a critical bio-stimulant that has often shown to enhance plant performance under cold stress. Melatonin application improved plant growth and tolerance to cold stress by maintaining membrane integrity, plant water content, stomatal opening, photosynthetic efficiency, nutrient and water uptake, redox homeostasis, accumulation of osmolytes, hormones and secondary metabolites, and the scavenging of reactive oxygen species (ROS) through improved antioxidant activities and increase in expression of stress-responsive genes. Thus, it is essential to understand the mechanisms of MT induced cold tolerance and identify the diverse research gaps necessitating to be addressed in future research programs. This review discusses MT involvement in the control of various physiological and molecular responses for inducing cold tolerance. We also shed light on engineering MT biosynthesis for improving the cold tolerance in plants. Moreover, we highlighted areas where future research is needed to make MT a vital antioxidant conferring cold tolerance to plants. [ABSTRACT FROM AUTHOR]

Based on prior knowledge and with the support of new methodology, solid progress in the understanding of seed life has taken place over the few last years. This update reflects recent advances in three key traits of seed life (i.e., preharvest sprouting, genomic imprinting, and stored-mRNA). The first breakthrough refers to cloning of the mitogen-activated protein kinase-kinase 3 (MKK3) gene in barley and wheat. MKK3, in cooperation with ABA signaling, controls seed dormancy. This advance has been determinant in producing improved varieties that are resistant to preharvest sprouting. The second advance concerns to uniparental gene expression (i.e., imprinting). Genomic imprinting primarily occurs in the endosperm. Although great advances have taken place in the last decade, there is still a long way to go to complete the puzzle regarding the role of genomic imprinting in seed development. This trait is probably one of the most important epigenetic facets of developing endosperm. An example of imprinting regulation is polycomb repressive complex 2 (PRC2). The mechanism of PRC2 recruitment to target endosperm with specific genes is, at present, robustly studied. Further progress in the knowledge of recruitment of PRC2 epigenetic machinery is considered in this review. The third breakthrough referred to in this update involves stored mRNA. The role of the population of this mRNA in germination is far from known. Its relations to seed aging, processing bodies (P bodies), and RNA binding proteins (RBPs), and how the stored mRNA is targeted to monosomes, are aspects considered here. Perhaps this third trait is the one that will require greater experimental dedication in the future. In order to make progress, herein are included some questions that are needed to be answered. [ABSTRACT FROM AUTHOR]

The rapid invasion of the non‐native Phragmites australis (Poaceae, subfamily Arundinoideae) is a major threat to native wetland ecosystems in North America and elsewhere. We describe the first reference genome for P. australis and compare invasive (ssp. australis) and native (ssp. americanus) genotypes collected from replicated populations across the Laurentian Great Lakes to deduce genomic bases driving its invasive success. Here, we report novel genomic features including a Phragmites lineage‐specific whole genome duplication, followed by gene loss and preferential retention of genes associated with transcription factors and regulatory functions in the remaining duplicates. Comparative transcriptomic analyses revealed that genes associated with biotic stress and defence responses were expressed at a higher basal level in invasive genotypes, but native genotypes showed a stronger induction of defence responses when challenged by a fungal endophyte. The reference genome and transcriptomes, combined with previous ecological and environmental data, add to our understanding of mechanisms leading to invasiveness and support the development of novel, genomics‐assisted management approaches for invasive Phragmites. [ABSTRACT FROM AUTHOR]

'Huangjinya' is a novel light-sensitive albino tea cultivar. Previously we have found that the leaves exhibit varying degrees of yellow coloration after being exposed to different light qualities, including red light (RL), blue light (BL), red + blue light (RB) and white light (WL). To understand the transcriptional response of different light qualities in leaf coloration of 'Huangjinya', RNA-seq analysis with three biological replicates was performed to identify differentially expressed genes (DEGs) involved in pigment metabolism pathways. With regard to porphyrin and chlorophyll metabolism, the protochlorophyllide oxidoreductase (POR) was upregulated and chlorophyllase (Chlase) was downregulated under RL compared with WL, which contributed to high chlorophyll content in 'Huangjinya'. The upregulated POR and magnesium chelatase H subunit (CHLH) were identified under RB. BL significantly promoted carotenoid biosynthesis, accompanied by the related upregulated genes. However, upregulated xanthoxin dehydrogenase (ABA2) reduced carotenoid content under BL. Zeaxanthin epoxidase (ZEP) was upregulated and ABA2 was downregulated under RB, resulting in high accumulation of carotenoid content. BL and RL upregulated expressions of genes involved in flavonoid biosynthesis in 'Huangjinya'. Differential expressions of genes involved in flavonoid biosynthesis was considered as the results of leaf color change. In addition, the genes related to photosynthesis were downregulated under RL, whereas these were upregulated under BL when compared with WL. In conclusion, the effect of light quality on the leaf coloration of 'Huangjinya' was mainly dependent on chlorophyll content by altering corresponding genes expressions. RL may promote the accumulation of chlorophyll in 'Huangjinya' leaves by upregulating the expression of genes related to chlorophyll synthesis (e.g., POR) and downregulating expression of genes related to chlorophyll degradation (e.g., Chlase). BL could upregulate the expression of genes related to both biosynthesis (e.g., POR, hemA) and degradation (e.g., Chlase) of chlorophyll, resulting in little change in leaf color of 'Huangjinya' under blue light when compared with white light. Upregulated genes involved in chlorophyll biosynthesis (e.g., POR, CHLH) induced greener color of 'Huangjinya' treated with RB. [ABSTRACT FROM AUTHOR]

Abstract: Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of ∼50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (∼100–200bp) and the usefulness of microarrays to study knockdown phenotypes. [Copyright &y& Elsevier]

Introduction:Vascular contributions to cognitive impairment and dementia (VCID) are a leading cause of dementia. An underappreciated, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), defined by elevated levels of plasma homocysteine, most often due to impaired B vitamin absorption in aged persons. Studies aimed at identifying neuropathologic features and gene expression profiles associated with HHcy have been lacking. Methods:Asubset of research volunteers from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort came to autopsy and had ante mortem plasma homocysteine levels available. Brain tissue and blood plasma drawn closest to deathwere used tomeasure homocysteine and relatedmetabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the HumanNeuroinflammation NanoString panel. Further analyses included an evaluation of plasma homocysteine effects on amyloid beta, tau, ionized calcium-binding adaptor molecule 1, and glial fibrillary acidic protein immunohistochemistry in the frontal and occipital cortices. Analytes and other study outcomes were evaluated in relation to ante mortem HHcy status:We identified 13 persons with normal ante mortem plasma homocysteine levels (<14 µmol/L) and 18 who had high plasma homocysteine levels (=14 µmol/L). Results: Participants with HHcy demonstrated increased levels of several plasma homocysteine cycle metabolites such as total cysteine, S-adenosyl-homocysteine, cystathionine, and choline. Inflammatory gene expression profiles showed a general downregulation in the setting of elevated plasma homocysteine. HHcy was associated with more and longer microglial processes, but smaller and fewer astrocytes, especially in participants of older age at death. HHcy in older participants was also associated with occipital cortex microhemorrhages and increased severity of atherosclerosis throughout the cerebral vasculature. Conclusions: Increased plasma homocysteine and older age were associated with the downregulation of inflammatory gene expression markers in association with significant glial and vascular pathology changes. Impaired immune function is a plausible mechanism by which HHcy increases cerebrovascular damage leading to impaired cognitive function. [ABSTRACT FROM AUTHOR]

Objective: Genetic variants spanning UBE2L3 are associated with increased expression of the UBE2L3‐encoded E2 ubiquitin‐conjugating enzyme H7 (UbcH7), which facilitates activation of proinflammatory NF‐κB signaling and susceptibility to autoimmune diseases. We undertook this study to delineate how genetic variants carried on the UBE2L3/YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. Methods: We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele‐specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture with quantitative polymerase chain reaction (3C‐qPCR), chromatin immunoprecipitation (ChIP)–qPCR, and small interfering RNA (siRNA) knockdown assays were performed on patient‐derived Epstein‐Barr virus–transformed B cells homozygous for the UBE2L3/YDJC nonrisk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. Results: Of the 7 prioritized variants, 5 demonstrated allele‐specific increases in nuclear protein binding affinity and regulatory activity. High‐throughput sequencing of chromosome conformation capture coupled with ChIP (HiChIP) and 3C‐qPCR uncovered a long‐range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and the downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3/YDJC risk haplotype, and correlated with the loss of CCCTC‐binding factor (CTCF) and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long‐range interaction between the 2 promoters and reduced UBE2L3 expression. Conclusion: The UBE2L3/YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1‐mediated interaction between the UBE2L3 and YDJC promoters. [ABSTRACT FROM AUTHOR]

SHANK2 (ProSAP1) is a postsynaptic scaffolding protein of excitatory synapses in the central nervous system and implicated in the development of autism spectrum disorders (ASD). Patients with mutations in SHANK2 show autism-like behaviors, developmental delay, and intellectual disability. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. In patient hiPSCs and derived neurons SHANK2 mRNA and protein expression was reduced. During neuronal maturation, a reduction in growth cone size and a transient increase in neuronal soma size were observed. Neuronal proliferation was increased, and apoptosis was decreased in young and mature neurons. Additionally, mature patient hiPSC-derived neurons showed dysregulated excitatory signaling and a decrease of a broad range of signaling molecules of the ERK-MAP kinase pathway. These findings could be confirmed in brain samples from Shank2 (−/−) mice, which also showed decreased mGluR5 and phospho-ERK1/2 expression. Our study broadens the current knowledge of SHANK2-related ASD. We highlight the importance of excitatory-inhibitory balance and mGluR5 dysregulation with disturbed downstream ERK1/2 signaling in ASD, which provides possible future therapeutic strategies for SHANK2-related ASD. [ABSTRACT FROM AUTHOR]

Epigenetic modifications are responsible for finetuning gene expression profiles to the needs of cells, tissues, and organisms. To rapidly respond to environmental changes, the activity of chromatin modifiers critically depends on the concentration of a handful of metabolites that act as substrates and co-factors. In this way, these enzymes act as metabolic sensors that directly link gene expression to metabolic states. Although metabolites can easily diffuse through the nuclear pore, molecular mechanisms must be in place to regulate epigenetic marker deposition in specific nuclear subdomains or even on single loci. In this review, I explore the possible subcellular sites of metabolite production that influence the epigenome. From the relationship between cytoplasmic metabolism and nuclear metabolite deposition, I converse to the description of a compartmentalized nuclear metabolism. Last, I elaborate on the possibility of metabolic enzymes to operate in phase-separated nuclear microdomains formed by multienzyme and chromatin-bound protein complexes. [ABSTRACT FROM AUTHOR]

Background: Treatment with immune checkpoint inhibitors (ICIs) targeting CTLA-4 and the PD-1/PD-L1 axis is effective against many cancer types. However, due in part to unresponsiveness or acquired resistance, not all patients experience a durable response to ICIs. HBI-8000 is a novel, orally bioavailable class I selective histone deacetylase inhibitor that directly modifies antitumor activity by inducing apoptosis, cell cycle arrest, and resensitization to apoptotic stimuli in adult T cell lymphoma patients. We hypothesized that HBI-8000 functions as an epigenetic immunomodulator to reprogram the tumor microenvironment from immunologically cold (nonresponsive) to hot (responsive).Method: Mice bearing syngeneic tumors (MC38 and CT26 murine colon carcinoma and A20 B-cell lymphoma were treated daily with HBI-8000 (orally), alone or in combination with PD-1, PD-1 L, or CTLA-4 antibodies. MC38 tumors were also analyzed in nanoString gene expression analysis.Results: HBI-8000 augmented the activity of ICI antibodies targeting either PD-1, PD-L1 or CTLA-4, and significantly increased tumor regression (p < 0.05) in the above models. Gene expression analysis of the treated MC38 tumors revealed significant changes in mRNA expression of immune checkpoints, with enhanced dendritic cell and antigen-presenting cell functions, and modulation of MHC class I and II molecules.Conclusions: These findings suggest that HBI-8000 mediates epigenetic modifications in the tumor microenvironment, leading to improved efficacy of ICIs, and provide strong rationale for combination therapies with ICIs and HBI-8000 in the clinical setting.Precis: As an HDACi, HBI-8000 plays an important role in priming the immune system in the tumor microenvironment. The current preclinical data further justifies testing combination of HBI-8000 and ICIs in the clinic. [ABSTRACT FROM AUTHOR]

Genomic amplification of OTUD7B is frequently found across human cancers. But its role in tumorigenesis is poorly understood. Lysine‐specific demethylase 1 (LSD1) is known to execute epigenetic regulation by forming corepressor complex with CoREST/histone deacetylases (HDACs). However, the molecular mechanisms by which cells maintain LSD1/CoREST complex integrity are unknown. Here, it is reported that LSD1 protein undergoes K63‐linked polyubiquitination. OTUD7B is responsible for LSD1 deubiquitination at K226/277 residues, resulting in dynamic control of LSD1 binding partner specificity and cellular homeostasis. OTUD7B deficiency increases K63‐linked ubiquitination of LSD1, which disrupts LSD1/CoREST complex formation and targets LSD1 for p62‐mediated proteolysis. Consequently, OTUD7B deficiency impairs genome‐wide LSD1 occupancy and enhances the methylation of H3K4/H3K9, therefore profoundly impacting global gene expression and abrogating breast cancer metastasis. Moreover, physiological fluctuation of OTUD7B modulates cell cycle‐dependent LSD1 oscillation, ensuring the G1/S transition. Both OTUD7B and LSD1 proteins are overpresented in high‐grade or metastatic human breast cancer, while dysregulation of either protein is associated with poor survival and metastasis. Thus, OTUD7B plays a unique partner‐switching role in maintaining the integrity of LSD1/CoREST corepressor complex, LSD1 turnover, and breast cancer metastasis. [ABSTRACT FROM AUTHOR]

DNA-binding One Zinc Finger (Dof) transcription factor (TF) family plays a pivotal role in various plant-specific biological processes. Sugar beet, Beta vulgaris L., is an important crop widely grown for sugar production, animal feed, and has many industrial and biotechnology applications. Although genome-wide analyses of the Dof family have been conducted in several plant species, information regarding Dof genes in sugar beet is extremely restricted. The present study analyzed 22 B. vulgaris Dof proteins (BvDofs) on the basis of conserved motifs, chromosomal locations, phylogenetic relationships, gene duplication, and expression patterns. A total of 15 conserved motifs were identified from all BvDof proteins and their distribution was supported by phylogeny. All genes were mapped on seven of the nine sugar beet chromosomes, except chromosomes 3 and 4. We showed that the BvDof family expanded mainly via ancient, segmental duplications; although more recent, tandem duplications were also involved. Comparative phylogeny using the Dof family members of Spinacia oleracea and Arabidopsis thaliana, divided Dof genes in Caryophyllales into seven major clades. Synteny analysis provided evidence for the substantial chromosome rearrangements during the evolution of the Caryophyllales genomes. Gene expression profiling of the BvDofs by RNA-seq analysis revealed differential expression of BvDofs during response to viral stress and between cultivars with distinct taproot traits, during different developmental stages. Taken together, these results offer an extensive resource for understanding the structure, evolution and function of Dof genes in sugar beet, which will help to improve crop productivity and biotechnological potential of this legume under stress conditions. [ABSTRACT FROM AUTHOR]

Isolation of an adequate quantity of high-quality RNA is a crucial step in gene expression analysis. Difficulties in sampling and RNA extraction of field-grown crops, especially from roots, are substantial obstacles to understanding actual gene expression in the field. We examined the effectiveness of the RNA extraction method (FRP) using pot- and field-grown soybean roots. High quantity and quality of total RNA could extract by the FRP method from both pot- and field-grown soybean roots at different growth stages. To determine the influence of root washing during the sampling process, roots were washed with water for 5 s, 1 min, and 5 min, then analyzed for aquaporin gene expression levels. Root washing for 5 min affected gene expression of some aquaporins. To obtain RNA that reflects the correct gene expression profile, it is necessary to minimize the effects of the sampling process. [ABSTRACT FROM AUTHOR]

Background: Lipid phosphate phosphatases (LPP) are critical for regulating the production and degradation of phosphatidic acid (PA), an essential signaling molecule under stress conditions. Thus far, the LPP family genes have not been reported in rapeseed (Brassica napus L.). Results: In this study, a genome-wide analysis was carried out to identify LPP family genes in rapeseed that respond to different stress conditions. Eleven BnLPPs genes were identified in the rapeseed genome. Based on phylogenetic and synteny analysis, BnLPPs were classified into four groups (Group I-Group IV). Gene structure and conserved motif analysis showed that similar intron/exon and motifs patterns occur in the same group. By evaluating cis-elements in the promoters, we recognized six hormone- and seven stress-responsive elements. Further, six putative miRNAs were identified targeting three BnLPP genes. Gene ontology analysis disclosed that BnLPP genes were closely associated with phosphatase/hydrolase activity, membrane parts, phosphorus metabolic process, and dephosphorylation. The qRT-PCR based expression profiles of BnLPP genes varied in different tissues/organs. Likewise, several gene expression were significantly up-regulated under NaCl, PEG, cold, ABA, GA, IAA, and KT treatments. Conclusions: This is the first report to describe the comprehensive genome-wide analysis of the rapeseed LPP gene family. We identified different phytohormones and abiotic stress-associated genes that could help in enlightening the plant tolerance against phytohormones and abiotic stresses. The findings unlocked new gaps for the functional verification of the BnLPP gene family during stresses, leading to rapeseed improvement. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 9 (CDK9), the kinase component of positive transcription elongation factor b (P-TEFb), is essential for transcription of most protein-coding genes by RNA polymerase II (RNAPII). By releasing promoter-proximally paused RNAPII into gene bodies, CDK9 controls the entry of RNAPII into productive elongation and is, therefore, critical for efficient synthesis of full-length messenger (m)RNAs. In recent years, new players involved in P-TEFb-dependent processes have been identified and an important function of CDK9 in coordinating elongation with transcription initiation and termination has been unveiled. As the regulatory functions of CDK9 in gene expression continue to expand, a number of human pathologies, including cancers, have been associated with aberrant CDK9 activity, underscoring the need to properly regulate CDK9. Here, I provide an overview of CDK9 function and regulation, with an emphasis on CDK9 dysregulation in human diseases. [ABSTRACT FROM AUTHOR]

Growth is one of the most important traits of aquaculture breeding programs. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Ruditapes philippinarum is an economically important marine bivalve. In order to gain insights into the molecular mechanisms to growth variability in marine shellfish, we conducted the transcriptome sequencing and examined the expression differences in growth-related gene and molecular pathways involved in growth trait of R. philippinarum. In this study, we investigated the molecular and gene expression differences in fast-growing and slow-growing Manila clam and focused on the analysis of the differential expression patterns of specific genes associated with growth by RNA-seq and qPCR analysis. A total of 61 differentially expressed genes (DEGs) were captured significantly differentially expressed, and were categorized into Ras signaling pathway, hedgehog signaling pathway, AMPK signaling pathway, p53 signaling pathway, regulation of actin cytoskeleton, focal adhesion, mTOR signaling pathway, VEGF signaling pathway, and TGF-beta signaling pathway. A total of 34 growth-related genes were validated significantly and up/downregulated at fast growing and slow growing of R. philippinarum. Functional enrichment analysis revealed the insulin signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway play pivotal roles in molecular function and regulation of growth trait in R. philippinarum. The growth-related genes and pathways obtained here provide important insights into the molecular basis of physiological acclimation, metabolic activity, and growth variability in marine bivalves. [ABSTRACT FROM AUTHOR]

Transcriptional dysregulation is central to many diseases including cancer. Mutation or deregulated expression of proteins involved in transcriptional machinery leads to aberrant gene expression that disturbs intricate cellular processes of division and differentiation. The subunits of the mediator complex are master regulators of stimuli‐derived transcription and are essential for transcription by RNA polymerase II. MED12 is a part of the CDK8 kinase module of the mediator complex and is essential for kinase assembly and function. Other than its function in activation of the kinase activity of CDK8 mediator, it also brings about transcription repression or activation, in response to several signalling pathways, a function that is independent of its role as a part of kinase assembly. Accumulating evidence suggests that MED12 controls complex transcription programs that are defining in cell fate determination, differentiation, and carcinogenesis. Mutations or differential expression of MED12 manifest in several human disorders and diseases. For instance, MED12 mutations are the gold standard for the diagnosis of several X‐linked intellectual disability syndromes. Further, certain MED12 mutations are categorised as driver mutations in carcinogenesis as well. This is a timely review that provides for the first time a wholesome view on the critical roles and pathways regulated by MED12, its interactions along with the implications of MED12 alterations/mutations in various cancers and nonneoplastic disorders. Based on the preclinical studies, MED12 indeed emerges as an attractive novel therapeutic target for various diseases and intellectual disorders. [ABSTRACT FROM AUTHOR]

Objectives. Chemotherapy is considered to be essential in the treatment of patients with colorectal cancer (CRC), but drug resistance reduces its efficacy. Many patients with advanced CRC eventually show resistance to 5-fluorouracil (5-FU) therapy. Synergistic and potentiating effects of combination therapy, using herbal and chemical drugs, can improve patients' response. Zerumbone (ZER), which is derived from ginger, has been studied for its growth inhibitory function in various types of cancer. Methods. The cytotoxic effects of ZER and 5-FU alone and their combination, on the SW48 and HCT-116 cells, were examined, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The mRNA and protein levels of β-catenin, survivin, and vimentin were measured in treated CRC cells, using qRT-PCR and western blot. Colony formation assay, scratch test, and flow cytometry were performed to detect the changes of proliferation, migration, and apoptosis. Key Findings. In HCT-116- and SW48-treated cells, the proliferation, the gene and protein expression levels of the markers, the migration, the colony formation, and the survival rates were all significantly reduced compared to the control groups, and the sharpest decline was observed in the 5-FU+ZER treatment groups. Conclusions. Combination therapy has shown promising results in CRC cells, especially in drug-resistant cells. [ABSTRACT FROM AUTHOR]

Background: Anastomosis group 1 IA (AG1-IA) of Rhizoctonia solani is the major agent of banded leaf and sheath blight (BLSB) disease that causes severe yield loss in many worldwide crops. MicroRNAs (miRNAs) are ~ 22 nt non-coding RNAs that negatively regulate gene expression levels by mRNA degradation or translation inhibition. A better understanding of miRNA function during AG1-IA infection can expedite to elucidate the molecular mechanisms of fungi-host interactions. Results: In this study, we sequenced three small RNA libraries obtained from the mycelium of AG1-IA isolate, non-infected maize sheath and mixed maize sheath 3 days after inoculation. In total, 137 conserved and 34 novel microRNA-like small RNAs (milRNAs) were identified from the pathogen. Among these, one novel and 17 conserved milRNAs were identified as potential virulence-associated (VA) milRNAs. Subsequently, the prediction of target genes for these milRNAs was performed in both AG1-IA and maize, while functional annotation of these targets suggested a link to pathogenesis-related biological processes. Further, expression patterns of these virulence-associated milRNAs demonstrated that theyparticipate in the virulence of AG1-IA. Finally, regulation of one maize targeting gene, GRMZM2G412674 for Rhi-milRNA-9829-5p, was validated by dual-luciferase assay and identified to play a positive role in BLSB resistance in two maize mutants. These results suggest the global differentially expressed milRNAs of R. solani AG1-IA that participate in the regulation of target genes in both AG1-IA and maize to reinforce its pathogenicity. Conclusions: Our data have provided a comprehensive overview of the VA-milRNAs of R. solani and identified that they are probably the virulence factors by directly interfered in host targeting genes. These results offer new insights on the molecular mechanisms of R.solani-maize interactions during the process of infection. [ABSTRACT FROM AUTHOR]

Copy number variation (CNV) may have phenotypic effects by altering the expression level of the gene(s) or regulatory element(s) contained. It is believed that CNVs play pivotal roles in controlling plant architecture and other traits in plant. However, the effects of CNV contributing to special traits remain largely unknown. Here we report a CNV involved in rice architecture by modulating tiller number and leaf angle. In the genome of Oryza sativa ssp. japonica cv. Nipponbare, we found a locus Loc_Os08g34249 is derived from a 13,002-bp tandem duplication in the nearby region of OsMTD1 , a gene regulating tillering in rice. Further survey of 230 rice cultivars showed that the duplication occurred in only 13 japonica rice cultivars. Phenotypic investigation indicated that this CNV region may contribute to tiller number. Moreover, we revealed that OsMTD1 not only influences rice tiller number and leaf angle, but also represses pri-miR156f transcription in the CNV region. Intriguingly, this CNV performs function through both the dosage and position effects on OsMTD1 and pri-miR156f. Thus, our work identified a CNV and revealed a molecular regulatory basis for its effects on plant architecture, implying this CNV may possess importance and application potential in molecular breeding in rice. [ABSTRACT FROM AUTHOR]

Summary: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Under various treatments, some patients still have a poor prognosis. Hence, it is necessary to find new valid targets for NB therapy. In this study, a comprehensive bioinformatic analysis was used to identify differentially expressed genes (DEGs) between NB and control cells, and to select hub genes associated with NB. GSE66586 and GSE78061 datasets were downloaded from the Gene Expression Omnibus (GEO) database and DEGs were selected. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were applied to the selected DEGs. The STRING database and Cytoscape software were used to construct protein-protein interaction (PPI) networks and perform modular analysis of the DEGs. The R2 database was used for prognostic analysis. We identified a total of 238 DEGs from two microarray databases. GO enrichment analysis shows that these DEGs are mainly concentrated in the regulation of cell growth, cell migration, cell fate determination, and cell maturation. KEGG pathway analysis showed that these DEGs are mainly involved in focal adhesion, the TNF signaling pathway, cancer-related pathways, and signaling pathways regulating stem cell pluripotency. We identified the 15 most closely related DEGs from the PPI network, and performed R2 database prognostic analysis to select five hub genes – CTGF, EDN1, GATA2, LOX, and SERPINE1. This study distinguished hub genes and related signaling pathways that can potentially serve as diagnostic indicators and therapeutic biomarkers for NB, thereby improving understanding of the molecular mechanisms involved in NB. [ABSTRACT FROM AUTHOR]

The experimental diets containing five levels of creatine supplementation, namely control (N = 0 g/kg creatine), B1 (1.00 g/kg glycine), B2 (0.40 g/kg creatine), B3 (0.80 g/kg creatine) and B4 (1.20 g/kg creatine) were tested to red tilapia up to apparent satiation 3 times a day for 56 days. After the feeding trial, fish were stressed by air exposure for challenge test against stress. The results described that creatine supplementation improved final body weight, feed conversion ratio (FCR) and protein retention. Creatine supplementation decreased blood glucose, cholesterol and triglyceride levels (p <.05), indicating more effective usage of non‐protein energy. Furthermore, creatine supplementation also increased plasma insulin levels and upregulated IGF‐1 and GLUT4 gene expressions in the liver significantly; it also improved robustness against stress, which was shown by higher superoxide dismutase, higher postchallenge survival and plasma cortisol, malondialdehyde and lower glucose levels. In conclusion, creatine supplementation at a dose of 0.289–0.389 g/kg diet escalated fish growth and improved robustness against stress via improving the SOD activity in red tilapia. [ABSTRACT FROM AUTHOR]

Fusarium fujikuroi is the pathogen of rice bakanae disease and has been intensively studied for gibberellin (GA) production. F. fujikuroi is phylogenetically subclassified into G- and F-groups, which differ in GA and fumonisin production. A higher amount of GA is produced by the G-group than the F-group. A previous study found that the GA production difference between the G-group strain Gfc0801001 and the F-group strain Gfc0825009 was due to the GA gene cluster. In this study, higher expression of P450–1 and P450–4 in the GA gene cluster were detected in G-group strains than in F-group strains. The nucleotide sequence of the bidirectional promoter region between P450–1 and P450–4 and expression of P450–1 and P450–4 were compared between G- and F-group strains. The promoter sequence was identical among G-group strains while nucleotide substitutions were detected at various locations in F-group strains. Most F-group strains did not share the same substitutions. These results combined with the ancestral position of the F-group in the phylogenetic tree suggest that the population carrying highly expressed sequence generates the G-group rather than the low expression of P450–1 and P450–4 in the F-group due to a specific mutation occured in G-group. [ABSTRACT FROM AUTHOR]