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Anthocyanins and proanthocyanins (PAs) are two end products of the flavonoid biosynthesis pathway. They are believed to be synthesized in the endoplasmic reticulum and then sequestered into the vacuole. In Arabidopsis thaliana, TRANSPARENT TESTA 19 (TT19) is necessary for both anthocyanin and PA accumulation. Here, we found that MtGSTF7, a homolog of AtTT19, is essential for anthocyanin accumulation but not required for PA accumulation in Medicago truncatula. MtGSTF7 was induced by the anthocyanin regulator LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1), and its tissue expression pattern correlated with anthocyanin deposition in M. truncatula. Tnt1-insertional mutants of MtGSTF7 lost anthocyanin accumulation in vegetative organs, and introducing a genomic fragment of MtGSTF7 could complement the mutant phenotypes. Additionally, the accumulation of anthocyanins induced by LAP1 was significantly reduced in mtgstf7 mutants. Yeast-one-hybridization and dual-luciferase reporter assays revealed that LAP1 could bind to the MtGSTF7 promoter to activate its expression. Ectopic expression of MtGSTF7 in tt19 mutants could rescue their anthocyanin deficiency, but not their PA defect. Furthermore, PA accumulation was not affected in the mtgstf7 mutants. Taken together, our results show that the mechanism of anthocyanin and PA accumulation in M. truncatula is different from that in A. thaliana, and provide a new target gene for engineering anthocyanins in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: DNBSEQ-T7 is a new whole-genome sequencer developed by Complete Genomics and MGI using DNA nanoball and combinatorial probe anchor synthesis technologies to generate short reads at a very large scale-up to 60 human genomes per day. However, it has not been objectively and systematically compared against Illumina short-read sequencers., Findings: By using the same KOREF sample, the Korean Reference Genome, we have compared 7 sequencing platforms including BGISEQ-500, DNBSEQ-T7, HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, and NovaSeq6000. We measured sequencing quality by comparing sequencing statistics (base quality, duplication rate, and random error rate), mapping statistics (mapping rate, depth distribution, and percent GC coverage), and variant statistics (transition/transversion ratio, dbSNP annotation rate, and concordance rate with single-nucleotide polymorphism [SNP] genotyping chip) across the 7 sequencing platforms. We found that MGI platforms showed a higher concordance rate for SNP genotyping than HiSeq2000 and HiSeq4000. The similarity matrix of variant calls confirmed that the 2 MGI platforms have the most similar characteristics to the HiSeq2500 platform., Conclusions: Overall, MGI and Illumina sequencing platforms showed comparable levels of sequencing quality, uniformity of coverage, percent GC coverage, and variant accuracy; thus we conclude that the MGI platforms can be used for a wide range of genomics research fields at a lower cost than the Illumina platforms., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes., (© 2015 American Society of Plant Biologists. All rights reserved.)

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Delayed implantation, considered a state of suspended animation, is widespread in mammals. Blastocysts under this condition remain dormant for an extended period but resume implantation competence upon favorable conditions. The underlying mechanism by which extended longevity of dormant blastocysts is maintained is not clearly understood. Using autophagy markers and the well-defined delayed implantation model in mice, we show that autophagy is important for the extended longevity of dormant blastocysts in utero during delayed implantation. However, prolonged dormancy leads to reduced developmental competency of blastocysts and cellular damage with compromised pregnancy outcome. Estrogen supplementation, which activates implantation of dormant blastocysts, induces the formation of multivesicular bodies in the trophectoderm in vivo. Collectively, our results suggest that autophagy is a critical cellular mechanism that is utilized for the prolonged survival of dormant blastocysts.

The patterning of initiating organs along specific axes of polarity is critical for the proper development of all higher organisms. Plant lateral organs, such as leaves, are derived from the shoot apical meristems located at the growing tips. After initiation, the leaf primordia of species such as Arabidopsis thaliana differentiate into a polarized structure consisting of a proximal petiole and a distal blade, but the molecular mechanisms that control proximal-distal pattern formation are poorly understood. The transcriptional activators BLADE-ON-PETIOLE1 (BOP1) and BOP2 are known to control Arabidopsis lateral organ differentiation by regulating gene expression along the adaxial-abaxial (dorsal-ventral) and proximal-distal polarity axes. Here, we demonstrate that the development of ectopic blade tissue along bop1 bop2 leaf petioles is strongly suppressed in a dosage-dependant manner by mutations in either of two closely related YABBY (YAB) genes, FILAMENTOUS FLOWER (FIL) and YAB3. Three KNOTTED-LIKE HOMEOBOX (KNOX1) genes also make lesser, and partially redundant, contributions to ectopic blade development in bop1 bop2 leaves. Mutation of these YAB and KNOX1 genes together causes nearly complete suppression of bop1 bop2 ectopic organ outgrowth at the morphological and cellular levels. Our data demonstrate that BOP1 and BOP2 regulate leaf patterning by controlling YAB and KNOX1 gene activity in the developing petiole.

Continuous organ formation is a hallmark of plant development that requires organ-specific gene activity to establish determinacy and axial patterning, yet the molecular mechanisms that coordinate these events remain poorly understood. Here, we show that the organ-specific BTB-POZ domain proteins BLADE-ON-PETIOLE1 (BOP1) and BOP2 function as transcriptional activators during Arabidopsis thaliana leaf formation. We identify as a direct target of BOP1 induction the ASYMMETRIC LEAVES2 (AS2) gene, which promotes leaf cell fate specification and adaxial polarity. We find that BOP1 associates with the AS2 promoter and that BOP1 and BOP2 are required for AS2 activation specifically in the proximal, adaxial region of the leaf, demonstrating a role for the BOP proteins as proximal-distal as well as adaxial-abaxial patterning determinants. Furthermore, repression of BOP1 and BOP2 expression by the indeterminacy-promoting KNOX gene SHOOTMERISTEMLESS is critical to establish a functional embryonic shoot apical meristem. Our data indicate that direct activation of AS2 transcription by BOP1 and BOP2 is vital for generating the conditions for KNOX repression at the leaf base and may represent a conserved mechanism for coordinating leaf morphogenesis with patterning along the adaxial-abaxial and the proximal-distal axes.

Context: Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by chronic oligo/anovulation, hyperandrogenemia, infertility, and metabolic alterations related to insulin resistance. These abnormalities in PCOS may have complex effects on pathophysiology of the endometrium, contributing to infertility and endometrial disorders., Objective: The objective of this study was to examine dysregulated signaling pathways in the endometrium of patients with PCOS (PCOSE) by analyzing expression profiles with a pathway-oriented method., Design: Microarrays, RT-PCR, laser capture microdissection, and immunohistochemistry were performed with endometrial tissues., Setting: This study was performed at a university hospital laboratory., Patients: This study comprised 12 regularly cycling women and 12 PCOS patients., Main Outcome Measure: Dysregulated signaling pathways in PCOSE were identified as a gene set., Results: Hierarchical clustering revealed distinct expression profiles for PCOSE and the endometrium of normal cycling women. Gene sets associated with androgen signaling were not enriched in PCOSE, although they affect ovarian physiology of PCOS patients. Several biological pathways including cell cycle, apoptosis, glycolysis, and integrin-Rho-cytoskeleton network were aberrantly down-regulated in PCOSE. Expression of genes constituting these gene sets enriched in normal cycling women was systemically down-regulated in PCOSE. Laser capture microdissection-coupled real-time RT-PCR and immunohistochemistry further demonstrated that cell proliferation in the stroma, but not the epithelium, is significantly reduced in PCOSE., Conclusions: Systemic down-regulation of various signaling pathways in PCOSE with extremely prolonged proliferative phase provides insight into the abnormal phenotypes that reflect pathophysiology of PCOS in the endometrium, possibly leading to increased risks of endometrial disorders.

The structure of 2',3'-didehydro-2',3'-dideoxynucleosides (d4Ns) was applied to design the novel bioisosteric 4'-seleno-d4Ns as potential inhibitors of human immunodeficiency virus reverse transcriptase (HIV RT). Conversion of 2',3'-dihydroxyl groups of 4'-selenoribofuranosyl pyrimidines into the olefin was accomplished by treatment of cyclic 2',3'-thiocarbonate with 1,3-dimethyl-2-phenyl-1,3,2-diazaphospholidine.

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.

To investigate the role of nuclear encoded genes in mitochondrial function during oocyte maturation and early embryogenesis we examined the expression pattern and function of the cytochrome oxidase (Cox) subunits, Cox5a, 5b, and 6b1 during oocyte maturation and early embryo development. Transcription of Cox5a, 5b, or 6b1 was observed in oocytes and during early development; their expression levels were abundant in mature oocytes (MII) and zygotes (1C), and lowest at the 2-cell stage (2C), gradually increasing from 4-cell to blastocyst stage. Immunocytochemical studies revealed that COX5A, 5B, or 6B1 proteins were expressed in all blastomeres of the blastocyst. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages, but disrupted mitochondrial distribution. Significantly higher apoptosis and lower cell numbers were observed in siRNA-treated blastocysts. Real time RT-PCR revealed that silencing of Cox5a, 5b, or 6b1 did not alter mRNA levels of Bcl-xL (Bcl2l1), but increased transcription levels of proapoptotic genes, Bax and caspase 3 (Casp3). Furthermore, mRNA and protein levels of E-cadherin (CDH1) were decreased in siRNA microinjected blastocysts. These results suggest that gene expression of the Cox subunits, Cox5a, 5b, and 6b1 is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst stage embryos.

BioSilico is a web-based database system that facilitates the search and analysis of metabolic pathways. Heterogeneous metabolic databases including LIGAND, ENZYME, EcoCyc and MetaCyc are integrated in a systematic way, thereby allowing users to efficiently retrieve the relevant information on enzymes, biochemical compounds and reactions. In addition, it provides well-designed view pages for more detailed summary information. BioSilico is developed as an extensible system with a robust systematic architecture.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.

Background: It has been reported that p38 and c-jun operate as mediators of cell proliferation and differentiation. Therefore, by studying the roles of c-jun and p38 in the proliferation and differentiation of normal human endometrial cells, we can better understand the mechanism of these processes in endometrial cells., Methods: Separation of glandular and stromal components was based on a modification of the work of Satyaswaroop et al. To confirm the purification of the endometrial cells and the expression of the transfected SV40 large T antigen, immunocytochemical analysis and western blot analysis were performed., Results: There were polygonal shapes in the stromal cells in the early passage 1-2, while the aged endometrial stromal cells were spindle shaped. To investigate passage-dependent molecular events in endometrial cells, the c-jun and pp38 levels were examined. Both c-jun and pp38 were significantly reduced with cellular aging and passages. To understand the role of c-jun, endometrial stromal cells were treated with SP600125 which is a specific inhibitor of c-jun. SP600125 induced morphological changes of young endometrial stromal cells with polygonal shape; the young cells appeared as aged endometrial cells with spindle shape. In addition, an immortalized endometrial cell line was established and shown to express activated c-jun, similiar to normal endometrial cells., Conclusions: These results suggest that the modulation of p38 and c-jun may play an important role in the differentiation and proliferation of human endometrial cells.

A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library. In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning). The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery. Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection. The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning. In the mutant, the stem and root were thinner than those in wild type. This phenotype was associated with reduced vascular development. The promoter activity of the VEP1 gene was detected preferentially in the vascular regions. We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.

The purpose of this study was to evaluate whether hydrosalpingeal fluid (HSF) is toxic to the mouse embryo as assessed by the blastocyst development rate (BDR) and by cell counting in vitro. HSF was collected from nine patients undergoing salpingoneostomy to correct hydrosalpinx. Two-cell embryos were obtained from superovulated ICR mice. T6 medium and T6 + 0.4% bovine serum albumin (BSA) were used as control media. T6 medium containing 10% or 50% HSF and 100% HSF from each patient were used as test media. Nine to 15 embryos were cultured in microdrops prepared from each of these media. The BDR was examined after 72 h of culture in these media. To assess the total cell number within each blastocyst, the blastocysts were fixed and stained with Hoechst 33342 to facilitate cell counting. The BDR was affected adversely only by 100% HSF and not in media containing 10% or 50% HSF. The mean BDR using T6 medium and T6 + BSA were 88.7% and 85.3%, respectively. The mean BDR using media containing 10% HSF or 50% HSF were 90.0% and 89.4%, respectively. Mean BDR using 100% HSF was 75.2% (P < 0.05). The overall mean cell counts (+/- SEM) using T6 medium and T6 + BSA were 86.9+/-3.2 and 91.0+/-3.8 respectively. Mean cells counts were decreased significantly only in blastocysts cultured in 100% HSF (63.3+/-4.6; P < 0.01) but not in blastocysts cultured in 10% or 50% HSF (90.8+/-4.2 and 81.9+/-6.1 respectively). Thus, it is concluded that HSF has no embryotoxic effect but has a mildly negative effect on embryonic growth and development.